Standard Enzymatic Methods Research Articles

Application of an assay for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine for the assessment of tobacco-related harm

BackgroundThe tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is formed from nicotine and related compounds during tobacco curing and is classified as a human carcinogen. Its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), is thought to be useful in the assessment of cigarette smoking harm minimisation strategies. MethodologyUrine samples were collected from 24 current Caucasian smokers participating in a smoking cessation study; before and four weeks after a quit attempt. Samples were spiked with NNAL-d3 internal standard, extracted with ethyl acetate using liquid–liquid extraction, reconstituted with deionised water and analysed using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Both free (unconjugated) and total NNAL was measured, with totals determined by cleavage of NNAL-glucuronide using standard enzymatic hydrolysis methods. ResultsFree NNAL levels (193.5±158.2pg/mL [mean±standard deviation]) measured in urine samples obtained at baseline (during ad lib smoking) were indicative of biological exposure to NNK. Free NNAL levels were significantly reduced four weeks after the quit attempt (64.5±77.6pg/mL; p<0.002). Assay performance met acceptance criteria with mean recovery of 59±23%, intra-day accuracy was 3.7%, 3.7 and 3.6% and precision was 11.3%, 5.1% and 5.3% at 5pg/mL, 20pg/mL and 100pg/mL levels respectively. Enzymatic hydrolysis of NNAL-glucuronide proved unreliable with complete loss of NNAL aglycone in some subject samples following the hydrolysis procedure. ConclusionLiquid-liquid extraction with UPLC–MS/MS is a convenient approach to measure low levels of NNAL in urine as a marker of biological NNK exposure, which can be used to assess the effectiveness of smoking reduction harm minimisation strategies. Enzymatic hydrolysis of NNAL-glucuronide and measuring the aglycone is not recommended.

Common Variants in Cholesterol Synthesis– and Transport–Related Genes Associate with Circulating Cholesterol Responses to Intakes of Conventional Dairy Products in Healthy Individuals

Background: Dairy intake has been associated with varying impacts on circulating cholesterol concentrations across nutritional epidemiology and intervention studies, with findings attributed mainly to differences in the nature of dairy products consumed or study designs. The contribution of the genomic architecture to such observations has yet to be revealed.Objective: We assessed the impact of multiple common genetic variations in cholesterol-related genes on responses of serum cholesterol to the recommended amount of dairy product intake in Canada.Methods: In a multicenter, randomized crossover design, 101 normolipidemic adults (n = 29 men and 72 women), with a mean ± SD age of 41.7 ± 16.7 y and a body mass index (BMI, in kg/m2) of 25.9 ± 4.3 consumed 3 servings/d of dairy [375 mL 1% milk-fat (MF) milk, 175 g 1.5% MF yogurt, and 30 g of 34% MF cheese] or energy-matched control products (juice, cashews, and cookies) provided within a prudent background diet for 4 wk each, separated by a 4- to 8-wk washout period. Serum lipid variables were determined by standard enzymatic methods by using an autoanalyzer. Candidate single nucleotide polymorphisms were assessed by TaqMan genotyping assay.Results: The responsiveness of serum total cholesterol (TC) and LDL cholesterol to the dairy compared with the control diet was associated with individuals’ genotypes. The cholesterol transport gene ATP-binding cassette subfamily G, member 5 (ABCG5) rs6720173-GG homozygotes had higher concentrations of TC (+0.18 mmol/L; P = 0.0118) and LDL cholesterol (+0.17 mmol/L; P = 0.0056) relative to C-allele carriers (−0.07 and −0.06 mmol/L, respectively). The bile acid synthesis gene cholesterol 7α-hydroxylase (CYP7A1) rs3808607-G-allele carriers had higher TC (+0.20 to +0.28 mmol/L; P = 0.0026) and LDL cholesterol (+0.19 mmol/L for GT genotype; P = 0.0260) relative to TT homozygotes (−0.11 and −0.03 mmol/L, respectively). In addition, the cholesterol synthesis gene 7-dehydrocholesterol reductase (DHCR7) rs760241-A-allele carriers had higher LDL cholesterol (+0.26 mmol/L; P = 0.0399) relative to GG homozygotes (+0.06 mmol/L).Conclusion: Genetic variations in ABCG5, CYP7A1, and DHCR7 may contribute to differing responses of serum cholesterol to dairy intake among healthy adults. This trial was registered at clinicaltrials.gov as NCT01444326.

