Effects of the active botanical blend "WKUP GT" on attention and cognitive functions after lunch in healthy volunteers.

OBJECTIVES: "WKUP GT", a low caffeine beverage consisting of carob, Guarana, Green Tea and Elderberry extracts was studied on attention and cognitive functions post-lunch in a pilot randomized double blind placebo controlled trial. METHODS: Thirty healthy volunteers were included in a crossover design trial, presenting five beverages randomly assigned to the following groups: placebo, "WKUP GT" (single, double or triple doses), or "caffeine" as an active control. Hemodynamic measurements were assessed as safety outcomes. The Cambridge Neuropsychological Test Automated Battery (CANTAB), was used to evaluate the patients when beverages were consumed 30 and 120 min after lunch (respectively Delta30 and Delta120 considering baseline). RESULTS: Drinking "caffeine" or "WKUP GT" after lunch, showed significant improvement (p < 0.05) in rapid visual information processing compared to placebo (Delta120 of "caffeine", "WKUP" single and double). In addition, improvement in Multitasking Test (Delta30 for "WKUP" double, and Delta120 for "caffeine" and "WKUP" triple compared to placebo) was observed. "WKUP" triple also showed significant improvement for "memory" when compared to placebo (Delta120). Compared to "caffeine", WKUP GT did not increase systolic blood pressure. CONCLUSION: "WKUP GT" showed improvements for attention, memory, psychomotor and executive function tasks after lunch without increase in pulse rate.

The Effect of Capsule-in-Capsule Combinations on In Vivo Disintegration in Human Volunteers: A Combined Imaging and Salivary Tracer Study.

Controlling the time point and site of the release of active ingredients within the gastrointestinal tract after administration of oral delivery systems is still a challenge. In this study, the effect of the combination of small capsules (size 3) and large capsules (size 00) on the disintegration site and time was investigated using magnetic resonance imaging (MRI) in combination with a salivary tracer technique. As capsule shells, Vcaps® HPMC capsules, Vcaps® Plus HPMC capsules, gelatin and DRcaps® designed release capsules were used. The three HPMC-based capsules (Vcaps®, Vcaps® Plus and DRcaps® capsules) were tested as single capsules; furthermore, seven DUOCAP® capsule-in-capsule combinations were tested in a 10-way crossover open-label study in six healthy volunteers. The capsules contained iron oxide and hibiscus tea powder as tracers for visualization in MRI, and two different caffeine species (natural caffeine and 13C3) to follow caffeine release and absorption as measured by salivary levels. Results showed that the timing and location of disintegration in the gastrointestinal tract can be measured and differed when using different combinations of capsule shells. Increased variability among the six subjects was observed in most of the capsule combinations. The lowest variability in gastrointestinal localization of disintegration was observed for the DUOCAP® capsule-in-capsule configuration using a DRcaps® designed release capsule within a DRcaps® designed release outer capsule. In this combination, the inner DRcaps® designed release capsule always opened reliably after reaching the ileum. Thus, this combination enables targeted delivery to the distal small intestine. Among the single capsules tested, Vcaps® Plus HPMC capsules showed the fastest and most consistent disintegration.

Effects of Low Doses of L-Carnitine Tartrate and Lipid Multi-Particulate Formulated Creatine Monohydrate on Muscle Protein Synthesis in Myoblasts and Bioavailability in Humans and Rodents.

