INTRODUCTION

Proximal femur fractures in the paediatric patients are very rare accounting for less than 1% of all paediatric fractures. Ninety percent of the fractures were due to significant trauma while ten percent were the results of low energy mechanism (Dial & Lark 2018). Low energy mechanism resulting in this type of fracture were usually pathological fracture (De Mattos et al. 2012). We present a case of proximal femur fracture in a paediatric patient after a trivial trauma which initially presented as referred pain in the knee. We found out that the patient had underlying undiagnosed bone cyst on the fracture site.  

CASE Report

A 6-year-old girl presented to the Emergency Department (ED) with the complaint of left knee pain, following a fall while playing with her older brother who was aged 12 years. They were playing horse riding where the patient was on the brother’s back in crawling position which was about 2 feet high. She lost her balance and fell on her left side, landed on the buttock while her brother was pulling her left leg. She complained of left knee pain especially over the supra-patella region which worsened with weight bearing. She also refused to walk because of the pain. 

The patient was born full term via spontaneous vaginal delivery (SVD) with a birth weight of 3.3 kg. There was no antenatal, intrapartum or postpartum complication. There was no prior history of admission. She had no past medical history and no history of fracture or bone disease before. Her developmental was according to the age with no significant delay. She was the third out of four siblings. There was no history of malignancy or bone disease in the family.

Knee radiograph did not show any evidence of fracture, subluxation, or dislocation (Figure 1). She was allowed home with analgesia and returned or reassessment after  3 days. 

At the time of reassessment, the patient was unable to bear weight, the left hip was held in flexion, abduction and external rotation position, while the left knee was in full flexion. She refused to move the knee and hip because of pain. On examination, the left knee did not have any swelling or tenderness. However, her left hip examination revealed decreased left range of motion and reproduction of pain on the patient’s left knee. Otherwise, the patient had no fever, the vital sign was stable, and blood investigations were normal. 

Radiographs of the pelvis and left hip were ordered based on the history and physical examination finding. The radiographs showed a comminuted intertrochanteric fracture of left femur with an underlying bone cyst (Figure 2). The patient was referred to the orthopedic team for further treatment and placed in a Thomas splint (Figure  3). The orthopedic team decided to treat with a hip-spica fiberglass cast of the left lower limb under general anesthesia. 

The fracture healed well (Figure 4) and after 3 months, she was able to walk without aids.

DISCUSSION

Proximal femur fracture in children and adolescents are of significance as the proximal femur fracture is prone to undergo osteonecrosis due to tenuous and changing blood supply to the femoral epiphysis. The prevalence of osteonecrosis after proximal femur fracture especially femoral neck fracture is very high at a rate of 29% out of 70 patients (Spence et al. 2016). Other than that, proximal femur fracture in paediatric population also can lead to coxa vara (30%), premature physeal closure (5% to 65%), non-union (1.6% to 10%), chondrolysis (uncommon) and infection (<1%) (Boardman et al. 2009). 

Proximal femur fracture in a paediatric patient are easily missed because of failure to perform radiological investigation at the initial presentation. A study done by Guly showed that the prevalence of the missed hip fracture were due to failure to radiograph was 6.8%; with the highest was 12% (wrist fracture) while the lowest was 2.5% (skull fracture). Guly showed that most common reason for failure to radiograph were; i) other injuries elsewhere; ii) underestimation of injury; iii) poor localization of injury (Guly 2001). The patient had presented with left knee pain which was the referred pain from the obturator nerve. Articular branches of the obturator nerve supply the hip and knee joints and hence pain produced in one joint can manifest as referred pain (Jacob 2008). There has been a case report of a 33-year-old male presenting with left knee pain following a grand mal seizure which turned out to be an impacted comminuted fracture of the left proximal femur (Sandoval 2011)

In a case of proximal femur fracture in the paediatric population as a result of a trivial trauma, pathological fracture should be highly suspected. Common causes of pathological hip fracture in children are osteomyelitis, simple and aneurysmal bone cyst, fibrous dysplasia, Langerhans cell histiocytosis, osteogenesis imperfecta, disuse osteopenia, metabolic bone disease, and malignancy (Biermann 2002; Boardman et al. 2009). In this case, we discovered that the patient had an underlying bone cyst based on the radiograph. Although the common location of solitary bone cyst is the proximal humerus, up to 21% are detected in the proximal femur. The prevalence is much lower in the iliac bone (7%), and ischial and pubic bones combined (2%) (Bloem & Reidsma 2012).

Reduction of hip fracture is best performed ideally within 24 hours after the injury to reduce the risk of osteonecrosis to the proximal femur by restoring blood flow through kinked but intact vasculature (Boardman et al. 2009). However, with regard to the development of osteonecrosis, the timing of reduction may be a less important factor compared to the fracture type and age at the time of injury (Moon & Mehlman 2006). According to the Delbet classification, type I, II, and III were 15, 6 and 4 times more likely to result in osteonecrosis compared to type IV fractures. Meanwhile, for the age factor, it is 1.14 time more likely to develop osteonecrosis in older children for each year of increasing age (Boardman et al. 2009).  

CONCLUSION

Proximal femur fractures in paediatric population are rare injuries. In comparison to adult proximal femur fractures, these injuries are associated with serious complication especially osteonecrosis. We recommend a thorough physical examination to avoid missing this fracture type for patients presenting with unspecific pain from a trivial trauma.

INTRODUCTION

Sacral injury is a rare injury encountered in trauma (Beckmann & Chinapuvvula 2017). A systematic method of approaching a trauma patient in Advanced Trauma Life Support (ATLS) protocol was taught to emergency doctors so that any life-threatening injury is not missed out and may be promptly attended if any such was found. Sacral fracture, although not life-threatening, could have serious complications which are neurological injury (Beckmann & Chinapuvvula 2017) and urological injury with concurrent pelvic fracture (Alfayez et al. 2016). However, due to its rarity, it is frequently overlooked and hence, not properly treated (Rodrigues-Pinto et al. 2017). Due to their rarity, they are frequently underdiagnosed and mistreated. Only 5% of sacral fractures occur in isolation. Injuries most often associated with sacral fractures include neurologic injuries (present in up to 50% of sacral fractures). There are less reports on the diagnosis and management (Rodrigues-Pinto et al. 2017). Only 5% of sacral fractures occur in isolation. Injuries most often associated with sacral fractures include neurologic injuries (present in up to 50% of sacral fractures, and treatment is mostly determined on a case-by-case basis. To be able to identify this injury clinically, it remains a challenge to emergency physicians, and they need to have a high index of suspicion from other injuries that are identified during the trauma survey so that the diagnosis is not missed out. Any trauma patient complaining of sacrococcygeal pain should be further investigated for sacral fracture (Yi & Hak 2012). Here, we present a case of sacral fracture where the only clue to the diagnosis was genitalia pain. 

CASE REPORT

A 43-year-old lady was involved in motor vehicle accident on the day of presentation. She was wearing a helmet and a pillion rider with a riding speed of 70 km/h during the incident. They were hit from their side by a car, but she could not remember the exact mechanism of fall. She denied any event of loss of consciousness.

Upon arrival to the Emergency Department, her vitals were stable with blood pressure of 117/56 mmHg, heart rate of 90 bpm, temperature 37˚C, respiratory rate 18 breaths/ minute and oxygen saturation was 98% under room air. She was able to speak and her airway was intact. She was not tachypnoeic and there was no paradoxical breathing. Apex beat was not displaced and trachea was centrally located. Chest spring was negative and breath sound were equal bilaterally. Her peripheries were warm. Pulse volume and perfusion was good. Her abdomen was soft and not distended, and pelvic spring was negative. There was no long bone deformity seen. Her GCS was full and pupils were equal and reactive to light bilaterally. She complained of pain over her genital area. However, inspection of the genital area and vaginal examination revealed no bruises or any external injuries. 

Upon log roll, a big abrasion wound was seen over the left side of gluteal region and left shoulder. There was tenderness over L5 area. Per rectal examination revealed tenderness upon inserting the finger but otherwise no bruise or external wound was seen. No bleeding seen from anal or genitalia area. Bulbocavernosus reflex and other lower limb reflexes were intact. Extended focused assessment sonography for trauma (EFAST) was negative. Her sensation over bilateral lower limbs were intact. However, the power of left lower limb was reduced due to pain at her lower back.

Subsequently, she was sent for radio-imaging. Her plain radiograph revealed a closed fracture of left inferior pubic rami and transverse fracture of sacrum. She was referred to orthopaedic team for further management. In view of multiple fractures at pelvic area, she was then subjected for a CT pelvic which revealed multiple fractures of the left-sided pelvis as below and these include comminuted fracture of the left inferior pubic ramus, fracture of left superior pubic ramus, comminuted fracture of left ilium, comminuted fracture of the S5 sacrum with sacrococcygeal displacement.

Her urinary bladder was catheterized and clear urine was seen. However, microscopically, there was presence of red blood cells. With regard to the blood results her haemoglobin level was 12.5 d/dL, white cell count was 16.3 X 109/L, platelet was 104 X 109/L. Her sodium level was 138 mmol/L, potassium 3.9 mmol/L, urea 3.2 mmol/L and creatinine 63.3µmol/L.  She was admitted to the Orthopaedic Ward and was started on regular painkiller for the pain control. Her injury was treated conservatively. Throughout the admission, there was no worsening in her injury and the neurology status. She was subsequently discharged on day 7 of admission. She was seen back in the clinic six weeks after discharged able to ambulate with walking stick.

DISCUSSION

Sacrum and coccyx form the lower part of the vertebral column. Sacral fracture is relatively rare and traumatic sacral fracture only occur at a reported rate of 2 per 100,000 (Beckmann & Chinapuvvula 2017). It is usually associated with pelvic fracture in trauma cases. The mechanism of injury might be able to give some clue for the attending physician to suspect the possibility of sacral fracture. However, it is still frequently missed in the primary survey due to the rarity of occurrence (Rodrigues-Pinto et al. 2017). In this patient, the index of suspicion of the possibility of sacral fracture was low because patient could not remember the exact mechanism of injury. The only clue to trigger a healthcare provider to order for a pelvic radiograph was the pain over the genitalia and L5 area, in which the genitalia and per rectal examination were unremarkable. 

Diagnosing sacral fracture clinically is not too easy, and it is usually associated with urinary tract injury. Our patient particularly complained of pain at genitalia area. Hence, a careful and thorough examination at the pelvic area may alarm the attending physician to the possibility of a sacral fracture. A careful digital rectal examination, along with vaginal examination should be conducted in any patient complaining of pain in the genitalia area. There is also a possibility of urinary bladder injury in patients with sacral injury. Gross hematuria is a hallmark sign of bladder injury in the presence of pelvic fracture in 16-27% of patients (Alfayez et al 2016). However, microscopic hematuria in the absence of overt signs of lower urinary tract injury is probably not indicative of bladder rupture (Figler et al. 2012), just as in this case, where there was no gross hematuria but microscopic examination of the urine did detect the presence of red blood cells. Cystography was not done after review by urology team. In females with gross hematuria, blood in introitus, and difficulty in placing a urethral catheter, a vaginal speculum examination, proctoscopy, or urethroscopy should be considered (Figler et al. 2012). These are clues that can be obtained from physical examination itself, which will guide the emergency physicians to the possibility of sacral fractures.

Besides clinical examination, radiograph also play a role in diagnosing sacral injury. Nevertheless, only 10-30 % of the fractures were identified on a plain pelvic radiograph (Beckmann & Chinapuvvula 2017). A large percentage of these are missed out, and the reasons could be the gas and bowel which overlies the sacrum, osteoporotic and osteochondrotic changes masking the fracture (Schicho et al. 2016). This leads to the use of computed tomography (CT) in diagnosing of this condition (Beckmann & Chinapuvvula 2017). However, if CT is not immediately available, especially in a rural hospital, the attending physician can order an inlet, outlet, and lateral views of the pelvic. Transverse process fractures of the L5 vertebra and pubic rami fractures are both commonly seen in association with sacral fractures and can be considered indirect evidence of a sacral fracture (Beckmann & Chinapuvvula 2017). The plain radiograph of this patient showed an obvious fracture of left inferior pubic rami and hence, she was subjected to CT scan.

The complication of sacral fracture is not limited to only urinary tract injury, but it also associated with neurological injuries (Rodrigues-Pinto et al. 2017) and this includes loss of ankle reflex, the external anal and bladder sphincter, and parasympathetic innervation of the detrusor muscle of the bladder which are all contributed by sacral plexus (Chiaruttini 1987). Fortunately, there was no neurological deficit in this case. Regular neurological examination is important so that the physician will be able to pick up any signs of neurological injury which was not initially apparent. When neurological deficit is present, these fractures are considered unstable. If the deficit is severe or persistent, patient may require treatment with decompression by sacral laminectomy (Chiaruttini 1987).

CONCLUSION

In conclusion, emergency physician have to have a high index of suspicion for sacral fractures due to its rarity and difficulty in detecting both clinically and radiologically on a plain radiograph which is part of the primary adjunct in trauma survey. Missing a sacral fracture will risk missing a urinary tract and sacral plexus injury, which is usually not immediately apparent. Hence, conducting a proper trauma survey with high index of suspicion may ultimately lead the physician to this important diagnosis that is frequently missed out. 

QUESTION

 A healthy 63-year-old male, presented to the Emergency Department with a complaint of left lumbar colicky pain for 4 days. It was associated with no bowel output for 3 days and abdominal distension for 2 days. He otherwise denied vomiting, fever, and constitutional symptoms. He has never experienced these symptoms before. Clinically, he was pink and not cachexic. He was tachycardic with a heart rate of 105 beats per minute. His abdomen was grossly distended and tender at left lumber region. Left kidney was slightly ballotable but limited due to pain. Biochemically, his renal function was deranged with a creatinine of 157.8 mol/L (eGFR of 41 ml/min/1.73m2). He had a total white cell count of 24 x 103/μL. Microscopic examination of the urine showed 5+ erythrocyte. Spot the radiological diagnosis and provide a brief management.

QUESTION

A 37-year-old lady with no known comorbidity complained of left hypochondrial pain for 6 months duration. It was associated with loss of weight and appetite with occasional carcinoid symptoms such as diarrhoea and sweating. She had no blood transfusion before. Clinically, she has no features of thalassemia and the abdomen was soft with a palpable vague mass on the left hypochondrial region. A computed tomography (CT) of the abdomen revealed a soft tissue mass measuring 8.9 x 10.3 x 9.0 cm in the enlarged spleen. A laparotomy was decided and the intraoperative findings are as in Figure 1 and 2. Spot the diagnosis and provide the outline of management.

Introduction

Charcot arthropathy is a devastating degenerative condition affecting one or more joints that is marked by joint instability and hypermobility resulted from peripheral nerve damage. It is a common complication seen in patients with diabetes mellitus as well as more uncommonly in those with spinal cord disorders.

There are few factors required for the arthropathy to occur and these include peripheral neuropathy, unrecognized injury, continuous stress on injury and an increased local blood flow as described by Rosenbaum et al. 2014. Based on Eichenholtz classification, stage 0 and 1 with clinical findings including swelling, erythema and warmth, the current management is aimed towards protected weight bearing or offloading. This is achieved by crutches or total contact casting. In stage 2, the clinical findings are reduced, and radiologically there is sclerosis, fusion of larger fragments, with resorption of debris. In this stage, total contact casting is still advocated, with CROW (Charcot restraint orthotic walker) a suitable substitute. Stage 3, which is the area of interest in our study, shows consolidation of the deformities. There is minimal inflammation at this stage, making surgery a good option for management. Surgical options in the absence of ulceration and infection, include deformity correction or joint fusion utilizing internal or external fixation depending on the deformity (Rosenbaum et al. 2014).

With increasing incidences of diabetic related foot complications such as neuropathy, ischemic foot ulcers, osteomyelitis especially in long standing diabetics, a treatment algorithm would be beneficial in managing these complex cases. Siebachmeyer et al. 2015 described an algorithm which is widely use in treating Charcot arthropathy as shown in Figure 1 (Siebachmeyer et al. 2015). We describe an algorithm which include a pharmacological treatment for management of Diabetic Charcot arthropathy as shown in Figure 2. There are various options for managing these diabetic foot problems, basically from wound debridement to amputation in more severe cases. Surgery is considered for patients with Charcot arthropathy with residual foot deformities. Depending on the areas of involvement, surgical fusion options include external fixation, which is either multi or uni-planar, internal fixation with nails or plates and screws. A study by Shah & De described that a nail fusion has less complications compared to external fixators (Shah & De 2011). 

However, there are still some controversies in managing complicated cases. Monitoring of surgical outcome is the utmost important for treating surgeons to improve their management plans and assist with decision making. Thus, we decided to conduct this study on treatment algorithm and functional outcome of hindfoot arthrodesis in Charcot Arthropathy in Universiti Kebangsaan Malaysia Medical Centre. 

The objective of the study was to review the feasibility of an algorithm in managing Charcot arthropathy and to assess functional, clinical and radiographic outcome of Charcot joints treated with hindfoot arthrodesis, of at least 6 months follow-up using standardized and internationally accepted scoring systems which are the American Orthopaedic Foot and Ankle Surgery (AOFAS) score, Short Form Survey Instrument (SF36) and radiographic assessment of foot and ankle.

Materials and Methods

The study was approved by Research Ethics Committee from The National University of Malaysia (UKM PPI/111/8/JEP-2016-437). The scoring for AOFAS and SF36 was done retrospectively, and the radiographs of the patients reviewed on follow up. Patients whose follow-up were not convenient, were contacted by telephone.

This was a retrospective study done in patients who were diagnosed with Charcot arthropathy and underwent surgery for hindfoot arthrodesis at the Universiti Kebangsaan Malaysia Medical Centre, from January 2011 until June 2016. Hindfoot arthrodesis surgery is the surgical fusion of the ankle and subtalar joints.

Patients with Charcot Arthropathy (according to Eichenholtz classification) treated with arthrodesis at least 6 months follow-up aged 18 and above were included in this study. Patients with medical conditions or trauma, which caused debilitating symptoms such as non-related fractures and cerebral vascular accidents were not included in this study. Patient defaulted from follow-up or unable to contact during study and aged below 18 years, were also not included in this study.

Functional status and health status were assessed using American Orthopaedic Foot and Ankle Society score, Short-Form 36 & radiographic evaluation. The Short Form (36) Health Survey is a survey of patient's health. The SF-36 consists of eight scaled scores, which are the weighted sums of the questions in their section. Each scale is directly transformed into a 0-100 scale on the assumption that each question carries equal weight. The validity and reliability have been widely studied and reported. It is also used in various studies done worldwide. The eight sections are vitality, physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, social role functioning and mental health.

The AOFAS has 4 modules of assessment which are 1st MPJ & ray, forefoot, rear and midfoot (used in study) and ankle. These areas are then divided in to a subjective and objective scoring system. The subjective aspect assessed are pain, appearance and function, while objective assessment included radiological and functional tests.

Radiographic evaluation aims to review the union of the arthrodesis as well as any evidence of infection including implant related. The evaluation was performed by a single orthopaedic ankle and foot consultant. 

For the ethical issues in this study, patients and family members are informed that their records and images would be reviewed for research purposes and they are granted the opportunity to forbid such use of their data.

Results

Upon reviewing the operative registry at Universiti Kebangsaan Malaysia Medical Centre, a total of 64 hind foot fusions were done between January 2011 until June 2016. The treatment algorithm that we used in this study was illustrated in Figure 2. From this total, upon reviewing case and operative notes, a total of 24 cases were diagnosed cases of Charcot arthropathy. A total of 6 patients were not contactable, 1 declined and 1 other patient passed away due to complications arising out of diabetes. The remaining 16 were included in the study. 

Data was analysed using commercially available software SPSS version 22.0 for Windows at the significant level p<0.05 (Table 1).

The number of male patients were 4 (25%) compared to 12 (75%) female patients (Table 2). The mean age was 58.1 years (20-71). The side of fusion was equal for both right and left (8/side). All patients reviewed were in Eichenholtz stage III during the timing of surgery. A total of 13 of the patients were diabetics, 2 had spina bifida and one patient had end stage renal failure. 

An 81% union rate was achieved in our patients, but 3 patients had non-union. Out of the 3 patients with non-union, 2 were attributed to deep infections (Table 3). There were also 2 cases of deep infection requiring removal of screw, as well as one case each of aseptic loosening of screws, superficial surgical site infection, and deep surgical infection. However, union was achieved in these patients. As the AOFAS and SF36 results were normally distributed, the paired T-test was used. The AOFAS (hindfoot) mean was 24.56 for pre- and 53.56 for post-operatively (Figure 3). The AOFAS (midfoot) showed similar results with 24.13 for pre- and 52.50 for post-operatively (Figure 4). The SF36 (mental) reported a significant improvement with p-value <0.05 in the mean scores 40.46 pre- and 49.91 post-operatively (Figure 5), while the SF36 (physical) reported no significant changes in pre-and post-operative period (35.11 & 38.04) (Figure 6).

Discussion

Charcot Neuroarthropathy (CN) has been attributed to an insensate limb. There are two postulated theories, which are neurovascular insufficiency and neurotraumatic. The former implies that the loss of autonomic supply results in an increased blood supply to the region, therefore increasing bone resorption. The latter suggests that a repetitive insult to the insensate limb causes microfracture. There is joint, soft tissue and bone inflammation with loss of architecture, which inevitably leads to instability, loss of joint congruency and bony destruction (Rajbhandari et al. 2002; Rogers et al. 2011; Kaynak et al. 2013). The diagnosis of this disease requires a high index of suspicion and a being able to identify neurological, vascular, and radiographic pathologies. Differentials to be considered are gout, deep vein thrombosis, rheumatoid arthritis and infections (Guven et al. 2013; Game et al. 2011). 

The management of CN should be divided into acute and chronic. Acute management in the early stages should focus on reduce the destructive properties of the disease. This is achieved by a non-weight bearing protocol or offloading with a total contact cast. The role of bisphosphonates is controversial with no long-term studies available yet to justify the use. The management of chronic CN aims to get a stable, plantigrade, painless, shoe able foot with avoidance of ulcer formation (Rajbhandari et al 2002; Rogers et al. 2011; Guven et al. 2013; Mittlmeier et al. 2009). Restoration of quality of life is important, as patients with CN are found to have a reduced level of physical activity when compared to more severe ailments (Mittlmeier et al. 2009). A study has shown that hyperbaric oxygen therapy can act as an adjunctive therapy to improve wound healing and at the same time helps to achieve a better quality of life especially in chronic diabetic foot ulcer (Bajuri et al. 2017).

Orthosis and surgical intervention are indicated, with arthrodesis with internal, external or combined fixations, exostectomies and reconstruction all options to achieve good outcomes. There is a report noted that complex reconstruction needs a bone graft as part of the fixation in order to maintain the structure of the reconstructed part. This helps the ossification to occur and later consolidation will take place (Muhammad Hafiz et al. 2018). Union rates for ankle and hindfoot arthrodesis are between 80-99% (Siebachmeyer et al. 2015; Zwipp et al. 2009) and this is despite the risk factors of vascular insufficiency and reduced immune status of patients. Limb amputation is reserved as the last resort (Guven et al. 2013). 

The usage of intermedullary fixed angle device with bone graft provides a safe and rigid fixation for patient with CN. While this holds true for patient with relatively good bone stock, those with talus insufficiency, a talectomy along with tibiocalcaneum fusion will be a good option (Ahmad & Raikin 2008). We did not encounter this in our series of cases.  The type of deformity dictates the correction required as well as site of correction. Malerba et al. (2015) report of no significant difference in both isolated tibiotalar and combined tibiotalar subtalar fusion. While isolated tibiotalar fusion does allow for some motion at the subtalar joint, there is associated morbidity of degenerative changes in this area (Malerba et al. 2015). Severe ankle and hindfoot conditions that cannot be adequately managed by other measures, can be treated using hindfoot fusion which act as a salvage option that offers a safe and reliable treatment (Rammelt et al. 2013).

We proceeded for surgery when our patients were in the 3rd stage of CN as the inflammatory markers now were normal. Simon et al., however, report a good clinical outcome in patients who underwent arthrodesis in Eichenholtz stage I. There was no infection and all 14 patients achieved union, with mean return to use of regular shoe was 27 weeks (Simon et al. 2000).

Schneekloth et al. 2016 found out that there is no direct comparison between the performance of surgery in the acute phase versus surgery in chronic phase but there is an encouraging result in surgical intervention done during acute phase (Schneekloth et al. 2016).

While we utilized the anteromedial and lateral approaches, Pelligrini et all reported a series of cases utilizing posterior Achilles Tendon-splitting approach with a union rate of 80%. However, the reported complications were significantly higher at 41% (Pellegrini et al. 2016). 

Based on a report, Ilizarov external fixator (IEF) can be used as an alternative treatment in treating the subtalar dislocation with unstable foot. This will allow a progressive soft tissue correction and at the same time provides the stability for arthrodesis (Bajuri et al. 2013). Fragomen et al. reported an 73% union rate in their review of using IEF for complex ankle fusions with CN as compared to 84%, overall. Non-smokers and non-CN patients had a 93% fusion rate (Fragomen et al. 2012). While IEF offers the advantage of earlier weight bearing, the authors delayed weight bearing in the CN group, worrying about failure of the construct. When comping the hindfoot nail to a uniplanar fixation though, the complication rate was significantly higher in the uniplanar method as well as a reduced union rate in which 100% by using nail & 66.7% with external fixation (Shah & De 2011). Complications associated with the uniplanar external fixation include pin tract loosening, pin site infection, and wound breakdown. 

Complications with arthrodesis with regards to infection vary from superficial surgical site infection (5- 49%) to deep seated osteomyelitis (up to 9%) (Zwipp et al. 2009; Pellegrini et al. 2016; Fragomen et al. 2012; Eschler et al. 2015). The presence of the hindfoot nail also invites the risk of stress fractures to the lower limb. Mansor et al. hypothesized that the crack path relies on the bone matrix microstructure in which the fracture toughness will increase with mode mixity (Mansor et al. 2015). Lidor et al. recognized thirteen stress fractures in one hundred and five ankle arthrodesis (12.8%). While the fractures can be treated, it still represents a short-term morbidity to the patient (Lidor et al. 1997).

One of the limitations pf the present study was the small sample size. Hence, studies with bigger sample size are advised in future.

Conclusion

The treatment algorithm currently used in our centre is acceptable and shows good outcomes. While the outcomes are of acceptable rates, efforts should now focus on reducing the complications as the burden of infection and non-union remains a known complication for most bone-related surgery.

AcknowledgEment

The authors would like to thank Professor Dr. Srijit Das and Dr. Kamarul Syariza Zamri for their indirect contribution and technical help in publishing this manuscript.

INTRODUCTION

Free radicals and oxidative stress are identified as the prime factors in causing cellular damage and biological ageing, leading to the shortening of lifespan (Yan et al. 2017; Harman 1956). The free radicals are capable of disrupting the cell membrane structure and cause damage to almost all cellular constituents like proteins, lipids and nucleic acids; interfering with the normal functions of the cell. To prevent these damages, there are endogenous and exogenous antioxidants defence system that can eliminate the free radicals. Examples of endogenous antioxidants include the enzymes catalase and glutathione peroxidases which reduce hydrogen peroxide (Khor et al. 2017), while the exogenous ones include vitamins C (Aumailley et al. 2016) and vitamin E (Zadeh-ardabili et al. 2017). 

The study of ageing and lifespan extension has been replicated in many different models: yeasts (Kwon et al. 2017), nematodes (Zhao et al. 2017), flies (Cao et al. 2017), rodents (Ke et al. 2017) and to some limited extent, humans (Qin et al. 2017). Baker’s yeast Saccharomyces cerevisiae is a unicellular, eukaryotic organism that can reproduce rapidly, (Toret & Drubin 2007; Scarfone et al. 2015) the bud neck easily manipulated and cultured in controlled environment (Williams et al. 2016). It has a shorter lifespan of a few days (Kwon et al. 2017) in comparison with other model organisms, enabling a shorter duration of research. More importantly, the genes in S. cerevisiae are highly homologous to those in animals and human (Liu et al. 2017; Karathia et al. 2011; Inoue et al. 1999). 

    S. cerevisiae lacking in antioxidants have many detrimental effects, among them is shorter lifespan  (de Sá et al. 2013; Demir & Koc 2010). Previous studies have demonstrated a few means to prolong the lifespan of S. cerevisiae. Among these are calorie restriction, introduce changes in mitochondrial metabolism (Barros et al. 2004), and regulating proteins involved in the ageing mechanism such as TOR (Powers et al. 2006; Pan et al. 2011) and sirtuin signaling pathways (Howitz et al. 2003; Anderson et al. 2003)Extension of lifespan via intervention of antioxidants and other compounds have also been proven (Nakaya et al. 2012; de Sá et al. 2013). However, the exact mechanism of their effect has not been elucidated yet. 