Graphene-titanium dioxide nanocomposite based hypoxanthine sensor for assessment of meat freshness

We report on the fabrication of a graphene/titanium dioxide nanocomposite (TiO2-G) and its use as an effective electrode material in an amperometric hypoxanthine (Hx) sensor for meat freshness evaluation. The nanocomposite was characterized by TEM, XRD, FTIR, XPS, TGA, BET, and CV using the redox couples [Fe(CN)6]−3/−4 and [Ru(NH3)6]+3/+2 respectively. The TiO2/G nanocomposite offered a favorable microenvironment for direct electrochemistry of xanthine oxidase (XOD). The fabricated Nafion/XOD/TiO2-G/GCE sensor exhibited excellent electro catalytic activity towards Hx with linear range of 20μM to 512μM, limit of detection of 9.5μM, and sensitivity of 4.1nA/μM. In addition, the biosensor also demonstrated strong anti-interference properties in the presence of uric acid (UA), ascorbic acid (AA) and glucose. Minimal interference of xanthine (Xn) was observed at ~7%. Moreover, the biosensor showed good repeatability (4.3% RSD) and reproducibility (3.8% RSD). The reported biosensor was tested towards the detection of Hx in pork tenderloins stored at room temperature for seven days. There was a good correlation (r=0.9795) between biosensor response and measurements obtained by a standard enzymatic colorimetric method. The TiO2-G nanocomposite is therefore an effective electrode material to be used in electrochemical biosensors to assess the freshness of meat.

Serum uric acid and lipid profile in diabetic retinopathy in rural Haryana, India

Background: Diabetic retinopathy is associated with severe ophthalmic morbidity. Present study aimed to analyse serum uric acid (UA) and lipid profile in newly diagnosed type-II diabetes mellitus (DM) who presented with and without retinopathy. Method: A total of 57 subjects were recruited for the present study and were divided in three groups. Sixteen type-II DM patients without retinopathy (group-I), 18 type-II DM patients with retinopathy (group-II) and 23 age and sex matched healthy controls (group-III). Five ml of fasting venous blood sample was collected under proper aseptic precautions from median cubital vein and analyzed for fasting blood sugar, HbA1c, UA, total cholesterol (TC), triglycerides (TG), high density lipoproteins (HDL) by standard enzymatic methods. Very low density lipoproteins (VLDL) and low density lipoptoteins (LDL) was calculated using Frieldwald’s formula. FBS, HbA1c, UA, total cholesterol, triglycerides, HDL, LDL and VLDL levels were compared between different groups. Statistical analysis was done using SPSS software and student‘t’ test. Results: A statistically significant lower value of mean HDL was found in group-I and II than group-III (p=0.01 and 0.00 respectively). Mean HbA1c and mean FBS significantly correlated with UA (r=-0.45, p=0.01 and r=-0.39, p=0.02 respectively) in group-I and II. A deranged HDL profile and a significant correlation between glycemic control and HbA1cand UA were thus found in diabetic subjects (with or without retinopathy). Conclusions: An insight into the deranged biochemical profile could assist in predicting onset of complications of diabetes.