The primary objective of this study was to investigate the potential synergy between low doses of L-carnitine tartrate and creatine monohydrate to induce muscle protein synthesis and anabolic pathway activation in primary human myoblasts. In addition, the effects of Lipid multi-particulates (LMP) formulation on creatine stability and bioavailability were assessed in rodents and healthy human subjects. When used individually, L-carnitine tartrate at 50 µM and creatine monohydrate at 0.5 µM did not affect myoblast protein synthesis and signaling. However, when combined, they led to a significant increase in protein synthesis. Increased AKT and RPS6 phosphorylation were observed with 50 µM L-carnitine tartrate 5 µM creatine in combination in primary human myoblasts. When Wistar rats were administered creatine with LMP formulation at either 21 or 51 mg/kg, bioavailability was increased by 27% based on the increase in the area under the curve (AUC) at a 51 mg/kg dose compared to without LMP formulation. Tmax and Cmax were unchanged. Finally, in human subjects, a combination of LMP formulated L-carnitine at 500 mg (from L-carnitine tartrate) with LMP formulated creatine at 100, 200, or 500 mg revealed a significant and dose-dependent increase in plasma creatine concentrations. Serum total L-carnitine levels rose in a similar manner in the three combinations. These results suggest that a combination of low doses of L-carnitine tartrate and creatine monohydrate may lead to a significant and synergistic enhancement of muscle protein synthesis and activation of anabolic signaling. In addition, the LMP formulation of creatine improved its bioavailability. L-carnitine at 500 mg and LMP-formulated creatine at 200 or 500 mg may be useful for future clinical trials to evaluate the effects on muscle protein synthesis.

L-Carnitine Tartrate Supplementation for 5 Weeks Improves Exercise Recovery in Men and Women: A Randomized, Double-Blind, Placebo-Controlled Trial.

L-carnitine tartrate has been shown to improve relatively short-term recovery among athletes. However, there is a lack of research on the longer-term effects in the general population. OBJECTIVE: The primary objectives of this randomized double-blind, placebo-controlled trial were to evaluate the effects of daily L-carnitine tartrate supplementation for 5 weeks on recovery and fatigue. METHOD: In this study, eighty participants, 21- to 65-years-old, were recruited. Participants were split into two groups of forty participants each, a placebo, and a L-carnitine Tartrate group. Seventy-three participants completed a maintenance exercise training program that culminated in a high-volume exercise challenge. RESULTS: Compared to placebo, L-carnitine tartrate supplementation was able to improve perceived recovery and soreness (p = 0.021), and lower serum creatine kinase (p = 0.016). In addition, L-carnitine tartrate supplementation was able to blunt declines in strength and power compared to placebo following an exercise challenge. Two sub-analyses indicated that these results were independent of gender and age. Interestingly, serum superoxide dismutase levels increased significantly among those supplementing with L-carnitine tartrate. CONCLUSIONS: These findings agree with previous observations among healthy adult subjects and demonstrate that L-carnitine tartrate supplementation beyond 35 days is beneficial for improving recovery and reducing fatigue following exercise across gender and age.

Comparison of protection and release behavior of different capsule polymer combinations based on L. acidophilus survivability and function and caffeine release.

Oral administration of active pharmaceutical ingredients, nutraceuticals, enzymes or probiotics requires an appropriate delivery system for optimal bioactivity and absorption. The harsh conditions during the gastrointestinal transit can degrade the administered products, hampering their efficacy. Enteric or delayed-release pharmaceutical formulations may help overcome these issues. In a Simulator of Human Intestinal Microbial Ecosystem model (SHIME) and using caffeine as a marker for release kinetics and L. acidophilus survivability as an indicator for protection, we compared the performance of ten capsule configurations, single or DUOCAP® combinations. The function of L. acidophilus and its impact on the gut microbiota was further tested in three selected capsule types, combinations of DRcaps® capsule in DRcaps® capsule (DR-in-DR) and DRcaps® capsule in Vcaps® capsule (DR-in-VC) and single Vcaps® Plus capsule under colonic conditions. We found that under stomach and small intestine conditions, DR-in-DR and DR-in-VC led to the best performance both under fed and fasted conditions based on the slow caffeine release and the highest L. acidophilus survivability. The Vcaps® Plus capsule however, led to the quickest caffeine and probiotic release. When DR-in-DR, DR-in-VC and single Vcaps® Plus capsules were tested through the whole gastrointestinal tract, including under colonic conditions, caffeine release was found to be slower in capsules containing DRcaps® capsules compared to the single Vcaps® capsules. In addition, colonic survival of L. acidophilus was significantly increased under fasted conditions in DR-in-DR or DR-in-VC formulation compared to Vcaps® Plus capsule. To assess the impact of these formulations on the microbial function, acetate, butyrate and propionate as well as ammonia were measured. L. acidophilus released from DR-in-DR or DR-in-VC induced a significant increase in butyrate and a decrease in ammonia, suggesting a proliferation of butyrate-producing bacteria and reduction in ammonia-producing bacteria. These data suggest that L. acidophilus included in DR-in-DR or DR-in-VC reaching the colon is viable and functional, potentially contributing to changes in colonic microbiota composition and diversity.