Vitamin E has eight isomers consisting of αα-, β-, γ-, δ-tocopherols (Matthäus & Musazcan Özcan 2015; Fu et al. 2017) and α-, β-, γ-, δ-tocotrienols (Lim et al. 2014; Black et al. 2000). Palm oil tocotrienol-rich fraction (TRF) contains major composition of α, β, γ, õ-tocotrienols and minor composition of α-tocopherols  (Lim et al. 2013; Makpol et al. 2011). The most prominent benefit of TRF as anti-oxidant is the effect in slowing down ageing process. Based on a human study (Chin et al. 2011),  a continuous supplementation of TRF for 6 months showed a reduction of protein carbonyl, an oxidative stress indicator, in a target group of more than 50 years. When oxidative stress indicator is reduced, an organism will face less oxidative stress and would have a longer lifespan (Aumailley et al. 2016). At the cellular level, human diploid fibroblast treated with TRF showed a reduction in senescence characteristics, which could be attributed to  the anti-oxidant effect of TRF (Makpol et al. 2013).

Ageing process of yeast can be observed in two aspects: chronological ageing and replicative lifespan. Replicative lifespan is defined as the total number of daughter cells generated by a mother cell (Sarnoski et al. 2017). Chronological ageing refers to the lifespan of S. cerevisiae which is measured by monitoring the mean and maximum survival times of populations of post-mitotic yeast cells that have stop replicating and continue to live the chronological lifespan (Kwon et al. 2017). It is established that there is a link between stress resistance and longevity with evidence for the conservation of the pathways that regulate lifespan in phylogenetically distant eukaryotes (Fabrizio & Longo 2003; Mirisola et al. 2014).

In this study, the effect of TRF on the viability of three S. cerevisiae strains in the initial stationary phase was determined. The three strains were the wild-type, CTT1Δ and GPx2Δ. The CTT1Δ∆strain lacked the gene encoding cytoplasmic catalase (Martins & English 2014); while the  GPx2Δ∆ strain lacked the gene encoding mitochondrial glutathione peroxidase (Tanaka et al. 2005).

MATERIALS AND METHODS

Yeast strains and media (yeast strains and culture conditions)

S. cerevisiae  strains used in this study were listed in Table 1. Yeast extract peptone dextrose (YPD) liquid medium (Difco, USA) contained 1% yeast extract, 2% peptone and 2% glucose, while YPD agar medium (Difco, USA) had similar composition but with additional 1.5% agar. 

Yeast growth measurement

One of the objectives of this study was to determine if TRF has the preventive effect of an antioxidant, thereby extending the lifespan of an organism. Keeping that in mind, the growth for all strains were measured to compare variations in growth patterns, and to determine the initiation of the stationary phase. Overnight culture was prepared by inoculating single colonies picked from those grown on YPD agar plate, into YPD broth agitated at 220 rpm in a shaking incubator (30oC)  (Jung et al. 2015). Biological triplicates were prepared from three different colonies. The overnight culture was transferred into 10 ml YPD broth to an initial density of 1-2 x106 cells/mL or OD600nm ≈ 0.2 (Fabrizio & Longo 2003). The culture was incubated at 30oC, at 220 rpm in shaking incubator (IKA KS 4000i contro). Absorbance of culture was measured by Shimadzu 2450 UV-visible Spectrometer at 600nm wavelength (Murakami et al. 2008),  and  appropriately diluted with Trypan Blue before counted manually by hemocytometer. The whole process was repeated every hour for a total of 15 hours. The cell concentration was calculated from hemocytometer cell count using the formula as follows:

Cell concentration =

Average cell count of the small box x Dilution factor

Volume of a small square (mL)

Determination of viability by colony-forming unit (CFU)

Cultures were diluted in sterile water with 105 dilution factor to achieve a cell density of 1 to 5 x 103 cells/mL. One hundred microliters of the diluted sample was spread onto YPD agar plates, incubated at 30oC, and colonies formed were counted at the end of 48 hours (Longo et al. 2012; Fabrizio & Longo 2003). 

    Chronological lifespan of the strains were determined by measuring the survival of non-dividing stationary phase cells (Mesquita et al. 2010).  All cultures were presumed to be 100% viable at 13th hour when the cells were in stationary phase, with subsequent CFU measurements normalized to the 13th hour CFUs. Averages and standard deviations for at least three biological replicates were calculated for each experiment (Murakami et al. 2008).

Preparation OF TRF

Stock solution of TRF was freshly prepared in the dark room by dissolving 1 g of Gold Tri E 70 (Sime Darby Bioganic Sdn. Bhd., Malaysia) in 1 mL of 100% ethanol (1:1) and kept at -20oC for not more than one month (Lim et al. 2013). The palm-derived TRF consisted of 80% tocotrienol (26.76% α-tocotrienol, 4.29% β-tocotrienol, 32.60% γ-tocotrienol, and 15.53% δ-tocotrienol) and 20% α-tocopherol. Subsequently, there were 159.5 mg α-tocopherol, 205.1 mg α-tocotrienol, 32.9 mg β-tocotrienol, 249.8 mg γ-tocotrienol, and 119 mg δ-tocotrienol in every gram of TRF. TRF was activated by incubating 10 μL stock TRF (1 g/1 mL) with 990 μL 100% ethanol overnight at 30oC (Khee et al. 2014). 

Dose determination of TRF 

The effects of various concentrations of TRF on S. cerevisiae viability were studied to determine the optimum dose on the wild type. Overnight culture of wild type was firstly prepared and then diluted in a new medium to achieve OD600nm≈ 0.2. The cells were then incubated for 13 hours to achieve stationary phase and ready to be treated with different concentration of TRF.  The final TRF concentration used were 0, 50, 100, 150, 200, 250 and 300 μg/mL (Lim et al. 2013). CFU was counted at time 0 hour and 24 hours of treatment. The CFU results at 24 hours were normalised to 0 hour. The optimum dose (300 μg/mL) was then applied to all strains of the yeast. 

Treatment with TRF

Overnight culture was prepared as described previously, and then diluted in a new YPD medium to achieve OD600nm≈ 0.2. The cells were then incubated for 13 hours to achieve stationary phase and treated with (300 μg/mL) of TRF.  

Statistical analysis

Each experiment was carried out in biological triplicates. Results were presented as mean + standard deviation (SD). Comparison between control and TRF treated groups were made by Student’s t-test (two-tailed). ANOVA test was used for comparing different TRF doses and TRF treatment on wild type and deletion strains, followed by Tukey’s honestly significant difference (HSD) post hoc test. P<0.05 was considered to be statistically significant.

RESULTS

All cultures had similar growth patterns. To determine whether the knockouts strains exhibit any different phenotype than the wild-type, the growth pattern of the strains were observed. All three strains showed similar growth patterns (Figure 1A), conforming to the normal phases of yeast growth; lag phase (0-2nd hour), log phase (2nd-12th hour) and stationary phase (12th hour onwards) (Salari & Salari 2017). Figure 1B confirmed the growth pattern by haemocytometer cell counting, where the wild type showed the highest significant cell concentration readings after six to nine hours (Figure 1B, p<0.05), compared to the other two strains. Between six to nine and 13 hours post culture, the wild-type strain has a higher number of cells (p<0.05) than the GPx2Δ∆ strain.  The number of cells of the CTT1Δ∆strain is significantly lower (p<0.05) than that of the wild-type  between six to nine hours post culture. Although, the graphs showed that the cultures are beginning the stationary phase at the 12th hour (Figure 1B), the TRF treatment started at the 13th hour, to ensure that all strain cultures have truly entered the stationary phase.

Optimal dose of TRF was 300 μg/mL. As there is no available report of S. cerevisiae treated with TRF, it was critical to determine the effect of TRF on the viability of S. cerevisiae, as well as determining the optimal working dose for this experiment. Figure 2 demonstrated wild-type S. cerevisiae viability after incubation with various concentration (0-300 μg/mL) of TRF for 24 hours. The viability was normalised to the viability of cells prior to treatment. Generally, there was increased viability of cells as the TRF dose was increased. Both doses of 250 μg/mL and 300 μg/mL showed significant increase in viability, as compared to the control group (p<0.05). Since the higher viability was achieved by 300 μg/ml of TRF, this dose was selected as the working dose.

Increased viability of the wild-type strain after treatment with TRF.  Stationary phase yeast cells of 13th hour culture were treated with 300 μg/mL of TRF for 24 hours. Shown in Figure 3, only the wild-type showed significant increase in viability compared to the control group (p<0.05). 

DISCUSSION

Conventionally, the concentration of yeast cells in a suspension is assayed by the means of spectrophotometry. The absorbance of yeast culture is measured at 600 or 660 nm, and repeated measurement of the same culture over the course of a series of time, enable researchers to monitor the growth of yeast cells (Barber & Lands 1973; Murakami et al. 2008; Salari & Salari 2017). However, this method allows measurement of absorbance of any solute and particles, without discriminating the live cells from the dead (turbidimetrically). Besides spectrophotometry, there are several methods available, that can assay the growth and viability of microbial cells. In this research, direct cell count (Figure 1B) and observation of CFU were used. Dilution of the cells prior to counting with haemocytometer allows only the non-viable cells to take-up the Trypan Blue, leaving the viable cells colourless (Tran et al. 2011). Due to this method of differentiation, the values in the cell counting method does not exactly match the absorbance of the growth. 

   ROS deactivation is crucial to ensure the survival of all aerobic life forms. When an overproduction of free radicals and ROS exceeds the capacity of antioxidant mechanism of the cell, the cell will experience oxidative stress. High level of ROS becomes toxic to cells, causing severe damage to cellular components such as proteins, DNA and lipids (Dezest et al. 2017). 

  CTT1 and GPx2 encode for cytoplasmic catalase (Martins & English 2014) and mitochondrial glutathione peroxidase in yeasts (Tanaka et al. 2005), respectively. Catalase and glutathione peroxidase are amongst the antioxidant enzymes to inactivate the ROS in cells. Catalase prevents cell damage by catalyzing the reduction of hydrogen peroxide to water and oxygen (Zeller & Klug 2004), while glutathione peroxidase catalyzes the reduction of hydrogen peroxide and other hydroperoxides to water or the corresponding alcohols, respectively, using glutathione as the reductant (Wang et al. 2017). Hence, cells lacking antioxidant enzymes are expected to have shorter lifespan and slower reproduction (de Sá et al. 2013; Demir & Koc 2010), as exhibited in Figure 1. In this study, the reduction of yeast cell count in CTT1Δ∆and GPx2Δ∆strains as compared to the wild type may suggest that CTT1Δ∆ and GPx2Δ∆ strains suffer a higher oxidative stress when approaching the stationary phase (Longo et al. 1996; Trancíková et al. 2004). However, previous studies have also shown that catalase (ctt1)-knockout S. cerevisiae, were able to survive without the enzyme, not even for H2O2 resistance (Okada et al. 2014; Martins & English 2014). Other studies showed that catalase is only needed when the cells are exposed to stressors like H2O2, heat-shock and ethanol (Du & Takagi 2007). The same effect was observed in glutathione peroxidase-knockout cells too, where the strain survived under normal condition, and even under peroxide (Inoue et al. 1999) and H2O2 assault (Okada et al. 2014). These abilities of the cells to survive could not be observed in our study model, since the cells were monitored at the initial stage of the stationary phase.

Chronological lifespan refers to the duration where the non-dividing, stationary phase cells maintain their viability. The stationary phase marks the ongoing depletion of source of glucose (Vasicova et al. 2015). Cells will begin to switch from fermentative metabolism to a respiratory metabolism (Williams et al. 2016). This change comes with increased ROS production (Trancíková et al. 2004; Watanabe et al. 2014). Results in Figure 3 indicates that TRF increases the viability of only the wild type strain in the initiation of stationary phase. This leads to the assumption that the presence of TRF cannot overcome the shortcomings of a knockout cell, lacking in catalase or glutathione peroxidase. Based on this observation, we postulated that TRF needs either catalase or glutathione peroxidase 2, or the presence of both enzymes, in order to increase the chronological lifespan at the initial stage. There is a lack of data as the effects of vitamin E on S. cerevisiae has not been widely explored, let alone the effects of vitamin E or TRF on its lifespan during the stationary phase. On the other hand, one research reported on the ability of  α-tocopherol reducing the replicative lifespan of S. cerevisiae (Lam et al. 2010).

Nevertheless, over the years, TRF was documented to exhibit many benefits. In one of the more recent studies, TRF was able to restore actin in cells of murine pre-implantation embryos, after been exposed to oxidative stress triggered by nicotine (Hamirah et al. 2017). It also has healing properties; such as protecting astrocytes against glutamate toxicity (Abedi et al. 2017), and reducing UV-induced skin redness (Yap 2018). Furthermore, TRF restored the lifespan of oxidative stress-induced Caenorhabditis elegans, even with increased DNA oxidation (Aan et al. 2013). TRF also inhibits melanogenesis in melanocyte cell culture, by reducing the expression of TYRP2 gene, resulting in reduced melanin content and tyrosinase activity (Suzana et al. 2009). 

    The benefits of TRF are attributed to the constituents, the various isoforms of vitamin E. Tocotrienols on their own, possess numerous health benefits. Although there are no documented findings of the effect of tocotrienol on S. cerevisiae, tocotrienols have been tested on many other organisms. A recent study reported how γ-tocotrienol counter radiation injury by instigating metabolic shifts (Cheema et al. 2017). Another study reported δ-tocotrienol causes cell cycle arrest in G1 and G2/M phase simultaneously, leading to an anti-prostate cancer activity (Sato et al. 2017). Furthermore, tocotrienol prolonged the lifespan of C. elegans, and increased resistance to infections (Kashima et al. 2012)

In S. cerevisiae, the α-tocopherol, another constituent of TRF; can prevent lipid peroxidation, owing to the hydroxyl group in domain I of the structure of tocopherol, as well as its lipophilic characteristic (Bitew et al. 2010). α-Tocopherol can also reverse detrimental effects caused by edelfosine (Bitew et al. 2010) and endosulfan (Sohn et al. 2004). A study by Raspor et al., (Raspor et al. 2005) demonstrate that treatment with Trolox, (α-tocopherol analogue) increased the cell viability, decreased intracellular ROS formation and suppressed DNA oxidation in Saccharomyces cerevisiae. In a human myoblast cell culture model, TRF treatment reduced the amount of intracellular ROS, reduced lipid peroxidation and decreased the number of cells undergoing late apoptotic events in senescent cells (Khor et al. 2017).

     Due to the limit of the duration of this particular research, it would be more advantageous to test the effect of TRF for longer hours in future. We conclude that the TRF is able to improve the viability of S. cerevisiae, but cannot replace the functions of absent antioxidant enzymes, to improve the chronological lifespan of S. cerevisiae at the initial stationary phase. 

CONCLUSION

The data showed that TRF increased the chronological lifespan of S. cerevisiae, at the stationary phase. The ability of TRF to increase the lifespan of an organism could be applied in other organisms as well.

Acknowledgement 

This research was funded by UKMMC Fundamental Research Fund (Project Code: FF-2015-209). The authors express their thanks to all the members of Department of Biochemistry, Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for their support and feedback. 

INTRODUCTION

The word “transgender” was first coined in the 1980s by Virginia Prince when referring to gender variant individuals (Prince 2005). Transgender is a term used when a person has discordant among their assigned gender at birth with their gender identity. Regarding the term ‘transsexual’, it is used for individuals who underwent or sought transition from male to female and vice versa mainly via cross-sex hormone management or sex reassignment surgery (Prince 2005; American Psychiatric Asociation 2013). The word ‘transvestite’ is a Latin word and it means cross-dresser was coined by Hirschfeld. It originally refers to the heterosexual cross-dresser, and it is a gender manifestation and not a sexual manifestation. In other words, a true transvestite is not a homosexual (Prince 2005). Many presumed ‘gender’ and ‘sexes’’ as the same entities, but it is not, and commonly leads to misconception and misuse of terms. Gender is used within the context to the role within the society whereas sex is used in the context of reproductive capacity. So, gender identity is more like a social identity and gender dysphoria is a descriptive term of an individual’s cognitive or affective discontent due to the incongruence of one’s allocated gender with their experienced gender (APA 2013). Another new term which emerged from transgender activism is cisgender which can be used to describe individuals who is on the same side (cis-) as their birth assigned sex whereas transgender is on the other side (trans-) of their birth-assigned sex. This term was used to prevent marginalization of transgender and it is thought to be a positive identification by using terms likes cisgender, ‘cisman’ or ‘ciswoman’ alongside with the usage of transgender, ‘transman’ or ‘transwoman’ (Aultman 2014). 

Previously, the term gender identity disorder was used according to Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) and now it has changed to gender dysphoria in the latest DSM-5 (APA 2013). The core features listed were the strong desire to be of opposite sex with preference to cross-dress and sturdy desire to have their sexual characteristics that matches one’s experienced gender. It is controversial whether to include gender dysphoria into DSM as there are a lot of debates, whether transgender is a pathological condition or it is just a natural variation. In DSM-5, the word ‘disorder’ was removed. Gender-related dysphoria is a major criterion for limiting the diagnosis to those who have significant distress for their cognitive and affective component. This is to focus on the dysphoria as the clinical misfortune instead than on the identity itself. Not all individuals are distressed with their inconsistency of assigned gender. This nomenclature approach is an attempt to reduce psychiatric stigma and discrimination on transgender population. The main challenge encountered by a physician is diagnosing, and treating individuals with gender identity disorder, or gender dysphoria (Coleman et al. 2012).

There are few formal epidemiological studies on the transgender population which makes it difficult to know the accurate prevalence of transgender. Estimation on the prevalence of transgender varies widely, and it also depends upon the definition of transgender and also the population being studied. It was reported that a child with gender identity disorder had diverse clinical presentation of gender identity/orientation disorder (Coleman et al. 2012; Zucker 1985) where homosexual/bisexual formed a majority of presentations, i.e. 46% (Figure 1). It is interesting to note that about 1/4 of the children studied in this long-term follow-up fall under a category of the uncertain group of neither homosexual/bisexual, transsexual, heterosexual nor transvestite.

Earlier researchers described the prevalence of transgender to vary from 1:12,000 to 1:45,000 for transgender females and approximately 1:30,000 to 1:200,000 for transgender males (De Cuypere et al. 2007). Few other studies showed even higher prevalence of transgender depending on the type of methodology used. From a population-based household probability study of 28000 adults in 2010 in Massachusetts showed that 1 in 215 people were identified as transgender (De Cuypere et al. 2007).

Cross-sex hormone treatment use feminizing hormones on individuals who are assigned as male at birth and vice versa with the use of masculinizing hormones on individuals who are assigned as female at birth (Coleman et al. 2012). The main reason for transgender motivating to seek for cross-sex hormone is to eradicate most of their secondary sexual characteristics and also to attain the characteristics of the opposite gender (Colton et al. 2011). However, data on the therapeutic approach is scarce and there is lack of consensus on the treatment approach for different countries (Coleman et al. 2012). So, uncertainties in this specialized field of transgender medicine may make it difficult for this group of individuals to get the opportunity to receive appropriate treatment which may predispose them to suffer from discrimination and harmful social conditions. In order to help these group of individuals, it is necessary to probe further into more research to support current guidelines. As it is their rights in these population to get treatment without prejudice. With more interventions developed for these vulnerable population may help to reduce further victimization, violence or stigma in these population (Chakrapani et al. 2019; Gonzales & Henning-Smith 2017). James et al. reported that about 58% of transgender individuals had experienced discrimination which included loss of job, eviction, bullying, physical harassment and even denial of access to medical services (James et al. 2017). Transgender individuals were found to be have higher unemployment rate and poverty issues compared to non-transgender counterparts. (Conron et al. 2012)

NEUROBIOLOGY IN TRANSGENDER

To date, there is paucity of data that provides credible information on etiology of transgenderism and no single factor has been proven with certainty that causes gender identity disorder (Cohen-Kettenis & Gooren 1999). Based on previous twin studies, it shows that there are strong genetic inheritance components of 62% for individuals with gender identity disorders in early development (Swaab 2004). Factors which influence the development of transgenderism are chromosomal disorders such as Klinefelter disorder, phenobarbital exposure during fatal developmental stage, and endocrine disorder (i.e. congenital adrenal hyperplasia, CAH) (Swaab 2004; Savic et al. 2010). However, only a small group of transgender have underlying endocrinology abnormalities (Swaab 2004; Savic et al. 2010). There were observations, which described possibility of abnormalities in the hypothalamus-pituitary-gonadal axis (Figure 2) in transgender as evidenced by a higher incidence of polycystic ovarian disease (PCOS) and menstrual cycle irregularities in female to male transgender (Swaab 2004). An earlier research found that males had more androgen staining (AR) in the hypothalamus, especially in the medial and lateral mammillary bodies (Swaab 2004).  Other nuclei within the hypothalamus such as the preoptic, periventricular and supraoptic nucleus were also shown to have the sex differences in AR staining, which is dependent on circulating androgen levels (Swaab 2004).  It was observed in animal studies that lesions in pre-optic area in hypothalamus caused shift in sexual orientations.

It has been hypothesized that atypical sexual differentiation results in gender dysphoria with the physical sexual characteristics growing in one direction and with the brain and gender identity growing in the opposite sexual directions (Savic et al. 2010). The basis behind this theory is that during the prenatal period, there is a different sensitivity window for sex hormone for the brain and sexual organs (Savic et al. 2010; Berenbaum & Beltz 2011). Sexual development for the genitals and brain occurred at different stages with the genital differentiation in the first two months of fetal development. Subsequently in the second and third trimester of pregnancy, the differentiation occurred independently and may result in transgenderism (Savic et al. 2010).  

Few studies showed that transgender’s brain is different from their natal sex member’s brain and more alike to the opposite sex (Savic et al. 2010; Swaab & Fliers 1985).  Transgender’s brain seemed to move more towards their experienced gender while receiving hormonal treatment (Gooren et al. 2015). This evidence showed that the brain organisation may be under influence of prenatal sex hormone, which plays a role in the development of the neural circuit (Berenbaum & Beltz 2011; Slabbekoorn et al. 1999). These organisational effects are permanent and may not depend on subsequent hormone influences (Slabbekoorn et al. 1999). 

Studies conducted over three decades described the human brain with the possibility of morphological sex differences (Swaab & Fliers 1985). Many observations failed to be replicated due to different methodology and sampling technique (Swaab & Fliers 1985). It has been observed that there are sexual differences in the shape of corpus callosum and suprachiasmatic nucleus (Swaab & Fliers 1985). The preoptic area, which is involved in gonadotrophin hormone release and sexual behaviour were found to be sexually dimorphic (Swaab & Fliers 1985). In this study, it was found that the sexually dimorphic nucleus was 2.5 +/- 0.6 times larger, and it contained 2.2+/-0.5 times as many cells in men (Swaab & Fliers 1985). Several studies also showed that sex hormone influenced verbal and spatial ability with androgen hormone favours visuospatial ability and oestrogen favours verbal fluency (Gooren et al. 2015; Slabbekoorn et al. 1999).

Few women who were subjected to diethylstilbestrol (DES) or children who have congenital adrenal hyperplasia (CAH) were observed to have masculinising effect. Such as a masculine pattern of lateralisation which shows right ear advantage in the dichotic listening task in DES-exposed women and masculine cognitive pattern is seen in women with CAH in both spatial and verbal tasks (Slabbekoorn et al. 1999). Studies stated that three children born were transsexuals, and a few of the children had gender dysphoria symptoms in a group of children who had in-utero exposure to diphantoin and phenobarbital (Swaab 2004; Dessens et al. 1999). 

Based on previous studies, there are significant relationships between sex-role identities with a serum testosterone level (Baucom et al. 1985). It has been shown that feminine-sex-typed women had lowest concentration of testosterone compared to masculine-typed women had been higher level of testosterone level (Baucom et al. 1985). Baucom et al. also noted that females with higher testosterone levels perceived themselves as more resourceful, independent and goal directed. Women with a lower testosterone level described themselves as traditional females being more cared, anxious and dejected mood (Baucom et al. 1985).

PHYSIOLOGY OF SEX HORMONE

Testosterone is cholesterol based steroid hormone, which has a diverse effect throughout the body as androgen receptors are widely distributed (Baucom et al. 1985; Zitzmann & Nieschlag 2001). Hence, testosterone can have both physical and psychological effects in the body. Interstitial cells of Leydig produce testosterone in the testicles (Hall 2015; Rhoades & Tanner 2003). Its production and the serum levels are being regulated by complex mechanism, which can be affected by both the endogenous and environmental factors (Hall 2015).

Figure 2 showed  hypothalamus liberates gonadotrophin releasing hormone (GnRH) to the vascular pituitary gland in order to stimulate the secretion of follicle stimulating hormone (FSH) and luteinising hormone (LH) (Hall 2015; Rhoades & Tanner 2003). LH signal Leydig’s cells to produce testosterone, which leads to development of male sexual characteristics (Hall 2015; Rhoades & Tanner 2003). However, the negative feedback mechanism and the complex interplay between the hypothalamic-pituitary-gonadal axis are still not fully understood due to its complexity (Baucom et al. 1985; Zitzmann & Nieschlag 2001; Rhoades & Tanner 2003). Testosterone has both androgenic and anabolic functions (Hall 2015). Regarding its androgenic function, it is mainly responsible for the development of masculine characteristics. During fetal development at the 7th week of embryonic life, XY chromosome causes the gonadal ridge to secrete testosterone and later the testes would take over the function of secreting testosterone (Hall 2015). Male body characteristics such as penis, scrotum, prostate gland, seminal vesicles and male genital ducts are thus formed. At the same time, it also acts to suppress the formation of female genital organs in the fetus (Hall 2015). 

Thereafter, testosterone is essentially not produced during childhood until about 10 to 13 years of age when the testosterone production starts again at puberty with the stimulus coming from the anterior pituitary gonadotrophic hormones (Hall 2015). Testosterone production continues throughout life until after 50 years of age, the level of testosterone drops to about 20 to 50% of the peak value reaches by 80 years of age (Hall 2015). 

At puberty, testosterone plays a major role in adult primary sexual characteristic development (Hall 2015; Rhoades & Tanner 2003). Testosterone can influence the testes, penis and scrotum to enlarge up to be eight-fold before the age of 20 years (Hall 2015; Rhoades & Tanner 2003). Apart from that, testosterone is also involved in secondary sexual characteristic’s development, which can distinguish a male from a female (Hall 2015; Rhoades & Tanner 2003). Body hair distribution in male is different compared to female such as growth of hair at the pubic region which goes up to the umbilicus along the linea alba of the abdomen, increased facial hair and hair on the chest or even the back (Hall 2015; Rhoades & Tanner 2003; Irwig 2017). Apart from that, testosterone also plays a role in hypertrophy of the laryngeal mucosa and enlargement of the larynx causing gradual changes into masculine voice (Zitzmann & Nieschlag 2001; Hall 2015; Irwig 2017). Testosterone also causes significant changes in increasing thickness of the skin, increased ruggedness of the subcutaneous tissue and increases sebaceous gland activities, which may contribute to the acne problem (Irwig 2017). Over time, the skin slowly adapts to testosterone to overcome the acne problem (Hall 2015). 

Regarding the musculoskeletal changes, testosterone is increased in muscle mass up to 50% more than that of the female counterparts (Hall 2015).  It also affects the bone growth by increasing the bone matrix and intensifies calcium retention, which ultimately increases the size and strength of the bone (Rhoades & Tanner 2003; Irwig 2017). Other specific changes related to testosterone hormone are narrowing and lengthening of pelvic outlet, which causes funnel-like shaped pelvis (Hall 2015). These changes are as a result of increased protein level due to anabolic function of testosterone, and also due to the calcium deposition (Hall 2015).

Testosterone also plays a role in basal metabolic rate, which probably is due to protein anabolism whereby surge in protein would increase enzymatic function and activities of all cells (Hall 2015). Other changes observed are increased in the number of red blood cells, blood volume and extracellular fluid volumes in males (Hall 2015).  

Basically, most sexual changes caused by testosterone are the effects of increased rate of protein synthesis in the target cells (Hall 2015).  Intracellularly, testosterone is mainly converted to dihydrotestosterone, and then it will induce DNA-RNA transcription to increase cellular protein production (Hall 2015).  Once physical maturity is achieved, basically testosterone plays a role in homeostatic function by sustaining secondary sexual characteristics, continuous spermatogenesis, sexual function and maintains the muscle bulk.

ESTROGEN

Regarding the female counterpart, estrogens are primarily secreted in the ovaries from cholesterol and acetyl coenzyme A (Hall 2015).  Primary function of estrogen is mostly on the reproductive system which is growth of the tissues and cellular proliferations on the reproductive organs (Hall 2015; Rhoades & Tanner 2003).  Main carrier proteins for estrogen are plasma albumin and specific estrogen binding globulins, and they are bound loosely, which enable them to be rapidly released to the target tissues quickly (Hall 2015; Rhoades & Tanner 2003). Liver is involved in estrogen degradation via conjugation, and most of its by-products are excreted in the bile and some in the urine (Hall 2015; Rhoades & Tanner 2003).