Covalent Immobilization of Lactate Oxidase onto Zirconia Coated Silica Nanoparticles/Chitosan Hybrid Film for Amperometric Determination of Lactate

An improved amperometric L-lactate biosensor was constructed based on covalent immobilization of lactate oxidase (LOx) onto zirconia coated silica nanoparticles (SiO2@ZrONPs)/chitosan (CHIT) hybrid film electrodeposited on the surface of a gold electrode (AuE). The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS), while SiO2@ZrONPs were synthesized by chemical reduction method and characterized by transmission electron microscopy (TEM), UV spectroscopy and X-ray diffraction (XRD). The biosensor showed an optimal response within 3s at pH 7.5 in 0.05M sodium phosphate buffer and 30°C, when operated at 20 mVs-1. The biosensor had a low detection limit of 0.2 nM with a wide working / linear range between 0.1 – 4000 μM. The biosensor was employed for measurement of L-lactic acid level in plasma of apparently healthy and diseased persons. Analytical recovery of added lactic acid (5.0 mM and 10.0 mM)) in plasma was 99% and 96.6% respectively. Within- and between-batch coefficients of variations were 1.79% and 2.89% respectively. There was a good correlation (R2=0.99) between plasma lactate values as measured by standard enzymatic spectrophotometric method and the present biosensor. The enzyme electrode was used 160 times over a period of 120 days, when stored dry at 4°C.

Anti Mullerian hormone (AMH) levels predict cardiovascular risk assessment in young women with PCOS

Serum levels of anti-mullerian hormone (AMH), a member of the TGFb family, are elevated in women with PCOS. There is conflicting information in women with and without PCOS regarding the association between low AMH levels and increased cardiometabolic risk. Young women with PCOS have an increased risk of metabolic syndrome and we hypothesized that serum AMH levels would predict risk of metabolic syndrome in this population. Retrospective cohort study. Women seen at the Penn PCOS center from 2010-2014 with complete metabolic work up were included in this study (n=469). Metabolic syndrome (Met Syn) was defined by modified NCEP-ATP III criteria (BMI≥30, sBP/dBP≥135/85mmHg or taking anti-hypertensives medications, fasting glucose ≥100mg/ml or taking medications for diabetes, TG≥150mg/ml, HDL-C ≤50mg/ml). AMH was measured using the Gen II ELISA, lipids were measured by standard enzymatic methods and total testosterone was measured by radioimmunoassay. Spearman correlation coefficients were used to determine relationships between continuous variables. Linear regression was used to model associations between AMH and lipids adjusting for confounders. AMH was dichotomized based on tertiles and univariate tests and logistic regression modeling were used to evaluate associations of selected variables with tertiles of AMH. The median AMH level in the entire cohort was 5.06ng/ml (IQR 3.08-7.97) . The mean age of the group was 27.6±6years, 42.1% were non-white and 8.1% were smokers. The overall prevalence of Met Syn was 19%. AMH levels positively correlated with total testosterone (p<0.008). On examination of individual components of Met Syn, AMH levels positively correlated with HDL-C (p<0.001) and negatively correlated with fasting glucose (p<0.004), total insulin (p<0.001), BMI (p<0.001), systolic and diastolic BP (p<0.003, p<0.005 respectively). AMH did not have a significant correlation with smoking, LDL-C, TG, CRP, DHEAS and SHBG. Total testosterone did not correlate with individual components of Met Syn. For every unit increase in AMH the odds of Met Syn decreased by 10% (OR 0.9 (0.83-0.97) in the univariate model (p=0.01) and in the multivariate model (p<0.02). The OR for Met Syn for women in the lowest AMH tertile compared to those in the highest AMH tertile was 2.3 (95% CI 1.1-4.6, p=0.02). Adjusted for age, race and testosterone the OR for Met Syn in the lowest AMH tertile was 2.1 (95% CI 1.01-4.3, p=0.04). Total testosterone did not predict risk of Met Syn (p=0.5). Our findings indicate that AMH levels, independent of age, race and testosterone are a strong predictor of Met Syn risk in young women with PCOS. Although the causal or temporal relationship for our findings is unclear, our study suggests that metabolic risk in young women with PCOS is influenced by the ovarian hormone AMH but not testosterone.

Serial measurement of lipid profile and inflammatory markers in patients with acute myocardial infarction.