Bioavailability of a Capsaicin Lipid Multi-particulate Formulation in Rats.

BACKGROUND AND OBJECTIVE: Because of the stomach-burning sensation it induces, capsaicin has been used at relatively low doses as a nutritional supplement, which has limited its bioavailability. The objective of this study was to investigate the serum bioavailability of capsaicin supplementation with or without a lipid multi-particulate (LMP) formulation. METHODS: Thirty-five rats were divided into five groups and administered capsaicin at either 0.2 or 1 mg/kg with or without the LMP formulation. Capsaicin bioavailability was assessed based on the area under the concentation-time curve (AUC), the time to peak concentration (Tmax), and the peak serum concentration (Cmax). RESULTS: For each formulation, the capsaicin Cmax was reached at 90 min and decreased thereafter. Serum capsaicin concentrations were greater in rats administered the higher dose of capsaicin (1 mg/kg) in the LMP formulation at all measurement times (P  ≤ 0.05). The AUC showed a significant increase, about 20%, when capsaicin was administered in the LMP formulation at the high dose (P = 0.002). The Tmax for oral capsaicin was similar whether or not administration was via the LMP formulation (P = 0.163). However, the Cmax of capsaicin increased in a dose-dependent manner (P < 0.05). Although the LMP formulation of the high dose of capsaicin resulted in a numerically higher Cmax, it was not statistically significantly higher (P = 0.068). CONCLUSIONS: The present work demonstrated that administration of capsaicin via the LMP formulation significantly impacted the pharmacokinetic parameters and the serum bioavailability of orally administered 1 mg/kg capsaicin in rats. The bioavailability of capsaicin in humans may also be increased by using the LMP formulation.

Effect of arabinogalactan on the gut microbiome: A randomized, double-blind, placebo-controlled, crossover trial in healthy adults.

OBJECTIVE: Promising evidence suggests beneficial health effects of arabinogalactan, but little is known about the effect of this non-digestible carbohydrate on the gut microbiota, a crucial mediator of human health. The objective of this study was to investigate the effect of an arabinogalactan product (ResistAid) on the fecal microbiome and short-chain fatty acids and gastrointestinal tolerance in healthy adults in a randomized, double-blind, crossover trial. METHODS: Thirty adults were randomly assigned to consume 15 g/d maltodextrin (control) or ResistAid for 6 wk. RESULTS: At week 6, compared to placebo, ResistAid supplementation led to a significant decrease in the ratio of fecal Firmicutes to Bacteroidetes, driven by an increase in Bacteroidetes and a decrease in Firmicutes. Moreover, the relative abundance of Bifidobacterium tended to increase with ResistAid supplementation. Additionally, ResistAid significantly decreased the α-diversity of the fecal microbiome. Predicted functional abundances based on 16S rRNA sequences showed that ResistAid supplementation increased the gene abundance of the gut microbiome for α-l-rhamnosidase, ß-fructosidase, and levanase, as well as tricarboxylic acid and vitamin B6 biosynthesis pathways. Fecal isovaleric, valeric, and hexanoic acids were significantly lower after ResistAid consumption. There were no statistically significant changes in bowel habit, stool consistency, gastrointestinal tolerance symptoms, chemistry profile, metabolic panel, or vitals, suggesting that consumption of 15 g daily ResistAid over 6 wk is safe. CONCLUSION: These results demonstrate that the gut microbiome composition and predicted functions can be modulated by ResistAid consumption, perhaps suggesting a mechanistic explanation on its reported benefits in metabolic parameters and the immune system.