In childhood, serum estrogen level is low and during puberty, the secretion of estrogen can be influenced by the anterior pituitary gonadotrophic hormones causing it to increase up to 20 folds or more (Hall 2015). This results in the enlargement of the reproductive organs and external genitalia and additionally there will be fat deposition in the mons pubis and labia majora (Hall 2015; Rhoades & Tanner 2003). Regarding the uterine endometrium, there is important development, which is crucial to prepare female individuals for pregnancy later on, which are developments of endometrial glands and proliferation of the endometrial stroma (Hall 2015).

The changes are also seen in the fallopian tube with increment of glandular tissue’s proliferation of the lining and also increase of ciliated epithelial cells, which are involved in propelling fertilized ovum to the uterus during pregnancy (Hall 2015). In addition to it, estrogen changes vaginal epithelium from the cuboidal cell epithelium in the pre-pubertal period to stratified type for it to be more resistant to trauma and infection (Hall 2015). 

Regarding the secondary sexual characteristics, estrogen is involved in development of breast tissues together with growth of the ductile system in the breasts (Hall 2015; Rhoades & Tanner 2003). Estrogen also leads to increase in fat deposition in the breasts (Hall 2015). Estrogen inhibits osteoclastic activity in the bones which stimulates skeletal growth (Hall 2015). During menopause when there is virtually no estrogen secretion, osteoporosis is a common condition seen leading to weakening of bones and risk of fractures (Hall 2015; Rhoades & Tanner 2003). 

When compared to testosterone, estrogen only causes a slight increase in protein position and basal metabolic rate (Hall 2015). Estrogen also causes an increase in fat deposition, which forms a characteristic female figure with fat deposition at the breast, subcutaneous tissues, thighs and buttocks (Hall 2015; Rhoades & Tanner 2003). Other effects of estrogen include the soft and smooth skin texture compared to the males (Hall 2015). 

PROGESTERONE

Progesterone is the most important progestin and it is produced normally by the corpus luteum at the second half of the menstrual cycle (Hall 2015). When progesterone is secreted, it is converted to other steroids quickly and liver plays a role in degradation of progesterone (Hall 2015; Rhoades & Tanner 2003).

The main function of progesterone in sexual development is secretory change in the uterine endometrium in order to prepare for fertilisation to take place (Hall 2015; Rhoades & Tanner 2003). Regarding the breast development, progesterone plays a role in alveolar cell proliferation to prepare the breast for its secretory function (Hall 2015; Rhoades & Tanner 2003). However, milk production only occurs with stimulation of prolactin hormone (Hall 2015; Rhoades & Tanner 2003).

CRITERIA TO START CROSS-SEX HORMONE

There are different criteria, which must be fulfilled before allowing an individual to start on cross-sex hormone therapy (Coleman et al. 2012; Irwig 2017). These criteria can be obtained from the World Professional Association for Transgender Health, Standard of Care, version 7, WPATH SOC 7, i.e. for obtaining informed consent and the initial visits, assessment and intervention (Cavanaugh 2016). WPATH recommends that cross-sex hormone therapy can be initiated once psychosocial assessment has completed by a qualified mental health professional unless the prescribing provider is also qualified to perform this type assessment. After that the individual is deemed to be an appropriate candidate to go for hormone therapy and after obtaining the informed consent for treatment (Unger 2017).  

It must be well documented that an individual has persistent gender dysphoria which was diagnosed by a mental health professional who is well versed in this field and the individual must have the capability to make an informed decision and give consent for the therapy. If the individual has significant medical or psychiatric comorbidity, it must be reasonably properly controlled (Coleman et al. 2012; Irwig 2017). There is also a minimum age for medical consent, and if the individual is a child or of adolescent age, there is a need for the individual’s parents or caretaker to give consent and to be involved in supporting the individual during the treatment process (Coleman et al. 2012). 

In the past four decades, real-life test (RLT) or real-life test experience (RLE) has been practiced which was likely derived from Dr. Harry Benjamin and subsequently it was reinforced by series of expert opinion through the revisions of Standards of Care (Levine 2009). However, there was lack of scholarly evidence or research ever done on it to support these practices and it was mainly based on level of expert opinion. So, there was lack of evidence to address its issues such as duration of real-life test, the purpose of test or assessment to use which sparks criticism and controversy of this practice. RLT is an extended period where individuals live as a member of desired sex full time prior to some irreversible social, medical or surgical step taken (Levine 2009). This practice gives the individual an opportunity to experience and to test their personal belief that life would be subjectively be better when they occupy their new gender role full time (Levine 2009). RLT would generally test the individual’s conviction, courage and adaptive challenge in their daily life. 

PHARMACOLOGICAL INFLUENCE AND INTERVENTION: CROSS-SEX HORMONE

Cross-sex hormone administrations of exogenous hormonal preparations, which lead to feminizing or masculinizing effects on the body. This treatment should be individualized based on individual’s needs with consideration on patient’s patient’s background medical co-morbid, psychosocial issues and risk-benefit weighting of the treatment to the patient (Coleman et al. 2012). There is a wide variation of hormonal treatment with a different diversity of doses available in the market (Coleman et al. 2012). Currently, with the newer transdermal preparations and the approach of using low doses may help to reduce adverse reactions, but it is still a concern for healthcare providers and patients (Coleman et al. 2012). To date, there is lack of study on the cross-sex hormone in transgender which is a major setback on gender affirmation treatment in transgender (Coleman et al. 2012). Another limitation noted is that it is different in countries and would vary in terms of availability of cross-sex hormone therapy. Fewer transgender even opted to take hormonal therapy without medical supervision as some parts of the world still have barriers in accepting transgender (Irwig 2017). Due to social stigma, lack of cultural acceptance and poor awareness on transgender, suboptimal transgender care is common (Coleman et al. 2012).

MASCULINISING HORMONE THERAPY (FEMALE TO MALE TREATMENT OPTIONS, FMTO)

TESTOSTERONE

Testosterone hormone comes in many different formulations such as oral, topical, transdermal and intramuscular form. The oral testosterone undecanoate can be given between 160 mg-240 mg OD, testosterone enanthate 50 mg to 200 mg weekly given intramuscularly or subcutaneously. For transdermal patch, the dosing can be between 25 mg to 75 mg daily and testosterone 1% gel at 25 mg to 100 mg daily. The main aim is to use the lowest possible dose to achieve masculinising effects in patients and have to balance it with its potential adverse reactions (Irwig 2017). This treatment generally is quite similar to treatment of hypogonadal males and the testosterone level should be increased to achieve normal male’s physiological range which is between 300 to 1000 ng/dL (Webb & Safer 2019).

There is no standard practice which can guide on the starting and maintaining dose. Usually, it is initiated at a low dose first and then gradually step up the dose with periodic monitoring of serum testosterone level to guide titration (Coleman et al. 2012). Testosterone therapy will change the body composition of fat and muscle bulk towards natal men (Colton et al. 2011). Usually, the agent of choice for testosterone would be intramuscular testosterone such as cypionate (Colton et al. 2011).

In the first year of treatment, patient is recommended to be monitored every 3 months to assess on the virilising effect and thereafter can be 6 monthly to yearly follow up. During each visit, it is recommended to monitor patient’s serum testosterone level, hematocrit and lipid profile with a baseline result prior to starting hormonal therapy. Other screenings that should be done are bone mineral density, pap smear and mammography for patients with cervixes and breasts (Coleman et al. 2012; Webb & Safer 2019).

OTHER AGENTS

Progestins may be used to help in cessation of menstruation (Hall 2015)

FEMINISING HORMONE THERAPY (MALE TO FEMALE TREATMENT OPTIONS, MFTO)

Feminising hormone therapy is slightly more complicated than the regimen for masculinizing hormone therapy as female transgender requires antiandrogen in addition to estrogen. The main goal of treatment would be to aim for female range of testosterone level which is less than 100 ng/dL and be cautious to avoid supra-physiological levels of estradiol which is to keep it below 200 pg/mL breasts (Coleman et al. 2012; Webb & Safer 2019; Gardner & Safer 2013).

It was recommended that female transgender to have regular follow up every 3 monthly for the first year of initiation of therapy to monitor the feminizing effect as well as to watch out for any adverse effects (Webb & Safer 2019). Baseline lipid profile, serum prolactin should be obtained prior to therapy and then repeated during every visit together with serum testosterone and estradiol with the aim to maintain it between the range of testosterone at 30 to 100 ng/dL and estradiol less than 200 pg/ml (Webb & Safer 2019; Gardner & Safer 2013). Other important surveillance would bone density test, screen for cancers, metabolic syndrome including monitoring of weight and also look for gallstones if clinically indicated (Webb & Safer 2019; Gardner & Safer 2013).

ESTROGEN

There are a few routes of administration, i.e. oral, transdermal form and parenteral estradiol valerate. However, oral estrogen, notably ethinyl estradiol is not advisable as there is an increased risk of venous thromboembolism (VTE) (Coleman et al. 2012). Due to safety concerns, transdermal is recommended and VTE is dose-related, so if possible, it is better to start at lower doses, particularly in individuals at risk to get VTE (Moore et al. 2003). Usually, the dose used for feminizing effect is about 2-3 times higher than the hormone-replacement therapy in post-menopausal women (Moore et al. 2003). After the age of 40 years, transdermal route was recommended as it bypass the first-pass metabolism which seemed to have better metabolic profiles (Unger 2017).

For oral estradiol it has been recommended to keep the dose between 2 to 4 mg OD, transdermal estradiol was between 0.1 to 0.4 mg biweekly and parenteral estradiol valerate was 5 to 30 mg once every fortnightly (Unger 2017).

ANTI-ANDROGEN

Primary objective of anti-androgen is to reduce the effect of endogenous testosterone activity to reduce masculine characteristics. Most studies suggested for combination of anti-androgen with estrogen for feminizing effects, and this combination may be the lower dosage of estrogen needed to reduce testosterone activity, which can reduce estrogen related adverse effects (Moore et al. 2003). So far, there are no consensus guideline on usage of anti-androgen (Unger 2017).

Anti-androgen came from a wide variety of different drug classes and below are the common anti-androgen drugs being used currently: (i) Spironolactone is one of the commonest drug being used which is a type of antihypertensive drugs that is known to directly inhibit testosterone secretion and binding of androgen to the androgen receptor (Aultman 2014). The dose being used commonly was between 100 to 200 mg OD and patients need to be monitored closely for hyperkalemia (Unger 2017), (ii) Cyproterone acetate is a synthetic compound of 17-hydroxyprogesterone and it was shown to have anti-androgen properties. It mainly acts as an androgen receptor antagonist (Wierckx et al. 2012)., (iii) GnRH agonists are neurohormones, which comes in injectable forms or implants are a more expensive form of treatment. The GnRH agonist inhibits the release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). It functions as a highly effective form of gonadal blocking (Coleman et al. 2012). GnRH agonist (leuprolide) can be given between 3.75 to 7.5mg intramuscularly every month (Unger 2017), (iv) 5-alpha reductase inhibitors, i.e. finasteride and dutasteride are known to inhibit testosterone from converting to its active form, which is the 5-alpha dihydrotestosterone (Coleman et al. 2012). Finasteride dosing commonly is 1 mg OD (Unger 2017). 

PROGESTIN

Progestin usage is rather controversial as its usage was found that it did not enhance growth of breast tissue or lower down testosterone level (Coleman et al. 2012). In addition to it, progestins have an increase in risk for pulmonary embolism, coronary artery disease, cerebrovascular accidents and breast cancer risk, especially when used in combination with estrogen (Coleman et al. 2012). The dose for oral Progesterone was between 20 mg to 60 mg OD (Unger 2017). Figure 3 shows the list of possible physical effects of cross sex hormones in transgender.

BIOLOGICAL INFLUENCES IN TRANSGENDER

Few researchers suggested that masculinizing treatment using testosterone or other androgenic sex steroids may further aggravate affective symptoms such as hypomania, mania or even psychotic symptoms in those individuals with co-morbid of psychiatric illness (Coleman et al. 2012; Seiger et al. 2016). With regard to the testosterone that may boost dopamine sensitivity in the brain as an adverse effect inducing psychotic disorders, studies in these areas of interest were contradictory and inconclusive (Elias & Kumar 2007; Trotman et al. 2013). On the contrary, estrogen may produce neuroprotective effect against psychotic disorders (James et al. 2016; Kirkbride et al. 2012; Weickert et al. 2016).

These adverse reactions seem to occur when there were higher doses or supra physiological serum testosterone level. However, evidence of such is limited and its risk is inconclusive. 

PSYCHOLOGICAL INFLUENCES IN TRANSGENDER WITH CROSS-SEX HORMONE

Cross-sex hormone was found consistently to bring positive effects to be transgender psychologically, which was replicated in many studies (Colton et al. 2011). Regarding female-to-male who are on testosterone therapy were observed to have improvement in quality of life, which was also seen for male-to-female counterparts (Gorin-Lazard 2012). 

MAJOR DEPRESSIVE DISORDER

A low level of estrogen was found to be associated with depression, which is seen in normal physiology of females during their menstrual cycle, postpartum period and menopausal period. It was found that transgender women had higher prevalence of depression with an estimated prevalence rate to range between 48% to 62% and when compared to general population in the United States, it was about 16.6% (Hoffman 2014; Budge et al. 2013). Estrogen was found to have a calming effect, and it was used to augment the effect of antidepressant in patients with depression (Khobzi  Rotondi 2011).

There is some evidence, which shows that androgen deprivation treatment in prostate cancer patients are linked to increased rate of depression, which may apply to male-to-female transgender taking anti-androgen treatment (Khobzi Rotondi 2011). Studies correlated the relationship between gonadal function and depressive episodes as it has been observed that hypogonadal men on testosterone treatment seemed to show improvement in the mood (Zitzmann & Nieschlag 2001). Cyproterone acetate usage has been observed to cause transient depressive symptoms during the first 6 months of hormonal treatment (Asscheman et al. 2011).

There are many risk factors apart from the hormonal effect which can predispose transgenders to depression, which are gender dysphoria, lack of social support, physical and verbal abuse, discrimination, being a sexual worker and socio demographic factors such as low education level and unemployment (Colton et al. 2011; Hoffman 2014). 

ANXIETY DISORDER

Regarding anxiety disorder in transgender, the rate can range from 26% to 38% (Budge et al. 2013). Gómez-Gil et al. reported findings of transgender on cross-sex hormone showed to have lower levels of social distress, anxiety and even depression when comparing with transgender who were not on cross-sex hormone therapy (Gómez-Gil et al. 2009).

MORTALITY RATE AND SUICIDE

Earlier researchers reported that mortality rate for male to female group was about 51% higher than the general population (Assheman et al. 2011). However, in the same study, regarding relation of female to the male group, there was no significant difference in the mortality rate in comparison to the general population. There was increased mortality rate up to 8-fold times in the males compared to the females and was mainly due to non-hormonal related such as illicit substance usage, suicide and acquired immune deficiency syndrome (AIDS) (Assheman et al. 2011).

Regarding suicide, there was six-fold increase in the male to female subjects, but it was pertinent to note that there were other confounding factors, which affected the results (Assheman et al. 2011). Individuals who were transgender, presented themselves to the Psychiatry Unit with history of suicidal attempts or substance abuse or affective disorder even prior to cross-sex hormone treatment. This was probably related to the psychological stress from gender dysphoria. Clements-Nolle et al. reported prevalence for suicide in transgender to be 32% (Colton et al. 2011; Clements-Nolle et al. 2006). 

PSYCHOLOGICAL AND COGNITIVE EFFECTS OF CROSS-SEX HORMONE THERAPY IN TRANSGENDER

Generally, cross-sex hormone therapy can alleviate overall well-being and, mainly it reduces gender dysphoria irrespective for transgender men or transgender women (Colton et al. 2011). In one longitudinal study, it was shown that hormonal therapy demonstrated positive effects in transgender. The study was conducted on the transgender before and after 12 months of hormonal therapy and it was found that their emotions were stable using Zung Self-rating Depression Scale (SDS) and Zung Self-rating Anxiety Scale (SAS) (Colizzi et al. 2014). In this prospective study, it showed that there are lower psychological distress and lesser functional impairment with hormonal therapy compared to one prior the hormonal treatment. The results showed that for anxiety before treatment, it was 50%, and it was only 17% following 12 months of hormonal treatment. The depressive psychopathology was 42% for pre-treatment group and 23% for those following 12 months of hormonal treatment. This study also used Symptom Checklist 90-R (SCL-90-R) to evaluate global psychological symptoms. The psychological distress is 24% at enrolment phase and 11% after being on hormonal therapy. Regarding functional impairment, it was 23% before hormonal treatment and 10% after hormonal treatment, respectively, using Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I) (Colizzi et al. 2014). Current evidence on cross sex therapy with regards to improving psychological well-being is quite encouraging especially for male-to-female transgender (Nguyen et al. 2018). 

In another study by Keo-Meier, Minnesota Multiphase Personality Inventory (MMPI-2) was used as it was believed that MMPI-2 results would remain stable even with intensive psychotherapy (Keo-Meier et al. 2015). This study showed that with 3 months of testosterone therapy, there was substantial improvement of transgender men in depression, hypochondria, hysteria and paranoia when compared to the female controls. The results showed that testosterone therapy in transgender men helped to improve multiple domains of psychological functioning.

The published data on effects of cross-sex hormone therapy to the person’s cognition is limited. Slabbekkoorn reported that there was a significant correlation with androgen treatment on spatial ability in female-to-male (FTM) transgender and these effects were not reversed after a five-week period of termination of androgen treatment (Slabbekoorn et al. 1999). With regard to testosterone treatment option, it has an activating effect which is not rapidly reversible on spatial ability performance. However, in this study, anti-androgen treatment did not seem to show a decline in spatial ability nor improved in verbal fluency in male-to-female (MTF) transgender. Most studies to date suggested that masculinizing therapy enhances in visual memory and 3-dimentional spatial memory tasks but worsens verbal memory (Nyugen et al. 2018). 

Studies showed that masculinizing therapy increases cortical thickness in the parieto-occipito-temporal regions may suggest possibility of some testosterone-induced structural connectivity. As for feminizing therapy, some research found that there was decreased in cortical thickness, decreased in subcortical volumetric measures and enlargement of ventricular system (Nyugen et al. 2018; Seiger et al. 2016; Spizzirri et al. 2018). Mueller et al. also found there was mean neuroanatomical volume for the amygdala, putamen and corpus callosum differed between transgender men and cismen in several brain structures including medial temporal lobe structures and cerebellum. He suggested that there is localized influence of sex hormones neuroanatomy (Mueller et al. 2017). 

There was also a relationship between estrogens and borderline personality where the latter may be adversely influenced by alterations in the estrogen levels, especially among women (Evardone et al. 2008). Estrogen regulation on the neurotransmitter systems in the CNS may explain the affective instability associated with borderline personality among the sample of women and men. Interestingly however, this relationship may also be greatly influenced by other variables, e.g. age, menopausal and genetic make-up and that this could potentially influence the interpretation of the results involved in this disorder (Mc Ewen 2001).

Generally, the studies to date on safety and effects of cross sex hormone towards the brain are very few to date with most studies limited to small sample size and most studies are cross-sectional in nature. So, future research should focus if these neuroanatomical changes gives impact to functional or cognitive changes as these domains are important in daily functioning in individuals on hormonal therapy. Interestingly, Mohammadi & Khaleghi suggested that there is relationship and interaction between culture, behavior and brain structures. Transgender will experience changes in lifestyle and beliefs and these changes in new culture and concepts may alter brain’s function and structure based on the culture-behaviour-brain loop model (Mohammadi & Khaleghi 2018).

AGGRESSION AND SEXUALITY

Animal studies showed that aggressive behaviour is found more predominantly in males, and some criminological studies showed that men use far more physical violence, and these studies linked testosterone to aggression. These include criminals who are involved in violent felonies were found to have higher testosterone levels compared to felons who were convicted for burglary or theft (van Goozen et al. 1995).

Regarding FTM treatment options, there are positive correlations of testosterone with aggression. In an earlier study by Slabbekoorn, it was shown that FTM who are on testosterone therapy have higher scores for anger proneness, which was replicated in a previous study by Van Goozen (van Goozen et al. 1995). In this same study, it was found that their increased sexual feelings measured by frequency of sexual activity after being on testosterone therapy for FTM and also general reduction in affect intensity. Interestingly, in an animal study where female rats were injected with testosterone, not only showed increased in aggressive behaviour but also showed an increase in the sexual behaviour (van Goozen et al. 1995). Based on these studies, it was suggested that females are more sensitive to minor variations in the circulating testosterone levels (van Goozen et al. 1995). However, there were conflicting results in which the administered exogenous supra-physiological levels of testosterone showed that there was no sufficient evidence to suggest link between testosterone with increased aggression (Anderson et al. 1992; O’Connor et al. 2002). 

We represented the summary of cross sex hormones having a role in the bio-psycho-behavioural domains in Figure 4.

ADVERSE EFFECTS FROM CROSS-SEX HORMONES

It has been reported that male to female transgender on hormonal treatment, experience more important adverse effects such as thromboembolism, cardiovascular event and osteoporosis. However, it has been found that female to the male transgender group on hormonal treatment seemed were relatively safer (Wierckx et al. 2012). It was reported that 12% of male to female transgender experiences thromboembolic or other cardiovascular event (Wierckx et al. 2012). Among the risk that comes with cross-sex hormone therapy are different for female to male group and male to female group. Another area to address is to look into possibility of sexual dysfunction in this population group as changes in level of hormones may interfere with sexual functioning. Validate questionnaire such as Female Sexual Function Index Questionnaire (FSFI) may be used during clinic visits (Tee et al. 2014; Hatta et al. 2007).

ADVERSE EFFECTS OF FEMALE TO MALE TREATMENT WITH REGARD TO HORMONAL THERAPY  

Testosterone is the main hormone used and its adverse reactions are weight gain, blunting in the insulin receptor sensitivity, worsening of lipid profile, and hematocrit elevation. These combinations of antagonistic reactions have raised the concern for possibly increasing the risk of cardiac and thrombo-hemolytic events. Polycythemia was also reported as one of its rarer adverse reactions. Other masculinizing hormones adverse effects are acne, androgenic baldness, sleep apnea and liver enzyme derangement (Cavanaugh 2016). So, surveillance on metabolic syndrome during each follow up with basic anthropometric measurement may be useful such as weight, waist circumference, BMI, blood pressure and heart rate on every clinic visit apart from regular blood investigations. Imaging or further test may be necessary if clinically indicated during follow up.

ADVERSE EFFECTS OF MALE TO FEMALE TREATMENT WITH REGARD TO HORMONAL THERAPY

Research more than a decade (Cavanaugh 2016), reported 20-fold increase in the venous thrombosis and also an increase in serum prolactin in a retrospective morbidity and mortality due to feminizing treatment. This may possibly increase the growth of prolactinomas (Cavanaugh 2016). The adverse effects are dose dependent and the risk further increased when other risk factors were present, e.g. Smoking, obesity, and advance age may increase the risk for cardiovascular event in transgender on feminizing treatment. So, regular follow-up is vital to watch out for these adverse effects by monitoring patient’s metabolic profile via anthropometric measurements and regular blood monitoring. Further investigations should be promptly initiated accordingly if patient has any early signs of any adverse effects (Figure 5). 

CONCLUSION

In summary, cross-sex hormone generally gives benefits to be transgender, but it may also bring deleterious effects such e.g. FTM gets reduced in affect intensity, and MTF experienced more tension, gloomy mood and easy fatigability. The present review showed that there are few heterogeneous results in some studies and one needs to take note on limitations in these literature. Inconsistent results may be due to limitations with methodology such as small sample size and issues with study design or sample distribution. Many definitive questions still remain unanswered due to lack of retrospective or prospective studies on cross-sex hormone. Generally, the evidence on cross-sex therapy is of low quality due to improper technique of randomization and control groups in the study performed. So, it is important to convey to patients regarding potential uncertainty and there is a need for individualised treatment approach based on risk and benefit ratio for each patient. Transgenderism is a complex disorder and treatment approach is not too easy, and it needs an integrated approach of physical, psychological, social and even cultural interventions. There are many issues to address on the care for transgender. Limited accesses for healthcare services for transgender and lack of trained mental health professionals are some of the important restrictions. Stigma and potential political discrimination to these special groups of individuals carry significant psychosocial issues to not only the transgender, but also pose a dilemma to the mental health professionals who are dealing with this special group of patients. So, it is important to educate the scientific community regarding treatment options that are available and also its latest evidence found. As it is important to mainstream transgender care among medical healthcare workers and published transgender medical treatment guidelines may provide a foundation for more generalized patient care and more assessible to for transgender population.

ACKNOWLEDGEMENT

The authors acknowledge the support by UKM (DLP-2014-009).

There are numerous studies over the past few decades that reiterate the many positive points of Obesity and Metabolic Surgery. It’s ability to provide a relatively more sustainable weight loss over a longer period of time, improvement in quality of life and weight-related comorbidities namely Type 2 Diabetes Mellitus are among a few to boast. However, as with any invasive procedure, Obesity and Metabolic Surgery is not free of possible complication risks, the main reason for which there has been a major push for improvements in efficacy and innovation for better and safer surgical weight loss options. In the era of “healthcare at your fingertips”, a simple Google search will be able to enlighten just anyone on the available options making it possible for an individual to “negotiate” or “bargain” with their surgeon before making a final decision. The four most well researched and documented weight loss procedures are Laparoscopic Sleeve Gastrectomy (LSG), Laparoscopic Roux-En-Y gastric Bypass (LRYGB), Biliopancreatic Diversion (BPD) and Laparoscopic Adjustable Gastric Banding (LAGB) (Buchwald et al. 2004). Look a little closer and you will not be able to turn away from being a tad bit curious about a few “new kids on the block” such as Laparoscopic Mini Gastric Bypass, Laparoscopic Sleeve Gastrectomy with Proximal Jejunal Bypass, Laparoscopic Banded Roux-En-Y Gastric Bypass and some of the less invasive endoluminal procedures including a recent FDA approved percutaneous aspiration device called Aspire Assist that has garnered much interest as well as criticism at the same time (Lee et al. 2014; Kumar 2016; Forssell & Norén 2015). Surgeons from all over the world, their institutions, and the supporting industry laud the excitement surrounding innovation in Obesity and Metabolic Surgery. A sentiment shared by consumers who perceive “new” as synonymous with improved.

However, the speed of which the “Innovation Train” has been clocking in recent years has made many weiry, causing them take a step back and look at the whole fiasco from a bird’s eye perspective. Would it not be more reasonable to delineate the mechanisms of existing bariatric procedures before new ones are pushed into the open? Better understanding of the relevant pathophysiology would allow design and redesign of future bariatric procedures to proceed and progress on a much more respectable and believable foundation. Walter Pories a world reknown, well respected and much loved Bariatric Surgeon with an flair for drawing brilliant cartoons about every day life as a surgeon, beautifully ilustrates this lack of understanding of the underlying mechanism of Obesity and Metabolic Surgery in a Cartoon (Figure 1).

While it is undeniable and irrefutable that surgery is a valid option for treatment of obesity and obesity related metabolic syndrome, our knowledge of how bariatric procedures work needs much more “work” in itself. Many of the so-called “published reports” pertaining to novel therapies boasts fantastic early outcome, with intermediate or long term data no where to be seen despite being in the market for more than 5 yrs. Poor outcome or unwarranted complications attributed to the rapid turnover of novel procedures especially those without rigorous or independent cross check mechanisms could pose a negative anddetrimental effect on health care access for patients suffering with obesity and obesity related diseases. For a tiny developing country in South East Asia country like Malaysia, the end effect of such a catastrophe could plummet our healthcare economy.

INTRODUCTION

Estimation of age at death of human is one of the four steps of biological identification (White et al. 2011). Documentation of age at death from unknown skeletons is very important because this data determines information about reason of death regardless of passed time after death (Kerley 1965). The investigation of estimation of chronologic age in cadaver, living human and skeletal remains has several methods (Ohtani & Yamamoto 2005; Ritz-Timme et al. 2000; Waite et al. 1999; Williams 2001) such as visual morphological method (Buckberry & Chamberlain 2002; Hanihara & Suzuki 1978; Listi & Manhein 2012; Stewart 1958; Watanabe & Terazawa 2006), histomorphometry method (Calixto et al. 2015; Lee et al. 2014; Stout & Paine 1992), CT-scan (El Morsi et al. 2015; Kellinghaus et al. 2010; Mühler et al. 2006; Tangmose et al. 2013), radiography (Bhise et al. 2012; Mateen et al. 2013; Pardeep et al. 2010), MRI (Tangmose et al. 2013; Tangmose et al. 2014; Vieth et al. 2014) and racemization of aspartic acid (Hare & Abelson 1968; Helfman & Bada 171975; Man et al. 1983; Masters et al. 1978; Yekkala et al. 2006). Radiology and visual morphological methods of dental and development of skeleton do not appropriate for adults (Ball 2002) because these methods have wide range of age (Ball 2002; Frost 1987; Ritz-Timme et al. 2000), large standard error of estimation (Frost 1987) and more closer biological age than chronological age (Ball 2002; Ohtani et al. 2002; Ritz-Timme et al. 2000).

