Glibenclamide-malonic acid cocrystal with an enhanced solubility and bioavailability.

OBJECTIVE: The objective of the work is to enhance the solubility, dissolution, and pharmacokinetic properties of glibenclamide (GLB) via cocrystallization technique. SIGNIFICANCE: Glibenclamide is an oral hypoglycemic agent used for treating non-insulin-dependent (type II) diabetes mellitus. It exhibits poor aqueous solubility and oral bioavailability, thereby compromising its therapeutic effect. Therefore, utilizing cocrystal approach for enhancing the solubility will modulate the physicochemical properties of GLB without altering its molecular structure. METHODS: Cocrystal was prepared by solution crystallization method using coformer malonic acid. The cocrystal was characterized by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Fourier transform infrared (FT-IR) studies. The prepared cocrystal was subjected to solubility, in vitro dissolution, and pharmacokinetic studies. RESULTS: The DSC endotherms, PXRD patterns, and the FT-IR spectra of the cocrystal established the formation of a cocrystal. The formation of eutectic mixture was refuted upon comparing the DSC endotherm and PXRD pattern of the cocrystal with that of the physical mixture. GLB showed a twofold enhancement in solubility and a significant improvement in the rate of dissolution (p < 0.05, independent t-test) after cocrystallization. The pharmacokinetic parameters on male Sprague Drawly rats showed 1.45 enhancement in AUC0-24 and 1.36-fold enhancement in the Cmax of GLB as compared to the pure drug. CONCLUSION: These findings demonstrate that cocrystallization technique was able to tailor the solubility and dissolution profile of GLB leading to an enhanced pharmacokinetic property.

Characterization of rotavirus strains isolated from children with acute gastroenteritis in Rawalpindi, Pakistan.

Diarrhea is the leading cause of childhood morbidity and mortality particularly in developing countries and rotavirus has been identified as the major pathogen associated with diarrheal infections. This study was conducted to detect genotypic distribution of predominant rotavirus strains circulating in children suffering from acute gastroenteritis in Rawalpindi, Pakistan. Stool specimens were collected from children ≤5 years of age, visiting Military Hospital, Rawalpindi, with signs and symptoms of acute gastroenteritis. Two hundred and eighty-four specimens were collected during the period from April 2017 to March 2018. Enzyme immunoassay was performed for detection of rotavirus and reverse transcription-PCR (RT-PCR) was carried out for amplification of VP7 and VP4 gene segments followed by multiplex PCR using genotype-specific primers. Out of 284 children, 71 were found rotavirus positive and among them, 54% were females and 46% males. Our findings showed 92% of infection among children ≤2 years of age, while, the peak age of rotavirus incidence was found to be 6-12 months. Although, rotavirus infection was observed throughout the year but frequency increased in winter. Subtype G1P[8] was more prevalent followed by G2P[4], G3P[8], and G4P[6] subtypes. The results of this study provide insight into the disease burden as well as information on rotavirus diversity which will be useful to develop future strategies to control and prevent diarrheal infections among children.

Pharmaceutical Cocrystal: A Novel Approach to Tailor the Biopharmaceutical Properties of a Poorly Water Soluble Drug.

BACKGROUND: The present study reports the formation of a cocrystal of candesartan with the coformer methyl paraben, its characterization and determination of its bioavailability. Candesartan is a poorly water-soluble drug having an anti-hypertensive activity. The recent patents on the cocrystals of the drugs Progesterone (US9982007B2), Epalrestat (EP2326632B1), Gefitinib (WO2015170345A1), and Valsartan (CN102702118B) for enhancement of solubility, helped in selection of the drug for this work. METHODS: Candesartan cocrystal was prepared by solution crystallization method. The formation of a new crystalline phase was characterized by Differential Scanning Calorimetry (DSC), Fourier Transform Infrared (FTIR) and Powder X-ray Diffraction (PXRD) studies. Saturation solubility studies were carried out in ethanol: water (50:50 % v/v) mixture. The dissolution studies were conducted in 900 ml of phosphate buffer at pH 7.4(I.P.) with 0.7% w/w of Tween 20 at 50 rpm, maintained at a temperature of 37±0.5°C in a USP type II dissolution apparatus. The pharmacokinetic behavior of candesartan and its cocrystal was thereof investigated in male Wistar rats. RESULTS: There was 6.94 fold enhancement in the solubility of candesartan after its cocrystallization. The dissolution profile of the cocrystal exhibited significant improvement in solubility at 60 and 120 minutes and it remained stable in ethanol: water (50:50%v/v) mixture for 48 h as confirmed by PXRD studies. The AUC0-24of the cocrystal was found to be increased by 2.9 fold in terms of bioavailability as compared to the pure drug. CONCLUSION: The prepared cocrystal was found to be relatively more soluble than the pure drug and also showed an enhanced oral bioavailability as compared to the pure drug.

