Hypertension seldom occurs in isolation of other risk factors to which it is metabolically linked. Antihypertensive drugs may increase coronary risk by their effect on metabolic risk factors of coronary heart disease. This study was performed to investigate the effects of 2 new antihypertensive drugs, irbesartan and valsartan, which are angiotensin II receptor blockers. A clinical study was carried out to assess the efficacy, clinical acceptability, hypotensive and adverse effect of irbesartan on known metabolic risk factors as well as laboratory parameters in the Malaysian hypertensive populations. Thirty-four mild to moderate hypertensive patients were included in a single blind study over a period of 3 months. Clinic blood pressures were normalized in 76.5% (n=26) of hypertensives patients, and 79.1 % (n=27) of hypertensive patients responded after treatment with 150mg and 300mg daily irbesartan. The proportion of reported adverse events after irbesartan therapy and placebo were 16% (n=8) and 26% (n=l3) respectively. Irbesartan had no significant effect on heart rate and body weight. An extended oral glucose tolerance test (OGTT) was performed at baseline and after 3 months of treatment in 7 hypertensive patients. It was noted that irbesartan had no influence on plasma glucose tolerance and insulin levels. Irbesartan increased plasma basal insulin levels with no effect on glucose levels in hypertensive patients. It also reduced total cholesterol, low density lipoprotein cholesterol and serum uric acid in hypertensive patients. Haematologically, irbesartan reduced erythrocyte count, and increased the mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and increased leucocyte counts. Irbesartan decreased aspartate aminotransferase levels. The noninvasive 24-hour ambulatory blood pressure monitoring showed that irbesartan significantly reduced mean 24-hour blood pressures but did not influence blood pressure variability. Irbesartan has an acceptable trough to peak effect (>50%) in hypertensive patients. In study to identify plasma total homocysteine (tHcy) levels, included 41 hypertensive patients (34 of them were receiving irbesartan at baseline) and 54 healthy normotensives. The potential effects of irbesartan on tHcy levels were also evaluated. The 95% confidence interval of tHcy levels for healthy normotensive and hypertensive subjects were 3.62 to 13.30?mol/L and 5.58 to 16.42?mol/L, respectively. Male normotensives had higher tHcy levels than their female counterparts. The tHcy levels were significantly higher in hypertensive patients compared to normotensive subjects. A positive association was observed between tHcy levels and diastolic blood pressure in normotensive subjects. Irbesartan produced significant reduction in tHcy levels after 3-month therapy in hypertensive patients. At baseline, there was an inverse relationship between tHcy and plasma insulin, but no significant correlation was observed after irbesartan therapy. Finally, an in vitro experiment was conducted to study the effect of glucose-induced insulin secretion using isolated rat pancreas by the perfusion technique. Concentration of valsartan used was based on the peak plasma concentration with a single standard oral dose of 80mg daily in human. Valsartan significantly increased insulin secretion at all concentrations (0.164mg/L or 1/10 of the peak, 1.64mg/L or at peak, and 16.4mg/L or 10 times of the peak).

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Ticlopidine is used as an anti-platelet drug in patients with ischaemic heart disease. An in vitro study suggested that ticlopidine inhibited CYP2D6 and the widely used antianginal metoprolol is metabolized by this polymorphic enzyme. The objective of this study to investigate the effect of ticlopidine treatment in patients maintained on chronic metoprolol therapy. The study was approved by the Ethics Committee of International Islamic University Malaysia (IIUM) and strictly adhered to Malaysian Good Clinical Practice (GCP) guidelines. This was an open labelled Case Controlled Study where all the patients were screened for the inclusion and exclusion criteria. CYP2D6 genotyping were performed for *3,*4,*5,*6,*9,*10, *14, *17 and duplication. Two weeks after the screening visit, blood for metoprolol was taken at timed intervals together with serial measurement of blood pressures and heart rates. Subsequently the patients were given a standard dose of ticlopidine 250 mg twice daily for a period of one month. At the end of study period, blood for metoprolol was repeated together with serial measurement of blood pressures and heart rates. Eighty seven patients completed the study. The frequency of predicted Poor Metabolizer (PM) was low at 2.6%, where both patients had homozygous *4/*4 and the majority (47.8%) carried the allele *10, predicted Intermediate Metabolizer (IM). After ticlopidine treatment, there were increasing trend within the metoprolol pharmacokinetic parameters among the different predicted phenotypes and different allele variations. Plasma Metabolic Ratio was significantly different (p< 0.05) between phenotypes and allele variations. The two Poor Metabolizers (PM) patients presented with bradycardia even at doses of 25 mg and 50 mg twice daily. There were wide variations among the CYP2D6*10 allele group for metoprolol pharmacokinetic parameters, suggesting the presence of a subgroup population that may overlap features with the Poor Metabolizer group. However, there were no significant change for both pre and post ticlopidine for blood pressure control and heart rate. After ticlopidine, there was a neutrophil count reduction in 32 patients and 6 patients had neutropenia with neutrophil count less than 2.5 x 10³ / μl. Concurrent use of ticlopidine with metoprolol may subject patients who are poor CYP2D6 metabolisers to have exaggerated response to beta blockade and blood dyscrasias may occur frequently.

