Comparison of serum levels of Cystatin-C and traditional renal biomarkers for the early detection of pre-hypertensive nephropathy.

OBJECTIVE: To compare serum Cystatin-C and serum creatinine levels along with estimated glomerular filtration rate of apparently healthy people of South Asian descent with pre-hypertension to determine which is better in detecting reversible renal dysfunction. METHODS: :The comparative cross-sectional study was conducted at the Army Medical College, Rawalpindi, Pakistan, in 2013-14, and comprised apparently normal healthy male and female volunteers. The subjects were divided into normotensive group 1 and pre-hypertensive group 2. Serum Cystatin-C levels were measured by sandwhich enzyme-linked immunosorbent assay technique whereas serum creatinine levels were measured by Jaffe's procedure. Glomerular filtration rate estimation was done by using standard equations. SPSS 20 was used for data analysis. RESULTS: Of the 78 subjects, 39(50%) were in normotensive group 1 and 39(50%) in the pre-hypertensive group 2. The mean age was 38.74 } 5.71 years in group 1 and 38.07 } 3.84 years in group 2. Serum Cystatin-C levels were higher in group 2 than in group 1(p= 0.0001), whereas serum creatinine levels manifested no statistical difference between the groups (p=0.106). Estimated glomerular filtration rate based on Cystatin-C significantly decreased in group 2 than in group 1 (p=0.0001). Serum Cystatin-C displayed a significant positive correlation and estimated glomerular filtration rate based on Cystatin-C negative correlation with the rising blood pressure values (p=0.0001).Serum Cystatin-C reflected a very high sensitivity and specificity at a cutoff value of 0.77 mg/l compared to serum creatinine. CONCLUSIONS: Serum Cystatin-C and Estimated glomerular filtration based on rate Cystatin-C appeared to be better renal biomarkers in the detection of pre-hypertensive nephropathy.

Effect of ascorbic acid and alpha tocopherol supplementation on acute restraint stress induced changes in testosterone, corticosterone and nor epinephrine levels in male Sprague Dawley rats.

BACKGROUND: Stress of various origins suppresses male reproductive functions through releasing stress hormones. Antioxidant like ascorbic acid (AA) and alpha tocopherol (AT) have been thought to protect the body against stress induced damage. Whether, these antioxidants confer protection against the stress induced increased levels of corticosterone and nor-epinephrine, and decreased testosterone secretion have been investigated in this study. METHODS: This quasi experimental study was carried out at the Department of Physiology, Army Medical College Rawalpindi in collaboration with National Institute of Health, Islamabad during March to September 2009. Eighty male Sprague Dawley rats were divided into five groups with sixteen rats in each group. Group-I served as the control without stress while group-II was exposed to restraint stress for 6 hours, group-III was administered AA, group-IVAT and group-V was supplemented with both the antioxidants along with standard diet for one month. All antioxidant supplemented groups were exposed to restraint stress for 6 hours. Immediately after the stress episode, blood sample was obtained for the assay of serum testosterone, serum corticosterone by EIA and plasma nor-epinephrine levels by ELISA. Data were analyzed on SPSS-13 and p-value less than 0.05 was considered significant. RESULTS: Acute restraint stress resulted in a statistically significant rise in corticosterone and nor-epinephrine levels and fall in serum testosterone levels. AA supplementation for one month revealed insignificant changes in stress induced hormonal parameters. AT alone and in combination with ascorbic acid prevented the fall in testosterone level as well as rise in corticosterone, however nor-epinephrine levels remained unchanged. CONCLUSION: Supplementation with AT alone or in combination with AA prevent reduction in testosterone and rise in corticosterone levels while keeping the nor-epinephrine levels unchanged after acute restraint stress in Sprague Dawley rats.

Adrenocortical response to 17-beta estradiol replacement in oophorectomized female sprague dawley rats.

OBJECTIVE: To determine the effect of estradiol treatment on serum corticosterone levels in Oophorectomized (OVX) female Sprague Dawley rats exposed to chronic restraint stress. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Physiology, Army Medical College, Rawalpindi and National Institute of Health, Islamabad, from January to December 2008. METHODOLOGY: A total of 90 female Sprague Dawley rats (age: 90 ± 10 days), were divided into three groups, each having 30 rats. Group-I comprised of healthy control female rats whereas group-II and III were experimental female rats exposed to chronic restraint stress after bilateral Oophorectomy and called estradiol treated and vehicle treated groups. Estradiol treatment of Oophorectomized rats was done once daily for 2 weeks. At the end of experiment, the rats were sacrificed and intracardiac blood sampling was done to measure serum corticosterone levels by enzyme linked immunosorbent assay (ELISA) kit. RESULTS: The restraint stress to estradiol treated rats for 2 weeks revealed that serum corticosterone levels were significantly increased (31.32 ± 5.46 ng/ml, p < 0.05) as compared to the healthy controls (17.48 ± 4.14 ng/ml). CONCLUSION: Chronic restraint stress results increases the serum corticosterone levels in Oophorectomized Sprague Dawley rats. Estradiol treatment increases the responsiveness of adrenal cortex of Oophorectomized female rats.