L-Carnitine Tartrate Downregulates the ACE2 Receptor and Limits SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for one of the worst pandemics in modern history. Several prevention and treatment strategies have been designed and evaluated in recent months either through the repurposing of existing treatments or the development of new drugs and vaccines. In this study, we show that L-carnitine tartrate supplementation in humans and rodents led to significant decreases of key host dependency factors, notably angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and Furin, which are responsible for viral attachment, viral spike S-protein cleavage, and priming for viral fusion and entry. Interestingly, pre-treatment of Calu-3, human lung epithelial cells, with L-carnitine tartrate led to a significant and dose-dependent inhibition of the infection by SARS-CoV-2. Infection inhibition coincided with a significant decrease in ACE2 mRNA expression levels. These data suggest that L-carnitine tartrate should be tested with appropriate trials in humans for the possibility to limit SARS-CoV-2 infection.

Trimethylamine N-Oxide in Relation to Cardiometabolic Health-Cause or Effect?

Trimethylamine-N-oxide (TMAO) is generated in a microbial-mammalian co-metabolic pathway mainly from the digestion of meat-containing food and dietary quaternary amines such as phosphatidylcholine, choline, betaine, or L-carnitine. Fish intake provides a direct significant source of TMAO. Human observational studies previously reported a positive relationship between plasma TMAO concentrations and cardiometabolic diseases. Discrepancies and inconsistencies of recent investigations and previous studies questioned the role of TMAO in these diseases. Several animal studies reported neutral or even beneficial effects of TMAO or its precursors in cardiovascular disease model systems, supporting the clinically proven beneficial effects of its precursor, L-carnitine, or a sea-food rich diet (naturally containing TMAO) on cardiometabolic health. In this review, we summarize recent preclinical and epidemiological evidence on the effects of TMAO, in order to shed some light on the role of TMAO in cardiometabolic diseases, particularly as related to the microbiome.

A Dose-Dependent Effect of Carnipure® Tartrate Supplementation on Endurance Capacity, Recovery, and Body Composition in an Exercise Rat Model.

The objective of this work is to investigate the effects of Carnipure® Tartrate (CT) supplementation with or without exercise on endurance capacity, recovery, and fatigue by assessing time to exhaustion as well as body weight and composition in rats. In addition, antioxidant capacity has been evaluated by measuring malondialdehyde (MDA) levels and antioxidant enzyme (superoxide dismutase, SOD; catalase, CAT; glutathioneperoxidase; GSHPx) activities. Fifty-six male Wistar rats were divided into eight groups including seven rats each. A control group did not receive CT nor exercise. Another control group received 200 mg/kg CT without exercise. The other six groups of rats went through an exercise regimen consisting of a 5-day training period with incremental exercise capacity, which was followed by 6 weeks of the run at 25 m/min for 45 min every day. CT was supplemented at 0, 25, 50, 100, 200, and 400 mg/kg per day during the 6 weeks. Rats submitted to exercise and supplemented with CT had a significant and dose-dependent increase in time to exhaustion and this effect seems to be independent of exercise (p < 0.05). Additionally, recovery and fatigue were improved, as shown by a significant and dose-dependent decrease in myoglobin and lactic acid plasma levels, which are two markers of muscle recovery. CT supplementation led to a dose-response decrease in body weight and visceral fat. These effects become significant at 200 and 400 mg/kg doses (p < 0.05). Additionally, the antioxidant capacity was improved, as shown by a significant and dose-dependent increase in SOD, CAT, and GSHPx. Serum MDA concentrations decreased in exercising rats with CT supplementation. CT supplementation led to a decrease in serum glucose, triglycerides, and total cholesterol concentrations with the lowest levels observed at 400 mg/kg dose (p < 0.05). These effects correlated with a significant dose-dependent increase in serum total L-carnitine, free L-carnitine, and acetyl-carnitine, which linked the observed efficacy to CT supplementation. These results demonstrate that CT supplementation during exercise provides benefits on exercise performance, recovery, and fatigue as well as improved the lipid profile and antioxidant capacity. The lowest dose leads to some of these effects seen in rats where 25 mg/kg corresponds to 250 mg/day as a human equivalent.

l-Carnitine Supplementation in Recovery after Exercise.