Hepatitis C infection in haemodialysis (HD) centres is still of utmost global concern to health care systems. Studying the molecular features of the virus throws light on its epidemiology and behavior in those undergoing prolonged HD. In this study, hepatitis C virus (HCV) infecting HD patients in Pahang were characterized and the prevalence of the various genotypes and subtypes determined and the full-length genome of the most prevalent genotype was elucidated. Viral RNA was extracted from sera of 40 HD patients. The molecular technique of reverse-transcription polymerase chain reaction (RT-PCR) was used to detect and amplify segments of the viral RNA at the 5’untranslated region (UTR) and the nonstructural 5B region (NS5B). These regions were well conserved among HCV genotypes thus making them useful for both virus detection and genotyping purposes. The base sequences of the amplicons were determined and later employed in phylogenetic analysis for genotype assignment of the isolates. The sequence pattern (SP) and the conformational features of domain III 5’UTR were also evaluated. The overall prevalence of HCV viremia in HD centres was 8.3 %, including one acute infection that occured during the course of study. For 33 of the 40 viremic patients, the HCV 5’UTR and NS5B base sequences were successfully determined. Five patients (15.2 %) were shown to be infected by more than one genotype. In the remaining 28 patients, genotypes 3, 1, 4, and 6 occurred in 19 (67.9 %), seven (25.0 %), one (3.6 %), and one (3.6 %) of them respectively. Four sequence patterns (SPs) were determined in domain III 5’UTR of genotype 3, a finding unique to this genotype. Overall, the secondary structure of the domain was not affected by nucleotide changes in the SP region. The complete base sequence of two genomes of the predominant subtype 3a isolates (MAL 22 & MAL 43) were elucidated and found to be consisting of 9430 and 9429 nucleotides. Genotype 3a HCV genome contained a long open reading frame capable of encoding a sequence of 3010 amino acid residues. In the phylogenetic analysis, both isolates clustered with known HCV-3a isolates (NZL1, K3a & CB), while it diverged from other genotypes by 47.1 - 41.8 %. Three hypervariable regions of the isolates were also revealed and described. The predominance of genotypes 3 and 1 was not unique to HD centres in Pahang as it was reported in normal population in Malaysia. Both the complete viral nucleotide sequence and the amino acid sequence of the coding region of the HCV genome provided the foundation data for future HCV studies in Malaysia including studies on epidemiology, novel diagnostic approaches and antiviral therapies. Despite the known high risk of chronic hepatitis C infection in HD patients, these infections, whether overt or occult and of single or mixed genotypes, were usually clinically silent. Thus, investigational viral nucleic acid based tests on HCV patients was recommended to be done periodically to monitor for the occurrence of these asymptomatic and sometimes even sero-negative infections.

BACKGROUND: Hydroxyapatite is widely used as bone graft substitute. Platelet-rich plasma (PRP) is enriched with growth factors. Hypothetically combination of PRP and hydroxyapatite would augment bone formation effect of hydroxyapatite and widen its application in spinal fusion. OBJECTIVE: To compare new bone formation between hydroxyapatite granules alone; hydroxyapatite granules in combination with PRP; and autograft as the control. METHODOLOGY: First phase of the study dealt with the production of PRP and characterization of platelets by platelet counts and platelet morphology. Second phase involved spinal fusion surgery in twenty-four adult New Zealand white rabbits. All the animals underwent single level bilateral intertransverse process fusion at L5-L6 vertebrae. One side of the animals received either hydroxyapatite granules alone (HA group) or combination of hydroxyapatite granules and PRP (HAPRP group) while the contralateral side received autograft (Autograft group). Four animals each in HA vs. Autograft and HAPRP vs. Autograft were assessed either at 6, 12 or 16 weeks by plain radiograph, computed tomography scan, undecalcified histology, histomorphometry and scanning electron microscopy. Using the histomorphometric data, the mean percentage of new bone areas over the corresponding fusion masses was compared between groups. RESULTS: Mean platelet count in whole blood and PRP were 363 x 103/3/uL (SD 112 x 103) and 1596 x 103/3uL (SD 512 x 103) respectively. Platelets showed minimal morphological changes. Autograft group showed significantly more new bone than HA and HAPRP at 6, 12 and 16 weeks (P=0.004, P<0.001 and P<0.001 respectively) but no significant difference between HA and HAPRP at 6, 12 and 16 weeks (P=0.154, P=0.929, P=0.487 respectively). Autograft, HA and HAPRP groups showed direct contact with new bone but Autograft demonstrated better integration than HA and HAPRP groups. CONCLUSION: Hydroxyapatite granules alone or in combination with PRP could not challenge autograft as bone graft substitute for posterolateral lumbar fusion. [305 words]

Nigella sativa seeds oil (NSO) and Murraya koenigii leaves (MKL) demonstrated robust antioxidant and anti-inflammatory activities in vitro and in vivo. Two vessel occlusion surgery (2VO) is an established rat model of Alzheimer’s disease (AD) which causes chronic cerebral hypoperfusion resulting in oxidative stress and neuroinflammation. These events lead to neurodegenerative brain changes especially within CA1 region of the hippocampus where spatial memory neurons are situated. This experimental intervention study was designed to assess the potential neuroprotective effect of the two herbal plants extracts when given orally to experimental animals ten days before 2VO surgery and continued until the tenth week post 2VO. The assessment was based on cognitive, histopathological and electron microscopical brain tissue analyses. 80 rats were equally divided into 4 main groups namely sham control, 2VO, NSO+2VO and MKL+2VO groups. Each one of them was further subdivided according to the experimental protocol of Morris water maze (MWM) test into long-term memory (LTM) subgroups which underwent acquisition MWM trials before 2VO and were retested for LTM performance on the 10th postoperative week, while the other subgroups were only introduced to MWM during the 10th postoperative week. These subgroups were tested by short-term memory (STM) and working memory test (WMT) protocols in a row. 2VO induced significant LTM, STM and learning impairment in 2VO rat group as compared to sham control as well as NSO+2VO groups, whereas the attenuation of 2VO induced memory impairment achieved by MKL treatment was significantly lower than that of sham control and NSO+2VO groups. The brain tissue analyses included histopathological as well as Transmission Electron Microscopy (TEM) examinations. Light microscope histopathology revealed significantly higher number of viable hippocampal cells in sham control and NSO treated rats groups as compared to untreated 2VO and MKL treated groups. Simultaneously, ultrastructural neurodegenerative changes were observed in CA1 hippocampal subfield of 2VO group which comprised necrosis and apoptosis of pyramidal neurons, astrocytic degeneration with microgliosis and thickening of endothelial basement membranes of hippocampal capillaries. These changes were substantially less demarcated in NSO treated group and were almost completely absent in sham control group. However, a great deal of similarity in ultrastructural deformities was found between untreated 2VO and MKL+2VO groups. It can be concluded that NSO, by virtue of its antioxidant and anti-inflammatory activity, was capable of preventing hippocampal neurodegeneration induced by 2VO, while MKL extract was only able to exert a modest preservation of memory without a neuroprotective effect on CA1 hippocampal neurons after cerebrovascular hypoperfusion initiated by 2VO surgery.