Serum concentration of lipids and lipoproteins changes during the course of acute coronary syndrome as a consequence of the inflammatory response. The objective of this study was to evaluate the effect of acute myocardial infarction (AMI) on the levels of lipid profile and inflammatory markers. We investigated 400 patients with AMI who were admitted within 24 h of onset of symptoms. Serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) were determined by standard enzymatic methods along with high sensitive C-reactive protein (hs-CRP) (latex enhanced immunoturbidimetric assay) and cytokines, interleukin (IL)-6 and IL-10 (quantitative ''sandwich'' enzyme-linked immunosorbent assay). The results indicate a trend of reduced TC, LDL, and HDL, and elevated TG levels, along with pro- and anti-inflammatory markers (p < 0.001), between day 1 and the day 2 serum samples of AMI patients. However, corrections in the serum levels have been observed at day 7. Our results demonstrate significant variations in the mean lipid levels and inflammatory markers between days 1, 2 and 7 after AMI. Therefore, it is recommended that the serum lipids should be assessed within 24 hours after infarction. Early treatment of hyperlipidemia provides potential benefits. Exact knowledge regarding baseline serum lipids and lipoprotein levels as well as their varying characteristics can provide a rational basis for clinical decisions about lipid lowering therapy.

(333) - Neutrophil Gelatinase-Associated Lipocalin (NGAL) in Heart Transplant Recipients After Conversion to Everolimus Therapy

Background: Due to the lack of nephrotoxic activity, proliferation signal inhibitors (PSI) such as everolimus are recommended for immunosuppression after heart transplantation, but the assessment of renal function in patients receiving PSI has led to conflicting results. We examined renal integrity and function using neutrophil gelatinase-associated lipocalin (NGAL) and conventional markers [plasma creatinine, cystatin C, urine albumin, a1-microglobulin (a1M)] in heart transplant patients, who underwent conversion to everolimus due to allograft vasculopathy, graft rejection episodes, or renal function deterioration, and in patients maintained on calcineurin inhibitors (CNI). Methods: This cross-sectional study included 121 consecutive heart transplant recipients: 44 patients received CNI-free immunosuppressive therapy with everolimus and 77 patients received CNI. Renal parameters were determined in plasma and urine samples using standard enzymatic or immunochemical methods. Results: Heart transplant recipients receiving everolimus therapy had significantly lower NGAL concentrations in plasma [median (95% CI): 128 (97–176) ng/mL vs. 252 (224–283) ng/mL, p < 0.001] and urine [median (95% CI): 6.4 (4.5–7.6) ng/g vs. 15.7 (10.2–25.9) ng/g creatinine, p < 0.001]. In contrast, no significant differences were observed between everolimusand CNI-treated groups with regard to creatinine and cystatin C, as well as urine albumin and a1M levels. Significant correlations were noted between plasma NGAL and creatinine (r = 0.42, p < 0.001), cystatin C (r = 0.44, p < 0.001), N-terminal brain natriuretic propeptide (r = 0.31, p < 0.01) and indicators of chronic inflammation [lipoprotein-associated phospholipase A2 (Lp-PLA2), r = 0.31, p < 0.01] and soluble CD40 ligand (sCD40L, r = 0.22, p < 0.05), and between urinary NGAL and a1M (r = 0.21, p < 0.05). Multiple regression analysis indicated that cystatin C and Lp-PLA2 were the best predictors of plasma NGAL. Conclusion: The present study documents reduced plasma and urinary NGAL levels in the absence of differences in conventional renal parameters in patients on CNI-free immunosuppressive therapy with everolimus. These results support favorable effects of everolimus on renal integrity in heart transplant recipients. 2015 Published by Elsevier Ltd on behalf of Japanese College of Cardiology.