In contrast, the histomorphometry method has been developed for the estimation of age at death (Ericksen 1991; Kerley 1965; Singh & Gunberg 1970). The histomorphometry method is a quantitative determination by using measurement of osteon morphology. Furthermore, age-estimating research based on various population groups with different bones has been extended. The sternal end of the fourth rib and anterior cortex of the femur were tested for use to as samples (Han et al. 2009; Kim et al. 2007) whereas, the previous study that conducted on clavicle, only focussed on Caucasian population (Stout & Paine 1992). Hence, we decided to review the histomorphometry method in clavicles.

HISTOMORPHOMETRY METHOD – A BRIEF HISTORY OF TIME

Histomorphometry, the quantitative study of histology, is a useful method to estimate age at death in the present and the ancient skeletal remains. This method has been used by physical anthropologists to determine skeletal age at death, health status and degree of preservation in modern (Kerley 1965) and archaeological (Ericksen 1991). This method reduces individual bias, reduces the level of experience for estimation of age (Frost 1987), is useful in constructing paleodemographic models and for forensic identifications when adult fragmentary skeletal remains are encountered (Stout & Paine 1992). Histological methods also provide smaller reported error of estimate values than morphological techniques. All histological aging methods are based upon the fact that the bone tissue is replaced or turned over throughout individual’s life. Living bone is a dynamic tissue that is constantly changing to meet daily demands.

In 1965, Kerley (1965) is the first researcher that determined age estimation method in human cortical bone from the microscopic examination by using cross-sections of long bone diaphysis. This method implies osteons counting, old osteons fragments and non-haversian canals and evaluation of the percentage of circumferential lamellar bone remaining in four circular visual fields located at the periosteal margin of the cortex. Several histological methods have been developed for estimation age at death in skeletons (Ericksen 1991; Kerley 1965; Kerley & Ubelaker 1978; Singh & Gunberg 1970; Stout & Paine 1992). The histological methods for age estimation get acceptant for useful in forensic and physical anthropology because these methods have greater accuracy of age estimation for older skeletal remains that age over 50 yrs than the conventional gross morphological methods.

Most current age estimation by histological methods depend on increasing the number of osteons and their fragments, osteon population density (OPD), with age (Stout & Paine 1992). In 1992, Stout and Paine (Stout & Paine 1992) described a method for estimating skeletal age from histology of the rib and clavicle, which may be more available for thin-sectioning since they are not typically used for standard anthropological estimations. Moreover, non-remodelled bone can predict relationship with advancing age (Maat et al. 2006). Young people have greater amount of lamellar (non-remodeled) bone and non-haversian canals than their older people. Increasing age, the non-remodeled bone in young persons is replaced with mature Haversian systems including osteons, lamellae and fragments of osteons. Thus, histomorphometric factors such as the percentage of non-remodeled bone (Maat et al. 2006) and the total number of osteons (Kerley 1965) present strong correlations with age (Kerley 1965; Singh & Gunberg 1970).

Several histological methods for age estimation have been developed in archeological and forensic skeletal remains (Crescimanno & Stout 2012; Keough et al. 2009; Kerley 1965; Lee et al. 2014) because these methods increase the number of intact and fragmentary osteons in the identified fields or per measured area with age. It is important to use the measurement of osteons for estimate age at death in elderly individuals who present high osteon densities. Numerous measurements of osteons have been studied in recent years (Britz et al. 2009; Crescimanno & Stout 2012; Dominguez & Crowder 2012; Han et al. 2009; Keough et al. 2009; Lee et al. 2014), and have been reported to differ with age. An age relate to decreasing of osteon size in human has been repeatedly observed in several researchers (Pfeiffer 1998; Singh & Gunberg 1970; Thompson 1980) and nonhuman primates, macaques, has been found the decrease in osteon size like a human (Burr 1992; Havill 2004).

However, several researchers attended to the relationship between osteon size and age, but few studies attended to the relationship between osteon shape and age. Moreover, osteon circularity (On.Cr) are studied. The results are limited by qualitative strain effect (Skedros et al. 1994) and location in cortex (Pfeiffer 1998). Currey (1964) reported that the osteons of elderly individuals are nearly circular shape, but younger individuals have more irregularly shape of osteons. Another study, Britz and colleagues (Britz et al. 2009) found that circularity of osteons of femur increased with age. Moreover, circularity of osteons has been used like a variable for evaluate species identification due to human osteons have less circular than non-human species (Crescimanno & Stout 2012; Dominguez & Crowder 2012). Moreover, Narasaki (1990) applied Thompson’s core technique to femoral samples of modern Japanese cadavers for testing the reliability and for establishing the Japanese population equations. In his study, the multiple correlation coefficients were not high, compared with other methods (Kerley 1965; Singh & Gunberg 1970; Stout & Paine 1992). It is claimed that age estimation equations for specific population should be established.

ANATOMY OF CLAVICLE AND ITS FUNCTIONS

The human clavicle (collar bone) is a paired-long bone and only set in horizontal plane (Moore et al. 2011) that starts from base of neck to the shoulder and locates on first rib (Scheuer et al. 2000). The shaft of clavicle has S-curved bone (Moore et al. 2011; Scheuer et al. 2000; White et al. 2011) that has medial end (sternal end) and lateral end (acromial end) (Moore et al. 2011; White et al. 2011). The medial half of clavicle is anteriorly convex and the lateral half is anteriorly concave (Moore et al. 2011). Medial end has rounded and diverged shaped like a trumpet and lateral end has flatten shaped in superoinferior (White et al. 2011). Medial end of clavicle articulates with clavicular notch of the manubrium of the sternum at sternoclavicular (SC) joint and lateral end of clavicle articulates with acromial process of scapular at acromioclavicular (AC) joint (Moore et al. 2011; Scheuer et al. 2000; White et al. 2011). The clavicle has several functions: (i) it is rigid base for muscle attachments, (ii) it is holding for glenohumeral joint and it increases range of motion of shoulder joint, (iii) it increases power of arm-trunk mechanism and (iv) it provides a protection for major vessels at the base of neck and vessels and nerves passing to the upper limb (Moseley 1968). Moreover, the roles of clavicle are transmission load along its long axis from upper extremity to the thorax (Harrington et al. 1993).

The clavicle has several clear benefits. First, the size determines the relatively high ratio of compact bone to spongy bone, so there is less possibility of damage to the clavicle during a fracture. Second, the bone is likely to be found entire preferably than in fragments. Third, the clavicle is not a weight-bearing bone, so few factors, including daily lifestyle, can affect the bone remodeling rate over time. Finally, the clavicle does not influence other anthropological tests (Stout & Paine 1992).

DEVELOPMENT OF CLAVICLE

In the human embryo, the clavicle and the mandible are the first bones that begin to ossification. Both bones begin in membrane and follow by secondarily develop growth cartilages. The development of bones and joints at prenatal period have been reviewed by Gardner (Gardner 1956). Human development of embryonic period was separated into 23 stages (the first 7 to 8 weeks of human embryonic development). A number of external and internal characteristics, shape and size, degree of differentiation and organization of several tissue and organs are used for identify each stages of human embryonic development. Thus, it can discuss and compare differentiation and growth in each embryo stage.

The early part of prenatal life, primary ossification in the human skeleton has been investigated by the best method that is histological examination of serial sections. Since ovulation, the human embryonic period composes of the first 7 to 8 weeks of development. At the end of this period, differentiation is almost completed, and the bones and the joints have a form and an preparation characteristic of adult (Gardner 1968).

The clavicle, like most membrane bones, initiates to form during the embryonic period. Firstly, the fibrocellular proliferation, blastema, which extends from the locality of the scapula in downward, forward and medially direction to the midline region. The blastema which forms clavicle seperates from the blastema which forms all other bones of the upper limb and sternum. The clavicle blastema forms in stages 17, 18 or 19 that form about five postovulatory weeks (O’Rahilly & Gardner 1972).

Soon after the blastema appears, an organic matrix is formed by blastema and is mineralized nearly as soon as it forms and bonds the connective tissue fibers together. This process shows intramembranous ossification. The bone forms continuously to grow like the cells that surround it become osteoblasts which continue to proliferate, forming fibers and matrix. During stages 18, 19 or 20 (about 5 post-ovulatory weeks on), the clavicle ossification usually begins like 2 separate centers, a medial and a lateral (O’Rahilly & Gardner 1972). The two centers of clavicle ossification are preceded by hypertrophy of the blastema cells in the beginning ossification areas and by increasing the amount of intercellular matrix. The two centers of clavicle ossification unify to form a long mass of bone that is more solid than trabeculae bone like other membrane bones (Andersen 1963; Gardner 1956; Hanson 1920).

Osteoblasts are the first cells in the two centers of ossification. These cells are formed by the cell that surround the centers. However, the cells on the acromial aspect of the lateral center and the cells on the sternum aspect of the medial center remain chondrogenous. The cartilage forming cells of the lateral center combine with densely packed mesenchymal cells area in which the forming of acromioclavicular joint. Similarly, the cartilage forming cells on the end of the medial center combine with densely packed mesenchymal cells are in which the forming of sternoclavicular joint. In the stages 20 to 21, the chondrogenous cells on lateral and medial ends of the clavicle have formed cartilage that are larger cells and less intercellular matrix than the hyaline cartilage and the chondrogenous activity at the sternal end of clavicle is more noticeable. The forming of blood, vascular elements, osteoblasts and osteoclasts are formed by immediate differentiation of incomplete cells in stages 20 to 21 too. The bone is destroyed, but a marrow cavity begins to be formed. Later, the clavicle grows like a long bone of the cartilage type. It increased in diameter by periosteal that is intramembranous ossification and increased in length through the activity of cartilaginous ends (Gardner 1968). By the end of the embryonic period, stage 23 (7 to 8 postovulatory weeks), the clavicle is surely S-shaped and is alike the form and relationships of adult. It has reached both the acromion and the sternum, usually has an early marrow cavity and grows like a typical cartilage bone (Gardner 1968).

The human foetal period, prenatal period, is started from the end of the 2nd month until term. The bones and joints are characterized by growth, maturation, remodeling and reconstruction process for a maintain its characteristic shape of the bone. In the normal skeleton, the increasing growth in personal bones bear accurate relationship to those skeletons as a whole. Ligaments become more collagenous, appearing of synovial fat pads, bursa development, tendinous attachments to bone move to adapt to growth and vascular epiphyseal cartilages (Gardner 1968).

Soon after the beginning of foetal period, the central invasive process in the clavicle reaches to the cartilaginous ends and cartilage cells calcify and form growth zones like the diaphysis aspects of epiphyseal plates. The clavicular growth zone, however, unusually shows the arrangement of zones like in other long bones. After beginning of epiphyseal ossification, articular cartilage form the growth zones. The hypertrophy of cartilage cells tends to be arranged irregularly more than longitudinal columns. The invaded cells from the marrow destroy the hypertrophied cartilage cells and place down bone around the fraction of calcified cartilage matrix masses. The majority forming of endochondral trabeculae is destroyed almost rapidly, so the relatively few are found in the marrow space (Gardner, 1968).

After beginning of bone remodeling, the pattern of growth of the clavicle is like other long bones. The bony surface of the shaft that near the growth zone is removed and the remnants of endrochondral can be found in the compacta. The remodeling in clavicle, however, is not noticeable like other bones because the diameters at the levels of the growth zones are not much greater than the center of the shaft. Throughout the foetal period, the clavicle is markedly S-shaped and has a thick, trabeculae compacta and a small marrow space (Gardner 1968).

The foetal period, the clavicle is vascularized by peripheral vessels. Hence, this vascularization begins formerly any occurring of secondary or epiphyseal ossification. Finally, the perichondrium which caps each cartilage becomes fibrocartilage and serves as articular cartilage. Corrigan (1960) described the neonatal clavicle that is usually only one development of epiphyseal center. The epiphyseal center of sternal end appears during adolescence and fuses with the shaft by the 3rd decade. The epiphyseal center of acromial end appears during adolescence and quickly fuses with the shaft.

THE PROCESS OF CLAVICLE OSSIFICATION

Bone information has two different processes, intramembranous or endochondral ossification, that derived from primitive mesenchymal tissue (Karsenty et al. 2009; Kronenberg 2003). Intramembranous ossification initiates from primitive foetal mesenchyme that differentiate to osteoblasts and forming bone. On the other hand, endochondral ossification needs the formation of a cartilage model, or anlagen, in a process known as chondrogenesis, followed by bone formation. These processes encircle a series of events that start from differentiation of mesenchymal cell to chondrocytes. Then, cells proliferate and change their phenotype by increasing in volume as hypertrophy. Afterward, hypertrophic chondrocytes synthesize a calcified matrix before they have apoptosis. Finally, osteoblasts enter this mineralized scaffold for further mineral deposition and tissue maintenance (Ballock & O’Keefe 2003; Stevens et al. 1999; Zuscik et al. 2008).

The clavicle is intramembranous ossification that is not preceded by hyaline cartilage model but bone develops from the condensation of mesenchyme that forms an ossification center. The mesenchymal cells differentiate into osteoblasts, which produce the surrounding osteoid matrix. Many ossification centers are formed, anastomose and produce a network of spongy bone that composes of thin rod, plates and spines called trabeculae. The hemopoietic tissue located between trabeculae. The osteoblasts are encircled by bone in the lacunae and become osteophyte. The osteocytes are trapped in the lacunae and they form a complex cell-to-cell connection through canaliculi. The other bones that are formed by intramembranous ossification are mandible, maxilla, and the most flat bones of skull (Telser et al. 2007).

The clavicle is a long bone, but it is difference from other long bones. The clavicle is the first bone that starts process of bone ossification but it is the last bone that completely fuses (Gardner 1968; Kumar et al. 1989; Ogata & Uhthoff 1990). The clavicle has not medullary cavity like other long bones and it is the first foetal bone to get ossification by only membranous ossification like other long bones (Kumar et al. 1989; Ogden et al. 1979; Scheuer & Blach 2004). The clavicle of human has been reported to complete fusing this bone that approximate 25 years old (Ogata & Uhthoff 1990). The ossification initially starts with two primary ossification centers from medial end and lateral end that fuse together in shaft of clavicle during 5th and 6th foetal week (Kumar et al. 1989; Moore et al. 2011; Ogden et al. 1979; Scheuer & Blach 2004). In 5th week, ossification center of clavicle presents primary center of ossification; medial and lateral centers; at shaft of clavicle and fuse in 6th week of development of embryo (Kumar et al. 1989; Ogden et al. 1979; Scheuer & Blach 2004). Then, secondary ossification begins develop at sternal end and acromial end from mesenchymal tissue. These cartilaginous masses permit growth by endochondral ossification. Endochondral ossification of medial cartilage mass at sternal end of clavicle maintains longitudinal growth of clavicle and intramembranous ossification of periosteum increase diameter of clavicle (Gardner 1968; Glenister 1976; Ogden et al. 1979). The secondary cartilage ossification shows configuration of epiphysis with different histological areas: reserve zone I, proliferation II, hypertrophy III, calcification IV, and vascular invasion V (Brighton 1984). Secondary ossification centers of longitudinal growth of clavicle show different pattern from other major long bones of the limb (Gardner, 1968; Ogata & Uhthoff 1990). Moreover, the ossification process of clavicle is slow maturation (Scheuer & Blach 2004). A secondary ossification center at the sternal end begins to fuse with the shaft of clavicle between age 18 and 25 years old and completely fused at age between 25 and 31 years old. The acromial end of clavicle may appear smaller scale-like epiphysis and it is not a fracture of clavicle. Failure of fusion of two ossification centers of clavicle cause bony defect of clavicle (Moore et al. 2011). Therefore, the clavicle is the first bone ossification and the last fused epiphysis bone.

The morphology of adult human clavicle (S-curve bone) is finished early in foetal period before birth (Black & Scheuer 1996). Length of clavicle in males and females create 80% of total length of clavicle in 12 and 9 years old, respectively (McGraw et al. 2009). The skeletal growth of clavicle has longer period, so clavicle may be response to several mechanical loading and shear stress. The various factors that may affect asymmetry clavicle feature are several mechanical loading, asymmetric vascularization, lateralized behavior, activity-induced changes or more stress loadings of the dominant hand side of the clavicle (Mays et al. 1999).

BONE REMODELING

The bone remodeling is a livelong process (Kini & Nandeesh 2012) that removes the part of old bone and replaces it with the newly bone including the matrix (Fernandez-Tresquerres- Hernández-Gil et al. 2006). In normal activity, this process repairs the microscopic damage of the bone and prevents accumulation of damage in the bone (Martin 1998; Turner 1998) Bone remodeling react to mechanical loading. Moreover, this process is important to bone strength preservation and mineral homeostasis (Kini & Nandeesh 2012). Bone remodeling cycle of normal bone, osteoclasts (bone resorption) and osteoblasts (bone formation) work respectively together (Martin et al. 1998). The average remodeling process is about 2 to 8 months, majority time is bone formation. The third decade of life has the maximum bone mass and balance of bone remodeling process until the 50 years old. Afterward, bone mass begins to decrease and more resorption process especially perimenopausal and early postmenopausal women (Kini & Nandeesh 2012).

REMODELING PHASES

Bone remodeling can be separate to the following six phases that are quiescent, activation, resorption, reversal, formation, and mineralization. The first step is activation followed by resorption, reversal and the last step is mineralization. This process is distributed randomly, but it targeted to repair areas (Burr 2002; Fernandez-Tresquerres-Hernández-Gil et al. 2006).

Firstly, the bone cells are lining in endosteal membrane that is the rest state (quiescent phase) follow by resorption of membrane lead to bone cells retraction (activation phase). Then, osteoclasts begin to dissolve the mineral matrix and decompose the osteoid matrix for resorb the bone (resorption phase). The resorption of osteoclasts creates irregular scalloped cavities on the trabecular bone surface, called Howship’s lacunae, or cylindrical Haversian canals in cortical bone. This phase of each bone remodeling cycle takes the time about 2–4 weeks. Afterwards, the bone resorption transition to bone formation (reversal phase) and osteoblast come into replace osteoclasts for bone formation (formation phase). The last phase, osteoid matrix is mineralized (mineralization phase) that occurs after osteoid deposition 30 days and complete at 90 and 130 days after osteoid deposition for trabeculae and cortical bone, respectively. This cycle begins the quiescent phase again. Finally, the amount of bone formation and bone resorption should equal when completed cycle of bone remodeling.

APPLICATION IN FORENSIC

For clavicle, the study of Stout and Paine in (1992) was the first study in clavicle. This study studied age estimation by histomorphometry of mid-shaft of left clavicle and middle third of the left sixth rib in whites, black, and unknown ethnics from 40 autopsy cases. The slides had 75μm thicknesses and were analyzed by microscopic analysis. The researchers measured cortical area, intact osteon density (pi), fragmentary osteon density (pf) and total visible osteon density (pi + pf). Age-predicting model for clavicle used total visible osteon density (pi + pf) as the independent variable. The results showed the differences between actual age and predicted age range from -8.1 to +20.6 years for the clavicle, -2.7 to +9 years for the rib, and -2.5 to +14.5years for the combined rib and clavicle formula. The mean difference were 1.1 (± 3.57) year for clavicle, 3.4 (± 1.06) year for rib, and 2.6 (± 2.20) year for combined rib and clavicle formula. The r-square value of age-predicting model (r2) were 0.699, 0.7211, and 0.7762, respectively. The benefit of clavicle and rib is available bone when the long bone cannot use for age estimation. The histimorphometry method can occur error from sampling sample so this study used two entire cross-sections of clavicle and rib and read every field per section or alternative reading of each field for avoid error. Moreover, the benefit of this procedure is ability to adjust location of microscopic field for reading any section. The combined clavicle and rib formula was recommended because this formula had slightly high standard error and r2. Therefore, the method of clavicle and rib should be appropriate for demographic studies and forensic identification.

In 2014, Lee and co-workers (Lee et al. 2014) studied age estimation by histomorphometry of clavicle in Korean. The study used right clavicle from 46 dissected cadavers and cut from the sternal end of the right clavicle (length 3 cm) and made two sequential slides (100-ìm thickness). The researchers measured ratio of the relative cortical area (RCA), osteon population density (OPD), and mean osteon area (OA) were microscopic measured and counted by using an image analysis program (Image J). The results showed negative correlation in RCA and OA and positive correlation in OPD from simple linear regression. The highest correlation between estimated age (R2 = 0.583) was OPD follow by RCA (R2 = 0.274) and OA (R2 = 0.100). The OPD and RCA were selected for the age-predicting equation from the multiple regression analysis using the stepwise method (R2 = 0.628). Difference of sex, RCA showed significantly different between sexes (p = 0.000). From the results, RCA should be measured by separate sexes.However, previous studies reported no significant relationship between bone composition and sex (Kerley 1965; Ericksen 1991), so this study do not separate data into male and female parts for equation, although sex was studied in RCA measurement. For OA, this study showed moderately unrelated with estimated age that contrast to the study of Osborne and co-workers (Osborne et al. 2004). OA was insufficient for reflect the remodeling rate. In further study, the researcher suggested that unremodeled area should be applied for age estimation by histomorphometry. Comparing result, this study had regression correlation result (R2 = 0.628) as well as Stout and Paine in 1992 (R2 = 0.699). However, difference of population group had different results. Equation for age estimation by histomorphometry in clavicle can be used for especially each population.

In 2015, Sobol and co-workers (Sobol et al. 2015) studied age estimation by histomorphometry of clavicle from fresh cadavers from autopsy in Poland. The study used fragments of shafts of left clavicles taken from 39 males and 25 females. They measured clavicle length (CL), clavicle width (CW), clavicle thickness (CT), number of osteons in the field of vision (ON), number of osteons with the Haversian canal of more than 70 μm (HC > 70 μm), average diameter of the Haversian canals (avg. ØHC), area occupied by interstitial lamellae (ILA %), area occupied by osteons (OA %), area occupied by fragments-remnants of osteons remain as irregular arcs of lamellar fragments (OFA %), average thickness of outer circumferential lamellae (avg. OCL, μm), and the relation of osteons with the Haversian canal of more than 70 μm in diameter to the total number of osteons (HC > 70 μm, %). The age of the bone remains was estimated using linear regression equation. The results showed the shaft of clavicle was the best field for analysis of bone tissue because shaft of the clavicle was most distant from the muscle and ligament attachment (Ingraham 2004) and affected by only subclavian muscle. This area showed the number of osteons with a large diameter (HC > 70 μm) increased with age because osteoclast activity of the resorption of osteon’s lamellae that had highest correlation (R2 = 0.87).

CONCLUSION

The histomorphometry methods can estimate age at death in skeletal remains based on morphology of osteon. Using different area of the same bone, different populations, including different procedures for section and analysis showed different results. Clavicle is the one of interesting bones for age estimation by using histomorphometry method because this bone is non-weight bearing bone that has only subclavian muscle affected mechanical loading. Moreover, it had less possibility of damage from fracture, found entire bone, and had few studies. Thus, the histomorphometry method from clavicle is very useful method for age estimation.

ACKNOWLEDGEMENTS

The authors are grateful to Excellence Center in Osteology Research and Training Center (ORTC), Chiang Mai University for their financial contribution and support. The authors also express their acknowledgement and gratitude to Prof. Srijit Das for the technical help in editing the manuscript.

INTRODUCTION

Osteoarthritis (OA) is a group of degenerative joint diseases (DJD) and one of the most common disorders worldwide which leads to pain and disability in the elderly (Arden & Nevitt 2006; Garstang & Stitik 2006). Osteoarthritis is considered by chronic loss of hyaline cartilage at the joint articulation and osteophytes formation in the affected joints which leads to progressive pain and limitation of movement and function in aged individuals (Gahunia et al. 1995; Hayashi et al. 2012; Henrotin 2012). The risk factors associated with OA include age, sex, history of joint injury, obesity, genetic factors, and mechanical factors, containing abnormal joint structure and malalignment (Garstang & Stitik 2006). The incidence of OA continues to increase with age and the prevalence of osteoarthritis is greater in men than in women in most joints before 50 yrs of age. Furthermore after 50 yrs, women are more often affected than men, especially in the hand, foot, and knee osteoarthritis (Van der Kraan 2012). Osteoarthritis can be defined by pathology of joint structure comprising chronic loss of hyaline articular cartilage, changes in the subchondral bone; the bone under the cartilage, development of osteophyte and increased bone sclerosis and thickness of the bone under the cartilage (Shane Anderson & Loeser 2010; Gahunia et al. 1995).

Not only change of joint structure, the connective tissue surrounding the joint can be affected. The synovial membrane has increase in 26inflammatory cytokines. The ligaments are often lax and there is muscle weakness. Currently, the diagnosis of OA comprises both clinical appearance and structural features. Plain film radiograph is widely used as a gold standard to define the damaged articular cartilage (Gahunia et al. 1995). The common radiographic features including decrease joint space, formation of osteophyte, subchondral thickness or sclerosis and abnormalities of bone density.

The etiology of osteoarthritis is not well understood and has multifactorial association (Garstang & Stitik 2006; Henrotin 2012; Hochberg 2012). Age is the most important risk factor of osteoarthritis and the relationship between aging and OA is well known but the mechanisms are still not fully understood (Loeser 2010). At the cell and tissue level, articular cartilage consist of few cells and highly surrounding extracellular matrix (ECM). Chondrocyte is present in articular cartilage and involved in maintaining the balance of matrix synthesis and degradation (Goggs et al. 2003). Many previous studies observed the chondrocyte death related to progressive loss of hyaline articular cartilage which report an increased amount of apoptotic program cell death (Aigner et al. 2001; Almonte-Becerril et al. 2010; Blanco et al. 1998; Dang & Kim 2009). Thomas investigated the incidence of chondrocyte apoptosis in equine articular cartilage specimens and observed the relationship between the process of chondrocyte death and the degree of cartilage degradation. They demonstrated that chondrocyte apoptosis is associated with degree of cartilage matrix damage and mechanical loading environment of the joint is a significant positive correlation between severity of OA and apoptosis (Thomas et al. 2007). Chondrocyte death has been observed during the development of OA but whether this is an early or late event is still unclear. Therefore, the breakdown of cartilage during the OA pathogenesis is related to chondrocytes death and the loss of ECM (Almonte-Becerril et al. 2010).

REACTIVE OXYGEN SPECIES (ROS)

Reactive oxygen species (ROS) are free radicals, which play an important role in the aging process in both human and animal models (Afonso et al. 2007; Ziskoven et al. 2010). There are group of free radicals oxygen molecules such as hydroxyl group (OH-), hydrogen peroxide (H2O2), superoxide anion (O-2) and nitric oxide (NO) (Henrotin et al. 2003; Lepetsos & Papavassiliou 2016). In the articular cartilage, chondrocytes generate several forms of ROS such as superoxide, hydrogen peroxide, reactive nitrogen species and nitric oxide. In normal process, there are various anti-oxidants which control the levels of ROS (Biemond et al. 1984). There are antioxidant system in the human body which include enzymatic and non-enzymatic antioxidants, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidise (GPX), glutathione (GSH), ascorbic acid (vitamin C), α-tocopherol (vitamin E) and carotinoids.

Oxidative stress can also disturbance in the antioxidant defenses mechanism against the level of ROS, which results in cellular damage and disruption of redox homeostasis leading to chondrocyte senescent by causing destruction of proteins, lipids, and DNA directly (Henrotin et al. 2005). Increase in ROS level is related to aging process and plays an important role in the progression of OA. There are various increasing inflammatory cytokines in OA such as IL-1, IL-6 and TNF-α, which encourage production of ROS as a result of increased level of MMPs (Loeser 2009; Loeser 2010).

