Correlation of insulin resistance with neutrophil to lymphocyte ratio and serum ferritin in male patients of metabolic syndrome.

OBJECTIVE: To determine the correlation of insulin resistance with neutrophil-to-lymphocyte ratio and serum ferritin, and to evaluate whether NLR and serum ferritin can predict insulin resistance in metabolic syndrome. METHODS: The cross-sectional analytical study was conducted at the University of Health Sciences, Lahore, Pakistan, from July 2016 to 2019, and comprised male patients of metabolic syndrome and healthy controls. The correlation involving insulin resistance, serum ferritin and neutrophil-to-lymphocyte ratio was determined. Data was analysed using SPSS 22. RESULTS: Of the 210 subjects, 160(76.2%) were cases with a median age of 45 years (interquartile range: 39-50 years), and 50(23.8%) were controls with a median age of 41 years (interquartile range: 35-50 years). Serum ferritin, alanine aminotransferase, total neutrophil count, lymphocyte count and neutrophil-to-lymphocyte ratio were significantly higher among the cases than the controls (p<0.05). Significant positive correlation of insulin resistance was observed with serum ferritin and neutrophil-to-lymphocyte ratio (p<0.05)) among the cases. Neutrophil-to-lymphocyte ratio significantly predicted insulin resistance among the cases (p<0.05). Conclusion: Neutrophil-to-lymphocyte ratio was fund to be a significant predictor of insulin resistance in metabolic syndrome.

Expression levels of MiRNA-16, SURVIVIN and TP53 in Preeclamptic and Normotensive women.

OBJECTIVE: To measure and compare micro ribonucleic acid-16, survivin and tumour protein p53-regulated apoptosis-inducing protein 1 expression levels in preeclamptic and normotensive pregnancies, and to check the correlation of micro ribonucleic acid-16 with messenger ribonucleic acid expression of survivin and tumour protein p53. METHODS: The observational cross-sectional comparative study was conducted at the Department of Physiology and Cell Biology, University of Health Sciences, Lahore, Pakistan, from 2016 to 2018, and comprised preeclamptic women in group A and normotensive women in group B. The preeclamptic patients were further categorised into early-onset preeclampsia subgroup A1and late-onset preeclampsia group A2. Expression of micro ribonucleic acid-16, messenger ribonucleic acid expression of survivin and tumour protein p53 in preeclamptic and normotensive pregnancies were analysed using real time polymerase chain reaction. Data was analysed using SPSS 22. RESULTS: Of the 54 patients, 27(50%) were in each of the two groups. Within group A, 14(52%) patients were in group A1 and 13(48%) in group A2. The expression of micro ribonucleic acid 16 showed significant increase in group A compared to group B (p<0.05). The difference was not significant between the subgroups A1 and A2. The levels of messenger ribonucleic acid expression of survivin and tumour protein p53 were deregulated in group A, with a decrease in survivin and an increase in tumour protein p53. The messenger ribonucleic acid expression of survivin and tumour protein p53 showed statistically significant differences across subgroups A1 and A2 (p<0.05). The micro ribonucleic acid-16 expression correlated negatively with messenger ribonucleic acid expression of survivin, but exhibited a positive correlation with tumour protein p53. CONCLUSIONS: Deregulated micro ribonucleic acid-16 along with differentially expressed apoptotic genes, survivin and tumour protein p53 might result in altered apoptosis implicated in the pathogenesis of preeclampsia.

TCF7-L2 rs7903146 polymorphism in metabolic syndrome with and without acute coronary syndrome.

