Interactive modeling for ongoing utility of pharmacogenetic diagnostic testing: application for warfarin therapy.

BACKGROUND: The application of pharmacogenetic results requires demonstrable correlations between a test result and an indicated specific course of action. We developed a computational decision-support tool that combines patient-specific genotype and phenotype information to provide strategic dosage guidance. This tool, through estimating quantitative and temporal parameters associated with the metabolism- and concentration-dependent response to warfarin, provides the necessary patient-specific context for interpreting international normalized ratio (INR) measurements. METHODS: We analyzed clinical information, plasma S-warfarin concentration, and CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) and VKORC1 (vitamin K epoxide reductase complex, subunit 1) genotypes for 137 patients with stable INRs. Plasma S-warfarin concentrations were evaluated by VKORC1 genotype (-1639G>A). The steady-state plasma S-warfarin concentration was calculated with CYP2C9 genotype-based clearance rates and compared with actual measurements. RESULTS: The plasma S-warfarin concentration required to yield the target INR response is significantly (P < 0.05) associated with VKORC1 -1639G>A genotype (GG, 0.68 mg/L; AG, 0.48 mg/L; AA, 0.27 mg/L). Modeling of the plasma S-warfarin concentration according to CYP2C9 genotype predicted 58% of the variation in measured S-warfarin concentration: Measured [S-warfarin] = 0.67(Estimated [S-warfarin]) + 0.16 mg/L. CONCLUSIONS: The target interval of plasma S-warfarin concentration required to yield a therapeutic INR can be predicted from the VKORC1 genotype (pharmacodynamics), and the progressive changes in S-warfarin concentration after repeated daily dosing can be predicted from the CYP2C9 genotype (pharmacokinetics). Combining the application of multivariate equations for estimating the maintenance dose with genotype-guided pharmacokinetics/pharmacodynamics modeling provides a powerful tool for maximizing the value of CYP2C9 and VKORC1 test results for ongoing application to patient care.

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 (GSH(T)) and cysteine (CYS(T)). 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.

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.

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.

Polyamines protect against radiation-induced oxidative stress.

Astronauts and cosmonauts are exposed to a wide variety of different hazards while in space that include radiation, which presents one of the most critical barriers to long-term missions. A major deleterious effect directly associated with ionizing radiation is the production of reactive oxygen species (ROS) such as peroxides and hydroxyl radicals. The free radicals generated by ultraviolet (UV) or ionizing radiation can attack cellular lipids, proteins and DNA. Endogenous free radical scavengers such as glutathione and the polyamines (e.g, spermidine and spermine) can inhibit the action of ROS. In particular, heme oxygenase-1 (HO-1), the enzyme involved in heme protein metabolism, can provide antioxidant protection through the production of the antioxidant bilirubin. Furthermore, polyamines have been shown to indirectly increase HO-1 content and antioxidant protection. The beta2-adrenoceptor agonist clenbuterol has been shown to stimulate polyamine synthesis and by extension, might provide a margin of antioxidant protection through increasing HO-1 content. However, it is unclear whether the polyamines are acting as a tertiary messengers for antioxidant protection in the be beta2-adrenoceptor signal transduction pathway. The purpose of this study was to study the role of the polyamine pathway in attenuating free radical-induced damage.

Effect of palm oil on oxidative stress-induced hypertension in Sprague-Dawley rats.

BACKGROUND: Oxidative stress, associated with increased plasma isoprostane (ISO) and reductions in plasma glutathione (GSH), has been shown to cause severe hypertension in normal rats. Palm oil (PO), with an unsaturated-to-saturated fatty acid ratio close to one and rich in antioxidant vitamins, has been investigated for its beneficial effects on arterial thrombosis and atherosclerosis. In this study, the effect of PO on oxidative stress induced by inhibition of GSH synthesis (using buthionine sulfoximine [BSO]) was examined. METHODS: Sprague-Dawley rats were separated into two groups and received either natural vitamin-rich PO (Carotino, 5 g/kg daily) or water by gavage. After 4 weeks, they were further divided between receiving either BSO (30 mmol/L/day in the drinking water) or drug-free water for an additional week. Mean arterial pressure (MAP), heart rate (HR), and body weight (BW) were measured before and weekly during the experiment. The levels of plasma ISO, nitric oxide (NO), prostacyclin (PGI2), and thromboxane A2 (TXA2) were determined by enzyme immunoassay, and plasma, heart, and kidney GSH by high-performance liquid chromatography. RESULTS: The PO reduced the age-dependent increase in MAP, and the pressor response to BSO, without changing the HR or BW compared to the BSO and control groups. It also elevated PGI2, NO, and aortic cGMP, but decreased TXA2 and aortic cAMP. In addition, the BSO-induced increase in ISO and TXA2, and the reduction in kidney GSH were attenuated by PO. However, the PO effect on NO, PGI2, cGMP, and TXA2 was partly counteracted by BSO. CONCLUSIONS: Palm oil reduces BSO-induced oxidative stress and attenuates hypertension by mechanisms involving changes in endothelium-derived factors.

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.

The role of oxidative stress in salt-induced hypertension.

