Effect of Defatted Dabai Pulp Extract in Urine Metabolomics of Hypercholesterolemic Rats.

A source of functional food can be utilized from a source that might otherwise be considered waste. This study investigates the hypocholesterolemic effect of defatted dabai pulp (DDP) from supercritical carbon dioxide extraction and the metabolic alterations associated with the therapeutic effects of DDP using 1H NMR urinary metabolomic analysis. Male-specific pathogen-free Sprague-Dawley rats were fed with a high cholesterol diet for 30 days to induce hypercholesterolemia. Later, the rats were administered with a 2% DDP treatment diet for another 30 days. Supplementation with the 2% DDP treatment diet significantly reduced the level of total cholesterol (TC), triglyceride, low-density lipoprotein (LDL), and inflammatory markers (C-reactive protein (CRP), interleukin 6 (IL6) and tumour necrosis factor-α (α-TNF)) and significantly increased the level of antioxidant profile (total antioxidant status (TAS), superoxide dismutase (SOD), glutathione peroxide (GPX), and catalase (CAT)) compared with the positive control group (PG) group (p < 0.05). The presence of high dietary fibre (28.73 ± 1.82 g/100 g) and phenolic compounds (syringic acid, 4-hydroxybenzoic acid and gallic acid) are potential factors contributing to the beneficial effect. Assessment of 1H NMR urinary metabolomics revealed that supplementation of 2% of DDP can partially recover the dysfunction in the metabolism induced by hypercholesterolemia via choline metabolism. 1H-NMR-based metabolomic analysis of urine from hypercholesterolemic rats in this study uncovered the therapeutic effect of DDP to combat hypercholesterolemia.

Alkalinization with potassium bicarbonate improves glutathione status and protein kinetics in young volunteers during 21-day bed rest.

BACKGROUND & AIMS: Physical inactivity is associated with lean body mass wasting, oxidative stress and pro-inflammatory changes of cell membrane lipids. Alkalinization may potentially counteract these alterations. We evaluated the effects of potassium bicarbonate supplementation on protein kinetics, glutathione status and pro- and anti-inflammatory polyunsaturated fatty acids (PUFA) in erythrocyte membranes in humans, during experimental bed rest. METHODS: Healthy, young, male volunteers were investigated at the end of two 21-day bed rest periods, one with, and the other without, daily potassium bicarbonate supplementation (90 mmol × d-1), according to a cross-over design. Oxidative stress in erythrocytes was evaluated by determining the ratio between reduced (GSH) and oxidized glutathione (GSSG). Glutathione turnover and phenylalanine kinetics, a marker of whole body protein metabolism, were determined by stable isotope infusions. Erythrocyte membranes PUFA composition was analyzed by gas-chromatography. RESULTS: At the end of the two study periods, urinary pH was 10 ± 3% greater in subjects receiving potassium bicarbonate supplementation (7.23 ± 0.15 vs. 6.68 ± 0.11, p < 0.001). Alkalinization increased total glutathione concentrations by 5 ± 2% (p < 0.05) and decreased its rate of clearance by 38 ± 13% (p < 0.05), without significantly changing GSH-to-GSSG ratio. After alkalinization, net protein balance in the postabsorptive state improved significantly by 17 ± 5% (p < 0.05) as well as the sum of n-3 PUFA and the n-3-to-n-6 PUFA ratio in erythrocyte membranes (p < 0.05). CONCLUSIONS: Alkalinization during long-term inactivity is associated with improved glutathione status, anti-inflammatory lipid pattern in cell membranes and reduction in protein catabolism at whole body level. This study suggests that, in clinical conditions characterized by inactivity, oxidative stress and inflammation, alkalinization could be a useful adjuvant therapeutic strategy.

Exploring the Lean Phenotype of Glutathione-Depleted Mice: Thiol, Amino Acid and Fatty Acid Profiles.

BACKGROUND: Although reduced glutathione (rGSH) is decreased in obese mice and humans, block of GSH synthesis by buthionine sulfoximine (BSO) results in a lean, insulin-sensitive phenotype. Data is lacking about the effect of BSO on GSH precursors, cysteine and glutamate. Plasma total cysteine (tCys) is positively associated with stearoyl-coenzyme A desaturase (SCD) activity and adiposity in humans and animal models. OBJECTIVE: To explore the phenotype, amino acid and fatty acid profiles in BSO-treated mice. DESIGN: Male C3H/HeH mice aged 11 weeks were fed a high-fat diet with or without BSO in drinking water (30 mmol/L) for 8 weeks. Amino acid and fatty acid changes were assessed, as well as food consumption, energy expenditure, locomotor activity, body composition and liver vacuolation (steatosis). RESULTS: Despite higher food intake, BSO decreased particularly fat mass but also lean mass (both P<0.001), and prevented fatty liver vacuolation. Physical activity increased during the dark phase. BSO decreased plasma free fatty acids and enhanced insulin sensitivity. BSO did not alter liver rGSH, but decreased plasma total GSH (tGSH) and rGSH (by ~70%), and liver tGSH (by 82%). Glutamate accumulated in plasma and liver. Urine excretion of cysteine and its precursors was increased by BSO. tCys, rCys and cystine decreased in plasma (by 23-45%, P<0.001 for all), but were maintained in liver, at the expense of decreased taurine. Free and total plasma concentrations of the SCD products, oleic and palmitoleic acids were decreased (by 27-38%, P <0.001 for all). CONCLUSION: Counterintuitively, block of GSH synthesis decreases circulating tCys, raising the question of whether the BSO-induced obesity-resistance is linked to cysteine depletion. Cysteine-supplementation of BSO-treated mice is warranted to dissect the effects of cysteine and GSH depletion on energy metabolism.