OBJECTIVE: To determine the frequency and association of single nucleotide polymorphism of transcription cell factor7-like2 rs7903146 (C>T) in metabolic syndrome patients with and without acute coronary syndrome. METHODS: The cross-sectional comparative study was conducted at the University of Health Sciences, Lahore, Pakistan, from July to December 2017. Patients of metabolic syndrome with and without acute coronary syndrome were selected from Sheikh Zayed Hospital, Lahore, and the Punjab Institute of Cardiology, Lahore. Healthy subjects were enrolled to act as controls. A fasting blood sample of 8ml was taken for deoxyribonucleic acid extraction and estimation of biochemical parameters. Single nucleotide polymorphism of transcription cell factor7-like2 rs7903146 C>T was determined using restriction fragment length polymorphism. SPSS 22 was used for data analysis. RESULTS: Of the 500 subjects, 200(40%) were group A patients without acute coronary syndrome, 100(20%) were in group B with acute coronary syndrome and 200(40%) were group C controls. Overall, 385(77%) were males and 115(23%) were females. The frequency of CC variant in group A was 35(17.5%) and in group C 22(11%), while CT was 32(16%) and 65(32.5%), and TT was 133(66.5%) and 113(56.5%), respectively. There was significant association of TT genotype with increased risk of metabolic syndrome (p=0.031), and CC genotype had no association (p=0.121). There was no significant difference of genotype frequency between groups A and B (p=0.246), but TT variant was significantly higher in group A compared to group B (p=0.009). CONCLUSIONS: TT genotype of transcription cell factor7-like2 rs7903146 C>T was found to be associated with increased risk of metabolic syndrome in patients without acute coronary syndrome compared to those with acute coronary syndrome and healthy controls.

Comparative gene expression analysis of Fas and related genes in preeclamptic and healthy women: A cross-sectional study.

BACKGROUND: Preeclampsia is a hypertensive disorder of pregnancy affecting about 2-10% pregnancies worldwide. mRNA expression of tumor necrosis factor alpha (TNF- α ), Fas, and FasL have been reported to be altered in placental bed in preeclamptic pregnancies. We hypothesized that the expression of these genes is also altered in peripheral blood mononuclear cells (PBMCs) in preeclampsia. OBJECTIVE: To compare the expression of Fas receptor and related genes in PBMCs of preeclamptic and normotensive pregnant women. MATERIALS AND METHODS: A cross-sectional comparative study comprising of 18 cases and 18 controls was designed. 5 ml of venous blood was drawn and collected considering aseptic measures. Buffy coat was separated by centrifugation and stored at -20°C. Favor Prep total RNA Isolation Kit (Favorgen, Taiwan) was used for RNA extraction. The mRNA expression of TNF- α , Fas, and FasL was measured by real-time polymerase chain reaction in PBMCs in preeclamptic and normal pregnancies. RESULTS: A significant increase in mRNA expression of TNF- α , Fas, and FasL (p ≤ 0.001) was observed in PBMCs of preeclamptic pregnancies compared to the control group (p ≤ 0.001). Moreover, a significant positive correlation was found between the TNF- α mRNA expression and Fas and FasL (p ≤ 0.001). CONCLUSION: The results lead to the conclusion that mRNA expression of TNF- α , Fas, and FasL in the maternal PBMCs is altered in preeclamptic pregnancies and might contribute to the pathogenesis of the disease.

Elevated Tumor Necrosis Factor (TNF)-α mRNA Expression Correlates with Nuclear Factor Kappa B Expression in Peripheral Blood Mononuclear Cells in Preeclampsia.

OBJECTIVE: To evaluate, compare and correlate the mRNA expression of nuclear factor kappa B (NF-kB) and tumor necrosis factor alpha (TNF-α) in peripheral blood mononuclear cells in preeclampsia and normotensive group. STUDY DESIGN: Cross-sectional comparative study. PLACE AND DURATION OF STUDY: The study was done in the Department of Physiology and Cell Biology, University of Health Sciences, Lahore, from November 2016 to November 2018. METHODOLOGY: Blood samples were collected and mRNA expression of NF-kB and TNF-α was measured quantitatively by real-time polymerase chain reaction in peripheral blood mononuclear cells in 27 preeclamptic and 27 normal pregnancies. The preeclamptic group was further divided into early and late onset preeclampsia. Statistical analysis was done using SPSS (version 22). RESULTS: The peripheral blood mononuclear cells mRNA expression of NF-kB and TNF-α differed within the two groups with an increase in expression in the diseased group (p <0.001). There was an increase of 2.79 fold in mRNA expression of TNF-α while the result for NF-αB was 2.28 fold. The difference in the expression of both NF-kB and TNF-α was significant within the two subgroups of preeclampsia (p <0.001). TNF-α was found to be strongly correlated with NF-kB (p <0.01).