Effect of Coronary Artery Disease risk SNPs on serum cytokine levels and cytokine imbalance in Premature Coronary Artery Disease.

BACKGROUND: Premature Coronary Artery Disease (PCAD) occurs almost a decade earlier in the South Asian population as compared to the West. Inclusion of genetic information can prove to be a robust measure to improve early risk prediction of PCAD. Aim was to estimate the genotypic distribution and risk allele frequencies of 13 Coronary Artery Disease (CAD) risk Single Nucleotide Polymorphisms in loci identified by the CARDIoGRAMplusC4D consortium namely MIA3 rs17465637; 9p21 rs10757274; CXCL12 rs1746048; APOA5 rs662799; APOB rs1042031; LPA rs3798220; LPA 10455872; MRAS rs9818870; LPL rs328; SORT1 rs646776; PCSK9 rs11591147; APOE rs429358; APOE rs7412 in Pakistani PCAD patients and controls. Moreover, the differential serum cytokine levels (IL-18, IL-10, IL-6, TNF-alpha, IL-18:IL-10 & TNF-alpha:IL-10 ratios) with respect to the genotypic distribution of these selected SNPs were determined. MATERIAL AND METHODS: The case-control study was carried out in National University of Sciences and Technology, Islamabad in collaboration with the Cardiovascular Genetics Institute, University College London, UK. Subjects (n=340) with >70% stenosis in at least a single major coronary artery on angiography were taken as PCAD cases along with 310 angiographically verified controls. ELISA was performed for measuring the concentrations of serum IL18, TNFA, IL6 and IL10. Genotyping was done using TAQMAN and KASPar assays. RESULTS: The risk allele frequencies (RAF) of APOE rs7412, CXCL12 rs1746048, 9p21 rs10757274, MIA3 rs17465637 and SORT1 rs646776 were significantly higher in the PCAD cases as compared to the controls. APOE rs429358 had the greatest influence among the selected GWAS/CARDIoGRAMplusC4D consortium CAD risk SNPs by significantly altering the serum levels of TNF-alpha, IL-10 and TNF-alpha:IL-10 ratio. It was followed by APOE rs7412 and CXCL12 rs1746048 which significantly altered the serum levels of IL-18; TNF-alpha and IL-18; IL-18:IL-10 ratio respectively. The cytokine imbalance denoted by IL-18:IL-10 was significantly higher in the risk allele carriers MIA3 rs17465637 and CXCL12 rs1746048 while TNF-alpha:IL-10 ratio was significantly raised in the risk allele carriers of APOE rs429358; MRAS rs9818870 and LPL rs328. CONCLUSION: The association of the selected SNPs with differential serum cytokine levels especially the cytokine imbalance points towards their potential causal role in the immune inflammatory pathogenic pathway of PCAD.

δ-Tocotrienol feeding modulates gene expression of EIF2, mTOR, protein ubiquitination through multiple-signaling pathways in chronic hepatitis C patients.