Given its pivotal role in fatty acid oxidation and energy metabolism, l-carnitine has been investigated as ergogenic aid for enhancing exercise capacity in the healthy athletic population. Early research indicates its beneficial effects on acute physical performance, such as increased maximum oxygen consumption and higher power output. Later studies point to the positive impact of dietary supplementation with l-carnitine on the recovery process after exercise. It is demonstrated that l-carnitine alleviates muscle injury and reduces markers of cellular damage and free radical formation accompanied by attenuation of muscle soreness. The supplementation-based increase in serum and muscle l-carnitine contents is suggested to enhance blood flow and oxygen supply to the muscle tissue via improved endothelial function thereby reducing hypoxia-induced cellular and biochemical disruptions. Studies in older adults further showed that l-carnitine intake can lead to increased muscle mass accompanied by a decrease in body weight and reduced physical and mental fatigue. Based on current animal studies, a role of l-carnitine in the prevention of age-associated muscle protein degradation and regulation of mitochondrial homeostasis is suggested.

Efficacy of a novel formulation of L-Carnitine, creatine, and leucine on lean body mass and functional muscle strength in healthy older adults: a randomized, double-blind placebo-controlled study.

BACKGROUND: Progressive decline in skeletal muscle mass and function are growing concerns in an aging population. Diet and physical activity are important for muscle maintenance but these requirements are not always met. This highlights the potential for nutritional supplementation. As a primary objective, we sought to assess the effect of a novel combination of L-Carnitine, creatine and leucine on muscle mass and performance in older subjects. METHOD: Forty-two healthy older adults aged 55-70 years were randomized to receive either a novel L-Carnitine (1500 mg), L-leucine (2000 mg), creatine (3000 mg), Vitamin D3 (10 µg) (L-Carnitine-combination) product (n = 14), L-Carnitine (1500 mg) (n = 14), or a placebo (n = 14) for eight weeks. We evaluated body mass by DXA, upper and lower strength by dynamometry, and walking distance by a 6-min walk test at baseline and after eight weeks of intervention. These measures, reflecting muscle mass, functional strength and mobility have been combined to generate a primary composite score. Quality of life, blood safety markers, and muscle biopsies for protein biomarker analysis were also conducted at baseline and the end of the study. RESULTS: The primary composite outcome improved by 63.5 percentage points in the L-Carnitine-combination group vs. placebo (P = 0.013). However, this composite score did not change significantly in the L-Carnitine group (P = 0.232), and decreased slightly in the placebo group (P = 0.534). Participants supplemented with the L-Carnitine-combination showed a 1.0 kg increase in total lean muscle mass (P = 0.013), leg lean muscle mass (0.35 kg, P = 0.005), and a 1.0 kg increase in lower leg strength (P = 0.029) at week 8. In addition, these increases were significant when compared to the placebo group (P = 0.034, P = 0.026, and P = 0.002, respectively). Total mTOR protein expression was increased in participants in the L-Carnitine-combination group at the end of the study compared to the baseline (P = 0.017). This increase was also significant when compared to the placebo (P = 0.039), suggesting that the increase in muscle mass and strength was due to new protein synthesis and mTOR pathway activation. CONCLUSIONS: The trial did reach its primary objective. L-Carnitine combined with creatine and L-leucine significantly improved the composite score which reflects muscle mass and strength, at the end of the study compared to placebo. The combination showed an increase in mTOR protein level, a driver for increased muscle mass which translated to an improvement in muscle strength. This new combination may provide a potential nutritional intervention to promote muscle growth and improved physical functioning in older adults.

L-Carnitine intake and high trimethylamine N-oxide plasma levels correlate with low aortic lesions in ApoE(-/-) transgenic mice expressing CETP.