Adrenergic receptor beta-3 rs4994 (T>C) and liver X receptor alpha rs12221497 (G>A) polymorphism in Pakistanis with metabolic syndrome.

The present study aimed to determine the association of adrenergic receptor beta-3 (ADRB3) rs4994 T>C and liver X receptor alpha (LXR-α) rs12221497 G>A polymorphism with metabolic syndrome (Met S) and the related traits in Pakistanis. Patients of Met S were recruited from the Endocrinology and Diabetic Clinic of Sheikh Zayed Hospital Lahore, over the time span of 6 months from July to December 2016. Single-nucleotide polymorphism (SNP) of ADRB3 was determined by restriction fragment length polymorphism and of LXR-α by amplification refractory mutation system polymerase chain reaction. The frequency of TT variant of ADRB3 T>C in Met S was 69 (34.5%) and in controls 89 (44.5%), frequency of TC 103 (51.5%) and 96 (48%), and of CC 28 (14%) and 15 (7.5%), respectively. In the recessive model (CC: TT + TC), CC genotype was found to be associated with the increased risk of Met S (P = 0.027; odds ratio [OR] = 2.09; confidence interval [CI] =1.08-4.03) and the association remained significant after controlling for the confounders such as age and sex. The frequency of GG variant of LXR-α G>A in Met S was 35 (17.5%) and in controls 15 (7.5%), GA 129 (64.5%) and 137 (68.5%), and AA 36 (18%) and 48 (24%), respectively. In the recessive model (GG: GA + AA), GG genotype was found to be associated with the increased risk of Met S (P = 0.004; OR = 2.52; CI = 1.33-4.80) and the association remained significant after controlling for the confounders such as age and sex. It was concluded that SNP of ADRB3 (190 T>C) and LXR-α (-115 G>A) were associated with the risk of Met S and might increase the susceptibility to the obesity-related traits.

Peroxisome proliferator activated receptor gamma 34C>G variant and anthropometric parameters in metabolic syndrome.

OBJECTIVE: To determine the frequency of 34 Cytosine >Guanine (proline 12 alanine) variant of peroxisome proliferator activated receptor gamma, and to associate it with metabolic syndrome, insulin resistance and anthropometric obesity parameters. METHODS: The cross-sectional comparative study was conducted at the University of Health Sciences, Lahore, Pakistan, from September 2016 to 2017, and comprised patients of metabolic syndrome and healthy controls. Blood pressure and anthropometric measurements of all the subjects were recorded. Fasting blood sample of 4ml was taken for biochemical parameter and deoxyribonucleic acid extraction. The frequency of genetic variant was determined by amplification refractory mutation system polymerase chain reaction. Data was analysed using SPSS 22. RESULTS: Out of 400 subjects, 200 (50%) each were patients and controls. Overall, there were 308 (77%) males and 92 (23%) females. Patients had significantly higher blood pressure, body mass index, waist circumference, waist-to-hip ratio, mid-arm circumference and triceps skinfold thickness compared to the controls (p<0.0001). Insulin resistance was also significantly higher in the patients (p<0.0001) and showed significant correlation with body mass index, waist circumference, waist-to-hip ratio, mid-arm circumference and triceps skinfold thickness (p<0.05).Waist circumference and triceps skinfold thickness were significant predictors of homeostatic model assessment for insulin resistance. Overall, the frequency of homozygous dominant genotype CC of PPAR2 34C>G was 291 (72.75%), heterozygous CG was 93 (23.25%) and homozygous recessive GG was 16 (4%).There was no significant difference in frequency of genotypes between the groups (p=0.216). However, waist circumference and body mass index were significantly lower in GG genotype compared to the CC (p=0.006 versus p=0.02). CONCLUSIONS: Waist circumference and triceps skinfold thickness were found to be the significant predictors of homeostatic model assessment for insulin resistance, while no association was found between 34 C>G variant of peroxisome proliferator activated receptor gamma and metabolic syndrome.