The search for new antimalarials is a continuing effort as the resistance to current antimalarial drugs occur one after another. Thus this study aims to discover a potential antimalarial in the Malaysian local plant. Plectranthus amboinicus is selected as a candidate for antimalarial study. There were four phases involved throughout this study. Phase one focused on the phytochemical screening of ethanolic extract and essential oil of P. amboinicus leaves. The phytochemical screening of the extracts was conducted according to the qualitative phytochemical screening test and Gas Chromatography Mass Spectrometry (GC-MS). Phytochemical screening revealed the presence of flavonoids (in the crude ethanolic extract), carvacrol (85.14 %), thymoquinone (1.65 %), terpinen-4-ol (0.7 %), octenol (0.62 %), thymol (0.23 %), and dioctylazelate (0.15 %) (in the essential oil). Phase two of the study focussed to evaluate the toxicity status of the experimenting extracts prior to conducting antimalarial study on mice infected with Plasmodium berghei. Acute oral toxicity test was conducted according to the OECD guidelines for the testing of chemicals, acute oral toxicity, limit test-acute toxic class method sections 423. Toxicity study with crude ethanolic extracts of P. amboinicus leaves revealed no toxic effect on the mice administered. The acute oral LD50 was found to be above 5000 mg/kg suggesting it to be safe for consumption. Testing with the essential oil also revealed no signs of toxicity and mortality although histopathology results showed the presence of glomerular apoptosis in kidney and evidence of central venous dilation in the liver. Thus, the LD50 was suggested as above 2000 µL/kg. Phase three focused to determine the antimalarial properties of the crude ethanolic extract and essential oil of P. amboinicus leaves. There were three important antimalarial tests that were performed on both the extracts prepared, namely prophylactic test, suppression test and curability test. An antimalarial study on the crude ethanolic extract showed no reduction of parasitemia of mice infected with P. berghei was seen in the suppressive and curative test, but pronounced reduction of parasitemia in the prophylactic test. The best dose of ethanolic extract was found to be 400 mg/kg which reduced parasitemia by 90.74 %. In contrast, the essential oil of P. amboinicus leaves not only possess some potential as a prophylaxis agent, but also a curative agent, but with a less parasitemia reduction compared to the ethanolic extract. The best prophylaxis dose was found to be 1000 µL/kg with 58.26 % reduction in parasitemia, while the best curative dose was also 1000 µL/kg with a reduction of parasitemia of 65.38 %. Similar to crude extract, no reduction of parasitemia was found in the suppression test of the essential oil. Phase four of the study focused on prophylactic test of partitioned fractions of crude ethanolic extract since the extract showed better reduction of parasitemia compared to an essential oil. The extract was partitioned with 3 different polarities of solvent to yield hexane, ethyl acetate and butane fractions. The prophylactic test on the partitioned fractions of the selective crude extract revealed hexane fraction (with 85.54 % chemo suppression) as the best prophylaxis agent, followed by the butane fraction (with 56.56 % chemo suppression) and ethyl acetate fraction (with 47.72 % chemo suppression). In all fractions 400 mg/kg was the most effective dose as prophylaxis agent. Based on these results, it can be concluded that crude extract shows better prophylaxis effect compared to fraction. P. amboinicus leaves extract shows promise as a new potential antimalarial candidate

Recently, hepatitis B virus antigen (HBsAg) level has been used as a cheaper marker than molecular methods, not only for indicating active hepatitis B infection but also for predicting the clinical and treatment outcomes. However, data correlating HBsAg level with other diagnostic markers in Malaysia are inadequate to investigate the legitimacy of using HBsAg level as a surrogate or complementary serological marker for chronic hepatitis B (CHB) patients. Therefore, the goals of this study were to quantify HBsAg level in CHB patients and investigate its correlation with immune status (exemplified by peripheral blood lymphocytes’PBL’ subset counts), HBeAg, anti-HBe, viral DNA load, HBV genotypes, and the presence of precore (G1896A) mutations.Methodology:A total of 50 CHB cases and 20 healthy controls were recruited for this cross-sectional study. Serum samples from cases were evaluated for both serological and virological parameters. The HBsAg concentrations for all patients were measured using Roche’s Elecsys HBsAg II assay. The immune status for both study populations were evaluated by estimating the percentage and count of PBL. Viral DNA from all patients’ sera was used for subsequent molecular tests such as PCR assay for HBV DNA detection and genotyping. Real time PCR with SYBR green assay was also performed for viral DNA quantification. Lastly, precore mutants of HBV were detected using high resolution melting analysis. The data were analysed to discover the relationship between HBsAg levels and the other parameters. Results: Viremic CHB patients exhibited narrowly higher mean levels of bilirubin and liver enzymes than the controls. The study of peripheral blood lymphocytes revealed that the percentage of CD4+& CD8+cells were significantly reduced in the patients’ while CD4+/CD8+ ratio has increased. The measurement of HBsAg titers showed that the first group which represents 56% of patients are those with HBsAg levels of >1000 IU/mL, while, second group with <1000 IU/mL represents 44% of CHB patients. Specific viral S small gene DNA was identified in most patients (n=35) and the phylogenetic tree analysis for these samples elucidated that the sequences were classified as genotypes C & B with prevalence rates 54.2% for genotype C and 45.7% for genotype B. In the viral load study, a total of 15 out of 35 patients have a high viral DNA load? 3×106genome copies/mL, while 7 patients were regarded having a low viral load < 3× 106 genome copies/mL. The remaining 13 patients were categorised as undetectable viral DNA load (3× 103 copies/mL). The correlation outcomes showed that a higher number of patients with low-HBsAg level are positive for anti-HBe antibodies. In addition, there is a positive correlation between surface antigen levels with elevated ALT enzyme, and significant positive and negative correlations have been found between HBsAg titres and the percentage of total T and NK cells respectively. The analysis of association between HBV genotypes and HBsAg levels revealed that patients with genotype C have the higher serum level of this protein than genotype B. Other finding has showed no significant relationship between viral DNA copy number and HBsAg level. The precore (G1896A) variants of HBV were detected in only 25% of patients who tested negative for HBeAg but did not illustrate any correlation with HBsAg titres. Conclusion:The overall outcomes presented in this study are conducive to the importance of quantitative HBsAg serum level as a clinical complementary laboratory biomarker for managing and predicting chronic hepatitis B virus infection progression in the Malaysian’ tested patients.