BACKGROUND: Impairment of endothelial function during hypertension is associated with increased production of superoxide radicals and reduced antioxidants. We investigated the involvement of oxidative stress in Dahl salt-sensitive (SS) and salt-resistant (SR) rats. METHODS: For a 2-week period, male rats were fed either high salt (HS; 8% sodium chloride) or low salt (LS; 0.3% sodium chloride) diets. Before and weekly on the diets, mean arterial pressure (MAP) and heart rate were measured by tail-cuff plethysmography. At the end of the experiment, plasma and tissue samples were collected for analysis of nitric oxide, prostacyclin, glutathione, and isoprostane. RESULTS: The MAP was increased in SS rats on HS diet, but not in those on a LS diet or in SR rats on either diet. Plasma levels of nitric oxide were reduced in SS rats on HS diet. Plasma prostacyclin levels in SS rats on either diet were lower than SR on LS diet. Increased dietary salt reduced plasma prostacyclin levels in SR, but not in SS rats. Plasma total 8-isoprostane was elevated in both SS and SR rats on HS diet compared with either strain on LS diet. Plasma levels of total glutathione were reduced in SS compared with SR rats, regardless of the level of dietary salt intake. The whole blood ratio of reduced-to-oxidized glutathione (GSH/GSSG) as well as the kidney total glutathione were lower in SS rats on HS diet. Aortic superoxide production in both strains on HS diet was increased compared with the animals on LS diet. CONCLUSIONS: These data suggest that HS diet may indirectly induce endothelial dysfunction through intermediate mechanisms that are associated with oxidative stress.

Relation of endothelial nitric oxide synthase gene to plasma nitric oxide level, endothelial function, and blood pressure in African Americans.

BACKGROUND: The role of eNOS gene polymorphisms on plasma nitrite or nitrate (NOx) level, endothelial function, and blood pressure (BP) remains unclear. METHODS: We estimated the relationship of eNOS polymorphisms (the T(-786)C in the 5'-flanking promoter region, T(-786)C; 27-bp repeat in intron 4, eNOS4; and Glu298Asp in exon 7, G894T) with plasma NOx level, brachial endothelial function assessed by ultrasound measure of brachial artery flow-mediated dilation (FMD), and BP in 60 healthy African Americans, 30 men and 30 women aged 18 to 73 years. RESULTS: Among them, 73.1%, 23.9%, and 3.0% carried TT, TC, and CC of T(-786)C, respectively, 14.5%, 27.5%, 53.6%, and 1.4% carried aa, ab, bb, and bc of eNOS4 polymorphism, respectively, and 70.4%, 23.9%, and 5.6% carried GG, GT, and TT of G894T, respectively. G894T and eNOS4 were observed in linkage disequilibrium. Mean values of age, plasma NOx, FMD, systolic and diastolic BPs were not significantly different (P >.05) by eNOS polymorphisms. Plasma NO(x) level was found to be associated with systolic BP (r = 0.51, P =.03), and diastolic BP (r = 0.41, P =.08), but not with FMD, in individuals with "a" allele of eNOS4 polymorphism after adjustment for age, body mass index, serum glucose, and smoking status. CONCLUSIONS: We reveal a positive association between plasma NOx level and BP in normotensive African Americans who carry the "a" allele of eNOS4. Because the frequency of the rare allele "a" is significantly higher in African Americans than in other ethnic groups, this finding may provide a clue to understanding the genetic susceptibility to hypertension in African Americans.

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.

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. METHODS: We analysed intact type I mouse collagens and AGE by conventional fluorescence spectroscopy, synchronous spectroscopy, high-performance liquid chromatography (HPLC), and borate quenching. RESULTS: The relative amount of each fluorophore depends on the collagen sample. Acid- or enzyme-hydrolysis results in loss of fluorescence intensity at 350-400 nm, and enhanced emission 400-500 nm which could be reproduced by controlled dopa oxidation. Borate buffer quenches fluorescence at lambda>400 nm from intact collagens, dityrosine, dopa, and oxidized dopa but does not quench tyrosine or AGE fluorescence. CONCLUSION: Collagen fluorescence depends on its source and previous history. The data indicate that collagen fluorescence is derived from tyrosine, dopa, interacting tyrosine residues, covalent dityrosine, and dopa oxidation product(s), but not AGE.

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.

Changes in muscle proteins and spermidine content in response to unloading and clenbuterol treatment.

Anabolic agents such clenbuterol (Cb) are useful tools for probing the mechanisms by which muscles respond to disuse. Cb was examined under different loading conditions with respect to its effects on muscle mass, protein (myofibrillar and cytosolic), and spermidine content in mature male rats. Compared with control treatment, Cb significantly increased loaded and unloaded soleus, plantaris, and extensor digitorum longus (EDL) mass. Likewise, Cb significantly increased loaded and unloaded soleus (24.8 and 21.6%, respectively), plantaris (12.1 and 22.9%, respectively), and EDL (22.4 and 13.3%, respectively) myofibrillar protein content. After unloading, cytosolic proteins significantly increased in the EDL but decreased in the soleus and plantaris. Cb significantly increased cytosolic protein levels in all loaded muscles, while only causing increases in unloaded soleus. When compared with controls, unloading caused significant reductions in spermidine levels in the soleus (40.4%) and plantaris (35.9%) but caused increases in the EDL (54.8%). In contrast, Cb increased spermidine levels in unloaded soleus (42.9%), plantaris (102.8%), and EDL (287%). In loaded muscles, Cb increased spermidine levels in all three muscles, but to a lesser degree than under unloading conditions. Nonlinear regression analyses indicated that the plantaris behaves like a slow-twitch muscle under unloading conditions and like a fast-twitch muscle when loaded. This suggests that the responses of these muscles to unloading and (or) Cb treatment might be influenced by factors beyond fiber type alone.