BACKGROUND: δ-Tocotrienol is a naturally occurring proteasome inhibitor, which has the capacity to inhibit proliferation and induce apoptosis in several cancer cells obtained from several organs of humans, and other cancer cell lines. Moreover, results of plasma total mRNAs after δ-tocotrienol feeding to hepatitis C patients revealed significant inhibition in the expression of pro-inflammatory cytokines (TNF-α, VCAM1, proteasome subunits) and induction in the expression of ICAM1 and IFN-γ after post-treatment. This down-regulation of proteasome subunits leads to autophagy, apoptosis of immune cells and several genes. The present study describes RNA-sequence analysis of plasma total mRNAs obtained from δ-tocotrienol treatment of hepatitis C patients on gene expression regulated by proteasome. METHODS: Pooled specimens of plasma total mRNAs of pre-dose versus post-dose of δ-tocotrienol treatment of hepatitis C patients were submitted to RNA-sequence analyses. The data based on > 1 and 8-fold expression changes of 2136 genes were uploaded into "Ingenuity Pathway Analyses (IPA)" for core analysis, which describes possible canonical pathways, upstream regulators, diseases and functional metabolic networks. RESULTS: The IPA of "molecules" indicated fold change in gene expression of 953 molecules, which covered several categories of biological biomarkers. Out of these, gene expression of 220 related to present study, 12 were up-regulated, and 208 down-regulated after δ-tocotrienol treatment. The gene expression of transcription regulators (ceramide synthase 3 and Mohawk homeobox) were up-regulated, and gene expression of 208 molecules were down-regulated, involved in several biological functions (HSP90AB1, PSMC3, CYB5R4, NDUFB1, CYP2R1, TNFRF1B, VEGFA, GPR65, PIAS1, SFPQ, GPS2, EIF3F, GTPBP8, EIF4A1, HSPA14, TLR8, TUSSC2). IPA of "causal network" indicated gene regulators (676), in which 76 down-regulated (26 s proteasomes, interleukin cytokines, and PPAR-ligand-PPA-Retinoic acid-RXRα, PPARγ-ligand-PPARγ-Retinoic acid-RARα, IL-21, IL-23) with significant P-values. The IPA of "diseases and functions" regulators (85) were involved with cAMP, STAT2, 26S proteasome, CSF1, IFNγ, LDL, TGFA, and microRNA-155-5p, miR-223, miR-21-5p. The IPA of "upstream analysis" (934) showed 57 up-regulated (mainly 38 microRNAs) and 64 gene regulators were down-regulated (IL-2, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-24, IL-27, IL-32), interferon ß-1a, interferon γ, TNF-α, STAT2, NOX1, prostaglandin J2, NF-κB, 1κB, TCF3, and also miRNA-15, miRNA-124, miRNA-218-5P with significant activation of Z-Score (P < 0.05). CONCLUSIONS: This is first report describing RNA-sequence analysis of δ-tocotrienol treated plasma total mRNAs obtained from chronic hepatitis C patients, that acts via multiple-signaling pathways without any side-effects. These studies may lead to development of novel classes of drugs for treatment of chronic hepatitis C patients.

Proteasome inhibitors modulate anticancer and anti-proliferative properties via NF-kB signaling, and ubiquitin-proteasome pathways in cancer cell lines of different organs.

BACKGROUND: Cancer is second most common cause of death in the United State. There are over 100 different types of cancer associated with different human organs, predominantly breast, liver, pancreas, prostate, colon, rectum, lung, and stomach. We have recently reported properties of pro-inflammatory (for treatment of various types of cancers), and anti-inflammatory (for cardiovascular disease and diabetes) compounds. The major problem associated with development of anticancer drugs is their lack of solubility in aqueous solutions and severe side effects in cancer patients. Therefore, the present study was carried out to check anticancer properties of selected compounds, mostly aqueous soluble, in cancer cell lines from different organs. METHODS: The anticancer properties, anti-proliferative, and pro-apoptotic activity of novel naturally occurring or FDA approved, nontoxic, proteasome inhibitors/activators were compared. In addition to that, effect of δ-tocotrienol on expression of proteasome subunits (X, Y, Z, LMP7, LMP2, LMP10), ICAM-1, VCAM-1, and TNF-α using total RNAs derived from plasmas of hepatitis C patients was investigated. RESULTS: Our data demonstrated that following compounds are very effective in inducing apoptosis of cancer cells: Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, amiloride, and quinine sulfate have significant anti-proliferation properties in Hela cells (44% - 87%) with doses of 2.5-20 µM, compared to respective controls. Anti-proliferation properties of thiostrepton, 2-methoxyestradiol, δ-tocotrienol, and quercetin were 70% - 92%. However, thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, and quinine sulphate were effective in pancreatic, prostate, breast, lungs, melanoma, Β-lymphocytes, and T-cells (Jurkat: 40% to 95%) compared to respective controls. In lung cancer cells, these compounds were effective between 5 and 40 µM. The IC50 values of anti-proliferation properties of thiostrepton in most of these cell lines were between doses of 2.5-5 µM, dexamethasone 2.5-20 µM, 2-methoxyestradiol 2.5-10 µM, δ-tocotrienol 2.5-20 µM, quercetin 10-40 µM, and (-) Corey lactone 40-80 µM. In hepatitis C patients, δ-tocotrienol treatment resulted in significant decrease in the expression of pro-inflammatory cytokines. CONCLUSIONS: These data demonstrate effectiveness of several natural-occurring compounds with anti-proliferative properties against cancer cells of several organs of humans. Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol and quercetin are very effective for apoptosis of cancer cells in liver, pancreas, prostate, breast, lung, melanoma, Β-lymphocytes and T-cells. The results have provided an opportunity to test these compounds either individually or in combination as dietary supplements in humans for treatment of various types of cancers.