OBJECTIVE: Dietary l-carnitine can be metabolized by intestinal microbiota to trimethylamine, which is absorbed by the gut and further oxidized to trimethylamine N-oxide (TMAO) in the liver. TMAO plasma levels have been associated with atherosclerosis development in ApoE(-/-) mice. To better understand the mechanisms behind this association, we conducted in vitro and in vivo studies looking at the effect of TMAO on different steps of atherosclerotic disease progression. METHODS: J774 mouse macrophage cells were used to evaluate the effect of TMAO on foam cell formation. Male ApoE(-/-) mice transfected with human cholesteryl ester transfer protein (hCETP) were fed l-carnitine and/or methimazole, a flavin monooxygenase 3 (FMO3) inhibitor that prevents the formation of TMAO. Following 12 week treatment, l-carnitine and TMAO plasma levels, aortic lesion development, and lipid profiles were determined. RESULTS: TMAO at concentrations up to 10-fold the Cmax reported in humans did not affect in vitro foam cell formation. In ApoE(-/-)mice expressing hCETP, high doses of l-carnitine resulted in a significant increase in plasma TMAO levels. Surprisingly, and independently from treatment group, TMAO levels inversely correlated with aortic lesion size in both aortic root and thoracic aorta. High TMAO levels were found to significantly correlate with smaller aortic lesion area. Plasma lipid and lipoprotein levels did not change with treatment nor with TMAO levels, suggesting that the observed effects on lesion area were independent from lipid changes. CONCLUSION: These findings suggest that TMAO slows aortic lesion formation in this mouse model and may have a protective effect against atherosclerosis development in humans.

The role of Ile87 of CYP158A2 in oxidative coupling reaction.

Both CYP158A1 and CYP158A2 are able to catalyze an oxidative C-C coupling reaction producing biflaviolin or triflaviolin in Streptomyces coelicolor A3(2). The substrate-bound crystal structures of CYP158A2 and CYP158A1 reveal that the side chain of Ile87 in CYP158A2 points to the active site contacting the distal flaviolin molecule, however, the bulkier side chain of Lys90 in CYP158A1 (corresponding to Ile87 in CYP158A2) is toward the distal surface of the protein. These results suggest that these residues could be important in determining product regiospecificity. In order to explore the role of the two residues in catalysis, the reciprocal mutants, Ile87Lys and Lys90Ile, of CYP158A2 and CYP158A1, respectively, were generated and characterized. The mutant Ile87Lys enzyme forms two isomers of biflaviolin instead of three isomers of biflaviolin in wild-type CYP158A2. CYP158A1 containing the substitution of lysine with isoleucine has the same catalytic activity compared with the wild-type CYP158A1. The crystal structure of Ile87Lys showed that the BC loop in the mutant is in a very different orientation compared with the BC loop in both CYP158A1/A2 structures. These results shed light on the mechanism of the oxidative coupling reaction catalyzed by cytochrome P450.

Effect of enhanced glycemic control with saxagliptin on endothelial nitric oxide release and CD40 levels in obese rats.

AIM: Endothelial cell (EC) dysfunction contributes to insulin resistance in diabetes and is characterized by reduced nitric oxide (NO) release, increased nitroxidative stress and enhanced inflammation. The purpose of this study was to test the effect of improved postprandial glucose control on EC function in insulin-resistant rats as compared to fasting glucose (FG) changes. METHODS: Obese Zucker rats were treated with 10 mg/kg/day saxagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, for 4 or 8 weeks and compared to lean rats. NO and peroxynitrite (ONOO(-)) release from aortic and glomerular ECs was measured ex vivo using amperometric approaches and correlated with FG, postprandial glucose, insulin, soluble CD40 (sCD40) and L-citrulline levels. RESULTS: Saxagliptin treatment improved NO production and reduced ONOO(-) release prior to any observed changes in FG levels. In untreated obese animals, NO release from aortic and glomerular ECs decreased by 22% and 31%, respectively, while ONOO(-) release increased by 26% and 40%. Saxagliptin increased aortic and glomerular NO release by 18% and 31%, respectively, with comparable reductions in ONOO(-) levels; the NO/ONOO(-) ratio, an indicator of NO synthase coupling, increased by >40%. Improved glycemic control was further associated with a reduction in sCD40 levels by more than ten-fold (from 300 ± 206 to 22 ± 22 pg/mL, p < 0.001). CONCLUSION: These findings indicate that enhanced glycemic control with DPP4 inhibition improved NO release and reduced inflammation in a manner not predicted by FG changes alone.

Applications of HILIC for targeted and non-targeted LC/MS analyses in drug discovery.