Globally, snakebite cases are estimated to be around 5 million annually affecting mainly the residents of poorer counties like Africa and Asia, and in 2009 WHO has categorised it as a ‘neglected tropical disease’. Currently the standard treatment for snake envenomation is the use of anti-snake venom (ASV) therapy. However this is expensive and not readily available in smaller hospitals in the developing world. Herbal medicine has been and is still in use in some cultures for the treatment of snakebite and one such plant is Tamarindus indica. This plant is found in many countries where snake envenomation is also prevalent. This study was conducted to evaluate the potential of using T. indica seed extract (TSE) to inhibit the effects of snake venom of three snakes; namely Naja kaouthia, Ophiophagus hannah and Daboia russelli. The testa of tamarind seed was used and it underwent ethanolic soxhlet extraction to obtain TSE. The inhibition of the activity of the following enzymes i.e phospholipase A2 (PLA2), proteinase and phosphomonoesterase (PME) in vitro by the three snake venoms with TSE was studied. SDS-PAGE experiment was conducted to observe the effects of TSE on venom proteins. In vivo acute subcutaneous (SC) toxicity of TSE in ICR mice was conducted. Study on the inhibition of lethality was conducted on each of the three snake venoms when SC TSE was injected into mice. Venom concentration and site were fixed but TSE concentration, time and site of injection were manipulated. Findings from venom enzymatic inhibition studies showed that, TSE was able to significantly reduce (p<0.05) all three venom enzymatic activities i.e PLA2, proteinase and PME. SDS-PAGE experiment showed that venom protein bands were disrupted when venom reacted with TSE. No signs of toxicity were observed over a period of 4 weeks when mice were exposed to SC TSE 60 mg/20 g body weight except for skin ulcers. Histological examination on liver, both kidneys and skin at the site of SC injection showed no changes compared to the control group injected with SC distilled water. TSE was able to increase the survival rate of ICR mice when exposed to each of the three snake venoms regardless of the site of injecting SC TSE. Mice injected with N. kaouthia or D. russelli venom, had increased 24 hour survival rate when SC TSE was given at 15 minutes; and of mice injected with O. hannah venom the 24 hour survival rate increased with higher TSE concentration when given sooner. In conclusion, SC TSE was safe to be injected up to 60mg/20 g and has the potential to delay the effects of venom from N. kaouthia, O. hannah and D. russelli.

Leptin is a multifunctional hormone produced mainly by the white adipocyte. For leptin to act, it needs to bind to its receptor. leptin and its receptor activity is correlated with breast carcinogenesis. The main aim of this study is to investigate the correlation between leptin/leptin receptor and the clinico-pathological features of breast cancer. Fifty one women with breast cancer were included in this study while the control group was 40 women with negative mammogram for breast cancer. Demograghic data, anthropometric measurements and blood samples for serum leptin measurement were taken from all the participants. Leptin level was compared in patients with that of the control. Pre and postoperative leptin levels were studied. We investigated the correlation of leptin and its receptor expression with the clinico-pathological features of the breast cancer through the immunohistochemistry. We localized leptin and its receptor intracellularly using transmission electron microscope (TEM) immunocytochemistry. This study investigates the role of leptin in breast carcinogenesis in many aspects and investigates the subcellular localization of leptin and its receptors in the breast cancerous cells and comparing it with that of normal breast tissue using TEM. No significant differences in leptin levels were obtained in the study group among marital status, parity and menopausal status. Leptin level was significantly high in the study group as compared with the control group; no significant difference was found in leptin levels between the preoperative and postoperative measurements. We found that the majority of the breast cancer cells studied, stained positively for leptin and leptin receptors with co-expression of leptin and its receptors. No significant correlation was found between leptin/leptin receptors expression with the race, menopausal status, lymph node metastasis, estrogen receptor expression, progesterone receptor expression, HER2 expression and tumor size. Majority of the patients with distant metastasis were associated with high leptin and leptin receptor expression. In our study, TEM showed that leptin gold particles are mainly present in the nuclear membrane of the cancerous cells and some particles appeared in the cytoplasm, while the adjacent breast epithelial cells showed that leptin gold particles are scattered all over the cell with much less than that of the cancerous cells. Leptin receptor gold particles are mainly present in the nucleus, nuclear membrane of the cancerous cells and some particles appeared in the cytoplasm as well as the cell membrane; the same pattern was seen in the adjacent breast epithelial cells. In conclusion; leptin may have an important role in the carcinogenesis of breast cancer and in its clinico-pathological features. Attempts to inhibit leptin levels and its expression might help the prevention of breast cancer and its aggressiveness.