THE DIAGNOSIS OF OSTEOARTHRITIS

Kellgren and Lawrence in 1957 were the first to use radiographic grading system using plain film radiograph. Nowadays it is the gold-standard for identifying the structural changes in joint structure in patient with OA (Altman & Gold 2007). Radiographic methods of knee OA have usually assessed the disease progression, its least expensive method for detecting joint structural change in patients with OA. The severity of disease normally determined by using the Kellgren and Lawrence grading system, the most widely used and accepted standard for diagnosis of radiographic OA. The grading system consists of 4 grade scales, for grade of 0 suggests that no structural changes of OA and grade 4 is defined as the severe one. The grading scale has been analyzed for illustrating the progression of OA comprising both osteophyte formation and decreased in joint space. PATHOGENESIS OF OSTEOARTHRITIS

Typical characteristics of osteoarthritis are the progressive loss and damage of hyaline articular cartilage in the articulation in the affected joint. The homeostasis of cartilage matrix synthesis and degradation are the main causes of disease progression (Aigner et al. 2001). The mechanism of matrix degradation is still unclear but associated with both environmental and genetic factors. There are many factors related the disease such as the change of metabolic, biochemical and metabolic homeostasis. In OA cartilage, not only the chondrocytes are produced many cytokines and growth factors but also the synovial tissue. On the anabolic process, the synthesis of extracellular matrix is stimulated by insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-b, fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) (Aigner et al. 2001). From the catabolic process, Matrix metalloproteinase (MMP) is increased and produced the inflammatory cytokines including interleukin (IL)-1, IL-17 and IL-18 and tumor necrosis factor (TNF)-a, which leads to decrease of aggrecan and matrix components, as a result of cartilage matrix degradation (Afonso et al. 2007; Blanco et al. 1998; Loeser 2010).

There are biochemical and structural changes in OA cartilage including The increasing of water content and the matrix macro molecules such as aggrecan can be decrease. The structure of the collagen is damaged, which leads to reduced stiffness and the cartilage more brittle.

BIOLOGICAL MARKERS ASSOCIATED OSTEOARTHRITISGLUTATHIONE (GSH)

Nowadays, many studies focused on damaging effects of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) in osteoarthritis (Valko et al. 2007; Ziskoven et al. 2010). Glutathione (GSH) is one of an endogenous important intracellular anti-oxidant, plays a role in the detoxification of oxygen free radicals. The increasing of ROS levels, the ratio of oxidized to reduced glutathione is changed (Loeser 2010). Glutathione is performed in the cytosol of various cell types and delivered to extracellular matrix and other organelles for example, mitochondria, endoplasmic reticulum and the nucleus (Bertrand et al. 2010; Blanco et al. 2004). GSH occurs not only the reduced state (GSH) but also oxidized state (GSSG). The ratio of GSH/GSSG is necessary to maintain normal functioning and is a good measure of oxidative stress of a tissue and organism (Regan et al. 2008). The protective effect of glutathione against the oxidative stress such as the glutathione is a importance co-factor of numerous enzymes for against oxidative stress, including glutathione peroxidase (GPx), glutathione transferase and others. There are previous studies about glutathione associated osteoarthritis as shown in Table 1. The glutathione represents the major cellular redox buffer and therefore is a representative indicator for the redox environment of the cell.

GLYCOSAMINOGLYCANS (GAGS)

Glycosaminoglycan (GAG) are one of the most important components of extracellular matrix (ECM) and play multiple roles in different tissues and organs, associated with other pathological conditions such as osteoarthritis, inflammation, diabetes mellitus, spinal cord injury and cancer. GAGs are polysaccharides, devided in five major groups including: chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS) and hyaluronan (HA) (Studelska et al. 2006). The mechanism of extracellular matrix degradation in joint diseases is not fully understood but the main factor associated is the reactive oxygen species (ROS). An increased level of ROS as a result of decreased levels of GSH leading to reduce the synthesis component of the articular cartilage, which are proteoglycan and hyaluronic acid. Inerot et al. (1978) investigated the structural change of proteoglycans of aging cartilage in the degenerated cartilage of osteoarthritis, found that proteoglycans from the degenerated cartilage were smaller than those from the same-age normal cartilage and had partially lost their ability to bind to hyaluronic acid and form aggregates (Inerot et al. 1978). The recent studies about glycosaminoglycans associated osteoarthritis as shown in Table 1. The structural and composition changes of glycosaminoglycan in OA cartilages could be the result of changes in both the catabolic and anabolic process of chondrocytes during the progression of the disease.

ADVANCED GLYCATION END PRODUCTS (AGES)

Advanced glycation end products (AGEs) are the macromolecules and formed in body during the process of normal aging, suggesting the association with development of osteoarthritis. AGEs produces from non-enzymatic glycation of proteins and lipids leading to formation of collagen cross-links (Nah et al. 2007; Yang et al. 2015) affects the structural properties of cartilage as a result of increased stiffness and brittle in the hyaline articular cartilage (Chen et al. 2005). Increasing of AGEs in cartilage involve in cellular activities such as bone remodeling process, which increase of abnormal proliferation, differentiation, and apoptosis of bone cells, such as osteoclasts (OCs), osteoblasts, and osteocytes (Yaffe et al. 2011; Yang et al. 2015). DeGroot et al. (2001) investigated the relationship between accumulation of AGE levels and chondrocyte turn over of proteoglycans in articular cartilage of human. They found that the accumulation of AGE increased with aged and its has negatively affects synthesis and degradation of proteoglycan as a result of development of OA (DeGroot et al. 2001). It has been suggested that, increased of advanced glycation end products (AGEs) in cartilage related to aging and responsible for decreased the ability of the cartilage to remodel its extracellular matrix, predisposing factor for the development of cartilage damage in OA (Viguet-Carrin et al. 2008). The previous studies of AGEs associated osteoarthritis as shown in Table 1.

CONCLUSION

Osteoarthritis is considered as a group of degenerative disorder, which leads to pain and limitation of functional movement in affected joint. The etiology and mechanism are still unclear but associated with many factors.In the cellular level, there are many cytokines and growth factors involve in the mechanism of osteoarthritis. In this review, we presented the literature review of osteoarthritis and biological marker associated the pathology.

INTRODUCTION

Iron is mandatory for normal foetal development, including the brain. Antenatal iron deficiency may have deleterious effects on intelligence and behavioral development of the offspring. Therefore, it is important to prevent foetal iron deficiency by preventing maternal iron deficiency. Besides, iron deficiency anemia is a risk factor for preterm birth and low birth weight (Milman 2006).

In Malaysia, routine antenatal iron supplementation is the norm but the practice is not evidence based due to lack of current data on the prevalence of iron deficiency. The most recently published data on anemia in pregnancy in Malaysia dates back 15 yrs ago and only surveyed rural areas of one of the poorer states (Zulkifli et al. 1997).

On the other hand, iron supplementation has a negative influence on absorption of other divalent metals and increases oxidative stress in pregnancy, for which reason the minimum effective iron dose should be advised in pregnancy (Milman 2006).

In many parts of Malaysia, urbanization has taken place and standards of education and living have improved. As such, the current socio-economic and health status have improved with declining prevalence of grandmultiparity and maternal mortality rates (Wong 1999). It is therefore possible that the country mayhave joined the ranks of developed nations in issues as basic as iron deficiency and anemia in pregnancy. The present study therefore aimed to assess the current antenatal iron status in Malaysia.

MATERIALS AND METHODS

A cross-sectional study was conducted over a six-month period on pregnant women who booked for antenatal care at an urban community health clinic located within a kilometer of a state hospital. A single blood sample was drawn from each subject and sent for laboratory assessment of full blood count and serum ferritin at the time of booking to assess for maternal anemia and iron status. All laboratory assays were conducted at the hematology and biochemistry laboratories of the state hospital.

Iron deficiency is defined as serum ferritin < 12 μg/l (Brittenham et al. 1981). Anemia is defined as hemoglobin < 11g/dl (World Health Organization 1968).

Based on Kish’s formula for prevalence study (Kish 1965)

where P = 0.212, D = 0.05 and Z1-α = Z0.95 = 1.96 (normal distribution table), the sample size required was 256.7, i.e. 257. Quota sampling was applied. Women with known chronic illness such as systemic lupus erythematosus, renal disease, thalassaemia or any other blood disorder, and those who refused to participate in the study, were excluded. All data obtained were analyzed using SPSS version 19.0.

RESULTS

A total of 250 pregnant mothers participated in the study, i.e. the response rate was 97.3% (250/257), of which 149 (59.6%) were Malays, 39 (15.6%) Chinese, 47 (18.8%) Indians, and 15 (6%) were of other ethnicities (Table 1). Out of the total number, 247 (98.8%) were married and 3 (1.2%) were single. Their age ranged from 20 - 49 yrs with a mean of 30.24 ± 1.16 yrs (mean ± standard deviation, SD). Most of the women (178, 71.2%) attained secondary school education, followed by 37 (14.8%) with tertiary education, 27 (10.8%) with primary education, and a minority (8, 3.2%) without any formal education. Most women (81.2%) were in the poverty bracket, with a family income of less than RM1000 per month. Majority of the women were primigravidae (33.6%), followed by gravidae 2, 3 and 4 (23.6%, 15.2% and 10.8% respectively), whilst 42 (16.8%) were grandmultiparae. Forty-two percent commenced antenatal care during the first trimester, 54.8% during the second trimester and 3.2% during the third trimester. Only 2% were multiple pregnancies and 1.6% vegetarian. Most women (77.6%) did not take vitamin supplements prior to pregnancy. Only 13.6% practiced contraception.

The overall prevalence of anaemia was 43.6% whilst the prevalence of iron deficiency was 31.6% (Table 2). The prevalence of anaemia and iron deficiency was highest among the Indians (46.8% and 42.5%, respectively), followed by the Malays (43.6% and 33.5%, respectively) and the Chinese (38.4% and 12.8%, respectively). Interestingly, overall, 19.1% of women with normal hemoglobin levels had iron deficiency (Table 2).

The overall mean hemoglobin was 11.00 ±±1.46 g/dl (mean ±±SEM) whereas the mean serum ferritin was 36.80 ±±2.26 μg/l. The mean serum ferritin was statistically significantly lower among Indians (27.17 ± 4.03 μg/l) compared to other ethnic groups (Table 1). Grandmultiparae had significantly lower serum ferritin (25.31 ±±3.99μμg/l) compared to women of lower parity. Late booking (in the third trimester) was associated with significantly lower serum ferritin (19.95 ±±6.80μμg/l) compared to earlier booking for antenatal care. Women with primary education had significantly lower serum ferritin (22.33 ±±7.85μμg/l) compared to other levels of education.

DISCUSSION

Anaemia and iron deficiency are most prevalent in Africa, the Middle East, Asia, and Western Pacific (de Benoist et al. 2008). In Africa, the prevalence of anaemia in pregnancy is about 57% (de Benoist et al. 2008). The exact prevalence of anaemia and iron deficiency varies from country to country, as does the cut-off values for hemoglobin and serum ferritin used in these countries. The contribution of iron deficiency to anaemia in each country also differs, depending on other locally prevalent causes (Berger et al. 2011). The prevalence of anaemia and iron deficiency in our population (43.6%) was similar to those found in the regions with the worst anaemia prevalence. In WHO terms, an anaemia prevalence of 40% and above, as seen in our study, is considered to be much severe public significance (World Health Organization 2012). It is obvious that the anaemia prevalence in Malaysia has not improved much for the past 15 yrs, as the prevalence amongst a rural population in Kelantan, one of the poorest states, was 47.5% in 1997 (Zulkifli et al. 1997). More recently published work on anaemia in Malaysia supports this perception, with prevalence rates ranging between 33% (Soh et al. 2015) and 57.4% (Nik Rosmawati et al. 2012). This was emphasized by Milman (2015) in his recent review on the seriousness of the situation in Malaysia and certainly calls for urgent remedial measures nationwide.

The cut-off value of serum ferritin used in our study (12 μg/l) was low compared to some other studies. This level correlates with iron-deficient erythropoiesis, just before anaemia sets in at serum ferritin <10μμg/l (Coad & Conlon 2011). The normal serum ferritin level is 40-160μμg/l, and levels below 25μμg/l indicate early negative iron balance, whereas levels below 20μμg/l indicate depletion of iron storage (Coad & Conlon 2011). Therefore, the statistics obtained in our study of 31.6% was certainly alarming.

The overall mean hemoglobin was 11.00 ±±1.46 g/dl, which means that the mean hemoglobin in this population of pregnant women just scrapes through the WHO criteria for anaemia (World Health Organization 1968). The mean serum ferritin was 36.80 ± 2.26 μg/l, which is well below the level corresponding to normal iron stores (Coad & Conlon 2011). Looking at the groups with the worst levels, the figures are much more worrying. For example, the mean values of serum ferritin among Indians and grandmultiparae almost touch the level indicative of early negative iron balance, and the serum ferritin level of women who book late in the third trimester without prior antenatal care is below the level indicative of storage iron depletion.

Is the high prevalence of antenatal anaemia a reflection of anaemia prior to marriage and embarking on child bearing? An interesting study by Chang et al. in (2009) showed that the prevalence of anaemia among apparently healthy adolescent girls in an urban Malaysian population was 28.3%, which was significantly lower than the matching prevalence among women of childbearing age within the same population, 41.7%. Nevertheless, both the figures are way above the anaemia prevalence in industrialized countries. Anaemia amongst adolescent girls is logically a strong predisposing factor for the subsequent occurrence and worsening, of anaemia in adult women of child bearing age. Correcting anaemia amongst adolescent girls is indeed vital towards reducing the magnitude of the problem amongst adult women.

Evidence from current medical literature advises caution in prescribing routine iron supplements in pregnancy because of its negative influence on gastrointestinal absorption of other divalent metals such as calcium, and increased oxidative stress (Milman 2006). Nevertheless, the existing guidelines suggest that iron supplements should not be given only if the serum ferritin exceeds 70 μg/l (Milman 2006). Therefore, the observations obtained in this study provide a strong basis in favor of routine iron supplementation in pregnancy among the urban poor in Malaysia. For serum ferritin levels of 30-70 μg/l, the recommended daily dose is 40mg ferrous iron, whereas for women with levels below 30 μg/l as in the high risk groups observed in this study, a much higher dose of 80-100 mg ferrous iron daily, is advised. A case could be made to call for routine assessment of serum ferritin amongst the group of pregnant women who are at especially high risk of having the worst iron status. Patient education on the importance of compliance to iron supplements cannot be overemphasized, as reluctance of women to consume routine iron supplements in pregnancy is common knowledge, as a result of an unwavering myth that it might lead to macrosomia and a difficult delivery. This myth must certainly be dispelled if a programme of routine antenatal iron supplementation is to succeed.

CONCLUSION

In conclusion, anaemia and iron deficiency in pregnancy are still serious issues amongst the antenatal population in Malaysia. Findings from this study calls for imperative measures to ensure continued provision of routine iron supplement in pregnancy. Routine checks on serum ferritin levels in certain high risk groups that recorded markedly low serum ferritin in this study should be considered, so that the appropriate higher daily dose of antenatal iron could be prescribed in order not to compromise the short and long term health of the mother and baby.

ACKNOWLEDGMENT

The authors would like to thank the Ministry of Health Malaysia for allowing recruitment of patients during the course of this study and for providing the laboratory services necessary for the tests that were performed.

Dental caries are multifactorial diseases involving complex biological interactions of acidogenic bacteria, fermentable carbohydrates and host factors such as the teeth and saliva (Selwitz et al. 2007). The acidogenic bacterial species, Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) have been considered as the main causative agents of dental caries. Higher incidences of dental caries are recorded when these two bacteria co-exist (Okada et al. 2005; Nurelhuda et al. 2010).

The interest on botanical medicine as a source of antimicrobial agents are increasing globally. The emergence and re-emergence of infectious diseases, the increase of chemotherapeutic failure and antibiotic resistance exhibited by pathogenic infectious agents has led to the screening of several medicinal plants for their potential antimicrobial activity (Kumar et al. 2015). Euphorbia tirucalli or known as Tetulang, belongs to Euphorbiaceae family and it is one of the most important trees known worldwide for its multiple uses. It is an evergreen shrub or a small tree endemic to tropical areas with pencil-41like branches containing white latex (Mwine & Van Damme, 2011).

Various studies indicated that this plant is a valuable source of medicinal compounds. Researches showed that active compound such as alkaloids, tannins and phenols in Euphorbia tirucalli was contributed their effectiveness in medicinal treatment (Sugumar et al. 2010). Researchers found that flavonoids in Euphorbia tirucalli effectively inhibited the bacteria growth due to its ability to form complex with extracellular proteins of cell wall and disrupt microbial membrane, whereas phenolic and polyphenols was toxic to micro-organisms. They also found that tannins in Euphorbia tirucalli able to inhibit bacteria by inactivate microbial adhesion, enzymes and cell envelope transport proteins (Upadhyay et al. 2010). Traditionally, Euphorbias are used as purgative, analgesic, anti-oxidant, anti-inflammatory, antipyretic, anti-microbial, anti-parasitic, anti-arthritic in the treatment of cough, asthma, rheumatism, cancer and other maladies as folk remedies (Prabha et al. 2008; Upadhyay et al. 2010; Sugumar et al. 2010; Jahan et al. 2011; Priya & Rao 2011; Rathi et al. 2012; Gupta et al. 2013). The stem of Euphorbia tirucalli is used to treat whooping cough, asthma, and infections of the spleen. The alcoholic extract of the stem of Euphorbia tirucalli was reported to possess broad-spectrum antimicrobial activity against Escherichia coli, Proteus vulgaris, Salmonella enteritidis, Bacillus subtilis, Staphylococcus aureus, S.epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Xanthomonascitri, Candida albicans, C. tropicalis, C.glabreta, Aspergillus niger, A. fumigatus, A. flavus and Fusarium oxysporum (Jadhav et al. 2010; Prasad et al. 2011; Rathi et al. 2012; de Araújo et al. 2014; Muthukumar et al. 2014).

Hence, the present study was conducted to evaluate the in vitro antibacterial activity of Euphorbia tirucalli stem extracts (methanolic, ethanolic and water) against dental caries-related bacteria (S. mutans and S. sobrinus) using the agar-well diffusion method.

MATERIALS AND METHODSBACTERIAL STRAINS

Two oral bacterial strains used in this study, i.e. S. mutans (ATCC 25175) and S. sobrinus (ATCC 33478) were commercially obtained from the American Type Culture Collection (ATCC, USA). The bacteria were grown anaerobically at 37°C with 5% CO2 in Brain Heart Infusion broth (Oxoid, UK) for 48 hrs. All bacteria growths were confirmed by microscopy and gram staining.

COLLECTION OF PLANT MATERIAL

Fresh stems of Euphorbia tirucalli were collected from the local area of Kubang Kerian, Kelantan Malaysia. The taxonomic identity of the plant was confirmed by botanist from the Forest Biodiversity Department, Forest Research Institute Malaysia (FRIM) and the voucher number was PID 440915-23. The stem was thoroughly washed under running tap water, air-dried and subsequently dried in hot air oven (Protech, Malaysia)at 60oC for 48 hrs.

PREPARATION OF EUPHORBIA TIRUCALLI STEM EXTRACTS

Euphorbia tirucalli stem extracts (methanol, ethanol and aqueous) were used for evaluation of antimicrobial properties. Dried stems of Euphorbia tirucalli were homogenized to a fine powder using a blender (Panasonic, Malaysia) and stored in an air tight bottle. One gm of the powdered stem was mixed with 30 ml of each solventin an incubator shaker (Jeio Tech, Korea) at 37oC for 24 hrs. The mixture was then centrifuged for 1400 x g for 5 mins. The resulting extracts were filtered using filter paper (Whatman No. 1, USA) and each filtrate was concentrated with a rotary evaporator (Heidolph, Germany) at 60oC under vacuum condition. The concentrated supernatant was then deep frozen at -20°C for 24 hrs and freeze-dried to powder. All extracts were kept at 4°C until use. The final concentrations of 10, 20 and 30 mg/ml were prepared by dissolving the powder in the same solvent. AGAR-WELL DIFFUSION METHOD

The dried plant extracts were dissolved in the same solvents, to final concentrations of 10, 20 and 30 mg/ml and were sterilized by filtration through 0.45 μm Millipore filters (Nalgene, UK). The antibacterial activity of various extracts of Euphorbia tirucalli stem was analysed in vitro by an agar-well diffusion method according to Jain et al. (2015) with some modifications. Using a sterile cotton swab, an overnight bacterial suspension was swabbed on the surface of sterile Mueller Hinton Blood agar (MHBA) (Thermo scientific, USA). The plates were dried for 5 mins to allow any excess moisture to be absorbed. Five wells of about 6.0 mm diameter were aseptically punched on MHBA plates by the sterile cork borer. Fixed volumes (50 μl) of different concentrations of methanolic, ethanolic and aqueous extract were added into each well. Commercially available amoxicillin (10 μg) was used as positive control while appropriate solvent was served as negative control. These plates were allowed to stand for 5 mins for the diffusion of extract to take place. The plates were then incubated at 37°C for 48 hrs. Antibacterial activity was evaluated by measuring the zones of inhibition (clear zone around each well) in millimeter (mm) using a digital calliper (Mitaka, Japan). The experiment was done in triplicates for each concentration and organism.

DETERMINATION OF RELATIVE PERCENTAGE INHIBITION

The relative percentage inhibition of the Euphorbia tirucalli stem extracts with respect to positive control was calculated by using the following formula (Ajay et al. 2003):

100 x (X – Y)

(Z-Y)

Where,

X: total area of inhibition of the test extract

Y: total area of inhibition of the solvent

Z: total area of inhibition of the standard drug

The total area of the inhibition was calculated by using area=ππr2; where, r=radius of zone of inhibition.

STATISTICAL ANALYSIS

Results were expressed as Mean ± standard deviation. Statistical analysis was done using a statistical package, IBM SPSS Statistics version 22 (IBM, Chicago, IL, USA) by applying mean values using non-parametric Kruskal-Wallis test followed by Mann-Whitney test to determine if there was a significant difference among different E. tirucalli extracts. A p value of less than 0.05 was considered significant.

RESULTS

The results of the antibacterial activities of Euphorbia tirucalli extracts are shown in Figure 1. The methanolic and ethanolic extracts of Euphorbia tirucalli showed inhibitory effects against the growth of S. mutans and S. sobrinus at a concentration of 20 mg/ml and 30 mg/ml. Methanolic extract of Euphorbia tirucalli exhibited slightly higher inhibitory effect against S. mutans and S. sobrinus compared to ethanolic extract of Euphorbia tirucalli. The aqueous extract of Euphorbia tirucalli did not show any inhibitory effect against S. mutans and S. sobrinus at all concentrations tested.

The antibacterial activity of Euphorbia tirucalli methanol, ethanol and aqueous extracts were compared with the positive control amoxicillin (standard drugs) for evaluating their relative percentage inhibition (Figure 2). E. tirucalli methanolic stem extract exhibited relative percentage inhibition against S. mutans (9.44%) and S.sobrinus (8.51%) at 20 mg/ml while relative percentage inhibition against S. mutans (14.96%) and S.sobrinus (13.5%) at 30 mg/ml. While the ethanolic stem extract of Euphorbia tirucalli showed relative percentage inhibition against S. mutans (7.14%) and S. sobrinus (6.96%) while relative percentage inhibition at 30 mg/ml extraction concentration against S. mutans (11.10%) and S.sobrinus (10.21%).DISCUSSION

Recent interest in exploring the antimicrobial potential of wide variety natural source has encouraged more studies done in this area. This study was aimed to evaluate and compare the efficacy of different Euphorbia tirucalli stem extracts against oral pathogens implicated in dental caries.

Methanolic and ethanolic extracts, more polar extracts exhibited inhibitory effects against S. mutans and S. sobrinus at a concentration of 20 and 30 mg/ml. At a concentration of 10 mg/ml, none of the tested strains were sensitive. This finding showed that high amountof bioactive antimicrobial compounds in the Euphorbia tirucalli was extracted with these solvents. Our findings are in agreement with the previous study that most of the bioactive antimicrobial compounds were extracted with methanol (Parekh & Chanda 2007; Sugumar et al. 2010). A methanolic extract of Euphorbia tirucalli showed slightly higher inhibitory activity against the selected bacterial strains than that of ethanolic extract. In our study, aqueous extract of Euphorbia tirucalli did not exhibit any antimicrobial effect against both bacteria. Our findings are in agreement with the previous reports (Parekh & Chanda 2007; Jadhav et al. 2010). Organic solvents were more effective compared to aqueous extract.This may be due to dissolution of plant metabolites in organic solvents as compared to aqueous solvent (Ahmad et al. 2001).

The differences in the antimicrobial properties in different extracts of Euphorbia tirucalli may be due to the biologically active phytochemical constituents. Some of the extracts may contain potential antibacterial constituents (Siddhartha et al. 2007). The presence of various phytochemicals such as alkaloids, tannins, polyphenol and triterpenes in Euphorbia tirucalli stems extracts which possess antimicrobial property may contribute to the formation of inhibition zone (Sugumar et al. 2010). Mechanism to fight bacteria was different, depending on the types of active compound. Alkaloid has been reported able to inhibit nucleic acid synthesis of bacteria, whereas the tannins able to give toxic to bacteria by increased their hydroxylation proses (Cushnie et al. 2014; Min et al. 2008). Another study found that polyphenol disturbs the growth of bacteria by inhibition of c-di-AMP that controls various functions in bacteria (Opoku-Temeng & Sintim 2016).

In this study, Euphorbia tirucalli extracts exhibited low percentage of inhibition as compared to control antibiotic at the tested concentrations, probably due to the differences in the bactericidal mechanism. While effective combinations between single natural products, with chemosynthetic or antibiotics have been reported, this phenomenon could also be applied to Euphorbia tirucalli. Although Euphorbia tirucalli extracts alone exhibited low antibacterial activity, drug synergism between antibiotics and bioactive plant extracts could be beneficial (synergistic or additive effects). On the other hands, cytotoxicity analysis of this compound need to be undertaken in order to expand its therapeutic indication so characteristics and potential can be gained. Furthermore, this experiment was conducted in vitro, which is considered a static system compared to in vivo tests, which may reflect the influence of various dynamic factors like systemic conditions, salivary flow, diet, and dental anatomy (Castro et al. 2000).

To the best of our knowledge, this is the first study to investigate the antibacterial activity of Euphorbia tirucalli stems extracts on oral bacteria. This in vitro study shows that Euphorbia tirucalli stems extracts have effect as an antimicrobial agent and implied that there are potential usages of this extract in the mouthwash and toothpaste.

CONCLUSION

In conclusion, methanolic and ethanolic stem extracts of Euphorbia tirucalli possessed antibacterial activity against S. mutans and S. sobrinus. Further phytochemical and pharmacological studies are required to identify the active principles of the extracts and their mechanism of actions.

ACKNOWLEDGEMENT

The authors wish to thank the staff of the Craniofacial Science Laboratory, School of Dental Sciences for their technical assistance and facilities provided. The authors declare that none of them has any conflict of interest in this study. The study was supported by Universiti Sains Malaysia Short Term Grant (304/PPSG/61312115).

INTRODUCTION

Parents experience stress when raising children. Nevertheless, bringing up children with disabilities is even more a gargantuan task with physical, psychological and mental effects. Parents often sacrifice their time, energy and financial resources in order to cope with their children’s impairments characterised by a wide range of symptoms. However, parents of children with Autism Spectrum Disorder (ASD) consistently reported higher levels of parenting stress compared to the parents of typically developing (TD) children and those with other neurodevelopmental disorders like Down Syndrome (Mancil et al. 2009). Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterised by deficits in communication skills, social interactions and restricted, repetitive and stereotyped patterns of behaviours. This condition is aggravated by the presence of comorbid conditions in which children with ASD are often associated with language disorders, cognitive delays, and anxiety and mood disorders. Brain development in ASD follows an abnormal pattern as shown in Magnetic Resonance Imaging (MRI) scans with accelerated unusual brain growth in early life that causes the enlargement of brain during childhood. Nonetheless, brain volume among adolescents and adults with ASD atypically decreases slightly in terms of structural volume and neuron number (Courchesne et al. 2010). Behavioural and emotional outbursts, are another core features among individuals with ASD in which there is an association between ASD and alexithymia (Szatmari et al. 2008). In turn, alexithymia is directly related to aggression (Konrath et al. 2012). Parents rated Disruptive Mood Dysregulation Disorder (DMDD) as the most prevalent among children with autism compared to children with Attention Deficit Hyperactivity Disorder (ADHD)-Combined and ADHD-Inattentive (Mayes et al. 2015). The mood swings and temper outbursts could be attributed to the higher levels of cortisol, serotonin and lower levels of oxytocin among individuals with ASD which lead to repetitive behavior (Yang et al. 2015).

There are limited studies that focus on parents of children with ASD or specifically parenting stress among these parents with contradictory results. Not all demographic factors were investigated. In 2007, Schieve, and colleagues compared the relative aggravation range among parents of children with and without ASD (Schieve et al. 2007); in 2013, Hassan and Inam studied the difference in parenting stress levels among fathers and mothers of children with ASD (Hassan & Inam 2013). In 2014, Zamora, and his team determined the difference in parental distress and parent-child dysfunctional interaction among parents of female and male children with ASD. The relationship of other demographic factors like number of siblings, age of children with ASD, gender of parents and ethnic groups with parenting stress were not clearly identified.