A Cost Effective Immobilization of Horseradish Peroxidase Nanoparticles on to Easy-To-Prepare Activated Plasticized Polyvinyl-Chloride Vial and Its Application

Horseradish peroxidase nanoparticles was immobilized covalently onto inner wall of polyvinyl chloride (PVC/plastic) vial through glutaraldehyde coupling with 64.13% retention of its initial activity and conjugation yield of 0.35mg/cm2. The vial bound enzyme nanoparticles showed optimum activity at pH-7.2, when incubated at 40oC for 5min. There was a linear relationship between immobilized peroxidase nanoparticles activity and H2O2 concentration in the range of 0.01mM to 100mM with apparent Km for H2O2 as 20mM. The vial bound enzyme nanoparticles was employed for enzymic colorimetric determination of H2O2 in milk and urine. Minimum detection limit of 0.01mM was obtained. Analytical recoveries of added H2O2 in urine (10mM and 20mM) were observed by 98.0% and 96.5% respectively. Within and between assays coefficients of variation (CVs) for H2O2 in urine determinations were detected by 5.3% and 4.5% respectively. A good correlation (r=0.997) was obtained between H2O2 values by standard enzymatic colorimetric method using free enzyme and the present method. The polyvinyl chloride vial was reused 150 times over a period of 4 months, when stored at 4oC. The method has the advantage that it provides the reusability of immobilized enzymes nanoparticles with enormous ease.

Neutrophil gelatinase-associated lipocalin (NGAL) in heart transplant recipients after conversion to everolimus therapy

BackgroundDue to the lack of nephrotoxic activity, proliferation signal inhibitors (PSI) such as everolimus are recommended for immunosuppression after heart transplantation, but the assessment of renal function in patients receiving PSI has led to conflicting results. We examined renal integrity and function using neutrophil gelatinase-associated lipocalin (NGAL) and conventional markers [plasma creatinine, cystatin C, urine albumin, α1-microglobulin (α1M)] in heart transplant patients, who underwent conversion to everolimus due to allograft vasculopathy, graft rejection episodes, or renal function deterioration, and in patients maintained on calcineurin inhibitors (CNI). MethodsThis cross-sectional study included 121 consecutive heart transplant recipients: 44 patients received CNI-free immunosuppressive therapy with everolimus and 77 patients received CNI. Renal parameters were determined in plasma and urine samples using standard enzymatic or immunochemical methods. ResultsHeart transplant recipients receiving everolimus therapy had significantly lower NGAL concentrations in plasma [median (95% CI): 128 (97–176)ng/mL vs. 252 (224–283)ng/mL, p<0.001] and urine [median (95% CI): 6.4 (4.5–7.6)ng/g vs. 15.7 (10.2–25.9)ng/g creatinine, p<0.001]. In contrast, no significant differences were observed between everolimus- and CNI-treated groups with regard to creatinine and cystatin C, as well as urine albumin and α1M levels. Significant correlations were noted between plasma NGAL and creatinine (r=0.42, p<0.001), cystatin C (r=0.44, p<0.001), N-terminal brain natriuretic propeptide (r=0.31, p<0.01) and indicators of chronic inflammation [lipoprotein-associated phospholipase A2 (Lp-PLA2), r=0.31, p<0.01] and soluble CD40 ligand (sCD40L, r=0.22, p<0.05), and between urinary NGAL and α1M (r=0.21, p<0.05). Multiple regression analysis indicated that cystatin C and Lp-PLA2 were the best predictors of plasma NGAL. ConclusionThe present study documents reduced plasma and urinary NGAL levels in the absence of differences in conventional renal parameters in patients on CNI-free immunosuppressive therapy with everolimus. These results support favorable effects of everolimus on renal integrity in heart transplant recipients.

A Modified Enzymatic Method for Measurement of Glycogen Content in Glycogen Storage Disease Type IV.