Decreased cerebral blood supply to the brain can generate a condition of chronic cerebral hypoperfusion, which is one of the physiopathological mechanisms of neuronal degeneration and cognitive impairment in neurodegenerative disorders including Alzheimer’s disease. Trigonella foenum graecum and Eurycoma longifolia Jack have been shown to have antioxidant and anti-inflammatory activities. Therefore, the aim of the current study was to evaluate the potential neuroprotective effect of ethanolic extract of Trigonella foenum graecum seeds (FS) and methanolic extract of Eurycoma longifolia roots (TA) on chronic cerebral hypoperfusion using rat animal model. Chronic cerebral hypoperfusion was induced by permanent bilateral ligation of the common carotid arteries in male Sprague–Dawley rats. The experimental groups were divided into four groups: a sham-operated group, a two-vessel occlusion (2VO) group, a 2VO group that was administered orally with the FS extract (100 mg/kg/day), and a 2VO group that was administered orally with the TA extract (100 mg/kg/day) from 3 days before the date of 2VO surgery and continued until the end of the 8th postoperative week. Spatial memory performance was assessed by the Morris water maze test. Serum malondialdehyde (MDA) content and superoxide dismutase (SOD), glutathione (GSH) activities, C-reactive protein (CRP) concentration were measured. The ultrastructural changes of the hippocampal area (CA1) were observed by transmission electron microscopic (TEM). Chronic cerebral hypoperfusion rats resulted in spatial memory impairments. This behavioural dysfunction was accompanied by decreasing SOD and GSH activities, increasing MDA content and increasing concentration of inflammatory marker (CRP) in serum. Similarly, ultrastructural neurodegenerative changes were observed in CA1 area of the 2VO group, which showed necrosis and apoptosis of pyramidal neurons, astrocytic degeneration with microgliosis, and thickening of endothelial basement membrane of hippocampal capillaries. Oral administration of FS extract significantly improved the memory impairment, enhanced antioxidant enzyme activities, decreased the content of MDA and the CRP levels to their normal levels as well as less ultrastructural changes in CA1 area of hippocampus. On the other hand, TA extract treatment showed a slight preservation of cognitive function, oxidative status and ultrastructural changes in CA1 region following chronic cerebral hypoperfusion. The potential activity offered by FS showed neuroprotective effect that may be beneficial in cerebrovascular type dementia. TA extract showed lesser neuroprotective effect. Further studies may be done to investigate the underlying mechanism.

A variety of evidences of genetic factors had been implicated in schizophrenia but the identification of specific gene has proven to be difficult. Based on the dopaminergic hypothesis in schizophrenia, most of the research conducted has focused on genes regulating dopaminergic function. Accumulating evidences suggest the role of DNA methylation in the patho-aetiology of schizophrenia and there are several confounding factors that may contribute to the DNA methylation. Therefore, the aims of the study are to assess the DNA methylation of COMT, DRD2 and DRD4 genes in the peripheral blood and their associations with the psycho-pathological symptoms, antipsychotic drugs treatment and BMI and the effect on the gene expression. The case control cross sectional study consisted of 138 schizophrenia patients from the Psychiatry Clinic, Hospital Kuantan Ampuan Afzan, Kuantan Pahang and 132 healthy controls from Kuantan district. The genomic DNA samples from the peripheral blood were bisulfite converted. The COMT, DRD2 and DRD4 DNA methylation levels were quantitatively measured by using the MethyLight Taqman® assay and normalized with the ALU reference control to give the percentage methylation ratio. The psycho-pathological symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS). The demographic data were calculated using descriptive statistics while parametric variables were compared using independent samples t-test or analysis of covariance, and the regression analysis was used for prediction. The independent-t test showed significant lower DNA methylation of COMT (p<0.001), DRD2 (p=0.001) and DRD4 (p=0.001) in schizophrenia patients. The lower methylations of each gene were also significant in males and females. There were inverse relationship between the DNA methylation of the dopamine associated genes and psycho-pathological symptoms. COMT, DRD2 and DRD4 DNA methylation were significantly correlated (p <0.002) with, and predictors of the Excitement and the Depressed subdomains of PANSS. In addition DRD4 DNA methylation was highly correlated (p=0.010) and significant predictor of the Disorganization subdomain. There were higher methylations of COMT and DRD2 in typical antipsychotics-treated patients (p?0.05). The overweight BMI schizophrenia patients showed higher COMT and DRD2 DNA methylation (p?0.05). In conclusion, this study strongly support the possible role of COMT, DRD2 and DRD4 DNA methylation could contribute in the patho-aetiology of schizophrenia. The relationship between the DNA methylation of dopamine-associated genes with the psycho-pathological symptoms and the antipsychotics used might indicate the epigenetic role of genes methylation in the manifestation of schizophrenia and their possible role in the mechanisms of action of antipsychotic drugs. The association of the DNA methylation of COMT, DRD2 and DRD4 with BMI support the theories that obesity in schizophrenia is multifactorial and DNA methylation could be one of the contributing factors.

