Natalia Silvestrov

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Effect of hindlimb suspension and clenbuterol treatment on polyamine levels in skeletal muscle.

Polyamines are unbiquitous, naturally occurring small aliphatic, polycationic, endogenous compounds. They are involved in many cellular processes and may serve as secondary or tertiary messengers to hormonal regulation. The relationship of polyamines and skeletal muscle mass of adductor longus, extensor digitorum longus, and gastrocnemius under unloading (hindlimb suspension) conditions was investigated. Unloading significantly affected skeletal muscle polyamine levels in a fiber-type-specific fashion. Under loading conditions, clenbuterol treatment increased all polyamine levels, whereas under unloading conditions, only the spermidine levels were consistently increased. Unloading attenuated the anabolic effects of clenbuterol in predominately slow-twitch muscles (adductor longus), but had little impact on clenbuterol’s action as a countermeasure in fast- twitch muscles such as the extensor digitorum longus. Spermidine appeared to be the primary polyamine involved in skeletal muscle atrophy/hypertrophy.

Rapid HPLC Procedure for the Quantitation of Phytochelatins in Plant Tissue Extracts

Abstract A simple HPLC procedure for the identification and quantitation of phytochelatins (PCs) in plant tissue extracts is described. The method, which does not require a derivatization step utilizes only 20 µL of sample volume. Linear quantitative response curve was generated for phytochelatin 3 (PC3) over a concentration range of 1.33 µmol/L–6.66 mmol/L. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.996. Limit of detection (LOD) and limit of quantitation (LOQ) values were 0.1 and 0.5 µmol, respectively. Phytochelatin 3 recovery using this method was relatively high (above 85%). Intra‐assay and inter‐assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of a wide range of PC concentrations in plant tissue extracts was demonstrated successfully.

Associations of Apelin, Visfatin, and Urinary 8-Isoprostane With Severe Hypertension in African Americans: The MH-GRID Study

BACKGROUND Apelin is an adipokine directly associated with adiposity, insulin resistance, and decreased blood pressure. Urinary 8-isoprostane is a marker of chronic oxidative endothelial stress. Visfatin, an adipokine that acts by binding and activating the insulin receptor, has been associated with hypertension. As severe hypertension (SH) is highly prevalent among African Americans (AA), we aimed to assess the association of these biomarkers with SH status. METHODS A sample of 250 AA participants (134 normotensive controls and 116 with SH (including 98 treatment controlled, SCH: severe controlled hypertension, and 18 treatment resistant, SRH: severe resistant hypertension)) from the Minority Health Genomics and Translational Research Bio-Repository Database (MH-GRID) in metro Atlanta had blood analyzed for apelin and visfatin and urine for 8-isoprostane. T-tests, sex-specific age-adjusted correlation coefficients, and multivariable logistic regression models were used to assess the association of biomarkers with hypertensive status. RESULTS Levels of apelin and 8-isoprostane were not statistically different between controls and SCH or SRH. Statistically significant differences were present in levels of visfatin between controls (1.03±0.84 pg/ml), SCH (1.34±1.14 pg/ml), and SRH (1.59±0.85 pg/ml). After multivariable adjustment, categorization in the middle 2 quartiles of urinary 8-isoprostane were associated with SH. In similar models, categorization into the highest quartile of visfatin was associated with SH (odds ratio = 2.80; 95% confidence interval: 1.02-7.02). A continuous association of visfatin with SH was present. CONCLUSION In our community sample of AA, there were increased odds of SH with increased levels of urinary 8-isoprostane and visfatin, but not with apelin.

VALIDATION OF A SOLILD PHASE EXTRACTION PROCEDURE FOR THE GC-MS IDENTIFICATION AND QUANTITATION OF COCAINE AND THREE METABOLITES IN BLOOD, URINE, AND MILK

A simple and widely used solid-phase extraction procedure (United Chemical Technologies Method Handbook) was applied for the GC-MS identification and quantitation of cocaine (COC), benzoylecgonine (BE), cocaethylene (COCE), and m-hydroxy- benzoylecgonine (HBE) in blood, urine, and milk. The method which utilizes BSTFA as a derivatizing agent yielded abundant diagnostic ions with high m/z values. Linear quantitative response curves were generated for the analytes of interest over a concentration range of 5–1000 ng/mL. Linear regression analyses of the standard curve in the three specimen types exhibited correlation coefficients ranging from 0.997 to 1.000. The LOD values for COC, COCE, and derivatives of BE, and HBE in the three specimen types ranged from 2.5 to 5.0 ng/mL. The LOQ values, however, ranged from 5.0 to 10.0 ng/mL. Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the detection and quantitation of COC, BE, COCE, and HBE was demonstrated successfully in human blood and urine samples, as well as blood samples obtained from cocaine-treated (subcutaneously) rats.