Genetic Association of Apolipoprotein A5-1131T>C Polymorphism with Traits of Metabolic Syndrome.

OBJECTIVE: To investigate the association of -1131T>C polymorphism of apolipoprotein A5 (APOA5) with metabolic syndrome and associated traits. STUDY DESIGN: A cross-sectional comparative study. PLACE AND DURATION OF STUDY: Department of Physiology, University of Health Sciences Lahore, from July 2016 to December 2017. METHODOLOGY: Study population included 200 cases of metabolic syndrome and 200 controls. Cases were selected from Sheikh Zayed Hospital, Lahore according to the International Diabetes Federation guidelines. Fasting blood sample of 8 ml was taken for biochemical and genetic analysis, as per objective. Demographics, waist circumferance and blood pressure (BP) were also recorded. RESULTS: Subjects with metabolic syndrome had significantly higher waist circumference, BP, serum lipid and glycemic parameters as compared to the controls (p<0.001). The observed frequency of APOA5-1131T>C genotype TT was 180 (45%), 202 (50.5%) for TC and 18 (4.5%) for CC. Minor C allele frequency of APOA5-1131T>C variant was significantly higher in metabolic syndrome as compared to the controls (0.33 vs. 0.26: p = 0.031*). In the Dominant genotype model (TC+CC vs. TT), 'TC+CC' genotype was significantly associated with the increased risk of metabolic syndrome (OR: 1.50, CI: 1.01-2.23, p = 0.044*). Waist circumference and fasting triglyceride levels were significantly higher in 'TC +CC' genotype as compared to the 'TT' in metabolic syndrome and controls (p = <0.001 and 0.043; p = 0.022 and 0.006). CONCLUSION: There was significant association of the 'TC+CC' genotype of APOA5 -1131T>C with the increased risk of Met S and the association remained significant after controlling for age and gender.

Altered expression of vascular endothelial growth factor, vascular endothelial growth factor receptor-1, vascular endothelial growth factor receptor-2, and Soluble Fms-like Tyrosine Kinase-1 in peripheral blood mononuclear cells from normal and preeclamptic pregnancies.

Preeclampsia (PE) is the leading cause of maternal and fetal morbidity and mortality. It complicates around 2%-10% pregnancies worldwide due to imbalance between proangiogenic and anti-angiogenic factors, leading to incomplete placentation, ischemia, and endothelial dysfunction. The study was aimed to analyze the mRNA expression of vascular endothelial growth factor (VEGF) and its receptors, i.e., VEGF receptor-1 (VEGFR-1), VEGF receptor-2 (VEGFR-2), and soluble Fms-like tyrosine kinase-1 (sFlt-1) from maternal peripheral blood mononuclear cells (PBMCs) of PE patients. This was a cross-sectional comparative study comprising 18 normotensive and 18 PE patients; the patients were further divided as early-onset preeclampsia (EOP) and late-onset preeclampsia (LOP). The expression level of VEGF, its receptors (VEGFR-1 and VEGFR-2), and sFlt-1 was investigated using real-time polymerase chain reaction. There was a significant change in the mRNA expression with a decrease in VEGF, VEGFR-1, and VEGFR-2 and an increase in sFlt-1 in PBMCs of PE and normal pregnancies (P < 0.001). sFlt-1 mRNA expression was increased by 2.95-fold in the PE group with an inverse correlation with expression of VEGFR-2 (Spearman's rho = 0.68). Based on these findings, we conclude that PE is associated with decrease in the mRNA expression of VEGF, VEGFR-1, and VEGFR-2 as compared to an increase in sFlt-1 in PBMCs.