There is a misassumption that children with ASD do not undergo puberty. Indeed, the reality is that all children undergo puberty despite Intelligence Quotient (IQ) and social skills. Children with ASD often experience puberty earlier than other typical peers of the same age. Hormone surges during adolescence lead to emotional outbursts such as unreasonable mood swings, aggression, arguing and defiance. Some may even include increased self-injurious behaviour. Logically, parents of adolescents with ASD should have a higher level of parenting stress compared to parents of children with ASD at infancy. However, this conclusion seems premature due to the relative lack of research on parenting stress among different ages of children with ASD. Identifying critical factors contributing to parenting stress enable the parents to handle ASD in their children better.

Raising children involved identification of ASD and proper handling of the children with this disorder. The current knowledge on ASD has enabled us to identify and embrace children with this disorder. At the same time, we should pay attention to parenting stress among the parents with ASD children. Hence, this study aimed to investigate the difference in parenting stress between parents of children with ASD and parents of TD children. The effects of demographic factors such as age of ASD children and gender of parents of children with ASD on the parenting stress were also investigated.

MATERIALS AND METHODSPARTICIPANTS

Participants included 30 parents of children with ASD younger than 12 yrs and 36 parents of physically and mentally healthy children without any neurodevelopmental disorders as the TD control group. The diagnosis of ASD were made clinically by the child psychiatrist or attending paediatrician according to DSM5. Children with comorbidities such as ADHD and any other neurodevelopmental disorders were excluded from the study. The TD group was regarded as the control group on the basis that they were attending mainstream education according to their age and not attending any doctors for any developmental problems.

MATERIALS

Parenting Stress Index (PSI), 3rd Edition Short Form was used to assess the parents. PSI by (Abidin 1995) is a psychometric clinical assessment that consists of 36 items used to analyse relative magnitude of parenting stress based on three scales which are, parental distress (PSI/PD), parent-child dysfunctional interaction (PSI/P-CDI) and difficult child (PSI/DC). Total stress accounts for the stress reported for all the 3 areas, including parental distress, stress originated from the relationships and interactions between parent and child and the child (Abidin 1995). Parental Distress (PSI/PD) determines the distress a parent is undergoing in his or her role as a parent as a function of personal factors that are associated with parenting. This includes inadequate social support, conflict with spouse, and stresses related to restrictions placed on other life roles (Abidin 1995). Parent-child dysfunctional interaction (PSI/P-CDI) highlights the parents’ perception that his or her child is a negative element in their lives as their children do not meet their expectation or they are not accepted by their children (Abidin 1995). The domain of PSI/DC portrays the difficulty experienced by parents in managing challenging behavioural characteristics of their children (Abidin 1995). The PSI Malay version was used so that it could be comprehended by those samples who are illiterate in English. The PSI Malay version was validated and it showed that it was a reliable questionnaire to investigate the parenting stress levels among caregivers of children with learning disabilities (Nazurah et al. 2015). Demographic data of samples were collected including age and sex for both parents and their children, ethnic group, marital status, education background, employment status, number of children, and presence of any caregiver. Parents undergoing intervention were also recorded.

PROCEDURE

After obtaining Universiti Kebangsaan Malaysia (UKM) Ethics Committee approval and written consent from the parents, samples were collected from the UKM Child Psychiatric Clinic (27%) and Tangkak Hospital (23%) while the parents were bringing their children for regular medical check-up. Some samples were also taken from the National Autism Society of Malaysia (NASOM) in Muar (20%) and Segamat (30%). All the participants completed the assessment in 15 and 20 mins. Samples referred to researcher when they encountered terms and statements that they could not understand. After completion, the scores for each of the domain were calculated. The results were recorded using Statistical Package for Social Science (SPSS) Version 21st by the IBM Corporation at New York, United States. A summary of social demographic variables was done using descriptive analysis of frequencies. The relationship between scores of various domains in PSI and sociodemographic variables as independent variables were analysed using cross-tabulation analysis. All the variables were transformed by labeling into categorical variables. Chi-square with Fischer-exact test was used to test for statistical significance. The scores were also compared using Whitney Mann U test if the variables were not normally distributed based on the Shapiro-Wilk Normality Test.

RESULTSSOCIO-DEMOGRAPHIC BACKGROUND

A total of 30 parents of children with ASD and 36 parents of TD children were recruited. The samples were collected from the UKM Child Psychiatric Clinic (27%), Tangkak Hospital (23%), NASOM in Muar (20%) and Segamat (30%). All the subjects recruited met the inclusion criteria and completed the PSI questionnaire. Subjects between the two groups were matched by age, sex of parents, ethnic group and marital status. The sociodemographic profiles are summarized in Table 1.

The Chi-square with Fischer-exact tests results of all the subscales of PSI between the ASD group and the TD control group are shown in Table 2. The score of PSI Defensive Responding (PSIDR) shows that most of the samples (95.5%) in this study did not show defensive responding while answering the PSI. There was a significant difference in total stress (p<0.001) between ASD group and TD control group as the ASD group showed extremely high levels of total parenting stress compared to TD group. Regarding the subscale of Parental Distress (PSI/PD), significant difference (p<0.001) was recorded with higher number of samples in the ASD group scored at the highest percentile (85 to 99th+ percentile) compared to TD control group. For the domain of Parent-Child Dysfunctional Interaction (PSI/P-CDI), there was also a significant difference (p<0.001) between these two groups in which there were higher number of samples in ASD group scored at the highest percentile compared to TD control group. Regarding the last subscale which is Difficult Child (PSI/DC), a significant difference (p<0.001) was shown with significantly higher number of samples in the ASD group scored at the highest percentile compared to TD group. Since all the 4 domains, Total Stress, PSI/PD, PSI/P-CDI, PSI/DC and PSI/DC were not normally distributed based on Shapiro-Wilk Normality Test, the scores were also compared using Mann Whitney U Test. From results in Table 3, it shows that Total Stress, PSI/PD, PSI/P-CDI, and PSI/DC in the ASD group was statistically significantly higher than the TD group (U=169.5, p=<0.001; U=81.0, p=<0.001; U=41.0, p=<0.001; U=51.0, p<0.001, respectively).

Parenting stress was also compared between sex of parents of children with ASD. For all the subscales of PSI/PD, PSI/P-CDI and PSI/DC, percentage of mothers scored at the highest percentile was slightly higher compared to fathers. However, there was no significant difference (p=1.000, p=0.833, p=0.767, and p=0.667) for all the subscales (Total Stress, PSI/PD, PSI/P-CDI, and PSI/DC respectively). Since all the 4 domains, Total Stress, PSI/PD, PSI/P-CDI, PSI/DC and PSI/DC were not normally distributed based on Shapiro-Wilk Normality Test, the scores were also compared using Mann Whitney U Test. From the results, it was shown that PSI/P-CDI, and PSI/DC in the mother group was slightly higher than the father group with no significance difference (U=100.0, p=0.767; U=84.5, p=0.667, respectively).

Parenting stress was also compared between parents of children with ASD of different age groups. The ASD children were divided into 2 age groups; a younger group of 1 to 7 years old and an older group of 8 to 12 years old. For the Total Stress and all the subscales of PSI/PD, PSI/P-CDI and PSI/DC, percentage of parents of older ASD children scored in the highest percentile was higher compared to parents of younger children with ASD. Similarly, there was no significant difference (p=0.651, p=0.700, p=1.000, and p=0.867) for all the subscales (Total Stress, PSI/PD, PSI/P-CDI, and PSI/DC, respectively). Since all the 4 domains, Total Stress, PSI/PD, PSI/P-CDI, PSI/DC and PSI/DC were not normally distributed based on Shapiro-Wilk Normality Test, the scores were also compared using Mann Whitney U Test. From the results, it shows that Total Stress, PSI/PD, and PSI/DC in the elder age group was slightly higher than the younger age group with no significance difference (U=97.5, p=0.651; U=87.0, p=0.700; U=98.5, p=0.867, respectively).

Parenting stress was also compared between parents of male children of ASD and TD group. Table 4 shows the relative Chi-Square with Fischer-exact tests of Total Stress, PSI/PD, PSI/P-CDI, and PSI/DC among parents of male children of ASD and TD group. There was a significant difference in Total Stress (p<0.001) between ASD group and TD control group as the ASD group showed extremely high levels of total parenting stress compared to TD group. Regarding the subscale of Parental Distress (PSI/PD), significant difference (p=0.001) was recorded with higher number of samples in the ASD group scored at the highest percentile (85 to 99th+ percentile) compared to TD control group. Regarding the domain of Parent-Child Dysfunctional Interaction (PSI/P-CDI), there was also a significant difference (p<0.001) between these two groups in which there were higher number of samples in ASD group scored at the highest percentile compared to TD control group. Regarding the last subscale which is Difficult Child (PSI/DC), a significant difference (p<0.001) was shown with significantly higher number of samples in the ASD group scored at the highest percentile compared to TD group. Since all the 4 domains, Total Stress, PSI/PD, PSI/P-CDI, PSI/DC and PSI/DC were not normally distributed based on Shapiro-Wilk Normality Test, the scores were also compared using Mann Whitney U Test. From results in Table 5, it shows that Total Stress, PSI/PD, PSI/P-CDI, and PSI/DC in the ASD group was statistically significantly higher than the TD group (U=15.0, p=<0.001; U=51.0, p=<0.001; U=22.5, p=<0.001; U=9.5, p<0.001, respectively).

Parenting stress was compared between parents of female children of ASD and TD group. Table 6 shows the relative Chi-Square with Fischer-exact tests of Total Stress, PSI/PD, PSI/P-CDI, and PSI/DC among parents of female children of ASD and TD group. For all the subscales of Total Stress, PSI/PD, PSI/P-CDI and PSI/DC, percentage of parents of female children of ASD group scored at the highest percentile was significantly higher compared to parents of female children of TD group. There was a significant difference (p<0.001, p=0005, p<0.001, and p<0.001) for all the subscales (Total Stress, PSI/PD, PSI/P-CDI, and PSI/DC, respectively). Since all the 4 domains, Total Stress, PSI/PD, PSI/P-CDI, PSI/DC and PSI/DC were not normally distributed based on Shapiro-Wilk Normality Test, the scores were also compared using Mann Whitney U Test. From results in Table 7, it shows that Total Stress, PSI/PD, PSI/P-CDI, and PSI/DC in the ASD group was statistically significantly higher than the TD group (U=12.0, p=<0.001; U=31.5, p=<0.001; U=20.0, p=<0.001; U=10.5, p<0.001, respectively).

DISCUSSION

Autism Spectrum Disorder (ASD) has been associated with higher levels of parenting stress in relation to parents of children with other disabilities and parents of TD children. Parenting stress is one of the dominant aspect of research nowadays as ASD has become more prevalent in the society. Based on the Centre of Disease Control and Prevention, 1% of the world population is afflicted by ASD. Researchers have shown that the nature of ASD requires extra caregiving demands from the parents that results in higher levels of parenting stress. A study done by Dumas et al. (1991), and his team showed that parents of children with ASD showed statistically higher levels of parenting stress compared to groups of parents of children with Down Syndrome and also parents of TD children. Studies using different instruments other than PSI also gave similar results in which the study done by Schieve et al. (2007) found, parents of ASD children scored highest levels of stress and aggravation range (55%) compared to parents of children with other developmental problems (44%), parents of children with special healthcare needs (12%) and parents of TD children (11%). A total of 89% of them also rated that their ASD children were facing emotions, concentration and behaviour impairments.

The scale of Defensive Responding indicates the extent of the respondents was presenting the most favourable impression of themselves to cover up the level problems or levels of stress in the relationship between parents and children. Those respondents who scored extremely low scores in PSIDR indicates that one of the following; the parent was trying to under report their stress symptoms, or the respondent was not fully involved in child care and thus he or she was not experiencing obvious parenting stress or the last possibility was that the sample handled parenting responsibilities well and maintained a good relationship with others, including his or her spouse (Abidin 1995). Since most of the samples (95.5%) exceeded the range of PSIDR, this indirectly indicated that the respondents reflected on themselves and their true feelings when answering the self-rated questionnaire.

When comparing parents of children with ASD and the TD control group, significantly higher scores of parental distress was recorded. This could be attributed by the lack of social support in the society in which acceptance and knowledge regarding ASD is still insufficient among the population. The contrast between the child’s appearance, which betrays no signs of disability, and his or her behaviour, which is perceived as abnormal and “strange”, frequently puts parents in very unpleasant and difficult conditions (Portway & Johnson 2005). Social disapproval for the child’s behaviour often leads to stigmatization, experience of shame for parents, and their exclusion from normal social activities (Farrugia 2009). Lack of knowledge and awareness about ASD cause the negative attitude of society towards children with ASD themselves and also their parents. Boyd (2002) finds that mothers can adapt with children with ASD with the assistance of informal social support. Unfortunately, this type of support is limited as the society often has the perception that parents are left to look after their children alone, deprived of help even from close family members if they encounter problems with the children’s functioning or disabilities. Parents’ social life is affected which takes a toll on rapport with relatives and friends due to obligation of child care (Farrugia 2009).

Parents of children with ASD also show elevated levels of dysfunctional interaction in between parents and children. In Asian countries, most of the parents prioritise obedience and academic excellence of children and having a child with disability is indeed a big challenge for most of the parents. Weak academic skills among children with mental health problems is one of the parents’ worries (Norhaniza et al. 2010). ASD children is characterised by cognitive impairments and their lower ability to learn and adapt new things definitely cannot meet expectations of most of the parents. ASD children have limited ability to initiate and maintain interaction with others (Volkmar et al. 2004). However, intellectual level of the child is not a determinant of deficits in language and communication skills as children with High Functioning Autism also experience significant problems in interpersonal relations. A study done by Davis and Carter (2008) on parents of newly-diagnosed children (ASD), parents were disturbed and burdened by impairments of social skills among their children. Similar results also shown by a research carried out (Tomanik et al. 2004) which reported that in mothers of children with pervasive developmental disorders consisting mainly of ASD, their children’s inability to participate in communication with others had led to significant stress among them. Communication impairments among ASD children is one of the main factors their parents seek professional assistance (Charman & Baird 2002).

Parents of children with ASD also reported higher scores in the subscale of Difficult Child compared to TD control group. Child behaviour problems is one of the factors that contribute the most to parenting stress (Bishop et al. 2007; Herring et al. 2006; Tomanik et al. 2004). Children with ASD with impaired adaptive functioning require extra care to assist them in daily tasks such as getting dressed, eating and daily hygiene. Besides, some ASD children have insomnia or sleeping difficulties in which they have problems in falling asleep, they have short sleeping time and they might wake up several times per night and this has brought their parents under extreme exhaustion due to their children’s circadian rhythm (Goodlin-Jones et al. 2008). They also possess self-injury, aggressive and other destructive behaviour (Richman 2009). With the accumulation of such demands along with other behavioural problems, it is no surprise that mothers evaluated taking care of children with autism to be much harder than raising most children of the same age (Montes & Halterman 2008).

When comparing the parenting stress between fathers and mothers of ASD children, there is no significant difference. Mothers recorded slightly higher levels of parenting stress compared to fathers. To date, very few research compared the parenting stress levels between fathers and mothers. Similar results were shown by the research carried out by Pisula (2007) in which mothers of children with ASD scored higher stress levels compared to mothers of children with Down Syndrome. In Asian countries, most of the responsibilities of taking care of children lie on mothers. Women are considered more emotional when dealing with stressful conditions (Hassan & Inam 2013). Having to deal with working stress and their children’s deficits could deteriorate the parenting stress level among mothers as some of the mothers have to work besides taking care of their children. Parenting stress in mothers was related to child’s social skills, while no such relationship was found in fathers (Baker-Ericzén et al. 2005).

Overall, parents of older ASD children recorded a higher total parenting stress compared to younger children of ASD. However, the parenting stress between parents of younger and older children with ASD was not significant. Like any other children, children with ASD also undergo puberty or adolescence. Impairments in social development and communication among youths with ASD become more apparent in adolescence. Adolescents with ASD often develop social anxiety due to their impairments socially and cognitively. Other than that, they also experience increase in hormone levels like other adolescents. The increase in androgen for males and estrogen for females indirectly leads to stress and anxiety and eventually aggravates the aggressive behavioural problems among them (Gillies & McArthur 2010). Parents may have to cope with these extra difficulties among their children with ASD as they grow older and this will stress them in their daily lives.

LIMITATIONS OF STUDY

There were few limitations that would affect the reliability and validity of the results. Due to time constraint, only a small sample size was used with a total of 66 parents recruited. Since the samples were taken only from few centres, we may not have generalized the results of this study to all Malaysian parents of children with ASD. This study could be further strengthened by using a larger sample size so that it can give better results and samples should be collected randomly throughout the country.

CONCLUSION

Parents of children with ASD showed significantly higher levels of stress compared to parents of TD children. Intervention towards ASD children should not only focus on minimizing the core symptoms but should also pay attention to the family, as well. Special attention should be tendered towards the parents in which their mental and psychological health should not be overlooked.

INTRODUCTION

Metabolic syndrome (MetS) is a constellation of medical conditions consisting of central obesity, hyperglycemia, hypertension, and dyslipidemia. Based on a joint interim statement, a person is diagnosed to have MetS with at least three of these conditions. The prevalence suffered by MetS is growing worldwide. It is associated with the increased morbidity and mortality following its complications which include heart disease (Kim et al. 2010). Previous studies suggested that consumption of fructose leads to the development of MetS (Elliott et al. 2002).

Though MetS may share the same features as Cushing’s disease, there 65are striking resemblances between these two conditions. In contrast to Cushing’s disease, plasma cortisol tends to be normal or lower in MetS. Nevertheless, rodent obesity models showed glucocorticoid plays an important role in the metabolic disease. The glucocorticoid reactivation is mediated by the enzyme known as 11β-hydroxysteroid dehydrogenase (11β-HSD). Amelung and his colleagues discovered the 11β-hydroxysteroid dehydrogenase enzyme (11β-HSD) in 1953 (Morton & Seckl 2008). This enzyme converts the inactive glucocorticoid hormone to active glucocorticoid hormone. Cortisone is an inactive form of glucocorticoid in human meanwhile 11-dehydrocorticosterone in rodents. Cortisol and corticosterone is the active form of glucocorticoid in human and rodents respectively (Cooper & Stewart 2009). The activity of 11β-HSD in cell and tissue was first explained by Monder and White in 1993 (Seckl et al. 2004). In 1980, Monder observed the activity of this enzyme in the liver was involved in the process of conversion of cortisone to cortisol by 11β-reductase (11β-OR) as well as conversion of cortisone to cortisol by 11β-dehydrogenase (11β-DH). Apart from liver, this enzyme was also found in placenta (Popovici et al. 2006) and kidney (Schnackenberg et al. 2013).

There are two isozymes of 11β-HSD which are 11β-HSD type 1 (11β-HSD1) and 11β-HSD type 2 (11β-HSD2). Both these enzymes are involved in the interconversion of active glucocorticoid to inactive glucocorticoid. Glucocorticoid hormones involved in stress adaptation, metabolism regulation and response to inflammation (Opperman 2006). The differences between these two isozymes are the co-factors, affinity and reaction of conversion (Draper & Stewart 2005). The 11β-HSD1 enzyme was discovered earlier than that of 11β-HSD2 enzyme (Seckl et al. 2004). This enzyme plays an important role in the liver and it is also involved in the development of obesity (Masuzaki et al. 2001), dyslipidemia (Cooper & Stewart 2009) and hyperglycemia (Andrews & Walker 1999). The 11β-HSD1 enzyme is widely distributed and dependent on nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. This enzyme has bi-directional action which acts as reductase (11β-OR) and dehydrogenase (11β-DH) (Opperman 2006). However, the function as 11β-OR is more dominant compared to 11β-DH. The 11β-HSD1 is widely expressed in human (Ricketts et al. 1998) and rodents (Moisan et al. 1990) including the liver (Bellemare et al. 2006), adipose tissue (Bujalska et al. 1997), lungs (Hundertmark et al. 1993), skeletal muscle, heart muscle and smooth muscle (Walker et al. 1991), anterior pituitary gland and brain (Moisan et al. 1990) and adrenal cortex (Shimojo et al. 1996). All these sites have glucocorticoid receptors except the heart and hippocampus (Whorwood et al. 1992).

Research studies showed the expression of 11β-HSD1 enzyme was mostly around the central vein and reduced radially (Brereton et al. 2001). The presence of expression of 11β-HSD1 enzyme on the adipose tissue and liver explained the pathogenesis of development of adiposity, carbohydrate metabolism and insulin sensitivity (Masuzaki et al. 2001). The development of central obesity was related to the 11β-HSD1 enzyme as shown by its expression which was higher in visceral adipose tissue than in subcutaneous adipose tissue (Bujalska et al. 1997). The activity of this enzyme induces the differentiation of pre-adipocyte to adipocyte by glucocorticoid hormone thus, leading to the development of central obesity (Cooper & Stewart 2009). Another research has showed that excessive expression of 11β-HSD1 enzyme in mature adipocyte leading to the development of metabolic syndrome which presented with central obesity, dyslipidemia and insulin sensitivity (Masuzaki et al. 2001).

To the best of our knowledge, there are limited studies on the expression and activity of 11β-HSD1 enzyme in the liver in MetS rat model following consumption of fructose drinking water. Therefore, the results of this study may contribute opportunities for development of therapeutic interventions through 11β-HSD1 inhibitor in the MetS area.

MATERIALS AND METHODSPREPARATION OF FRUCTOSE IN DRINKING WATER

Pure fructose >99% (Syarikat Systerm Malaysia) was used. The preparation of fructose drinking water (FDW) was done fresh every alternate day (Sánchez-Lozada et al. 2007) and based on weight/volume formula (Shahraki et al. 2011). To prepare fructose 20% drinking water, 20 gm of fructose was diluted in 100 mL of tap water. The bottles were then covered with aluminium foil to prevent fermentation (Abdulla et al. 2011). The FDW was given daily for eight weeks as ad libitum.

ANIMAL PREPARATION

All procedures were carried out in accordance with the institutional guidelines for animal research of the Universiti Kebangsaan Malaysia (UKMAEC FP/ANAT/2012/FARIHAH/18-JULY/453-JULY-2012-AUGUST-2013). Twelve male Wistar rats weighing between 200 and 300g were housed individually in a temperature controlled room (24oC) with adequate ventilation and illuminated for 12 hrs daily (lights on from 0700 to 1900) with free access to standard rat chow (Gold Coin, Klang, Selangor, Malaysia) and tap water ad libitum. The rats were acclimatised for 1 week. The rats were randomly divided into two groups. Each group comprised six rats. The F20 group was given 20% fructose in drinking water while the Control group (C) received only tap water.

EXPERIMENTAL PROTOCOL

After eight weeks, the rats were sacrificed by diethyl ether inhalation (Ahmad et al. 2011). The liver of each rat was sampled into 1-mm cubes and fixed immediately in 10% formalin for 48 hrs to measure the expression of the enzyme by using the immunohistochemical (IHC) staining. The liver samples were sectioned into approximately 1gm weight and was immediately covered with aluminium foil and kept in the refrigerator at -70˚C for measurement of the activity of the 11β-HSD.

EXPRESSION OF 11β-HSD1 ENZYME IN LIVER

The immunohistochemical staining was performed with LSAB-2 HRP system. After fixation with formalin 10% and processed, the liver samples were sectioned at 5 μm and placed on a silanized glass slide and kept dry for 24 hrs. Then, the liver tissues on the slides underwent dewaxing with xylene and rehydrated with series of alcohol to water. The antigen was retrieved by placing the slides in the antigen retrieval solutions. The endogenous peroxidase activity was blocked with peroxidase blocking solution containing H2O2. The sections were then incubated with primary antibody. To abolish cross-reactivity, the biotinylated LINK was placed on the sections. Then, the sections were incubated with streptavidin conjugated with horseradish peroxidase (streptavidin-HRP) and followed by incubation with substrate chromogen solution. Finally, the sections were counterstained with Mayer’s Hematoxylin and dehydrated with series of alcohol followed by dewaxed with xylene. The slides were then mounted with DPX and viewed under light microscope.

The measurements of the expression of the enzymes were measured qualitatively under light microscope under magnification x20. The immunopositive cells were observed around the central vein. The score was given according to the intensity of the staining of the granules in the hepatocyte cytoplasm. Score 0 was given if there was no staining, score 1 for weak intensity staining followed by score 2 with mild intensity staining and final score 3 with the highest intensity of the staining of the granules. The scoring was done using double-blinded method (Haque et al. 2006).

ACTIVITY OF 11β-HSD1 ENZYME IN LIVER

The liver samples were first kept cooled under room temperature. The sample was then put in the 10 mL test tube with 5 ml of phosphate buffer solutions (PBS) added. The homogenization process was done in ice. The samples were then centrifuged at 1000 rpm at 4˚C for 10 mins. The top layer of the solutions was taken and placed in appendorf tube for further analysis.

The measurement of the activity of 11β-HSD1 in the liver was performed using the ELISA kit. All ELISA kit components and samples were taken out from refrigerator and kept under room temperature just before used. The procedure were done according to the company standard procedure. For the assay procedure, determination of diluted standard wells, blank and samples were done. Wells with standard, blank and samples were added with 100 μl of dilution. Then, removal of water was done following two hours of incubation at 37°C. Each well was added with 100 μl Detection Reagent ‘A’ working solution. Again, incubation of the kit was done for 1 hr at 37°C. Following incubation, 350 μl wash solution were used to wash the kit for 3 times. The kit was keep for incubation for 30 mins at 37°C following adding the 100 μl of Detection Reagent B working solution to each well. Then it was washed for 5 times. Subsequently,each well was added with 90 μl of Substrate Solution and followed by incubation for 20 mins at 37°C. Then, each well was added with 50 μl of Stop Solution.

Finally, the reading of the enzymes activity were done by using spectrophotometry technique at 450 nm wave on microplate reader. The values of optic density (O.D) were taken as a value of the activity of the enzyme. STATISTICAL ANALYSIS

All data were tested for normal distribution and presented as Mean±standard error of mean (SEM). The comparisons between groups were analysed by a one-way Analysis of Variance (ANOVA) followed by Tukey test for multiple group comparisons. The level of significance was taken as p < 0.05. All statistical analysis was conducted using SPSS version 20 software.

RESULTS EXPRESSION OF 11β-HSD1 ENZYME IN LIVER

The immunohistochemical staining to measure the expression of the enzyme was shown in Figure 1. The granules of 11β-HSD1 in the hepatocytes cytoplasm were stained brown which was highest around the central vein and reduced radially in all the three groups. In the group that was given fructose in drinking water showed greater intensity of discoloration around the central vein as compared to the C group. The statistical analysis showed that the scoring was significantly higher in F20 compared to C group (Figure 2A).

ACTIVITY OF 11β-HSD1 ENZYME IN LIVER

Following consumption of fructose in drinking water for eight weeks, the activity of 11β-HSD1 enzyme in the liver was significantly increased in F20 group compared to C group (Figure 2B).

DISCUSSION

The 11β-HSD enzyme was first discovered by Amelung and his friends in 1953 (Seckl et al. 2004). This enzyme involved in conversion of inactive glucocorticoid which is cortisone (in human) and 11-dehydrocorticosterone (in rats) to active glucocorticoid which is cortisol (in human) and corticosterone (in rats) (Cooper & Stewart 2009). The activity of 11β-HSD enzyme in cells and tissues was first described by Monder and White in 1993 (Seckl et al. 2004). There is activity of this enzyme in the liver and it is involved in conversion of cortisol to cortisone by 11β-(DH) as well as conversion of cortisone to cortisol by 11β-(OR). Thus, it has been postulated that this enzyme is involved in the pathway of glucocorticoid. The excess of glucocorticoid can lead to the differentiation of the adipocytes (Hauner et al. 1987; Hauner et al. 1989). This differentiation is mediated by 11β-HSD1 enzyme. Hence, the long term usage of glucocorticoid may cause MetS (Asensio et al. 2004; Rosmond 2005).

The expression of 11β-HSD1 is also widely distributed in human and rodents tissues including liver (Ricketts et al. 1998; Moisan et al. 1990). Having the knowledge that this enzyme is present in the liver, prompted us to study the effect of FDW on 11β-HSD1 in liver. Research done by Brereton et al. (2001) on expression of 11β-HSD1 in the liver by using immunohistochemical technique has showed that the concentrations of the enzyme is highest around the central vein and reduced radially.

The present study was done using the full blown MetS rat model induced by fructose drinking water for eight weeks (Mamikutty et al. 2014). To utter surprise, the results indicated that with consumption of 20% fructose in drinking water for eight weeks had increased the expression as well as the activity of 11β-HSD1 enzyme in the liver. The distribution of this enzyme was consistent with previous study which was highly distributed around the central vein and reduced radially (Brereton et al. 2001).

There are three mechanisms that we hypothesized for the development of MetS following consumption of fructose in drinking water. Firstly, consumption of fructose does increase the caloric intake, secondly by the increment of expression and activity of 11β-HSD1 enzyme and lastly by the lipogenic property of fructose.