Deficiency of glycogen branching enzyme in glycogen storage disease type IV (GSD IV) results in accumulation of less-branched and poorly soluble polysaccharides (polyglucosan bodies) in multiple tissues. Standard enzymatic method, when used to quantify glycogen content in GSD IV tissues, causes significant loss of the polysaccharides during preparation of tissue lysates. We report a modified method including an extra boiling step to dissolve the insoluble glycogen, ultimately preserving the glycogen content in tissue homogenates from GSD IV mice. Muscle tissues from wild-type, GSD II and GSD IV mice and GSD III dogs were homogenized in cold water, and homogenate of each tissue was divided into two parts. One part was immediately clarified by centrifugation at 4°C (STD-prep); the other part was boiled for 5min then centrifuged (Boil-prep) at room temperature. When glycogen was quantified enzymatically in tissue lysates, no significant differences were found between the STD-prep and the Boil-prep for wild-type, GSD II and GSD III muscles. In contrast, glycogen content for GSD IV muscle in the STD-prep was only 11% of that in the Boil-prep, similar to wild-type values. Similar results were observed in other tissues of GSD IV mice and fibroblast cells from a GSD IV patient. This study provides important information for improving disease diagnosis, monitoring disease progression, and evaluating treatment outcomes in both clinical and preclinical clinical settings for GSD IV. This report should be used as an updated protocol in clinical diagnostic laboratories.

Inverse association of legume consumption and dyslipidemia: Isfahan Healthy Heart Program.

Dietary intervention for improving serum lipids emphasizes on dietary fiber, plant protein, and flavonoids. This study was performed to examine whether regular legume consumption could alter serum lipids level and prevalence of dyslipidemia. This cross-sectional study was performed among 9660 randomly selected Iranian adults in 3 districts in the central part of Iran, using data collected in the Isfahan Healthy Heart Program in 2007. Dietary behavior including frequency of legume consumption was assessed by 48-item food frequency questionnaire. Fasting serum lipids were measured by standard enzymatic methods. We applied analysis covariance test to compare adjusted mean of serum lipids across legume consumption quartile and logistic regression test was used to determine odds ratio 95% confidence interval of dyslipidemia based on legume consumption in unadjusted and 4-adjusted models. Adjusted mean triglyceride significantly reduced and high-density lipoprotein cholesterol (HDL-C) enhanced by increasing legume consumption (P=.04). The frequency of legume consumption associated with occurrence of all kinds of dyslipidemia except for hypercholesterolemia, especially for those who had consumed legume more than 1 time per day in adjusted models (hypertriglyceridemia: 0.82 [0.68-0.98]; high low-density lipoprotein [LDL-C]: 0.76 [0.60-0.97], and low HDL-C: 0.81 [0.71-0.92]). We found an inverse dose-response association between the frequency of legume consumption and the risk of hypertriglyceridemia, high LDL-C, and low HDL-C. Therefore it is suggested that increasing legume intake may be an important part of a dietary approach for primary prevention of cardiovascular disease.

Association of lipoprotein subclasses and carotid intima-media thickness in the Chinese population

Aims: To determine whether lipoprotein subclasses are associated with the carotid intima-media thickness (IMT) in Chinese population. Patients & methods: Total cholesterol (TC), triglycerides, high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) were measured by standard enzymatic methods. The subclasses of LDL were determined by 1D gradient gel electrophoresis and subclasses of HDL were determined by 2D gradient gel electrophoresis in 342 participants. Carotid IMT was measured by means of high-resolution vascular ultrasound. Results: In multivariate analysis, TC and LDL-C levels were positively correlated with the carotid IMT (p < 0.001). In stepwise multiple regression analysis, small dense LDL was found to be significantly associated with IMT after adjustment for other metabolic risks and traditional lipids such as triglycerides, TC and HDL-C. Additionally, HDL subclasses were not correlated with IMT. Conclusion: Small dense LDL was a stronger predictor for carot...