Role of Cytokine Gene Score in Risk Prediction of Premature Coronary Artery Disease.

AIMS: Pro- and anti-inflammatory cytokines play a significant role in early atherosclerosis. Linkage disequilibrium patterns differ between ethnic groups pointing toward the need to develop population-specific gene risk scores. Our objective was to investigate the role of a cytokine gene score in the risk prediction of premature coronary artery disease (PCAD). METHODS: A case-control study was performed at the National University of Sciences and Technology (NUST) in collaboration with the Cardiovascular Genetics Institute, University College London, United Kingdom. Three hundred forty subjects with >70% stenosis in at least one coronary vessel on angiography were labeled as PCAD cases and compared with 310 angio-negative controls. Genotyping of the rs187238 (interleukin [IL]-18), rs1800795 (IL-6), rs1800629 (tumor necrosis factor [TNF]-alpha), rs1800871 (IL-10), and rs1946519 (IL-18) SNPs was performed using KASPar and TaqMan assays. RESULTS: The odds ratio for cytokine gene score was significantly higher for PCAD (p = 0.025) when adjusted for age, sex, and ethnicity. There was a highly significant difference in gene risk allele frequency between Pakistanis and Caucasians (Northwick Park Heart Study II [NPHSII]) for rs187238 (IL-18), rs1800795 (IL-6), rs1800629 (TNF-alpha), and rs1800871 (IL-10) (p < 0.01). CONCLUSIONS: A cytokine gene score has significant discriminatory ability and potential in the risk prediction of PCAD. Cytokine gene risk allele frequencies differ significantly between Pakistanis and Caucasians supporting the need to develop population-specific gene scores.

Evaluation of Pharmacokinetics, and Bioavailability of Higher Doses of Tocotrienols in Healthy Fed Humans.

BACKGROUND: Tocotrienols has been known to lower serum lipid parameters below 500 mg/d, while increase lipid parameters at higher dose of 750 mg/d. δ-Tocotrienol has a novel inflammatory property of concentration-dependent inhibition and activation. Therefore, inhibition (anti-inflammatory) property of tocotrienols at low doses is useful for cardiovascular disease, whereas, activation (pro-inflammatory) property using high dose is found effective for treatments of various types of cancer. We have recently described plasma bioavailability of 125 mg/d, 250 mg/d and 500 mg/d doses of δ-tocotrienol in healthy fed subjects, which showed dose-dependent increases in area under the curve (AUC) and maximum concentration (Cmax). Hence, in the current study, higher doses of tocotrienols have used to analyze its effect on plasma pharmacokinetic parameters. AIMS: To evaluate the safety and bioavailability of higher doses (750 mg and 1000 mg) of annatto-based tocotrienols in healthy fed subjects. All four isomers (α-, ß-, γ-, δ-) of tocols (tocotrienols and tocopherols) present in the plasmas of subjects were quantified and analyzed for various pharmacokinetic parameters. STUDY DESIGN: An open-label, randomized study was performed to analyze pharmacokinetics and bioavailability of δ-tocotrienol in 6 healthy fed subjects. All subjects (3/dose) were randomly assigned to one of each dose of 750 mg or 1000 mg. Blood samples were collected at 0, 1, 2, 4, 6, 8 h intervals and all isomers of α-,ß-,γ-,δ-tocotrienols, and tocopherols in plasmas were quantified by HPLC. RESULTS: Oral administration of 750 and 1000 mg/d of tocotrienols resulted in dose-dependent increases in plasmas (ng/ml) AUCt0-t8 6621, 7450; AUCt0-∞ 8688, 9633; AUMC t0-∞ 52497, 57199; MRT 6.04, 5.93; Cmax 1444, 1592 (P<0.05), respectively, of δ-tocotrienol isomer. Moreover, both doses also resulted in plasmas Tmax 3.33-4 h; elimination half-life (t1/2 h) 2.74, 2.68; time of clearance (Cl-T, l/h) 0.086, 0.078; volume of distribution (Vd/f, mg/h) 0.34, 0.30; and elimination rate constant (ke; h-1) 0.25, 0.17, respectively of δ- tocotrienol isomer. Similar results of these parameters were reported for γ-tocotrienol, ß- tocotrienol, α-tocotrienol, δ-tocopherol, γ-tocopherol, and ß-tocopherol, except for α- tocopherol. CONCLUSIONS: This study has described pharmacokinetics using higher doses of 750 mg/d and 1000 mg/d of δ-tocotrienol. These results confirmed earlier findings that Tmax was 3-4 h for all isomers of tocotrienols and tocopherols except for α-tocopherol (6 h). These higher doses of tocotrienols were found safe in humans and may be useful for treatments of various types of cancer, diabetes, and Alzheimer's disease.