Fluorescence of putative chromophores in Skh-1 and citrate-soluble calf skin collagens

Background/purpose: Fluorescence of Skh‐1 hairless mouse and calf skin acid‐soluble type I collagens are envelopes of several bands putatively due to tyrosine (excitation/emission peak at 275/300 nm), dihydroxyphenylalanine (dopa; 280/325 nm), tyrosine aggregate (285/360 nm), dityrosine 325/400 nm), and advanced glycation end (AGE) product (370/450 nm), respectively. As these fluorophores can be markers of pathological conditions, we wish to present further evidence for or against these assignments.

Effects of cholecalciferol supplementation on serum and urinary vitamin D metabolites and binding protein in HIV-infected youth

Vitamin D insufficiency is widespread in HIV-infected patients. HIV and/or antiretroviral therapy (ART), particularly efavirenz (EFV), may interfere with vitamin D metabolism. However, few data from randomized, controlled trials exist. Here, we investigate changes in vitamin D metabolites and binding protein (VDBP) after 6 months of supplementation in a randomized, active-control, double-blind trial investigating 2 different monthly cholecalciferol (vitamin D3) doses [60,000 (medium) or 120,000 (high) IU/month] vs. a control arm of 18,000 IU/month in 8-25year old HIV-infected youth on ART with HIV-1 RNA <1000 copies/mL and baseline 25-hydroxycholecalciferol (25(OH)D3) ≤30ng/mL. A matched healthy uninfected group was enrolled in a similar parallel study for comparison. Changes after 6 months were analyzed as intent-to-treat within/between groups [control group (low dose) vs. combined supplementation doses (medium+high)]. At 6 months, 55% vs. 82% of subjects in control and supplementation groups, respectively, reached 25(OH)D3 ≥30ng/mL (P=0.01) with no difference between medium and high doses (both 82% ≥30ng/mL). There were few differences for those on EFV vs. no-EFV, except serum VDBP decreased in EFV-treated subjects (both within- and between-groups P≤0.01). There were no significant differences between the HIV-infected vs. healthy uninfected groups. The major finding of the present study is that cholecalciferol supplementation (60,000 or 120,000 IU/month) effectively raises serum 25(OH)D3 in the majority of HIV-infected subjects, regardless of EFV use. Notably, response to supplementation was similar to that of uninfected subjects.

Toward Understanding the Influence of Soil Metals and Sulfate Content on Plant Thiols

Plants respond to increased concentrations of metals by a number of mechanisms, including chelation with phytochelatins (PCs). Soil specimens and plants (Veronica anagalis-aquatica, Typha domingensis, Cynodon dactylon, Chenopodium album, Rumex dentatus, Amaranthus gracilis, Chenopodium murale, Inula viscosa) leaves were collected from two sites in northern Jordan and subsequently metals (cadmium, copper, and lead), sulfate, and PC (from leaves) levels were determined. One of these sites was contaminated with metals and the other served as a control site. The contaminated site had elevated cadmium, copper, lead, and sulfate levels. This increase of metal and sulfate levels in the soil at the contaminated site correlated with a rise in plant total glutathione (GSHT) and cysteine (CYST). These increases were not attributed to an elevation in total phytochelatin levels. However, a significant increase in the ratio of short-chain phytochelatins to the total phytochelatin stores was observed. The individual effects of metals and sulfate on glutathione, short-chain PCs and long-chain PCs levels were dissimilar.

P-322: Effect of losartan on oxidative stress-induced hypertension in Sprague-Dawley rats

BACKGROUND Hypertension induced by oxidative stress has been demonstrated in normal rats. In the current study, we investigated the effect of the oral AT(1) receptor blocker losartan (10 mmol/kg/day) on oxidative stress, induced by glutathione (GSH) depletion (using buthionine-sulfoximine, BSO, 30 mmol/L/day in the drinking water), in Sprague-Dawley rats. METHODS Mean arterial pressure (MAP) was measured by tail-cuff plethysmography and the plasma levels of total 8-isoprostane, nitric oxide, prostacyclin, thromboxane A(2), angiotensin II, aldosterone, and aortic cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were determined by enzyme immunoassay. Plasma, heart, and kidney GSH were analyzed by high-performance liquid chromatography. Aortic and renal superoxide production was determined by fluorescence spectrometry. RESULTS In the BSO-treated group, MAP, angiotensin II, isoprostane, thromboxane A(2), and superoxide were elevated; whereas prostacyclin, GSH, cAMP, and cGMP were reduced, compared to control. Losartan alone reduced MAP, and increased renal GSH, plasma nitric oxide, angiotensin II, aldosterone, and aortic cGMP. When administered concurrently with BSO, losartan reversed the BSO-induced elevation of MAP, superoxide, and thromboxane A(2) as well as the reduction in prostacyclin and aortic cAMP levels, but did not significantly alter the reduction in GSH or the elevation in angiotensin II and aldosterone. CONCLUSIONS Losartan attenuates BSO-induced hypertension, which appears to be mediated, in part, by angiotensin II and the prostanoid endothelium-derived factors.