Effect of cholecalciferol and levo carnitine on plasma glucose, plasma insulin and insulin resistance in type 2 diabetic rats.

OBJECTIVE: To compare the effects of combined and individual supplementation of cholecalciferol and levo carnitine on plasma glucose, plasma insulin and insulin resistance in type 2 diabetic rats. METHODS: The randomised controlled trial was conducted at the Department of Physiology, Army Medical College, Rawalpindi, between October 2010 and April 2011. It comprised 80 healthy Sprague Dawley rats who were divided into four groups (n = 20 each). Rats were fed high-fat diet for 2 weeks followed by an intraperitoneal injection of streptozocin to induce type 2 diabetes mellitus. group I served as diabetic control; group II was given cholecalciferol; group III; levo carnitine; and group IV was administered cholecalciferol and levo carnitine together. After 6 days of supplementation, terminal intracardiac blood extraction was done and samples were analysed for fasting plasma glucose and plasma insulin. Insulin resistance was calculated by homeostatic model assessment for insulin resistance. SPSS 17.0 was used for statistical analysis. RESULTS: Fasting plasma glucose levels were significantly decreased (p < 0.001) in the combined supplementation group compared to the diabetic control and individual supplementation groups. Combined supplementation showed a significant increase in fasting plasma insulin levels when compared with diabetic control and levo carnitine groups (p < 0.001), and the effect of combined supplementation on ameliorating insulin resistance was significantly better (p < 0.001) as compared to the individual supplementation of cholecalciferol and levo carnitine. CONCLUSIONS: The combined supplementation of cholecalciferol and levo carnitine for 6 days markedly improved the glycaemic control, insulin secretion and insulin resistance in type 2 diabetic rats on high-fat diet A prolonged supplementation by both the compounds along with caloric restriction may yield a more promising outcome.

Serum levo-carnitine levels and skeletal muscle functions in type 2 diabetes mellitus in rodents.

OBJECTIVE: To study serum levo-carnitine (l-carnitine) levels and isometric contraction, force frequency relationship and fatigue of rodent skeletal muscles in type 2 diabetes mellitus. STUDY DESIGN: Randomized controlled trial. PLACE AND DURATION OF STUDY: Physiology Department, Army Medical College, Rawalpindi, from January 2009 to January 2010. METHODOLOGY: Sixty Sprague-Dawley rats were randomly divided into two groups; group I (control), fed on normal diet ad libitum and Group II (diabetic), fed on high fat diet and administered streptozocin to induce type 2 diabetes mellitus (T2DM). At 21st day, plasma glucose and TG/HDL ratio were measured to confirm the development of T2DM in group II. At 28th day, blood was drawn by intracardiac puncture to estimate serum levo-carnitine levels. Contractile functions of skeletal muscles were assessed by using iWorx AHK/214 physiological data acquisition unit. Simple muscle twitches, maximum isometric twitch tension (MITT), time-to-peak twitch tension (TPTT) and time-to-relax to 50% of the peak twitch tension (1/2RT) of extensor digitorum muscles were recorded. Muscles were stimulated at higher frequencies to determine maximum fused tetanic tension (MFTT), maximum fused tetanic tension after fatigue protocol (TTFP) and recovery from fatigue (RF). RESULTS: Serum levo-carnitine level decreased significantly in the diabetic group. Both groups had similar MITT, TPTT and 1/2RT but decline in MFTT, TTFP and RF was significant in the diabetic rats. CONCLUSION: T2DM adversely affected serum levo-carnitine levels and the contractile functions of rodent skeletal muscle at high frequency stimulation.

Levo-carnitine reduces oxidative stress and improves contractile functions of fast muscles in type 2 diabetic rats.