Tracking of cardiovascular risk factors from childhood to young adulthood — the Pune Children's Study

Tracking of cardiovascular risk factors from childhood to young adulthood — the Pune Children's Study

An amperometric uric acid biosensor based on chitosan-carbon nanotubes electrospun nanofiber on silver nanoparticles

A novel amperometric uric acid biosensor was fabricated by immobilizing uricase on an electrospun nanocomposite of chitosan-carbon nanotubes nanofiber (Chi-CNTsNF) covering an electrodeposited layer of silver nanoparticles (AgNPs) on a gold electrode (uricase/Chi-CNTsNF/AgNPs/Au). The uric acid response was determined at an optimum applied potential of -0.35V vs Ag/AgCl in a flow-injection system based on the change of the reduction current for dissolved oxygen during oxidation of uric acid by the immobilized uricase. The response was directly proportional to the uric acid concentration. Under the optimum conditions, the fabricated uric acid biosensor had a very wide linear range, 1.0-400μmolL(-1), with a very low limit of detection of 1.0μmolL(-1) (s/n = 3). The operational stability of the uricase/Chi-CNTsNF/AgNPs/Au biosensor (up to 205 injections) was excellent and the storage life was more than six weeks. A low Michaelis-Menten constant of 0.21mmolL(-1) indicated that the immobilized uricase had high affinity for uric acid. The presence of potential common interfering substances, for example ascorbic acid, glucose, and lactic acid, had negligible effects on the performance of the biosensor. When used for analysis of uric acid in serum samples, the results agreed well with those obtained by use of the standard enzymatic colorimetric method (P > 0.05).

Microenzymatic Evaluation of Oat (Avena sativa L.) β‐Glucan for High‐Throughput Phenotyping

ABSTRACTOats (Avena sativa L.) have received significant attention for their positive and consistent health benefits when consumed as a whole grain food, attributed in part to mixed‐linkage (1‐3,1‐4)‐β‐d‐glucan (referred to as β‐glucan). Unfortunately, the standard enzymatic method of measurement for oat β‐glucan is costly and does not provide the high‐throughput capability needed for plant breeding in which thousands of samples are measured over a short period of time. The objective of this research was to test a microenzymatic approach for high‐throughput phenotyping of oat β‐glucan. Fifty North American elite lines were chosen to span the range of possible values encountered in elite oats. Pearson and Spearman correlations (r) ranged from 0.81 to 0.86 between the two methods. Although the microenzymatic method did contain bias compared with the results for the standard streamlined method, this bias did not substantially decrease its ability to determine β‐glucan content. In addition to a substantial decrease in cost, the microenzymatic approach took as little as 6% of the time compared with the streamlined method. Therefore, the microenzymatic method for β‐glucan evaluation is an alternative method that can enhance high‐throughput phenotyping in oat breeding programs.

Comparative study of serum lipid levels in normotensive and pre-eclamptic Nigerian women

Background : Pre-eclampsia is a common syndrome that occurs in the second half of pregnancy and often manifest with hypertension and significant proteinuria. It occurs in up to 10% of pregnancies and is the second cause of maternal mortality worldwide accounting for 17% of maternal deaths in Nigeria. Aim : This study was performed to compare the serum lipid levels of pregnant normotensive and pre-eclamptic women. Methods : It was a case control study involving 50 normotensive and 50 preeclamptic women at the University College Hospital, Ibadan, making a total of 100 participants. Their venous blood (5mls) was collected after an overnight fast of 8-12 hours and this was analysed for cholesterol, triglyceride, high density lipoprotein-cholesterol (HDL-C), very low density lipoprotein-cholesterol (VLDL-C) and low density lipoprotein-cholesterol (LDL-C) using standard enzymatic methods. Results : The study showed that there was a significantly higher levels of triglyceride, VLDL-C and HDLC in the pre-eclamptic compared to the normotensive group (P Conclusion : Lipids therefore have an important role in the aetiopathogenesis of pre-eclampsia and could be estimated during the conservative management of patients with preeclampsia. Key Words : Serum lipids; hypertension; pre-eclampsia; normotensive women; Nigerian women

Subclinical Atherosclerosis and Markers of Immune Activation in HIV-Infected Children and Adolescents