Sepsis is common in the ICU worldwide and contributes to high mortality. However, timely diagnosis, outcome prediction and antibiotic monitoring in sepsis remains challenging. In Chapter Three, the diagnostic value of model-based insulin sensitivity (SI) for sepsis was studied in 38 non-diabetics on their ICU admission in a cross-sectional study. The findings indicated that baseline SI was significantly lower in sepsis (n = 18) versus non-sepsis (n = 20) (0.996 ± 1.269 versus 5.012 ± 4.930 × 10-4 L/mU/min, P = 0.002), with clinically valid diagnostic performance (AUC 0.814). In Chapter Four, similar methodology was applied to a mixed cohort of 86 diabetic and non-diabetic patients newly admitted to ICU. Although baseline SI was significantly lower in sepsis (n = 41) versus non-sepsis (n = 45) (0.560 ± 0.676 versus 1.097 ± 1.473 × 10-4 L/mU/min, P = 0.037), the biomarker failed to diagnose sepsis in this cohort. Hence, model-based SI may be a useful diagnostic test of sepsis when specifically applied to the non-diabetic ICU patients. In Chapter Five, the prognostic value of a combination of biomarkers in sepsis was explored in a prospective cohort study of 159 ICU patients. It was found that a prediction equation utilizing baseline total leukocytes count, procalcitonin, interleukin-6 and arylesterase activity of paraoxonase-1 predicted 30-day mortality with a remarkable performance (AUC 0.814). Therefore, a multi-marker approach using these biomarkers may be a useful predictor of mortality in sepsis. In Chapter Six, the utility of point-of-care procalcitonin (POCT) to guide duration of antibiotic in the ICU was examined in a randomized-controlled trial. Eighty patients were allocated to either the POCT-guided arm (n = 40) or control arm (n = 40). The mean duration of antibiotic was 6.3 ± 2.1 days in the POCT-guided arm versus 9.1 ± 4.7 days in control arm (P = 0.001), while there was no significant difference in 30-day mortality. Thus, POCT guidance reduced antibiotic duration without compromising mortality in our patients.

Introduction: Chlorpyrifos (CPF) is an organophosphate (OP) that is widely used as pesticide in agriculture. Epidemiological studies reported that the incidence of chronic kidney disease (CKD) of unknown cause was increasing among agricultural workers who were exposed to OPs during their working life through dermal contact. However, there is little information on the effects of chronic subcutaneous low dose of OP CPF on kidney in experimental animals. To date, the mechanism of OP-induced kidney damage is not fully elucidated. Objective: The aim of this study was to assess the effects of chronic subcutaneous low dose of OP CPF exposure on the kidney by investigating the structure and function of kidney and to assess the possible mechanisms of OP-induced kidney damage. Methodology: Eighteen male Sprague Dawley rats were randomly divided into three groups, with six rats in each group. Group 1 served as control group, while groups 2 and 3 received subcutaneous vehicle (3% dimethyl sulfoxide + 97% v/v soy oil) and CPF (18.0 mg/kg) respectively, every other day for 180 days. Blood was taken for biochemical analysis while kidney tissues were harvested for histology, immunohistochemistry (IHC) and selective gene expression. Cystatin C, acetylcholinesterase (AChE), advanced glycation end products (AGEs) and malondialdehyde (MDA) levels were measured using quantitative sandwich enzyme immunoassay. Results: Biochemical parameters (urea, creatinine, uric acid, glucose), cystatin C, AGEs and MDA levels were significantly increased (p < 0.05), while AChE activity and electrolytes levels were significantly decreased (p < 0.05) in the CPF-exposed rats. Structural damage of kidney, including diffuse global glomerular hypercellularity and diffuse necrosis of proximal tubular cells were observed in CPF-exposed kidney. IHC revealed strong immunostaining of polyclonal anti-AGEs in glomeruli and polyclonal anti-MDA in the proximal tubular cells of CPF-exposed rats. The expression of genes involved in glucose metabolism (Ager), oxidative stress (Sod3, Cat, Gsr, Pon1 and Nos2) and cell death pathways (Cycs, Casp3, Casp, 8, Casp, 9, Ripk1, Ripk3, Cst3, Havcr1 and Lcn2) showed downregulation trend. Conclusion: Chronic subcutaneous low dose of CPF caused renal dysfunction as evidence by increased endogenous glomerular filtration markers and reduced electrolytes levels and structural kidney damage. The downregulation of these genes could be due to selective exhaustion of pathways that were persistently activated during the prolonged chronic OP-mediated injury. In brief, chronic subcutaneous low dose of CPF caused nephrotoxicity. The process could be facilitated by the effects of CPF on glucose metabolism and oxidative stress.

Acute myocardial infraction (AMI) is the most common clinical manifestation of coronary artery disease (CAD). Young age is no longer considered a protective factor since the incidence of young adults with AMI is increasing. Hypertension is an important risk factor for CAD in young adults. Prehypertension without proper management is also associated with an increased risk of CAD. Hence, the identification of CAD biomarkers in young hypertensive and prehypertensive adults is necessary to improve risk stratification of premature AMI in these cohorts. The main objective of this study was to compare protein expression profiles of young adults with AMI to control subjects for the identification of proteins (candidate biomarkers) that are differentially expressed in AMI patients. This study also aimed to determine the plasma concentrations of the candidate biomarkers in young adults with normotension, prehypertension, hypertension and AMI and evaluate the relationship between AMI and potential CAD biomarker/s in young hypertensive and prehypertensive subjects. This study comprised of two phases; discovery and verification. In the discovery phase, proteins in the pooled plasma samples from young male adults (10 AMI patients and 10 controls) aged 18 to 45 years were separated by two-dimensional gel electrophoresis (2-DE). The protein spots that were differentially expressed in AMI patients relative to the controls were identified via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. In the verification phase, the plasma concentrations of the identified proteins were measured using enzyme-linked immunosorbent assay (ELISA) in 40 plasma samples of control, prehypertensive, hypertensive and AMI groups. In the discovery phase, haptoglobin (Hp), apolipoprotein AI (Apo AI) and apolipoprotein AIV (Apo IV) were significantly upregulated in AMI patients in comparison to the controls (p < 0.05). Meanwhile in the verification phase, the plasma concentration of Hp was significantly higher in AMI patients in comparison to the control, prehypertensive and hypertensive subjects (290.63±99.90 vs. 170.02±108.11 vs. 175.05±108.11 and vs. 208.47±112.97 ng/ml, p < 0.006) respectively. The plasma concentrations of Apo AI and Apo AIV were also elevated in AMI patients, yet the increases were not significant compared to the other groups (p > 0.05). Plasma concentration of Hp was significantly associated with young AMI (OR: 1.019, 95% CI: 1.006-1.033, p = 0.003) after adjusting for other known CAD risk factors. There was also a significant association between AMI and plasma concentration of Hp in hypertensive and prehypertensive subjects (OR: 0.985, 95% CI: 0.973-0.997, p = 0.017 and OR: 0.981, 95% CI: 0.969-0.993, p = 0.002) respectively, independent of other known CAD risk factors. Plasma Hp concentration was significantly correlated with high sensitivity C-reactive protein hs-CRP (r = 0.370, p < 0.001). In Conclusion, consistent upregulation of Hp in discovery and verification phases reflect its potential role as a biomarker of CAD in young adults. Hp is also a potential CAD biomarker that could be utilized as AMI predictor in young adults with hypertension and prehypertension.The significant correlation between Hp and hs-CRP indicates the potential role of these proteins as inflammatory markers in the establishment of CAD in young adults.