BACKGROUND: Metabolic derangements in type 2 diabetes mellitus (T2DM) are likely to affect skeletal muscle contractile functions adversely. Levo-carnitine improves muscle contractile functions in healthy humans and rats and corrects metabolic derangements in T2DM. Therefore, it is likely to improve muscle contractile functions in T2DM as well. This study was designed to determine the effect of levo-carnitine on serum levo-carnitine levels, oxidative stress and contractile parameters of fast muscle in T2DM. METHODS: Ninety Sprague-Dawley rats were randomly divided into three equal groups. Healthy rats served as the controls, while T2DM was induced in diabetic and carnitine groups. The carnitine group was administered levo-carnitine 200 mg/kg/day intraperitoneally for 6 days. At 28th day, extensor digitorum longus muscles were removed and their functions were assessed using iWorx data acquisition unit (AHK/214). Blood obtained by intra-cardiac sampling at 28th day was used for estimation of serum malondialdehyde (MDA) and levo-carnitine levels. RESULTS: Maximum isometric twitch tension, time-to-peak twitch tension and time-to-relax to 50% of the peak twitch tension were not significantly different amongst the groups. Carnitine group showed significant improvement in maximum fused tetanic tension, maximum fused tetanic tension after fatigue protocol and recovery from fatigue after 5 minutes of rest period compared to the diabetic group. Serum MDA levels were reduced, while serum levo-carnitine levels were elevated significantly in carnitine group as compared to the diabetic group. CONCLUSION: Levo-carnitine supplementation increases serum levo-carnitine levels which decreases oxidative stress. This action improves contractile force but delays fatigue in fast muscles of diabetic rats.

Effect of levocarnitine on endurance capacity in type-2 diabetic rats.

BACKGROUND: Carnitine is an essential cofactor for the enzymes transporting long chain fatty acids across mitochondrial membranes for beta oxidation and also modulates the intra-mitochondrial acylCoA/CoA ratio. This study was conducted to determine the effect of levo-carnitine on endurance capacity, skeletal muscle fatigue characteristics and glycogen stores in diabetic rats. METHODS: This laboratory based experimental study was conducted in department of Physiology, Army Medical College, Rawalpindi, in collaboration with National Institute of Health (NIH), Islamabad, from June 2009 to July 2010. The study was carried on 60 healthy male Sprague-Dawley rats. Serum creatine phosphorkinase (CPK) levels were measured to exclude skeletal muscle disorder. Rats were fed high fat diet (2 weeks) followed by intra-peritoneal injection of streptozocin (35 mg/kg). On 21st day, after confirmation of type 2 diabetes by measuring plasma glucose and TG/HDL ratio, rats were divided into 2 equal groups; group I (Diabetic) and group II (Carnitine). Group II was administered 1-carnitine (200mg/kg) for 6 days. Both groups were further subdivided into 2 equal groups- a (swim group) and b (non-swim group). At end of 4th week, the rats of swim group were subjected to swimming test. The extensor digitorum muscle (EDL) of rats of non-swim group was dissected for evaluation of skeletal muscle fatigue characteristics. The glycogen content of EDL muscle and serum free carnitine (FC) levels of all groups were measured. RESULTS: Carnitine treated rats exhibited improvement in swim time as well as skeletal muscle glycogen stores (p < 0.001). Significant improvement was also observed in skeletal muscle fatigue characteristics (p < 0.05). Serum free carnitine levels were also significantly raised in carnitine groups; the swim groups showed a lower FC levels as compared to their respective non-swim groups (p < 0.001). CONCLUSION: Levocarnitine increases the glycogen stores and improves the skeletal muscle fatigue characteristics, leading to improvement in endurance capacity in type 2 diabetic rats.

Cut-off values of anthropometric indices to determine insulin resistance in Pakistani adults.

OBJECTIVE: To determine the cut-off values of anthropometric indices to indicate insulin resistance and correlation of these indices with insulin resistance in Pakistani adults. METHODS: The cross-sectional study was conducted at the Military Hospital and Army Medical College, Rawalpindi, Pakistan, from June 2010 to November 2011. The study measured 209 adults for body mass index, waist circumference, waist-to-hip ratio, waist-to-height ratio and conicity index. Receiver operating characteristics curve analyses were done to determine the predictive values of these anthropometric measures and their cut-off values for insulin resistance by triglyceride/high density lipoprotein ratio. RESULTS: Overall mean age was 51.5 +/- 1.16 years (range: 28-73) and there were 136 (65%) males and 73 (35%) females. Body mass index had the maximum predictive value for insulin resistance followed by waist circumference and waist-to-height ratio in males (p < 0.0001), sensitivity and specificity being 68% and 62% respectively with cut-off value of 25.04 kg/m2. ROC curve analyses showed the maximum predictive value of conicity index for insulin resistance followed by waist circumference and waist-to-height ratio in females (p < 0.08), sensitivity and specificity being 65% and 50% respectively with cut-off value of 1.39. CONCLUSION: In Pakistani male adults, BMI is the best indicator of insulin resistance, while in female adults, conicity index is the best indicator.This is the first study in Pakistan reporting predictive values of anthropometric indices as a non-invasive method in determining insulin resistance.