HIV-infected adults display increased cardiovascular disease, probably driven by inflammation and immune activation. These relationships have not been addressed in vertically HIV-infected children and adolescents, a population at very high risk for long-term non-AIDS complications. Carotid intima media thickness (IMT) was measured in a cohort of HIV-infected children and adolescents and healthy controls. C-reactive protein and markers of immune activation (CD38⁺HLA-DR⁺) and immune senescence (CD28⁻CD57⁺) were determined. One hundred fifty HIV-infected patients and 150 controls were included, 64.8% female. IMT was thicker in HIV-infected patients (0.434 mm ± 0.025 vs. 0.424 mm ± 0.018, P < 0.001). After adjustment by age, sex, body mass index, and smoking status, HIV infection was independently associated with thicker IMT (odds ratio, 2.28; 95% confidence interval: 1.25 to 4.13; P = 0.007). Among HIV-related variables, a low CD4 nadir was related to an increased IMT. Although HIV-infected subjects presented higher frequencies of activated CD4⁺ and CD8⁺ T cells (P = 0.002 and P = 0.087, respectively), no relation was found between IMT and inflammation, immune activation, or senescence. Structural changes of the vasculature present early in vertically HIV-infected subjects as well as immune activation and senescence. These patients should be carefully monitored for the prompt detection and early treatment of cardiovascular disease.

Selection of optimumTrichodermaspecies for cellulase activity and glucose production from wheat crust

Cellulase enzyme and glucose are important industrial bioproducts which can be obtained from cheap agrowastes. The aim of the present study was to select optimum Trichoderma species for cellulase activity and production of glucose from wheat crust. Three different strains of this fungus i. e. T. koningi 5139, T. longibrachiatum 5140 and T. reesei 5142 were selected. The conditions were optimized by using wheat crust and culturing Trichoderma species with solid state fermentation method. Different incubation times (3–8 days), temperatures (26, 28, 30, 32 and 34°C) and pHs (3.0–9.0) were experimented for the production of cellulase and glucose. Cellulase activity, glucose and protein were measured using standard filter paper activity, enzymatic and Lowry methods in raw enzymatic solution of the cultures, respectively. Cellulase activity and produced glucose were higher in T. reesei compared with two other strains in the same condition (P<0.05). Our results demonstrated that T. reesei was the optimum strain. Also, best culture conditions were 96 h of incubation at 30°C and pH 6.0 for giving highest cellulase activity and produced glucose.

Joint Linkage and Association Analysis with Exome Sequence Data Implicates SLC25A40 in Hypertriglyceridemia

Hypertriglyceridemia (HTG) is a heritable risk factor for cardiovascular disease. Investigating the genetics of HTG may identify new drug targets. There are ~35 known single-nucleotide variants (SNVs) that explain only ~10% of variation in triglyceride (TG) level. Because of the genetic heterogeneity of HTG, a family study design is optimal for identification of rare genetic variants with large effect size because the same mutation can be observed in many relatives and cosegregation with TG can be tested. We considered HTG in a five-generation family of European American descent (n = 121), ascertained for familial combined hyperlipidemia. By using Bayesian Markov chain Monte Carlo joint oligogenic linkage and association analysis, we detected linkage to chromosomes 7 and 17. Whole-exome sequence data revealed shared, highly conserved, private missense SNVs in both SLC25A40 on chr7 and PLD2 on chr17. Jointly, these SNVs explained 49% of the genetic variance in TG; however, only the SLC25A40 SNV was significantly associated with TG (p = 0.0001). This SNV, c.374A>G, causes a highly disruptive p.Tyr125Cys substitution just outside the second helical transmembrane region of the SLC25A40 inner mitochondrial membrane transport protein. Whole-gene testing in subjects from the Exome Sequencing Project confirmed the association between TG and SLC25A40 rare, highly conserved, coding variants (p = 0.03). These results suggest a previously undescribed pathway for HTG and illustrate the power of large pedigrees in the search for rare, causal variants.