Differential Expression of Placental Growth Factor, Transforming Growth Factor-ß and Soluble Endoglin in Peripheral Mononuclear Cells in Preeclampsia.

OBJECTIVE: To measure the peripheral blood mononuclear cells (PBMCs) mRNA expression of placental growth factor (PlGF), Transforming growth factor beta (TGF-ß), and soluble Endoglin (sEng) in the blood of preeclamptic and normotensive pregnant women. STUDY DESIGN: Cross-sectional analytical study. PLACE AND DURATION OF STUDY: Department of Physiology and Cell Biology, University of Health Sciences, Lahore, from November 2016 to April 2018. METHODOLOGY: The study included 50 normotensive and 57 preeclamptic patients (18-40 years of age), all in the third trimester of pregnancy. The preeclamptic group was further divided into early-onset preeclampsia (EOP) and late-onset preeclampsia (LOP). Blood samples from patients and healthy controls were collected and mRNA expression was measured (18 patients and 18 controls) by real time PCR. Statistical analyses were done using SPSS (version 22). The values were considered significant at 0.05 level of significance. RESULTS: The PBMCs mRNA expression of PlGF, TGF- and sEng were significantly different between the preeclampsia and control group (p<0.001). A significant decrease in expression of TGF- was observed in LOP group compared to controls (p<0.001); whereas, the difference in the expression of EOP compared to controls was not significant (p=0.12). Similar to TGF-, the expression of PlGF was significantly decreased among EOP and LOP compared to controls. Detailed analysis of sEng showed significantly increased expression in both EOP and LOP as compared to healthy group (p<0.001). CONCLUSION: There is a significant difference in extra-placental expression of PlGF, and sEng in preeclampsia.

Serum profile of cytokines and their genetic variants in metabolic syndrome and healthy subjects: a comparative study.

AIM: To identify genetic variants in promoter areas of IL-6 -174 G>C and TNF-α -308 G>A in metabolic syndrome (Met S) and controls and associate them with Met S and serum cytokine levels.It was a cross-sectional study, including 224 cases of Met S and 200 controls. A fasting blood sample was taken and biochemical parameters including serum glucose, insulin, lipid profile, interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were measured. Restriction fragment length polymorphism was used to identify the genetic variants of IL-6 and TNF-α. Serum levels of IL-6 and TNF-α and insulin resistance were significantly higher in cases than the controls. IL-6 showed significant positive correlation with HOMA-IR and TNF-α. CC genotype of IL-6 was associated with the increased risk of Met S (P=0.016, OR for CC vs GC+GG = 2.33, CI: 1.15-4.71). There was no significant difference of TNF-α genotypes between the cases and the controls. Serum TNF-α and IL-6 levels were significantly higher in AA and CC genotypes of TNF-α (-308 G>A) and IL-6 (-174 G>C) as compared with the GG (P=0.00 and P=0.001). Significant correlation of IL-6 with TNF-α and insulin resistance was observed that may provide us a therapeutic target for preventing metabolic derangements from insulin resistance.

Women Diagnosed with Endometriosis Show High Serum Levels of Diethyl Hexyl Phthalate.