Gender differences of heart rate variability in healthy volunteers.

OBJECTIVE: To identify the basic values of heart rate variability in Pakistani population and to verify our hypothesis that there are gender differences in cardiovascular autonomic modulation. METHODS: The descriptive cross sectional study based on convenience probability sampling was conducted at Armed Forces Institute of Cardiology/National Institute of Heart Diseases (AFIC/NIHD) Pakistan. The duration of the study was from December 2009 to July 2010. It involved 24-hour holter monitoring of 45 healthy individuals using holter electrocardiography (ECG) recorder. Heart rate variability was analysed in time (SDNN, SDANN, SDNNi, rMSSD, pNN50) and frequency domains (power, VLF, LF, and HF). RESULTS: The time domain indices; SDNN (male = 140 +/- 36ms vs. females = 122 +/- 33ms; p = 0.09), SDANN (male = 123 +/- 34ms vs. females = 111 +/- 34ms; P = 0.23), SDNNi (male = 64 +/- 19ms vs. females = 52 +/- 14ms; P = 0.03), and pNN50 (male = 14 +/- l0ms vs. females = 12 +/- 7ms; P = 0.43) were decreased in female volunteers when compared with males. Comparison of frequency domain indices; Total power (male = 4041 +/- 3150ms(2) vs. females = 2750 +/- 1439ms(2); P = 0.07), VLF (male = 2912 +/- 2675ms(2) vs. females = 1843 +/- 928ms(2); P = 0.06), LF (male = 788 +/- 397ms(2) vs. females = 556 +/- 346ms(2); P = 0.04) and HF (male = 318 +/- 251ms(2) vs. females = 312 +/- 277ms(2): P = 0.94) amongst males and females showed attenuated heart rate variability in females. Of all the observed values, SDNNi and LF were found significantly (p < 0.05) decreased in women. CONCLUSION: In healthy population, heart rate variability is low in women than men. It reflects sympathetic dominance in women in our population.

Correlation of serum alanine aminotransferase and aspartate aminotransferase levels to liver histology in chronic hepatitis C.

OBJECTIVE: To assess the correlation of serum alanine aminotransferase and aspartate aminotransferase levels to severity of disease on liver biopsy in patients of chronic hepatitis C. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Department of Gastroenterology at Military Hospital, Rawalpindi, from January 2006 to February 2007. METHODOLOGY: One hundred and seven diagnosed non-cirrhotic chronic hepatitis C adult patients were included. Serum alanine aminotransferase and aspartate aminotransferase levels were determined. Knodell histopathological Index was determined on liver biopsy. The correlation and regression value between serum alanine aminotransferase levels and liver histology and serum aspartate aminotransferase and liver histology in chronic hepatitis C patients was determined using Pearson correlation analysis. RESULTS: Patients of chronic hepatitis C had raised serum alanine aminotransferase and levels with the mean baseline level of 93 International units per litre (IU/L) with a range of 13-383 IU/L. Serum aspartate aminotransferase levels were also elevated with mean baseline level of 59.65 IU/L ranging from 18-370 IU/L. On liver biopsy based on Knodell histopathological Index, 47.7% of patients had mild, 39.9% had moderate and 13.1% had severe disease. There was significant association between serum alanine aminotransferase levels severity of the disease on liver biopsy (p < 0.03) with weak positive correlation between the two (r = 0.217). There was also significant association between serum aspartate aminotransferase levels and severity of the disease on liver biopsy (p < 0.001) with weak positive correlation between the two (r = 0.32). CONCLUSION: The serum alanine aminotransferase and serum aspartate aminotransferase levels do not indicate the severity of the disease on liver biopsy in chronic hepatitis C patients.

Correlation of eosinophil derived neurotoxin with airway resistance in asthmatics.