Effect of peer counselling by mother support groups on infant and young child feeding practices: the Lalitpur experience.

OBJECTIVE: Our primary objective was to evaluate the effect of peer counselling by mother support groups (MSG's) in improving the infant and young child feeding (IYCF) practices in the community. METHODS: We conducted this repeated-measure before and after study in the Lalitpur district of Uttar Pradesh, India between 2006 and 2011. We assessed the IYCF practices before and after creating MSG's within the community. The feeding practices were reassessed at two time points-2 (T1) and 5 years (T2) after the intervention and compared with that of the pre-intervention phase (T0). RESULTS: The total population covered by the project from the time of its initiation was 105000. A total of 425 (T0), 480 (T1) and 521 (T2) mother infant pairs were selected from this population. There was significant improvement in the following IYCF practices in the community (represented as %; adjOR (95% CI, p) such as initiation of breast feeding within 1 hour at both T1 (71% vs. 11%); 19.6 (13.6, 28.2, p =  <0.0001)and T2 (62% vs. 11%); 13.3 (9.4, 18.9, p =  <0.0001); use of prelacteal feeds at both T1 (67% vs. 15%); 12.6 (CI: 9.0, 17.6, p<0.0001) and T2 (67% vs. 5%); 44.4 (28.8, 68.4, p = <0.0001); rates of exclusive breast feeding for 6 months at both T1 (50% vs. 7%); 13.6 (7.6, 25.0, p =  <0.0001) and T2 (60% vs. 7%); 20.5 (11.3, 37.2, p =  <0.0001); initiation of complementary feeding at T1 (85% vs. 54%); 5.6 (3.6, 8.7, p =  <0.0001) and T2 (96% vs. 54%); 22.9 (11.8, 44.1, p =  <0.0001) and complementary feeding along with continued breast feeding at both T1 (36% vs. 4.5%); 6 (1.15, 31.4, p = 0.033) and T2 (42% vs. 4.5%); 8.06 (1.96, 49.1, p = 0.005) as compared to pre-intervention period (T0) after adjusting for important social and demographic variables. CONCLUSIONS: Peer counseling by MSG's improved the IYCF practices in the district and could be sustained.

Prospective validation study of transorbital Doppler ultrasound imaging for the detection of transient cerebral microemboli.

BACKGROUND: Transient cerebral microemboli are independent biomarkers of early risk of ischaemic stroke in acute carotid syndromes. Transcranial Doppler imaging (TCD) through the temporal bone is the standard method for detection of cerebral microemboli, but an acoustic temporal bone window for TCD is not available in around one in seven patients. Transorbital Doppler imaging (TOD) has been used when TCD is not possible. The aim of this study was to validate the use of TOD against TCD for detecting cerebral microemboli. METHODS: The study included patients undergoing elective carotid endarterectomy; all had confirmed temporal and orbital acoustic windows. Subjects gave written informed consent to postoperative TCD and TOD monitoring, which was performed simultaneously for 30 min by two vascular scientists. RESULTS: The study included 100 patients (mean(s.e.m.) age 72(1) years; 65 men). Microemboli were detected by one or both methods in 40·0 per cent of patients: by TOD and TCD in 24 patients, by TOD alone in ten and by TCD alone in six. For detecting microemboli, TOD had a sensitivity of 80·0 per cent, specificity of 86·1 per cent, positive predictive value of 71·6 per cent and negative predictive value of 91·2 per cent. Bland-Altman analysis revealed no significant bias (bias 0·11 (95 per cent c.i. -0·52 to 0·74) microemboli; P = 0·810) with upper and lower limits of agreement of +6 and -6 microemboli. CONCLUSION: TOD appears a valid alternative to TCD for detecting microembolic signals in patients with no suitable temporal acoustic window.