BACKGROUND: Endometriosis is one of the common causes of infertility with very diverse etiology. In modern lifestyle, humans are exposed to several endocrine-disrupting chemicals (EDCs) which may lead to reproductive disturbances. Diethyl hexyl phthalate (DEHP) is one of the common EDCs to which women are exposed by the use of cosmetics, perfumes, food packaging, medicine, and insecticides. AIM: The aim of this study was to measure the levels of DEHP in women diagnosed with endometriosis and healthy females and to compare these levels among different stages of endometriosis. SETTING AND DESIGN: This descriptive study was conducted at a tertiary care hospital. SUBJECTS AND METHODS: This study comprised of fifty women diagnosed with endometriosis, aged 20-40 years, and fifty age-matched healthy, fertile women without endometriosis. Females on any medication/comorbid conditions were excluded from the study. Serum DEHP concentrations were measured by high-performance liquid chromatography (HPLC). The results were expressed as means ± standard deviation (SD) (ng/ml of DEHP). STATISTICAL ANALYSIS: Data were analyzed using IBM-SPSS version 20. Two-sample Student's t-test and one-way ANOVA were applied for the analysis. RESULTS: The mean ± SD value of DEHP in cases was 65.29 ± 21.69 ng/ml; however, the mean ± SD value of DEHP in controls was undetectable. Comparison of DEHP among stages of endometriosis revealed an increasing trend with advanced stages (III and IV). The values were not different between the two age groups of women with endometriosis (20-30 and 31-40 years). CONCLUSION: High levels of DEHP in women diagnosed with endometriosis suggest a role of phthalates in the etiology of endometriosis and permit careful use of such compounds.

Association of TP53 codon 72 polymorphism in women suffering from endometriosis from Lahore, Pakistan.

OBJECTIVE: To investigate TP53 gene codon 72 polymorphism in women with endometriosis and compare it with healthy samples. METHODS: This case-control study was carried out at Jinnah Hospital, Services Hospital and Sheikh Zayed Hospital, Lahore, Pakistan, from 2014 to 2016, and comprised patients with endometriosis and healthy controls. SPSS 21 was used for statistical analysis. RESULTS: Of the 176 participants, 88(50%) were healthy controls and 88(50%) were endometriosis patients. The observed genotype frequencies for controls and patients were 14(15.9%) and 31(35.3%) for proline/proline, 46(52.3%) and 35(39.8%) for proline/arginine, and 28(31.8%) and 22(25%) for arginine/arginine, respectively. The association of different genotypes was not significant in patients with moderate-to-severe endometriosis (p=0.574). The presence of pro/pro genotype enhanced the chances/odds of getting the disease (p<0.05). However, the risk further increased with the advancement of age, particularly in the 27-46 age group (p<0.05). CONCLUSIONS: In Pakistani women the association of TP53 gene codon 72 arginine/proline polymorphism was present..

Serum vitamin D levels and gene polymorphisms (Fok1 and Apa1) in children with type I diabetes and healthy controls.

OBJECTIVE: To compare the pattern of Vitamin D receptor (VDR) polymorphisms (Apa I and Fok I) in Type I Diabetes mellitus (T1DM) as cases vs healthy population as control and to investigate the association of VDR polymorphism with vitamin D levels in cases and controls. METHODS: The hypothesis of the study was "VDR gene polymorphisms (Fok 1 and Apa 1) and vitamin D levels are associated with the T1DM". The case-control study was carried out on 44 cases and 44 controls. Clinically diagnosed unrelated cases were recruited from the Diabetic Clinic of Jinnah Hospital, Lahore during Aug. 2012 to Jan 2013. Unrelated controls with normal glucose levels and no first-degree family history of T1DM were selected by convenient sampling. Vitamin D levels of both cases and controls were measured using Enzyme Linked Immunosorbant Assay (ELISA). Genotyping was performed by Restriction Fragment Length Polymorphism (RFLP)-PCR method and the data were analyzed statistically with IBM-SPSS 21. RESULTS: Our results demonstrated suboptimal vitamin D levels in whole of our sample population, whether control or cases (p = 0.529). There was no statistically significant difference in 25-Hydroxyvitamin D3 levels between cases (11.351 ± 5.92) and controls (12.335 ± 6.64). VDR polymorphism was not associated with susceptibility to T1DM in our sample population. Similarly, no association between VDR polymorphism and vitamin D levels was observed i.e. FokI p=0.507 and p=0.543 and ApaI p=0.986 and p=0.307 for cases and controls respectively. CONCLUSIONS: There is an overall deficiency of Vitamin D levels in cases and control subjects while SNPs association studies suggested that in our sample population there was no association of VDR gene polymorphisms Fok I and Apa I with TIDM.