OBJECTIVE: To determine the correlation of EDN with lung function tests (FVC, FEV1, FEV1/FVC and FEF25-75%) in asthmatics and to compare these with matched controls. METHODS: This study was carried out at Army Medical College & Military Hospital, Rawalpindi, Pakistan. Forty four asthmatic patients and equal number of matching controls were selected. Lung function tests were done by using compact spirometer. Severity of asthma was graded according to the American Thoracic Society (ATS) "Asthma severity code and classification chart." Venous blood was used for estimation of eosinophil count while serum was used for estimation of EDN by ELISA. Correlation between EDN and various lung functions were calculated. RESULTS: The asthmatic patients had significantly more eosinophil count (p<0.001) and Serum EDN levels (p<0.001) than the controls. A significant correlation (r = 0.934, p<0.001) was found between eosinophil count and EDN. A significant correlation between absolute eosinophil count and some of the lung functions [% predicted FEV1 (r = -0.908, p<0.001) and FEV1/FVC (r = -0.830, P<0.001)] was found. A similar significant correlation was found between EDN and some of the lung functions [% predicted FEV1 (r = -0.855, p<0.001) and FEV1/FVC (r = -0.814, P<0.001)]. Absolute eosinophil count and EDN increased significantly (p<0.001) with increasing severity of asthma. CONCLUSION: Serum EDN has significant correlation with changes in lung functions and severity of asthma.

Interferon-alpha induced and ribavirin induced thyroid dysfunction in patients with chronic hepatitis C.

Chronic hepatitis C (CHC) is one of the commonest infectious diseases of the liver and may lead to cirrhosis or hepatocellular carcinoma. Combination therapy with pegylated interferon (PEG-IFN) and Ribavirin is the treatment of choice for CHC. Combination therapy is thought to act by means of antiviral mechanisms and immunomodulation. Thyroid dysfunction is the most common autoimmune adverse effect associated with combination therapy; hypothyroidism is more common than hyperthyroidism. Antithyroid antibodies and female sex have a predictive value in the development of interferon induced thyroid disease (IITD). Patients with CHC should be informed of the possibility of side effects on the thyroid gland. Screening for antithyroid antibodies and thyroid function tests should be performed in patients with CHC before the commencement of antiviral treatment, and during and after it. This article reviews different aspects of IITD, including its pathogenesis, clinical manifestations, association with treatment regimens and treatment response and the outcome of thyroid dysfunction.

Association of response to combined interferon alpha-2b and ribavirin therapy in patients of chronic hepatitis c with serum alanine aminotransferase levels and severity of the disease on liver biopsy.

BACKGROUND: Raised serum alanine aminotransferase (serum ALT) levels indicate active liver disease while liver biopsy has been considered the 'gold standard' for assessing the severity of disease in patients of chronic Hepatitis C. The response of these patients to standard treatment regimen of interferon (INF)-alpha-2b and ribavirin for 24 weeks have been studied. OBJECTIVE: The objective of this study was to evaluate the association of response to combined INF alpha-2b and ribavirin therapy in patients of chronic hepatitis C with serum ALT levels and severity of the disease on liver biopsy. METHODS: This quasi experimental study-was conducted in Department of Physiology at Army Medical College and Military Hospital, Rawalpindi from January 2006 to February 2007. One hundred and seven diagnosed non cirrhotic chronic hepatitis C patients were studied. Prior to the commencement of treatment, qualitative assay of Hepatitis C Virus (HCV) Ribonucleic Acid (RNA) was done by Polymerase chain reaction (PCR). Knodell Histopathological Index (HPI) was determined on liver biopsy. The standard treatment of INF-alpha-2b, 3 million units 3 times a week subcutaneous, and Ribavirin 800-1200 mg per day was given for 24 weeks. Serum ALT levels were determined before the start of treatment and later at weeks 12 and 24. Qualitative assay of HCV RNA was done by PCR at the end of treatment to determine the response to treatment. Statistical analysis was done on SPSS 15. RESULTS: Out of 107 patients of chronic hepatitis C, 92 (69 males, 23 females) patients (84%) responded to INF-alpha-2b and ribavirin therapy and revealed negative qualitative assay of HCV RNA by PCR at the end of 24 weeks of treatment while serum ALT levels were normal in 88% of patients at 12 weeks and in 97% at the end of 24 weeks of treatment. Knodell HPI revealed mild, moderate and severe disease in 47.7%, 39.9% and 13.1% of patients respectively. No association was established between response to treatment and severity of the disease on liver biopsy (p < 0.11) and serum ALT levels (p = 0.09). CONCLUSION: Response to Interferon alpha-2b and ribavirin therapy in patients of chronic hepatitis C is not associated with the levels of serum ALT and the severity of the illness graded on liver biopsy.