Consumption of fructose drinking water for eight weeks in male Wistar rats does increase the total caloric intake (Mamikutty et al. 2014). Hence, more calories were kept in the form of triglyceride (TG). Triglycerides are stored in the adipose tissue and act as an energy reservoir (Zhou & Qin2012). The storage of TG in adipose tissue increased the deposition of adipose tissue in the abdomen and thus formed the central obesity. In obesity, the adipose tissue tends to expand (Zhou & Qin 2012). The abdominal adipose tissue is the important component in the development of dyslipidemia, hyperglycemia and hypertension (Kershaw & Flier 2004). Hypertriglyceridaemia causes reduced insulin sensitivity which leads to the development of hyperglycaemia.

The abdominal adipose tissues also activate the sympathetic nervous system (SNS) and renin angiotensin (Kotsis et al. 2010) and it is able to compress the kidney (Davy & Hall 2004). These lead to the increase of the cardiac output (CO) and further hypertension. Angiotensinogen which is formed by adipose tissue is converted to angiotensin I. The angiotensin converting enzyme converts the angiotensin I to angiotensin II. Angiotensin II is a potent vasoconstrictor as well as causes retention of sodium and water, thus resulting in hypertension.

Apart from increased calorie intake by consumption of fructose drinking water, the development of MetS might also be established through excessive expression and activity of 11β-HSD1 enzyme in liver. Previous studies showed that differentiation of pre-adipocyte to mature adipocyte by 11β-HSD1 enzyme resulted in the development of central obesity (Rabbitt et al. 2002). The mature adipocyte underwent hypertrophy, which leads to obesity (Yau et al. 1995; Tomlinson & Stewart 2001). In our study, we showed that following consumption of FDW resulted in hypertrophy of central adipocytes in obese Wistar rats (Mamikutty et al. 2014). Excessive fat will initially accumulate in adipose tissue, followed by the deposition in ectopic organs such as liver and heart (Britton & Fox 2011). Thus, it may lead to the complication of MetS which include non-alcoholic fatty liver disease (NAFLD). This condition can deteriorate further to liver cirrhosis and liver carcinoma. Consumption of FDW 20% in male Wistar rat has proven to cause NAFLD stage 1 (Mamikutty et al. 2015).

In the development of dyslipidemia, the conversion of 11-dehydrocorticosterone to corticosterone induces the process of lipogenesis (Cooper & Stewart 2009). Thus, more lipids are formed and increasing the triglyceride (TG) level, thereby leading to hypertriglyceridemia. The 11β-HSD1 enzyme also plays a role in the development of diabetes mellitus and insulin resistance (Andrew & Walker 1999). The conversion of 11-dehydrocorticosterone to corticosterone by 11β-HSD1 enzyme resulted in gluconeogenesis hence causing hyperglycemia. This is consistent with our previous result that proved consumption of FDW20% for eight weeks leads to hyperglycemia in Wistar rats (Mamikutty et al. 2014).

CONCLUSION

In summary, we observed an increase in the expression and activity following FDW consumption suggesting the involvement of 11β-HSD1 enzyme in the possible mechanism of development of in our study was the lack of details in the molecular level concerning the 11β-HSD1 enzyme related with obesity and metabolic syndrome. Further study is required to investigate at the molecular level to further explain the detailed pathway involved in this MetS rat model.

ACKNOWLEDGEMENT

This research was supported by Universiti Kebangsaan Malaysia research grant FF-429-2012 and UKM-DLP-2011-065. The authors gratefully acknowledge the staff of Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur. We also thank Professor Dr. Srijit Das for the technical help.

INTRODUCTION

Acute myeloid leukaemia (AML) is a group of clonal haematopoietic stem cell disorders, where failure to differentiate into mature cells and excessive proliferation in the bone marrow stem cells compartment, result in the accumulation of myeloblasts (Stone et al. 2004). In Malaysia, leukaemia is the seventh most common cancer, with an incidence of 2.9 per 100,000 population accounting for all leukaemias (Meng et al. 2013).

It should be noted that cytogenetics is the most powerful prognostic factor for predicting remission rate, relapse and overall survival (OS) in AML 82patients (Kumar 2011). Cytogenetic abnormalities can be found in up to 60% of newly diagnosed AML cases (Kumar 2011). These cytogenetic aberrations permit patients’ risk to be categorised as favorable, intermediate, and adverse risk groups (Estey & Döhner 2006) depending on the presence or absence of specific chromosomal abnormalities (Kumar 2011). Patients with t(8;21), t(15;17), and inv(16) were allocated to the “favorable risk” group; patients lacking any of these abnormalities and established to have abn(3q), del(5q), -5/-7, or complex karyotype were assigned to the “adverse risk” group; and patients with normal karyotype and numerical or other structural abnormalities, comprised the "intermediate risk" group (Grimwade et al. 2010). Cytogenetically normal AML (CN-AML) patients constitute the largest (approximately 45%) group of all patients (Foran 2010). Rearrangements and mutations of several genes such as Fms-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), and CCAAT enhancer-binding protein-αα(CEBPA) were identified in this group of patients and shown to be important prognostic factors (Swerdlow et al. 2008, Foran 2010).

The Musashi (Msi) gene family members, Msi1 and Msi2, are located on chromosome 12 and 17, respectively (Byers et al. 2011). They encoded for a group of RNA-binding proteins that bind, via 2 ribonucleoprotein-type RNA recognition motifs in their N-terminal, to consensus motifs in mRNAs to inhibit translation (Sakakibara et al. 2002; Barbouti et al. 2003; Kawahara et al. 2008). In haematopoietic stem cells (HSCs), Msi2 is the predominant form expressed and its knockdown leads to reduced engraftment and depletion of HSCs in vivo (Kharas et al. 2010). Also, its expression is associated with up-regulation of cell cycle genes (RAS, mitogen-activated protein kinase, cyclin D1, and MYC) and with homeobox genes, including MEISI, HOXA9, and HOXA1 (Byers et al. 2011). Recently, Park et al. (2015) demonstrated that Msi2 directly maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting with and retaining efficient translation of HOXA9, MYC, and IKZF2 mRNAs. Furthermore, Msi2 is highly expressed in human myeloid leukemia cell lines in which its depletion via inhibiton of Ki-67 expression leads to decreased proliferation and increased apoptosis (Kharas et al. 2010; Park et al. 2015; Han et al. 2015).

Kharas et al. (2010) studied Msi2 expression data gained from 436 AML patients. The expression of Msi2 was significantly associated with specific cytogenetic and molecular genetic aberrations. Patients with inversion of chromosome 16 showed lower Msi2 levels comparative to other leukaemia subgroups, whereas Msi2 expression was higher in subjects harboring monosomy 7. Likewise, high Msi2 expression was associated with FLT3 internal tandem duplications and NPM1 mutations but not with mixed-lineage-leukemia 1 partial tandem duplication (MLL-PTD) and CEBPA (Kharas et al. 2010).

Byers et al. (2011) measured Msi2 protein level by immunohistochemistry (IHC) in 120 AML patients. Their study concluded that Msi2 is highly expressed in myeloblasts which associated with decreased survival in AML, suggesting its use as a new prognostic marker (Byers et al. 2011).

The objective of the study was to measure the expression of Msi2 protein in AML patients by IHC technique and to demonstrate the correlation between the Msi2 protein with several known prognostic parameters such as age of patients, white blood cell (WBC) count at diagnosis, French-American-British (FAB) subtypes, cytogenetic categories as well as the remission status post induction and finally to correlate the expression of Msi2 with OS. Few previous studies have suggested that Msi2 protein is a useful new prognostic biomarker where high expression of this protein is associated with decreased survival in patients with AML. The knowledge gained not only to evaluate the effect of this protein in our local AML population, but it is also hoped to contribute to the strategies for the treatment of AML in order to improve our patients’ survival rate.

MATERIALS AND METHODSSAMPLE SELECTION

All the available cases with histological diagnosis of AML in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for 6 yrs were selected by using universal sampling. A total of 64 cases of newly diagnosed AML were collected. Bone marrow (BM) trephine sections with at least 20% leukaemic blasts were selected as a part of the inclusion criteria to maintain adequacy of the specimen for evaluation. Meanwhile, all the hematoxylin and eosin (H&E) slides of BM trephine biopsy were reviewed and the paraffin embedded tissues were retrieved for whole tissue section and IHC for Msi2 expression.

IMMUNOHISTOCHEMISTRY

Primary antibody rabbit monoclonal [EP1305Y] antibody specific to Msi2 (Code No: ab76148, Abcam England) was diluted to optimal dilution 1:2000 using Dako Antibody Diluent (Code No. S080983, Dako Denmark). The whole BM trephine biopsy sections were cut onto SuperfrostTM Plus coated slides (Thermo ScientificTM, USA). Furthermore, the staining was performed using the protocols from staining kit Dako REALTMEnvisionTM Detection System, Peroxidase/DAB+, Rabbit/Mouse (Code No. K5007, Dako Denmark). Washing steps between each reagent were performed using Lab Vision™ Tris-Buffered Saline and Tween™ 20 (20x) (Thermo Scientific™ USA). The DAB Substrate Working Solution was prepared by diluting the 50x concentrated Dako REALTM DAB+ chromogen with Dako REALTM Substrate Buffer (Code No. K5007, Dako Denmark).

The tissue sections were initially incubated on a slide warmer at 65ºC for 30 mins. Next, tissue sections were dewaxed using 2-steps Xylene (Merck Germany) and rehydrated using decreasing alcohol solutions (100%, 80% and 70%) prepared from 100% Surgipath Reagent Alcohol (Leica Microsystems USA). Tissue sections were subsequently incubated with peroxidase blocking reagent H2O2 for 6 mins. Furthermore, tissue sections were pretreated using heat-induced antigen retrieval Citrate Buffer Solution, pH 6.1 (DAKO) in the Pascal Pressurized Chamber (Dako Cytomation, USA) at 96°C for 40 mins. Next, tissue sections were left to cool down at room temperature for 20 mins. Slides were then incubated with primary antibody for 60 mins at room temperature, followed by secondary antibody incubation with Dako REALTM EnvisionTM/HRP, Rabbit/Mouse (ENV) (Code No. K5007, Dako Denmark) for 30 mins. Tissue sections were then incubated with DAB Substrate Working Solution for 7 mins. After the procedures have been completed, slides were counterstained with Hematoxylin 2 (REF 7231, Thermo Scientific USA) for 5 seconds followed by dehydration step with increasing alcohol solutions (80%, 90% and 100%) and 2-steps Xylene. Finally, the slides were mounted and cover slipped using DPX mounting medium (Cat. No.: 100579, Merck Milipore Germany).

Normal placental tissue was used as a positive control and included with each batch of sections to confirm the consistency of the analyses. It showed strong staining in trophoblast cells (Figure 1).

ANALYSIS OF IMMUNOSTAINED SLIDES FOR MSI2 EXPRESSION

The whole BM trephine biopsy sections immunostained for Msi2 were reviewed by two independent pathologists and scored qualitatively and quantitatively at x4 and x40 magnification. The discrepancies between the reviewers were resolved by consensus.

PERCENTAGE OF POSITIVE BLAST CELLS

Msi2 expression was detected in the cytoplasm and nucleus of the blast cells. The percentage of positive cells was scored semi-quantitatively. Regarding the cytoplasmic staining, the scoring method was as follows: 0 = no expression, 1 = cytoplasmic staining in less than 10% of the blast cells, 2 = cytoplasmic staining in 10-25% of the blast cells, 3 = cytoplasmic staining in 26-50% of the blast cells and 4= cytoplasmic staining in more than 50% of the blast cells. The representative images for each scoring are shown in Figure 2. The percentage of positive blasts for the nuclear staining was much less than the cytoplasmic thus the scoring method was as follows: 0 = no expression, 1 = ≤10% of the blast cells and 2 = >10% of the blasts.

STAINING INTENSITY

The definition of staining intensity was based on report published by Byers et al. (2011). The staining intensity, for both cytoplasmic and nuclear which indicates the density of staining, was scored as follows: 0 = no expression, 1+ = weak expression, 2+ = moderate expression and 3+ = strong expressions. The representative images for each intensity for both cytoplasmic and nuclear are shown in Figure 3 and 4, respectively.

The expression of Msi2 for percentage positive staining and staining intensity was divided into two subgroups i.e. low Msi2 expression and high Msi2 expression. The AML cases where the cytoplasmic staining scoring for Msi2 was scored as 1 and 2 were considered as low Msi2 expression. Whereas, the AML cases with cytoplasmic staining scoring of 3 and 4 were considered as high Msi2 expression. For the cytoplasmic staining intensity, the weak expression (1+) was regarded as low Msi2 expression, whereas, the AML cases with moderate (2+) and strong (3+) Msi2 expression were considered as high Msi2 expression.

CYTOGENETICS

Cytogenetic analysis using G-banding technique was performed on metaphases from bone marrow aspirates taken at diagnosis with the use of standard procedures and all cases were performed at UKMMC cytogenetics laboratory.

STATISTICAL ANALYSIS

Data collected was entered into a computer file and statistically analysed using IBM Statistical Package for Social Sciences (SPSS) version 21.0 statistic software. Descriptive analysis of the data was performed in numbers and percentages for Msi2 staining as well as for demographic variables. Chi-square test was conducted to analyse the relationship of Msi2 expression to AML, and also to correlate Msi2 expression with clinicopathological parameters and OS. Finally, Kaplan-Meier survival analysis was performed to measure the outcome with Msi2 expression. A P value of <0.05 was considered significant.

RESULTSDEMOGRAPHIC DATA OF PATIENTS AND CLINICAL PARAMETERS

A total of 64 patients were included in this study. All patients included received intensive chemotherapy according to standard AML protocols. Patients’ age ranged from 1-81 yrs with two peaks incidence, the first peak between 10-15 yrs of age and the second peak between 35-45 yrs of age (median = 30 yrs). Follow-up data for 52 patients were available. Out of the 52 patients 38(59.4%) succumbed to their malignancy early on the course of follow-up before reaching 5 yrs and 14 patients (21.9%) were still alive at the end of the follow-up period. Patient demographics are summarized in Table 1.

EXPRESSION OF MSI2 IN AML ON BONE MARROW TREPHINE SECTIONS

The positive cases for Msi2 were 61 cases (95.3%), whereas, 3 cases (4.7%) were negative. Total cellular positivity (cytoplasmic and nuclear staining) was demonstrated in 46 cases (71.9%) and 15 cases (23.4%) showed only cytoplasmic positivity. There were no cases that showed only nuclear staining.

CYTOPLASMIC STAINING SCORING

Out of 64 cases of AML, 11 cases (17.2%) showed score 1, 15 cases (23.4%) showed score 2, 15 cases (23.4%) showed score 3 and 20 cases (31.3%) showed score 4. Total absence of Msi2-positive cells (score 0) was observed in 3 cases (4.7%).

CYTOPLASMIC STAINING INTENSITY SCORING

In 64 cases of AML, Msi2 showed variability in staining pattern (representative images at each intensity for cytoplasmic intensities is shown in Figure 5). There were 19 cases (29.7%) showed weak expression (1+), 30 cases (46.9%) showed moderate expression (2+), 12 cases (18.8%) showed strong positivity (3+) and 3 cases (4.7%) showed negative staining for Msi2.

NUCLEAR STAINING SCORING

The majority of the cases (39.1%) showed score 2 for nuclear staining and 21 cases (32.8%) showed score 1, whereas, 18 cases (28.1%) were negative for nuclear staining.

NUCLEAR STAINING INTENSITY SCORING

Out of 64 cases of AML, 18 cases (28.1%) showed no nuclear staining and only three cases (4.7%) showed strong positivity (3+). There were 27 cases that (42.2%) showed weak expression (1+) and 16 cases (25%) that showed moderate expression (2+).

CORRELATION OF MSI2 EXPRESSION WITH CLINICAL PARAMETERS

Correlation of Msi2 expression with the clinical parameters and OS for the cytoplasmic staining and total cellular staining were performed, whereas, nuclear staining was not performed as there were no cases showed only nuclear staining. Finally, Msi2 expression of the total cellular positivity was correlated with the clinical parameters and OS. CORRELATION OF THE CYTOPLASMIC STAINING SCORING AND CYTOPLASMIC STAINING INTENSITY OF MSI2 EXPRESSION WITH CLINICAL PARAMETERS

Regarding the correlation between the cytoplasmic staining scoring of Msi2 expression and the patients' age, low expression of Msi2 was observed in 25 (14.0%) cases and 36 (59.0%) cases showed high Msi2 expression. There was no significant correlation between the two age groups at any level of staining (P = 0.4). For WBC count, there was no significant correlation between the low and high Msi2 expression for the cytoplasmic staining scoring in the two groups (P = 0.3). Similarly, there were no significant correlations between the two subgroups of Msi2 expression and the eight FAB subtypes (P = 0.3), the three cytogenetic subtypes (P = 0.2), the two groups of the remission status post induction chemotherapy (P = 0.7) and the two groups of OS (P = 0.8). The correlation between the cytoplasmic staining scoring of Msi2 expression with clinical parameters is summarized in Table 2. For the correlation between the cytoplasmic staining intensity of Msi2 expression and the clinical parameters, our results showed there were no significant correlations at any level of staining. The results are summarized in Table 3.

CORRELATION OF THE TOTAL CELLULAR STAINING OF MSI2 EXPRESSION WITH CLINICAL PARAMETERS

The information regarding patients’ age was available in 61 cases. Out of 61 cases 46 (75.4%) cases showed total cellular staining and 15 cases (24.6%) showed only cytoplasmic staining of Msi2. There was no significant correlation between the two groups (P = 0.9). Similarly, there were no significant correlations between both total cellular or cytoplasmic staining with WBC count or FAB subtypes (P = 0.7 and P = 0.1, respectively). Interestingly, our results showed significant correlation between the intermediate cytogenetic category and the Msi2 expression in the total cellular staining (P = 0.04). Neither total cellular nor cytoplasmic staining were significantly correlated with the remission status post induction chemotherapy or OS (P = 0.8 and P = 0.5, respectively). The correlation of the total cellular staining of Msi2 expression with the clinical parameters is summarized in Table 4.

KAPLAN-MEIER SURVIVAL ANALYSIS

Neither cytoplasmic staining scoring, nor cytoplasmic staining intensity of Msi2, nor total cellular staining were significantly associated with outcome by Kaplan-Meier analysis for values low or high expression of Msi2 (P = 0.7, P = 0.5, and P = 0.4, respectively). Because our results showed significant correlation between the cytogenetic categories and the expression of Msi2 in cases with total cellular positivity, further; Kaplan-Meier survival analysis was performed within the cytogenetic subgroups. There was no significant correlation between the three groups (P = 0.1).

DISCUSSION

Acute myeloid leukaemias (AMLs) are aggressive haematological neoplasms that require urgent treatment (Munker 2007). Despite intensive treatment with chemotherapy, only a minority of patients can be cured at present (Munker 2007). Hope et al. (2010) mentioned that, for the development of novel targeted therapies aimed at reigning in leukaemic stem cells, the understanding of the molecular machinery that controls HSC self-renewal is critical. Msi2 protein is a protein recently identified as a regulator of the HSCs compartment and of leukaemic stem cells (Kharas et al. 2010), and a poor prognostic biomarker of survival, both in mRNA and protein expression studies (Back et al. 2013). This knowledge has led its use as a novel prognostic marker and proposed as a new target for therapy in AML (Back et al. 2013; Park et al. 2015). This is particularly important in CN-AML, where CN-AML is the largest cytogenetic subgroup of the disease and is marked by heterogeneous survival (Foran 2010).

The present study showed that the incidence of AML is high in young population, i.e. 60 yrs and younger. The median age of all cases was 30 yrs. Though the literature shows that the incidence of AML is higher in elderly (>60 yrs age), and the median age at presentation usually between 60 and 70 yrs.

It should be recalled that the majority of patients (68.8%) had WBC count more than 10x10⁹/L at diagnosis in keeping with the literature review. The FAB subtypes of patients in our study showed that the majority of patients belong to M4 (29.7%) followed by M5 (20.3%). None of the patients in this study belong to M0 or M1 subtype. Kumar (2011) reported higher incidence of M2 (25%) followed by M4 (20%). Similarly, Byers et al. (2011) showed a higher percentage of patients with M2.

The diagnostic karyotype provides the framework for risk-stratification schemes in AML (Grimwade et al. 2010). The majority of patients in this study showed normal cytogenetic finding with normal karyotype. The cytogenetic findings in this study showed that the majority of patients (43.8%) belong to the intermediate cytogenetic subgroup. These data were comparable to that previously reported in the literature. Moreover, there were only 39 BM aspirates from patients with AML available for assessment of the remission status post induction as 25 of them had succumbed to their illness early on the course of follow-up before detecting their remission status. Meanwhile, the analysis from these groups showed that the data were comparable between the group of patients who had achieved remission versus the group that did not achieve remission (29.7% vs 31.3%, respectively).

In the present study, we identified the OS of patients with AML related to the MSi2 expression. Follow-up data on OS was performed in only 52 patients as 12 patients defaulted treatment. The majority of our cases (59.4%) succumbed to their malignancy early on the course of follow-up (<5years). The most common cause of death was neutropenic sepsis (48.4% of cases). Hämäläinen et al. (2009) mentioned that severe sepsis is an important cause of treatment-related morbidity and mortality in AML and demonstrated in his study that severe sepsis was found in 13% of AML patients. He also found that severe sepsis was associated with 27% mortality in AML patients (Hämäläinen et al. 2009). The reason being that during chemotherapy, patients with AML have a great risk of life-threatening infections because of neutropenia due to the chemotherapy and the disease as well, combined with damage to the mucosal barrier and impairment of the innate immune system induced by chemotherapy (Hoffbrand et al. 2011).

The identification of Msi2 as poor prognosticator by its strong association with outcome in cases with AML was demonstrated by Kharas et al. (2010) and Byers et al. (2011). Their findings prompted us to investigate the expression of Msi2 in our own population of AML. Byers et al. (2011) reported immunohistochemically demonstrable Msi2 expression in both the cytoplasm and nucleus of 70% of the blast cells in all AML examined.

It should be mentioned that this work studied 64 cases of AML for expression of Msi2. Positivity for Msi2 were demonstrated in 61 cases (95.3%), whereas, 3 cases (4.7%) were negative. In 46/64 (71.9%) cases the blast cells showed total cellular positivity (cytoplasmic and nuclear staining), 15/64 (42.2%) cases showed only cytoplasmic staining. There were no cases showed only nuclear staining. However, Byers et al. (2011) reported that some of their cases showed nuclear staining only.

The present study observed that the blast cells in some of the cases showed interstitial pattern of staining, whereas, in others; the blast cells showed positive staining in sheets. Haematopoietic cells such as myeloid, erythroid and megakaryocytes were negative. Additionally, the cytoplasmic staining was more frequent than the nuclear staining for all levels of staining. These findings were consistent with previous report by Byers et al. (2011).

The results of the present study showed that the majority of the cases had high expression of Msi2 for cytoplasmic staining. In contrast to Byers et al. (2011) revealed a lower percentage of positivity in most of the cases. The present study did not find significant correlations between Msi2 expression, neither the cytoplasmic (staining scoring nor staining intensity) nor the total cellular staining, and the clinical variables recorded (age, WBC count at diagnosis, FAB subtypes, remission status).

The results of the present study showed that the only clinical parameter correlated with Msi2 expression was the cytogenetics. The study demonstrated significant correlation between the total cellular staining and the intermediate cytogenetic subgroup (P = 0.04). In contrast to Byers et al. (2011) which did not show significant correlation between Msi2 and cytogenetics, however; their study demonstrated significant correlation between total cellular staining as well as the nuclear staining and FAB subtypes. No significant correlation was found between Msi2 expression with OS in the present study. This result could be due to limited data available (OS for 52 patients only).

Finally, the Kaplan-Meier overall survival analysis, showed that neither cytoplasmic (staining scoring nor staining intensity) nor total cellular staining were significantly associated with overall survival. Furthermore, we performed Kaplan-Meier survival analysis within the cytogenetic subgroups, but our results, also showed that there was no significant correlation with the outcome. These results were in contrast to Byers et al. (2011) study which demonstrated that strong staining (3+) of Msi2 expression was associated with poor outcome for both total cellular and nuclear positivity using Kaplan-Meier survival analysis. Additionally, their study showed significant correlation between the favorable cytogenetic subgroup and the outcome (Byers et al. 2011). Another study by Back et al. (2013), using Kaplan-Meier survival analysis, recognized that elevated levels of both HOXA9 and Msi2 were associated with a shorter survival time and, on the other hand, single protein expression for either HOXA9 or Msi2 was not significantly associated with survival.

We humbly admit few limitations of the study. Our sample size was small. We did not compare the results using any other image analysis system. Due to constraint in the budget, we did not perform any additional marker needed for mutational study which is usually employed by World Health Organization (WHO) to classify AML. We admit that there was no correlation seen between the intermediate cytogenetic subgroup of AML and the OS, but we think that with a larger sample size and using additional molecular analysis could result in significant findings.

CONCLUSION

The present study found that Msi2 protein expression was demonstrated in the majority of our AML patients whereby it is beneficial in daily practice for newly diagnosed AML patients. Furthermore, the majority of the cases had high expression of Msi2 and showed variability in staining pattern. This variability in the staining pattern prompt pathologists to be aware of that during interpretation of immunostaining result. The study also showed that the total cellular staining of Msi2 correlated with the intermediate cytogenetic subgroup. However, our study did not find any correlation between Msi2 expression and OS and other clinicopathologic variables recorded. These debatable results provide new area for researchers to conduct further researches to determine the role of Msi2 positive blast cells in AML and their association with prognosis and outcome.

ACKNOWLEDGMENTS

This study was supported by Universiti Kebangsaan Malaysia (UKM) Fund FF-115-2012. The study was approved by the UKM Medical Centre Medical Research Committee. The authors gratefully acknowledge the assistance of Professor Adnan Mansoor in the preparation of the manuscript.

INTRODUCTION

Wound is one of the most common form of morbidity, afflicting millions of individuals daily. Normal wound healing process involves three phases which are inflammation phase, proliferation phase and remodeling phase. Each phase is continuous and overlapping (Witte & Barbul 1997). The objective in wound management is to heal the wound in the shortest time possible, with minimal scarring and to reduce discomfort to the patient.

Sea cucumbers, belonging to the class Holothuroidea, are marine invertebrates. Generally, sea cucumber has soft, slimy body, mouth, an anus and tube feet. Sea cucumber has long been appreciated in Asia and Middle Eastfor nutritional and pharmacological value (Bordbr et al. 2011). While in China, sea cucumbers are health products that are very valuable and popular based on the value of health and delicious taste (Kiew & Don 2012). Even without scientific proof, sea cucumbers have long been known, particularly in Malaysia as a traditional food that is effective in treating hypertension, asthma, rheumatism, cut and burns (Chen 2003; Wen et al. 2010). The effectiveness of sea cucumbers in the therapeutic effect can be attributed to the presence of many bioactive substances such as glycoprotein, phenolic peptides and essential fatty acid. These compound provide a variety of benefits in terms of pharmaceutical and therapeutic health and does not cause side effects (Bordbr et al. 2011).

Stichopus chloronotus (Black Knobby or green fish) is one of the common species that can be found around indo-Pacific Ocean such as Taiwan, Singapore, Thailand and Malaysia. It is the second most common species found in Malaysia (Choo 2008). It has been reported that Stichopus chloronotus has composition of fatty acid which is important and play a key role in wound healing process (Fredalina et al. 1999). Therefore, we conducted a scientific evaluation of optimum dose concentration of Stichopus chlronotus aqueous extract mixed with emulsifying ointment for wound healing.

MATERIALS AND METHODSAQUEOUS EXTRACTION OF STICHOPUS CHLORONOTUS

The sea cucumber (Stichopus chloronotus) used in this study was collected from Bidong Island, Merang, Terengganu, Malaysia. The visceral organs of the animals were removed and kept in -70°C. Before extraction, the sea cucumber was thawed and washed using distilled water. The sea cucumber body was then dried in oven at 60°C until no change of mass. The method used is according to Fredalina et al. (1999) with minor modification. The dried sea cucumber was cut into smaller pieces and homogenized without water. Then, distilled water was added and soaked overnight. The filtered water was collected (flask A), and sea cucumber soak again at 4 hrs and followed by centrifugation at 3000 rpm for 20 mins. The resulted supernatant was collected (flask B). Both water in the flask A and B were mixed and stored at -20°C. After 24 hrs, the sample was lyophilized by freeze dryer to produce a powdery-like extract.

PREPARATION OF AQUEOUS EXTRACT EMULSIFYING OINTMENT OF STICHOPUS CHLORONOTUS

Total of 0.5 g of aqueous extract of Stichopus chloronotus powder was added on 99.5 g Cetamacrogol emulsifying ointment (paraffin ointment). The mixture was mixed well until homogenous. The mixture was applied topically to the wound area once daily.