Nutritional Supplement-5 with a Combination of Proteasome Inhibitors (Resveratrol, Quercetin, δ-Tocotrienol) Modulate Age-Associated Biomarkers and Cardiovascular Lipid Parameters in Human Subjects.

BACKGROUND: Age-associated altered redox imbalances and dysregulated immune function, contribute to the development of a variety of age associated diseases. Inflammatory markers and lipid profiles are useful prognostic indicators of a variety of age-associated and cardiovascular diseases. We have previously studied the impact of several proteasome inhibitors on several markers of inflammation and lipid profiles in vitro, in vivo, in cell lines, animal models, and in human subjects. The current study represents an extension of this work. Our main hypothesis is that a combination of various naturally-occurring proteasome inhibitors, which inhibits nitric oxide (NO), and C-reactive protein (CRP) mediated inflammation, will have better efficacy in the prevention and treatment of age-associated disorders including cardiovascular disease. METHODS: Two double blind, randomized, placebo-controlled cross-over trials were conducted to determine the impact of a mixture of NS-5 (resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid) on serum NO, CRP, γ-glutamyl-transferase (γ-GT) activity, total antioxidant status (TAS), total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides levels. Healthy seniors (Group-1; n = 32) free-living (A, B; 16/group), and hypercholesterolemic (Group-2; n = 64) subjects on AHA-Step-1-diet were divided into two groups (C, D; 32/group). Baseline levels were established for parameters as mentioned above. Groups A, C were administered 4-capsules/d of NS-5 and groups B, D, placebo (starch) for 6-weeks. Groups were crossed-over, followed by a 2-week wash-out period. Groups A, C were given 4-capsules/d of placebo and groups B, D, 4-capsules/d of NS-5 for 6-weeks. Groups C, D were continued on AHA-Step-1-diet. RESULTS: All the subjects completed each phase in both studies without any complaints. There were significant ( P < 0.01 - 0.05) decreases in the serum levels of NO (30%, 26%), CRP (29%, 21%), γ-GT activity (14%, 17%), and blood pressure (systolic/diastolic, 3/6%, 3/3%) of Groups A and B, respectively, of free-living healthy seniors without affecting the total, HDL-, LDL-cholesterol or triglycerides compared to their respective baseline values. However, serum levels of NO (36%, 43%), CRP (31%, 48%), γ-GT (17%, 20%), total cholesterol (19%, 15%), LDL-cholesterol (28%, 20%), triglycerides (11%, 18%) of Groups C and D were significantly ( P < 0.01-0.05) decreased with NS-5 treatment of hypercholesterolemic subjects compared to baseline values, without affecting the serum HDL-cholesterol levels. The serum levels of total antioxidant status (TAS) were increased (10%, 14%; P < 0.05) in Groups A and B, increased (19%, 24%; P < 0.02), and blood pressure (systolic/diastolic, 5/6%, 3/5%) in Groups C and D with NS-5 treatment, compared to respective baseline values. CONCLUSIONS: The consumption of NS-5 mixture decreased significantly serum NO, CRP and γ-GT levels, improved TAS and lipid profiles at risk cardiovascular and hold promise for delaying onset of age-associated diseases.

Pesticide exposure and endocrine dysfunction in the cotton crop agricultural workers of southern Punjab, Pakistan.

This study evaluated pesticide effects on reproductive and thyroid hormones of cotton farmers of southern Punjab, Pakistan. A total of 88 cotton farmers (42 spray applicators and 46 cotton pickers) were randomly included with an equal number of age- and sex-matched controls. Sampling was done in high spraying and peak picking seasons. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, prolactin, thyroid-stimulating hormone (TSH), total triiodothyroxine (TT3), and free thyroxine (fT4) were carried out by enzymatic immunoassay. Plasma cholinesterase (PChE) levels were measured by Ellman's method. Serum FSH, LH, and testosterone levels were significantly high in spray applicators (P < .01).Serum FSH and testosterone levels were significantly raised in cotton pickers (P < .01). Serum prolactin was decreased significantly in both groups (P < .01).Serum fT4 was significantly reduced in cotton pickers (P < .01). Pesticide exposure is associated with thyroid and reproductive hormone levels disturbance.