Hypertension is emerging as the most prevalent risk factor of ischemic heart disease in young adults, but awareness is low in this age group. The prevalence of prehypertension in this population is also high, putting them at higher cardiovascular risk. The pathophysiology of essential hypertension has yet to be fully understood, and epigenetic modifications have been proposed to play some role. To date, very few epigenetic studies were done in young adults with prehypertension and hypertension. The aim of this study was to compare the level of DNA methylation in the promoter of implicated genes in young adults with normotensive blood pressure, prehypertension and hypertension. An observational cross-sectional study was conducted among 240 subjects age 18 to 45 years in Kuantan, Pahang, Malaysia. Eighty subjects were recruited for each blood pressure group; normotension, prehypertension, and hypertension as defined by the Ministry of Health Malaysia Clinical Practice Guidelines 4th edition. MethyLight analysis was performed to determine DNA methylation levels of IL-6, ADD1 and AGTR1 gene promoter in the blood. Differentially methylated genes in prehypertension and/or hypertension group were followed by gene expression study (n = 10 per group). There was no significant difference in IL-6 methylation between hypertensive and normotensive. IL-6 predicted prehypertension in males (p = 0.014), but not females. Hypertensive and prehypertensive males, and prehypertensive females, had lower ADD1 methylation than their respective normotensive counterparts. After adjusting for other covariates, ADD1 methylation predicted prehypertension and hypertension in males (p = 0.002 and p = 0.034 respectively). There was no significant difference in AGTR1 methylation between the three groups in both sexes. There was no significant association between IL-6 and ADD1 methylation level and gene expression level. DNA methylation of IL-6 and ADD1 are independent predictors of prehypertension and/or hypertension in males hence has potential as an adjunct biomarker for risk stratification or disease progression. This is the pioneering study of IL-6, ADD1 and AGTR1 methylation in prehypertensive and hypertensive young adults. Further study to delineate potential mechanisms linking DNA methylation to disease development is warranted.

Hypertension is the leading chronic cardiovascular disease (CVD) affecting 5.8 million Malaysians. The prevalence of hypertensive disorders of pregnancy (HDP) in Malaysia is approximately 23.3 per 1000 live births. HDP can cause both maternal and foetal morbidity and mortality. It is also an independent risk factor of CVD with endothelial dysfunction postulated as the main pathophysiology. Endothelin-1 (ET-1), a potent vasoconstrictor, has been identified as a pivotal mediator in HDP. Prolonged disturbances in nitric oxide (NO) bioavailability and imbalance of angiogenic factors such as soluble FMS like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) found in endothelial dysfunction may increase susceptibility to CVD and other major adverse cardiac events (MACEs). This study aims to determine serial ET-1, NO and other angiogenic factors in patients with HDP and its association in persistent endothelial dysfunction. Thirty-six pregnant women from the following categories (i) normal pregnant women (Control) (ii) chronic hypertension during pregnancy (CH) and (iii) gestational hypertension (GH) participated in this study. Blood pressure indices measurements and sample collection was done at antepartum (32 weeks) and postpartum (8 weeks and 12 weeks). ET-1 and serum NO was measured using the Human ET-1 (Endothelin-1) ELISA Kit and Nitric Oxide (total) detection kit respectively. sFlt-1, PlGF and VEGF were measured using commercially available kits. A competitive NO antagonist, asymmetric dimethylarginine (ADMA), was measured using high performance liquid chromatography (HPLC). Serum ET-1 was significantly higher in patients with CH (55.3 pg/ml) and GH (35.6 pg/ml) compared to control (11.8 pg/ml) during antenatal until 12 weeks postpartum (CH 38.3 pg/ml, GH 29.5 pg/ml, Control 1.9 pg/ml); accompanied by significantly high levels of sFlt-1 in HDP subjects. Conversely, subjects with CH and GH had lower levels of serum NO, PlGF and VEGF. Persistently higher levels of ET-1 and lower levels of NO up to 12 weeks postpartum in patients with history of HDP results in unmitigated vasoconstrictive effects of ET-1 despite BP normalisation in GH subjects. Long term NO/ET-1 imbalance and non-physiological levels of angiogenic factors in persistent endothelial dysfunction may account for the increased CVD risk.