ANIMAL HANDLING

Total of 12 male Sprague-Dawley rats weighing between 250-300 gm were used as experimental animals, supply from Laboratory Animals Resource Unit, Universiti Kebangsaan Malaysia. The rats were then divided randomly into 3 groups of control: Normal control group (NO), Positive control group (PC) and Negative control group (NC) and 3 groups of aqueous extract emulsifying ointment of Stichopus chloronotus (SC): SC 0.1%, SC 0.5% and SC 1.0%. NO group comprised non treated animals, PC group was animals treated with Flavine, NC group was animals treated with Cetamacrogol emulsifying ointment only.

After general anaesthesia, four round 6 mm diameter, full-thickness wound was made on the dorsal of each rat using disposable punch biopsy. Wounds were then treated daily with emulsifying ointment mixture and measurement of wound size were done on day 1, 3, 6, 8 and 10.

MACROSCOPIC OBSERVATION

Wound area reduction was measured using a calliper and photographs were taken. The percentage changes in wounds area was calculated using the following equation:

Wound size reduction (%) =

(W0 - Wt) / W0 X 100

Where, W0 =index wound area, Wt = wound area after time interval

Data were analysed using SPSS version 20. Results were presented as mean ± SEM. Mixed ANOVA and one-way ANOVA were used to analyse and compare data with p < 0.05 as the limit of significant.

This experiment was approved by the Animal Ethics Committee of the Faculty of Medicine of Universiti Kebangsaan Malaysia (FP/FAR/2013/ISA/20-MARCH/503-MARCH-2013-MAY-2014).

RESULTS WOUND REDUCTION ANALYSIS

All treatments demonstrated a reduction in the wound at each time interval (Figure 1). However, the SC group demonstrated a reduction in wound more rapidly compared to other groups starting on day 6.

Using SPSS Mixed ANOVA analysis, there were significant differences between the percentage in wound reduction and time. This interaction was directly proportional to the percentage reduction of wound increased with increasing time intervals.

On day 6 after wound creation, wounds treated with 0.5% aqueous extract Stichopus chloronotus emulsifying ointment (SC 0.5%) demonstrated a significant wound reduction advantage when compared to all other groups (p<0.05). Figure 2, SC 0.5% shown significant difference in percentage of wound reduction on day 3 and day 6 when compared to normal control group (NO) and also on day 3 until day 8 after wound creation when compare to positive control group (PC).

Macroscopic observation also demonstrated the effects of SC 0.5% was better than the other groups starting on day 6 after wound creation (Figure 3).

DISCUSSION

Gamat has been used for many decades to heal minor wounds by local Malay population and this study attempts to validate that use. There are three basic mechanisms for wound healing: connective tissue matrix deposition, contraction and epithelialization (Diegelmann & Evans 2004).

Our results demonstrated clear wound healing advantage with wounds treated with aqueous extract Stichopus chloronotuse emulsifying ointment mixture. Wound healing was significantly better when compared to gold standard for minor wound treatment like flavine. This effect could be due to the large number of therapeutic properties of sea cucumber, which includes antioxidant (Althunibat et al. 2009), antimicrobial (Kumar et al. 2007), anti-inflammatory (Bordbr et al. 2011), antiviral and other wound healing properties (Aydin et al. 2011; Fredalina et al. 1999).

In this study, we used aqueous extract of Stichopus chloronotus. According to Althunibat et al. (2009), aqueous extract of Stichopus chloronotus exhibited superior antioxidant activity compared to its organic extract by about 80%. The majority of sea cucumber antioxidant property was contributed by its hydrophilic components. According to Fredalina et al. (1999), fatty acid compound in Stichopus chloronotus plays an important role in wound healing process. The study also demonstrated that the fatty acids contained in the aqueous extract of Stichopus chloronotus are palmitic acid 2.2%, stearic acid 9.71%, linolenic acid 12.59%, oleic acid 7.50%, eicosapentaenoic acid 27.84, decosahexaenoic acid 57.55% & Arachidonic acid 1.46%.

Furthermore, Ridzwan et al. (2001) reported that methanol, ethanol and aqueous extract of Stichopus chloronotus showed positive anti-fungal effects. In another study using a different species of sea cucumber, Holothuria polii, both the methanolic and aqueous extracts showed anti-fungal activities. Based on these studies, it was concluded that both the aqueous and methanolic extracts of sea cucumber contained antimicrobial components (Fredalina et al. 1999; Ismail et al. 2008). The antimicrobial potential of sea cucumber extract can also be ascribed to the presence of steroidal sapogenins antimicrobial agents (Abraham et al. 2002). These antimicrobial effects of sea cucumber could reduced inflammation and infection to promote wound healing.

Another factor affecting wound healing is nutritional status. Carbohydrates and lipids are the primary source of energy in wound healing process. Glucose, the basic form of sea cucumber is rich in polyunsaturated fatty acids (PUFA) especially arachidonic acid (AA), the precursor of eicosanoids, which supports the growth and blood clotting processes to expedite wound healing. These nutrients are found in fresh sea cucumber, which contains moisture, protein, lipids, ash and carbohydrates. The compositions of these nutrients vary from 82.0% to 92.6%, 2.5% to 13.8%, 0.1% to 0.9%, 1.5% to 4.3% and 0.2% to 2.0%, respectively (Aydin et al. 2011). However, Chen (2003) demonstrated that fully dried sea cucumber may still contain protein as high as 83%. This all can contribute to the effectiveness of the Stichopus chloronotus in the wound healing process.

CONCLUSION

In conclusion, 0.5% aqueous extract of Stichopus chloronotus emulsifying ointment mixture demonstrated the best concentration for wound healing in a rat model.

ACKNOWLEDGEMENTS

The authors would like to thank Universiti Kebangsaan Malaysia for providing the grant Galakan Pensyarah Muda (GGPM-2011-103).

INTRODUCTION

Diagnosis of primary spontaneous pneumothorax is made in young patients with no underlying lung disease. However, there is increasing incidence of bullous lung disease particularly among smokers. A giant bulla can mimic a pneumothorax, hence differentiation between the two is a challenge (Isha et al. 2016). Chest tube insertion in a tension pneumothorax is life-saving. However, chest tube insertion into giant bullae can be harmful. Physicians are sometimes reluctant to request for CT thorax that would differentiate these two due to time and cost restraints.

CASE REPORT

An 18-year-old, thin built gentleman presented with sudden onset right sided chest pain while playing video game arcade. Chest pain was aggravated with movement and deep inspiration. This was associated with shortness of breath. The patient was a chronic smoker and had symptoms of upper respiratory tract infection 3 days prior. No other history was significant.

Generally, he appeared tachypnoeic with the respiratory rate of 35 breaths/ min. No wheeze or stridor was heard. There was no cyanosis. Blood pressure was 136/90 mmHg, heart rate was 80 beats/min and oxygen saturation on room air was 98%. Lungs examination revealed reduced air entry on the right and hyper-resonant percussion on the 93left. Trachea was slightly deviated to the left. Chest radiograph showed a right pneumothorax (Figure 1a). A total of 2500 ml air was aspirated via needle thoracocentesis. The procedure was stopped as soon as the patient started to cough. Chest radiograph was done post-procedure to assess resolution.

Post-procedural chest radiograph revealed expansion of right pneumothorax (Figure 1b). In response to this, chest tube was inserted. Chest radiograph was taken post-chest tube insertion and there was substantial right lung haziness (Figure 2). The impression at that moment was re-expansion pulmonary oedema. One hour after chest tube insertion, dyspnoea started to worsen. Mild pallor was noted. Chest tube drained a total 1.2 litre of blood. Patient developed tachycardia with the heart rate of 120 beats/min, nevertheless the blood pressure and the saturation was stable. At this point Class II hemorrhagic shock was suspected.

CT thorax showed right haemopneumothorax with contralateral mediastinal shift. The right lung was collapsed. There were multiple bullaes seen within the collapsed right upper, middle and lower middle lobes, as well as the left apical segment of left upper lobe, largest seen at the right upper lobe measuring 1.6 x 1.7 x 2.3 cm (Anterior-Posterior x Width x Cranial-Caudal). The impression from the CT thorax was massive right haemopneumothorax with increasing attenuation value of the pleural fluid upon contrast administration suggestive of slow oozing into the pleural cavity.

Patient was pushed to the operation theatre for emergency thoracotomy. Intra-operative findings were collapsed right lung with multiple bullae on the upper lobe. There were adhesion bands between apex chest wall and upper lobe. Haemorrhage was secured from one of the ruptured bullae of upper lobe adjacent to the chest wall. Total blood removed was 1250 ml. Stapler bullectomy was done and two drains were inserted at the apex and diaphragm. Patient was later admitted to ICU and finally discharged home well.

DISCUSSION

A bullae is defined as a distended air space in the lung more than one centimeter in diameter. A giant bullae is a bullae that occupies more than 30% of a hemithorax. Giant bullae develops as a complication of cigarette smoking. However, other causes are idiopathic. Patients with giant bullae can have no symptoms. Acute bleeding into the bulla will present with hemoptysis. On physical examinations patients may have barrel-shaped chest, decreased breath sounds, and hyper-resonant percussion. This makes it more difficult to distinguish from a pneumothorax.

Giant bullae can be identified on plain chest radiograph, and it is always confused with pneumothorax. The characteristics of the pleural line on the plain chest radiograph can aid in differentiating a pneumothorax from a giant, thin-walled bulla. In large bulla, the pleural line usually concave (open angle) compared to the lateral chest wall, while in pneumothorax, the pleural line is usually convex (narrow angle) compared to the lateral chest wall. On CT thorax, majority of the giant bullae are subpleural and located at the upper lobes.

A primary spontaneous pneumothorax (PSP) is a pneumothorax that occurs without a precipitating event in a person that with no known underlying lung disease. However, majority of patients with PSP have underlying undiagnosed lung disease, and the rupture of the subpleural bleb was the cause of the pneumothorax (Bense et al. 1993). Predisposing factors to PSP include smoking, Marfan syndrome, family history, thoracic endometriosis, and homocystinuria. Cigarette smoking is an important risk factor for PSP. The postulated pathophysiology is that cigarette smoke induces the influx of macrophages and neutrophils that leads to degradation of the lung elastic fibres. The degradation results an imbalance in the oxidant-antioxidant and protease-antiprotease systems. Small airways obstruction secondary to the inflammation increases alveolar pressure, thus causing an air leak into the lung interstitium. Subsequently, the air moves to the hilum, resulting in pneumomediastinum and causing rupture of the mediastinal pleura when the mediastinal pressure increases and results in pneumothorax (Ohata & Suzuki 1980).

The use of lung ultrasound is important in the diagnosis of pnuemothorax in unstable patients. However, it was reported that giant bullae can mimic ultrasonic findings of a pneumothorax with giving an absent sliding sign and a ‘bleb point’ that mimics a lung point (Gelabert & Nelson 2015).

CONCLUSION

Giant bullae can mimic a pneumothorax. Plain radiograph alone is inadequate to distinguish a giant bullae from a pneumothorax. This is imperative because the management is different. If the patient is haemodynamically stable, and underlying lung disease cannot be ruled out, it is advised to proceed with CT thorax to exclude bullous lung disease.

INTRODUCTION

Orbital cellulitis, an infection affecting the soft tissues of the orbit, includes the fat and muscles within the bony orbit posterior to the orbital septum (Mallika et al. 2011). Delayed treatment may result in blindness, cavernous sinus thrombosis, meningitis, cerebral abscess and even death (Bergin & Wright 1986). Early diagnosis coupled with immediate and aggressive medical treatment is important in preventing these devastating outcomes of the disease (Reza et al. 2013).

CASE REPORT

A 30-year-old gentleman sustained multiple superficial facial injuries and a conjunctival laceration wound in the right eye following a motor vehicle accident. Dressing was done on his facial wounds but he failed to receive antibiotic eye drops for the conjunctival wound. Four days later, he presented to the eye clinic complaining of right eye swelling and redness, associated with blur vision, diplopia and mild pain on eye movement. Examination revealed a mechanical ptosis of the right eye, mild proptosis, conjunctival injection and chemosis. A linear conjunctival laceration wound was found with surrounding slough, necrotic tissue and pus collection within the wound (Figure 1). Extraocular muscle movements were restricted in all gazes (Figure 1). However, optic nerve functions were preserved. Visual acuity in the right eye was 6/24 correctable with pinhole to 6/18, while the left eye was 6/6. There was no relative afferent pupillary defect (RAPD). Fundus examination normal and the optic disc was not swollen. An urgent computed tomography (CT) scan of the orbit and paranasal sinuses revealed thickening of the pre-septal soft tissue and sclera, dilated superior ophthalmic vein and a possible slow flow carotid-cavernous fistula. There was also sinusitis involving both ethmoidal, maxillary and the left frontal sinus. There was no orbital fracture or evidence of cavernous sinus thrombosis (Figure 2). He was started on intravenous Ceftriaxone 2 gm daily with intravenous Metronidazole 500 mg three times daily for one week. Excision of the necrotic conjunctival tissue was done few days later. His condition improved with treatment and he was discharged with oral Cefuroxime and Metronidazole for another week. Upon follow up a week later, the conjunctival chemosis resolved with only mild injection of the conjunctiva. A repeat CT scan brain and orbit showed improvement of proptosis with normal calibre of ophthalmic vein and no evidence to suggest a slow flow carotid-cavernous fistula.

DISCUSSION

Orbital cellulitis is the infection and inflammation of the orbital soft tissues extending beyond the orbital septum. Risk factors for this condition include the spread of infection from the sinus cavity, eyelids, face, dental abscess, foreign bodies or distant sources by hematogenous spread (Chaudhry et al. 2012). There are no reports of orbital cellulitis from direct inoculation from an untreated conjunctival laceration wound as seen in this patient. Anatomically, the conjunctiva is a loosely adherent layer of mucous membrane onto the underlying episcleral layer. At the conjunctival fornices, it deflects onto itself to cover the inner side of the eyelid. A laceration wound on the conjunctiva might have allowed infection to track through the loose adherence and spread into the orbital space. It is also possible that the infection may have extended from sinusitis in the ethmoidal sinuses. However, this is less likely in this patient as there was no evidence of a breach in the lamina papyracea.

Common early presenting symptoms of orbital cellulitis include eyelid oedema, pain with eye movement, and ophthalmoplegia (Lee & Yen 2011). Late signs like decreased vision and proptosis indicate vision threatening complications from ischaemic, compressive or infective optic neuropathy (Bluestone et al. 2002). Intracranial extension is a lethal sequelae leading to meningitis and cavernous sinus thrombosis which may result in bilateral blindness (Thakar et al. 2000). Determining the source of infection with imaging is also important as drainage is sometimes necessary to expedite recovery especially in cases with intraorbital abscess collection (Bluestone et al. 2002).

The classification of orbital cellulitis was initially set forth by Hubert in 1937 and was later modified by Chandler et al. in 1970 into five stages (Thakar et al. 2000). Stage I is described as preseptal cellulitis with inflammatory oedema of the eyelid. Stage II is a true orbital cellulitis with diffuse oedema of the orbital contents. Stage III is the presence of subperiosteal abscess between the periorbita and the bony orbital wall, usually at the medial or superolateral aspect of the orbit. Stage IV is when orbital abscess occurs with abscess collection within the orbital tissues. Cavernous sinus thrombosis is seen in stage V (Thakar et al. 2000).

Medical therapy is the mainstay of treatment (Thakar et al. 2000). Choosing the appropriate antibiotics is crucial according to the sensitivity pattern. A combination therapy with good penetration into the blood-brain barrier such as a third generation Cephalosporin and Metronidazole for greater coverage on gram-negative anaerobes is preferred in trauma cases as the underlying organism is likely to be of multiple types (Thakar et al. 2000).

CONCLUSION

Immediate and aggressive therapy is important in treating orbital cellulitis to prevent devastating sequelae. Thus, it is important for primary care and emergency physicians to identify the predisposing factors as early referrals to an ophthalmologist can save a patient’s vision and life. Sometimes, a small conjunctival laceration can be missed as a simple subconjunctival haemorrhage. Therefore, a thorough eye check should be done to exclude any possible external ocular soft tissue injury and an ophthalmology assessment might be sought for suspicious cases.

INTRODUCTION

Adult Attention Deficit Disorder (ADD) is a relatively common, often unrecognized condition. In United States, it is estimated to affect about 4.4% of adult population (Kessler et al. 2006). Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) (American Psychiatric Association, 2013) defined ADD under ADHD chapter. It define four out of the nine ADHD symptoms of inattention relating directly to problems with organization and planning (i.e. loses things, is forgetful, has difficulties organizing tasks, and fails to finish task. Adults with ADD live with the symptoms and suffer effects of the illness which attribute to their own shortcomings (Brook et al. 2013).

Methylphenidate and atomoxetine are currently the most prescribed drugs for adult ADHD. However, it was found that even though it was effective but it was not always well tolerated. One of proposed alternatives treatment for ADHD was the antidepressants, particularly those with noradrenergic activity (Buoli et al. 2016). There are multiple reports on the use of such antidepressants for the treatment of ADHD in adult populations.

CASE REPORT

A 23-year-old man was referred by a general practitioner (GP) for being had poor concentration and gradual decline in his study in computer science in Europe. The parents noted that he was not able to pay attention in his class and he spent time playing computer games.

He was easily distracted by irrelevant tasks like playing with his computer game while reading his book, bounced from one activity to another, or became bored quickly. His problem of his mood and study was sometimes overlooked because they are less overtly disruptive. He had symptoms of inattention and concentration difficulties which included extreme distractibility while focusing in his study, struggling to complete his reading tasks, tendency to overlook details, and leading to errors or incomplete work. For the past 2 yrs, occasionally he felt low in his mood for an episodic period of 2 – 3 months because he was not able to focus in his study and also absent from his class. Sometimes, he was anxious. However, he denied any psychopathology amounting to a diagnosable entity of anxiety and major depressive disorder or dysthymia.

He was seen by the psychiatrist and diagnosed to have ADD and started with Buproprion 150 mg daily to control his anxiety and mood symptoms. He consumed his medication without significant side-effects. Fifteen days following medication, he subsequently felt better. Regarding his task in focusing on his study, he was also prescribed with Tablet Ritalin 10 mg PRN. After decline in his study in computer science in Europe. The parents noted that he was not able to pay attention in his class and he spent time playing computer games.

He was easily distracted by irrelevant tasks like playing with his computer game while reading his book, bounced from one activity to another, or became bored quickly. His problem of his mood and study was sometimes overlooked because they are less overtly disruptive. He had symptoms of inattention and concentration difficulties which included extreme distractibility while focusing in his study, struggling to complete his reading tasks, tendency to overlook details, and leading to errors or incomplete work. For the past 2 yrs, occasionally he felt low in his mood for an episodic period of 2 – 3 months because he was not able to focus in his study and also absent from his class. Sometimes, he was anxious. However, he denied any psychopathology amounting to a diagnosable entity of anxiety and major depressive disorder or dysthymia.

He was seen by the psychiatrist and diagnosed to have ADD and started with Buproprion 150 mg daily to control his anxiety and mood symptoms. He consumed his medication without significant side-effects. Fifteen days following medication, he subsequently felt better. Regarding his task in focusing on his study, he was also prescribed with Tablet Ritalin 10 mg PRN. Aftera month with both daily intake of his medication, he felt much better and was able to concentrate on his study.

DISCUSSION

This case report highlighted a scenario of an adult gentleman who was recently diagnosed to have adult attention deficit disorder, which was missed during the early stage of his life. This situation also happens in Europe as many cases happen to be never been diagnosed or treated when there were children (McCarthy et al. 2009). In this case, he presented at age of 23 yrs and happened to be undiagnosed during childhood.

There were not much difference in the diagnosis of ADHD in adult. In this case, as inattention was a predominant. Patient fullfilled the criteria such as not being able to pay or sustaining attention to his study, often easily distracted from his task, appeared to have tendency for incomplete task and possibility of leading to error. All these criteria lead to the diagnosis of ADHD (Katragadda & Schubiner 2007).

Anxiety is defined as the feeling of being very worried about something that may happen or may have happened. A person is conscious of the unpleasant emotional state of threat or danger which can affect one’s behaviour and physiological system. Depression is defined as feeling of sadness that may makes one think that there is no hope for the future (Ho et al. 2007). A patient suffering from this illness has difficulty to complete daily tasks. Prolonged untreated causes distress and anxiety to the patient. It showed that ADHD in adulthood has higher risk to get anxiety disorder and depression as it happened in this patient (Biederman et al. 2010; Schatz & Rostain 2006).

Treatment in adult ADHD could be either pharmacotherapy or non-pharmacotherapy (Hamedi et al. 2014). Patient with ADHD may benefit from cognitive behaviour therapy. As in pharmacotherapy, a stimulant is often chosen as first line of ADHD treatment (Epstein et al. 2014; Buoli et al. 2016). In this case, patient presented with anxiety and mood symptoms which was common presentation in adult with ADHD (Biederman et al. 2010). It is supported that an antidepressant showed an evidence to be effective in management. Bupropion is one of the antidepressants that showed effectiveness in treating ADHD with comorbid depression and anxiety (Daviss et al. 2001; Buoli et al. 2016). It is important to identify the symptoms

CONCLUSION

Currently there were very limited database regarding alternative management for adult ADHD. More evidence base study were needed to prove and explained the effectiveness of treatment for ADHD with the used of antidepressant. As in this case, the outcome proved that Bupropion as an antidepressant should be considered for pharmacological management of adult ADHD especially in case of anxiety and depression presenation.

INTRODUCTION

Plasma cell myeloma is a neoplastic clonal disease associated with plasma cell infiltration in the medullary space. It causes clonal proliferations of immunoglobulin detectable as monoclonal protein peak or M-protein on serum or urine protein electrophoresis studies. This condition is also known as monoclonal gammopathy and the proteins are mostly IgG M-protein type in more than 50% of cases and IgA in 20% of cases (Swerdlow et al. 2008). Monoclonal gammopathy can also produce only monoclonal light chains in 20% of cases (Swerdlow et al. 2008) and fewer than 2% of cases produce monoclonal IgD, IgE, or IgM (Reece et al. 2010).

Biclonal gammopathies are rare group of disorders characterized by simultaneous production of two distinct monoclonal proteins. These two distinct monoclonal proteins can be the result of proliferation of two clones of plasma cells, each producing a different monoclonal immunoglobulin, or it may be due to the production of two monoclonal proteins by a single clone of plasma cells (Kyle et al. 1981). The most common combination of these biclonal gammopathies are IgG and IgA (53%), followed by IgM and IgG combination (24%) (Kyle et al. 1981). Other reports have described combinations of biclonal gammopathies, including IgD/IgG, IgG/IgM, IgA/IgG, and kappa/lambda light chain biclonal gammopathies (Huppmann et al. 2010; Kim et al. 2011). The clinical presentations and the response to therapy in such cases is said to be similar to monoclonal gammopathy type of plasma cell myeloma.

Here, we report a rare case of plasma cell myeloma with IgG (lambda) and IgA (lambda) type of biclonal gammopathy detected by appearance of two bands in the lambda region on serum protein electrophoresis. The classes were further confirmed by immunofixation. We present our data to disseminate knowledge on this rare presentation of plasma cell myeloma.

CASE REPORT

A 76-year-old male was referred to the haematology team for unexplained persistent anaemia with reverse albumin to globulin ratio. He was otherwise asymptomatic. Physical examination was unremarkable except for pallor. Full blood count showed anaemia (haemoglobin count of 7.7g/dL) with lymphocytosis (5.6x109/L). The blood film exhibited hypochromic microcytic anaemia with presence of rouleaux formation (Figure 1). Bone marrow biopsy showed presence of 50% abnormal plasma cells which exhibited multinucleated forms, flame-shaped plasma cells and vacuolated cytoplasm. Occasional immature forms were also present which exhibited more dispersed chromatin, low nuclear cytoplasmic (N/C) ratio with some having prominent nucleoli (Figure 2). All other haematopoietic cells were markedly reduced. The trephine biopsy reported hypercellular marrow composed of malignant plasma cells that were positive to CD138 with lambda light chain restriction.

The immunophenotypic analysis of the bone marrow aspirate sample showed an abnormal cells population gated at CD38 bright with low side scatter (Figure 3). These cells expressed CD138, CD56 with lambda light chain restriction. They were negative for CD19, CD117 and CD79a which was consistent with plasma cell myeloma with aberrant expression of CD56.

Bone survey showed no lytic lesion. The biochemical parameters showed normal renal profile with no evidence of hypercalcaemia. Urinalysis showed moderate proteinuria (2+) with the presence of Bence Jones protein. The serum protein electrophoresis (Figure 4a) showed biclonal paraprotein in the gamma region with paraprotein quantitation of 60.4 g/L. Urine protein electrophoresis (Sebia) also indicated similar findings of tubular proteinuria with the presence of Bence Jones protein with protein quantitation of 65.8 mg/L. The corresponding serum immuno-fixation electrophoresis (Sebia) revealed biclonal gammopathy with the presence of two distinctly separate bands in the IgG lambda and IgA lambda region whilst the urine immuno-fixation exhibited band only in the IgG lambda region (Figure 4b).

Based on the above findings, the diagnosis of plasma cell myeloma stage II (The International Staging System) was made. The patient had been started on chemotherapy regime consisting of cyclophosphamide, thalidomide and dexamethasone but eventually died after completed 1st cycle due to neutropenic sepsis.

DISCUSSION

The present case highlights a rare expression of two distinct monoclonal proteins in a plasma cell myeloma patient. To the best of our knowledge, this is the first case reported in our institution. Plasma cell myeloma is characterized by heavily mutated variable region (VH regions) with no intraclonal variation produced by the immunoglobulin. This results in clonal proliferations of plasma cells which produce monoclonal immunoglobulin heavy chain (IgH) or monoclonal immunoglobulin light chain (IgL). However, previous studies reported up to 2% of myelomas cases which secrete two different IgH isotypes or subclasses (Kyle et al. 1981). This condition which is also called biclonal gammopathies is characterized by the production of two distinct monoclonal proteins with most being IgG/IgA (53%) or IgG/IgM (26%) (Kyle et al. 1981). Our patient in this case report belongs to the group of presence of two distinctly separate bands in the IgG lambda and IgA lambda region. There are other reports showing the different combination of monoclonal proteins. Banerjee et al., (2016) reported very rare pattern of IgA with Lambda and IgG with Kappa light chains M bands. Kumar et al. (2014) has even described rarer variety of biclonal gammopathy of IgA kappa variant. Similarly other combination of biclonal IgA kappa and IgA lambda (Al-Riyami et al. 2015) were also reported.

Biclonal gammopathy can occur as a result of proliferation of two clones of plasma cells, each producing an unrelated monoclonal immunoglobulin, or it may result from production of two monoclonal proteins by a single clone of plasma cells (Kim et al. 2011). Although these biclonal gammopathies can be due to proliferation of two different clones of unrelated plasma cells and monoclonal proteins, it has been postulated that some biclonal pairs may result from a transformation event in a cell undergoing a variable region switch from one heavy chain class or subclass to another. If this was the case, it would be predicted that the variable regions of the biclonal pair would be identical and it might be possible to find some plasma cells producing both monoclonal proteins (Kyle et al. 1981).

Plasma cell myeloma with monoclonal IgG subtype has been shown to have clonotypic Cμ transcripts in the bone marrow of 68% of patients which proves that these cells could be the clonogenic origin of the tumor clone. However, no extensive study has been done on cases of biclonal plasma cell myeloma in order to identify what abnormality can be detected at the RNA level (Decaux et al. 2008).

Based on a study done by Decaux et al. (2008) on clinical and biological features of biclonal gammopathies review of 203 cases, it was reported that about 40% of the patients with biclonal gammopathies have been found to be symptomatic and most of them were more frequently associated with lymphoproliferative diseases rather than plasma cell myeloma (Chen et al. 2013). This is quite contradictory to our case in which the patient was asymtomatic with normal serum calcium level, normal renal profile and no lytic lesion seen on the skeletal survey which is typical presentation of smouldering myeloma.

In term of treatment response, Kyle et al. (1981) in his study on 57 cases of biclonal gammopathies showed that the response to therapy and survival were much the same as in patients with plasma cell myeloma with monoclonal protein in most of the cases. Nevertheless, there are cases of plasma cell myeloma with biclonal IgD and IgM reported showing disease aggressiveness and chemo-resistant (Chen et al. 2013). In our case, although the patient was started on chemotherapy regime but eventually died due to neutropenic sepsis which is a main concern especially when using thalidomide for the treatment of frail patients (Palumbo et al. 2012).

CONCLUSION

We reported a rare case of plasma cell myeloma with a combination of IgG lambda and IgA lambda biclonal gammopathy. Further extensive study on molecular events responsible for this exceptional combination may be helpful to determine their clonal origin, pathogenesis and may unfold whether they belong to a truly biclonal population or rather a single neoplastic clone.