Suppression of Nitric Oxide Production and Cardiovascular Risk Factors in Healthy Seniors and Hypercholesterolemic Subjects by a Combination of Polyphenols and Vitamins.

BACKGROUND: Dysregulated immune function associated with ageing has been implicated in a variety of human diseases. We have demonstrated the anti-inflammatory properties of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavinin a variety of experimental animal models, and determined that these compounds act by inhibiting proteasome activity. AIMS: To determine whether serum nitric oxide (NO) levels increase with age in humans, and whether the combined cholesterol-lowering and inflammation-reducing properties of resveratrol, pterostilbene, Morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid would reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes. METHODS: Elderly human subjects were stratified into two groups based on total serum cholesterol levels. Initial total serum cholesterol levels were normal and elevated in Group 1 and 2 subjects, respectively. Baseline serum NO, C-reactive protein (CRP), γ-glutamyltransferase (γ-GT) activity, uric acid, total antioxidant status (TAS), total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides levels were established over a four week period. Group 1 subjects subsequently received nutritional supplementation with one of two different combinations (NS-7 = 25 mg of each, resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid, morin hydrate or NS-6 = morin hydrate replaced with quercetin, 50 mg/capsule). Group 2 subjects also received these nutritional supplements (two capsules/d), but an AHA Step-1 diet was also implemented. After these interventions were administered for four weeks, the above parameters were re-measured and changes from baseline levels determined. Nitric acid (NO) levels in children, young adults, and seniors were also compared. RESULTS: The key results of the current study were: 1) that serum NO levels were significantly increased in seniors compared to both children (~80%) and young adults (~65%); 2) that the intake of two capsules/d of NS-7 or NS-6 for four weeks significantly (P < 0.05) decreased serum NO (39%, 24%), CRP (19%, 21%), uric acid (6%, 12%) levels, and γ-GT activity (8%, 6%), respectively in free-living healthy seniors; 3) that serum NO (36%, 29%), CRP (29%, 20%), uric acid (6%, 9%) γ-GT activity (9%, 18%), total cholesterol (8%, 11%), LDL-cholesterol (10%, 13%), and triglycerides (16%, 23%) levels were significantly (P < 0.02) decreased in hypercholesterolemic subjects restricted to AHA Step-1 diet plus intake of SN-7 or SN-6 (two capsules/d), respectively; 4) that TAS was increased (3%, 9%; P < 0.05) in free-living healthy seniors receiving NS-7 or NS-6 alone, and in hypercholesterolemic subjects plus AHA Step-1 diet (20%, 12%; P < 0.02) with either of the combinations tested. CONCLUSIONS: Serum NO levels are elevated in elderly humans compared to children or young adults. Diet supplementation with combinations of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.

Early detection of cardiac dysfunction by BNP in beta-thalassaemia major patients.

BACKGROUND: Cardiomyopathy is the leading cause of mortality in beta-thalassaemia major (BTM) patients. Brain natriuretic peptide (BNP) has been used for latent cardiac dysfunction in heart patients other than thalassaemic patients. The objective was to determine its role in subclinical detection of iron-induced cardiotoxicity in BTM patients. METHODS AND RESULTS: EDTA plasma was taken from 33 thalassaemic patients and 29 healthy controls, stored at -20 degrees C until analysis. The median (range) age of thalassaemic major children was 10 (6-21) years and mean serum ferritin levels were 3956 (1929-6979) microg/L. The BTM children had significantly (P < 0.01) higher BNP median (range) 83.94 (45.93-196.80) pg/mL as compared to controls 55.62 (32.58-99.84) pg/mL. Mitral E-wave velocities were found higher in patients rather than controls (132.12 +/- 29.40 vs. 117.70 +/- 24.81; P < 0.05). The E/Ea ratio was significantly higher in BTM patients than in the control group (16.35 +/- 6.01 vs. 19.26 +/- 4.67; P = 0.001). We found a significant positive correlation between BNP and E/Ea ratio (r = 0.53; P < 0.01). BNP at a cut-off value of 84.39 pg/mL was highly accurate in ruling out diastolic dysfunction (E/Ea < 8) with a sensitivity of 80% and a specificity of 88%. CONCLUSION: BNP has a good predictive value in detecting latent LV dysfunction in BTM patients.

Serum gamma glutamyl transferase: a novel biomarker for screening of premature coronary artery disease.