Chronic exposure to inorganic arsenic has been linked with multiple medical conditions, which shifted the use of inorganic to the organic-based herbicide, monosodium methyl arsenate (MSMA). However, with increasing numbers of chronic kidney disease of unknown causes (CKDu), chronic exposure to herbicide is believed to be one of the potential explanation. To date, studies on the effects of organic arsenic exposure on the kidney are limited. Therefore, this study aimed to investigate the effect of chronic oral organic arsenic exposure on the rat’s kidney. Thirty-six Sprague Dawley rats (N=36) were randomly divided into MSMA exposed, and its corresponding control groups for 2-,4- and 6-month, each with six animals per group. The exposed groups were given oral MSMA at 63.20 mg/kg body weight, while control groups received distilled water. At the end of each duration, the serum was collected for the creatinine level. The kidney tissues were harvested for arsenic level measurement, histopathological, immunohistochemistry, real-time PCR analysis and ultrastructural analysis. Genes expressions were done for kidney injury marker gene (KIM-1), oxidative stress genes (Catalase, GSR, NOS1), apoptosis genes (Tp53, Caspase-3 and Caspase-9) and inflammatory genes (Interleukin-6 and Interleukin-8). Serum creatinine was not significantly different between exposed and control groups. Tissue arsenic level was significantly higher in exposed groups as compared to that of the control group. All gene expression markers were downregulated at 2-month and upregulated at 4-month except for Catalase which remained downregulated. At 6-month, only KIM-1, GSR and Caspase-3 remained upregulated. Histological, immunohistochemistry and ultrastructural findings showed chronological changes in the glomeruli and proximal tubules with increased expressions of malondialdehyde (MDA) staining, Caspase-3 and TUNEL staining with the duration of exposure. Therefore, chronic oral exposure to low dose organic arsenic has demonstrated evidence of kidney injury in rats possibly due to oxidative stress.

The treatment of osteomyelitis is still a major challenge in orthopaedics. A study of osteomyelitis and infection requires the use of a suitable animal model. The New Zealand White Rabbit (NZWR) is an acceptable experimental model that can be used for local delivery of antibiotics in osteomyelitis treatment as it can mimic the disease process in humans. The objectives of this study were to create osteomyelitis in the rabbit femurs and to analyse the treatment given via gentamicin impregnated with biomaterials beads. Thirty-six (36) NZWRs were used in the study. They were divided into two groups [Hydroxyapatite (HA) and calcium sulphate (CaSO4)] with four subgroups 3-, 6-, 12-, and 26-weeks intervals. There were two surgeries performed for each NZWR. The first was to induce the osteomyelitis by inoculating Staphylococcus aureus in the distal of the animal, and the second surgery was for debridement and biomaterial impregnated antibiotics implantation. The responses of the treatments (gentamicin impregnated with HA and CaSO4) to were evaluated through gross appearance, radiograph, micro-CT, microbiological, and histological examination. The rabbits were sacrificed accordingly to evaluate the healing process of the affected bone. The results showed osteomyelitis changes in all rabbits after the inoculation of the bacteria at 3 weeks. The rabbits’ weights reduced after three weeks of bacteria inoculation following the treatment with biomaterial impregnated antibiotics, they showed significant increase in weights at 12 and 26 weeks in both groups. The microbiology analysis at 26 weeks showed that no bacteria were isolated. All the defects at the drilled site of the distal femurs of the NZWRs were united at 12 weeks interval. The histological examination revealed healing of the infected area with the appearance of a new bone formation at 6 to 26 weeks. The micro-CT results revealed increased the trabeculae numbers with the treatments. The biomaterials containing CaSO4 disappeared by 26 weeks. There was completed bone healing at 26 weeks of interval for both groups. The results of the gentamicin impregnated with HA and CaSO4 for all parameters are comparable. Hence, the antibiotics impregnated with biomaterials are proven effective in the treatments of osteomyelitis. In conclusion, the results of this study show that gentamicin impregnated with biomaterial has a great potential to be utilised for the treatment of osteomyelitis.

Calcium phosphate is an ideal bone substitute material that is widely used for bone repair due to its excellent biological properties including biocompatibility and osteoconductivity. In order to improve the properties of calcium phosphate materials for clinical use, a new injectable self-hardened synthetic bone cement (Osteopaste) was developed. Osteopaste consists of tetra-calcium phosphate (TTCP) and tricalcium phosphate (TCP) powder. It was intended for the treatment of bone fracture or reconstruction of bone defects. The objective of this study was to compare bone formation between Osteopaste and commercialized synthetic bone grafts; JectOS (calcium phosphate) and MIIG-X3 (calcium sulphate) at three different assessment periods. The first phase of the study was to establish the critical size defect in New Zealand White rabbit model. The second phase involved the implantation of Osteopaste, JectOs and MIIG-X3 in critical size defects. Thirty-nine New Zealand White rabbits were divided into four groups (Osteopaste, JectOs, MIIG-X3 and sham). Each group was further divided into three subgroups according to the assessment period either at 6, 12 or 24 weeks. Each subgroup consisted of four rabbits except the sham group which consisted of only one rabbit. A critical size defect of approximately 4.5 mm (width) X 9.0 mm (length) was created at the proximal tibial metaphysis of rabbit's right leg and then implanted with either Osteopaste, JectOs or MIIG-X3. At each assessment period, plain radiograph and computed tomography (CT) scan were performed before the animals were sacrificed for undecalcified histology, histomorphometry and scanning electron microscopy assessments. Using the histomorphometric data, the mean percentage of new bone areas and the length of unbridged defects were compared between groups. In this study, a simple and safe method for performing critical size defect at proximal tibial metaphysis was established. The Osteopaste group exhibited radiographic density in between JectOS and MIIG-X3. The critical size defect in Osteopaste group was bridged by new bone at 12 weeks. In MIIG-X3 group, the defect was bridged at 24 weeks whereas in JectOS group, the defect was not bridged at all assessment periods. New bone area was the largest in MIIG-X3 group followed by Osteopaste and JectOS groups. Osteopaste had formed direct bonding with host bone without intervening soft tissue compared to JectOS and MilG-X3. There were significant differences in new bone area percentages between Osteopaste, JectOs and MIIG-X3 at 6, 12 and 24 weeks post-surgery (P<0.0001 ). In conclusion, the performance of Osteopaste to promote new bone formation is in between JectOS and MIIG-X3.