RP-HPLC has largely been the analytical method of choice in the pharmaceutical industry for many years because of the poor aqueous solubility and hydrophobicity of most small molecule drug candidates. RP-HPLC coupled to MS has provided an excellent analytical tool for qualitatively and quantitatively determining levels of drug molecules or drug metabolites for studies such as purity assessment and pharmacokinetic profiling. Quantitation of endogenous metabolites is an emerging field in drug discovery, gaining popularity for evaluating pharmacokinetic-pharmacodynamic relationships and targeting activity and efficacy. While RP-HPLC-MS is suitable for a range of applications, many endogenous molecules, especially those found in urine, are small polar compounds that do not retain well by RP-HPLC. This has made hydrophilic interaction LC (HILIC) an attractive alternative and useful approach to polar molecule analysis. Additionally, because HILIC is routinely used with traditional RP organic solvents such as ACN and methanol, it can be easily coupled to MS. This paper will review selected examples from the current literature as well as discuss some new applications from the author's own laboratory focusing on the applicability of HILIC to quantitative and qualitative profiling of endogenous metabolites in drug discovery.

The role of protease inhibitors in the pathogenesis of HIV-associated lipodystrophy: cellular mechanisms and clinical implications.

Metabolic complications associated with HIV infection and treatment frequently present as a relative lack of peripheral adipose tissue associated with dyslipidemia and insulin resistance. In this review we explain the connection between abnormalities of intermediary metabolism, observed either in vitro or in vivo, and this group of metabolic effects. We review molecular mechanisms by which the HIV protease inhibitor (PI) class of drugs may affect the normal stimulatory effect of insulin on glucose and fat storage. We then propose that both chronic inflammation from HIV infection and treatment with some drugs in this class trigger cellular homeostatic stress responses with adverse effects on intermediary metabolism. The physiologic outcome is such that total adipocyte storage capacity is decreased, and the remaining adipocytes resist further fat storage. The excess circulating and dietary lipid metabolites, normally "absorbed" by adipose tissue, are deposited ectopically in lean (muscle and liver) tissue, where they impair insulin action. This process leads to a pathologic cycle of lipotoxicity and lipoatrophy and a clinical phenotype of body fat distribution with elevated waist-to-hip ratio similar to the metabolic syndrome.

Binding of two flaviolin substrate molecules, oxidative coupling, and crystal structure of Streptomyces coelicolor A3(2) cytochrome P450 158A2.

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

Functional variant of CYP4A11 20-hydroxyeicosatetraenoic acid synthase is associated with essential hypertension.

BACKGROUND: The CYP4A11 arachidonic acid monooxygenase oxidizes endogenous arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE), a metabolite with renovascular and tubular functions. Mice with targeted disruption of Cyp4a14, a murine homologue of CYP4A11, have severe hypertension. We combined molecular and biochemical approaches to identify a functional variant of the CYP4A11 20-HETE synthase and determine its association with hypertensive status in 2 independent human populations. METHODS AND RESULTS: A thymidine-to-cytosine polymorphism at nucleotide 8590 resulted in a phenylalanine-to-serine substitution at amino acid 434. Expression of cDNA with serine 434 resulted in a protein with a significantly reduced AA and lauric acid metabolizing activity. In a population of 512 whites from Tennessee, the age, body mass index, and gender-adjusted OR of having hypertension attributable to the 8590C variant was 2.31 (95% CI 1.41 to 3.78) compared with the reference 8590TT genotype. In subjects from the Framingham Heart Study, the adjusted ORs of hypertension associated with the 8590C variant were 1.23 (CI 0.94 to 1.59; n=1538) in all subjects and 1.33 (CI 1.01 to 1.77; n=1331) when subjects with diabetes were excluded. No association of the variant with hypertension was detected in a population of 120 blacks. CONCLUSIONS: We identified a variant of the human CYP4A11 (T8590C) that encodes for a monooxygenase with reduced 20-HETE synthase activity. The association of the T8590C variant with hypertension supports its role as a polygenic determinant of blood pressure control in humans, and results obtained from the large population database suggest that the relevance of the variant may vary according to hypertension comorbidity.