BACKGROUND: We aimed to elucidate the association between gamma glutamyl transferase (GGT) activity with prevalence of premature coronary artery disease (CAD) in young Pakistani patients undergoing diagnostic coronary angiography. METHODS: A total of 218 young adults (age ≤ 45 years) underwent diagnostic angiography. Serum samples were taken from all the patients and analyzed for serum GGT activity, cholesterol and triglycerides. RESULTS: Coronary artery disease patients had significantly increased GGT activity (P = .001) and exhibited a significant positive correlation with blood pressure, cholesterol, blood glucose, and smoking and negative correlation with total antioxidant status (P < .01). CONCLUSION: The study revealed good diagnostic accuracy at cutoff of 35 U/L with a sensitivity of 92%, specificity of 81%, and diagnostic odds ratio of 48 in estimation of premature CAD in young Pakistanis.

Deferiprone challenge test for monitoring iron overload in hepatitis positive thalassemic major patients.

Viral hepatitis is common among beta-thalassemia major (BTM) children in Pakistan. Transfusional iron overload in BTM is usually monitored by serum ferritin. But its levels are falsely raised in viral hepatitis and do not reflect the true iron body burden in thalassemic patients. The objective of the study was to develop a test for monitoring iron overload in 'Hepatitis B&C' positive BTM patients by urinary iron excretion (UIE) after oral deferiprone chelation as compared to serum ferritin. We recruited 130 BTM patients from the registry of Thalassaemia centre at Rawalpindi, Pakistan. The patients were grouped into Hepatitis positive and Hepatitis negative based on ELISAtest. Serum ferritin levels were analyzed by kit on Access II. Each patient was given 75mg/kg of deferiprone at morning. Baseline UIE before deferiprone, and 4, 8 12 hours (hrs) UIE after deferiprone were analyzed on Selectra E. One hundred and thirty BTM patients aged 3 to 23 years comprising of Hepatitis positive (n=69) and Hepatitis negative (n=61) participated in the study. Hepatitis positive thalassemic patients had significantly high serum ferritin median (IQ) 4349 (2782-5927) mug/Lthan 3338 (2189-5506) mug/Lin the Hepatitis negative (p=0.001). We did not find any significant change in UIE at 4, 8, and 12 hours between two groups after Deferiprone intake (p=NS). We observed significant positive correlation between serum ferritin and 4 hours UIE in Hepatitis negative patients (r=0.57; p=0.01) but not in the Hepatitis positive patients (r=0.16; p=NS). Deferiprone challenge with measurement of 4 hours UIE is cost effective and non-invasive test rather than serum ferritin for monitoring iron overload in Hepatitis' positive BTM patients.

Adverse effects of pesticides residues on biochemical markers in pakistani tobacco farmers.

Tobacco is an important cash crop of Pakistan and tremendous amount of irrational pesticides are being used to control insect growth. The frequency of plasma pesticide residues above acceptable daily intake (ADI) and its correlation with biochemical markers for assessment of adverse health effects in the tobacco farmers at district Sawabi, Pakistan was determined. Total 109 adult males consisting of 55 tobacco farmers exposed to pesticides and 54 controls were included. Pesticides residues in blood were analyzed on HPLC and GC-NPD. Plasma butyrylcholinesterase (BChE) was analyzed by Ellman's method. Biochemical markers including serum calcium, phosphorus, urea, creatinine, bilirubin and liver enzymes were measured on Selectra-E auto analyzer. The tobacco farmers had multiple pesticides residues above ADI in their blood consisting of 35 (63%) methomyl; 31 (56%) thiodicarb; 34(62%) cypermethrin; 27 (49%) Imidacloprid; 18 (32%) Methamidophos and 15 (27%) endosulfan. BChE activity was significantly decreased in the pesticides exposed farmers as compared to controls (P<0.001). Plasma biochemical markers including ALT, AST, CK, LDH and phosphate were significantly raised in the pesticides exposed farmers as compared to control group (P<0.001). Total pesticides residues revealed a significant positive correlation with AST (r=0.42), LDH(r= 0.47), ALT (r=0.20) and phosphorus (r=0.51). Excessive exposure to pesticide caused cytotoxic changes in the hepatic and renal biochemical markers which were positively correlated with pesticide residue. Hence these biomarkers might be used in addition to BChE activity for monitoring of adverse effects of pesticides on the health of farm workers.