Metabolic activation of furan moiety makes Diosbulbin B hepatotoxic.

Diosbulbin B (DIOB), a furanoid, is a major constituent of herbal medicine Dioscorea bulbifera L. Exposure to DIOB caused liver injury in humans and experimental animals. The mechanisms of DIOB-induced hepatotoxicities remain unknown. The present study demonstrated that DIOB induced hepatotoxicities in a time- and dose-dependent manner in mice. H&E stained histopathologic image showed the occurrence of necrosis in the liver obtained from the mice treated with DIOB at dose of 200 mg/kg. Pretreatment with KTC protected the animals from hepatotoxicities and hepatic GSH depletion induced by DIOB, increased area under the concentration-time curve of blood DIOB, decreased urinary excretion of GSH conjugates derived from DIOB, and increased urinary excretion of parent drug. Pretreatment with BSO exacerbated DIOB-induced hepatotoxicities. In order to define the role of furan moiety in DIOB-induced liver toxicities, we replaced the furan of DIOB with a tetrahydrofuran group by chemical hydrogenation of the furan ring of DIOB. No liver injury was observed in the animals given the same doses of tetrahydro-DIOB. The furan moiety was essential for DIOB-induced hepatotoxicities. The results implicate the cis-enedial reactive metabolite of DIOB was responsible for the observed toxicities. The observed modest depletion of hepatic GSH in DIOB-treated animals suggests the actions of one or more reactive metabolites, and the hepatic injury observed could be due at least in part to reactions of these metabolites with crucial biomolecules. Cytochrome P450 3A enzymes are implicated in DIOB-induced hepatotoxicities by catalyzing the formation of the reactive metabolite of DIOB.

Correlative analysis of metabolite profiling of Danggui Buxue Tang in rat biological fluids by rapid resolution LC-TOF/MS.

In this work, the metabolite profiles of Danggui Buxue Tang (DBT) in rat bile and plasma were qualitatively described, and the possible metabolic pathways of DBT were subsequently proposed. Emphasis was put on correlative analysis of metabolite profiling in different biological fluids. After oral administration of DBT, bile and plasma samples were collected and pretreated by solid phase extraction. Rapid resolution liquid chromatography coupled to time-of-flight mass spectrometry (RRLC-TOFMS) was used for characterization of DBT-related compounds (parent compounds and metabolites) in biological matrices. A total of 142 metabolites were detected and tentatively identified from the drug-containing bile and plasma samples. Metabolite profiling shows that rat bile contained relatively more glutathione-derived conjugates, more saponins compounds and more diverse forms of metabolites than urine. The metabolite profile in plasma revealed that glucuronide conjugates of isoflavonoids, dimmers, acetylcysteine conjugates and parent form of phthalides, as well as saponin aglycones were the major circulating forms of DBT. Collectively, the metabolite profile analysis of DBT in different biological matrices provided a comprehensive understanding of the in vivo metabolic fates of constituents in DBT.

In vitro formation of quinoid metabolites of the dietary supplement Cimicifuga racemosa (black cohosh).

Botanical dietary supplements containing Cimicifuga racemosa (Actaea racemosa; black cohosh) are used commonly by women to assuage menopausal symptoms including hot flashes and sleep disorders. Despite the popularity of such supplements, little is known about the metabolism or possible toxicity of many compounds that could be concentrated therein. The aim of this study was to selectively identify phase I metabolites resulting from metabolic bioactivation of constituents of black cohosh in vitro and to determine whether evidence of such metabolites could be found in the urine of perimenopausal women taking black cohosh oral supplements. A variation of an ultrafiltration mass spectrometric assay devised previously was used to screen an extract of black cohosh for the formation of electrophilic phase I metabolites that had been trapped as GSH conjugates. Mercapturates (N-acetylcysteine conjugates) corresponding to the GSH conjugates identified during screening were synthesized and characterized using LC-MS/MS with product-ion scanning. During a phase I clinical trial of black cohosh in perimenopausal women, urine was collected from seven subjects, each of whom took a single oral dose of either 32, 64, or 128 mg of the black cohosh extract. These urine samples were analyzed for the presence of mercapturate conjugates using positive-ion electrospray LC-MS and LC-MS/MS. On the basis of their propensity to form GSH adducts following metabolic activation by hepatic microsomes and NADPH in vitro, a total of eight electrophilic metabolites of black cohosh were detected, including quinoid metabolites of fukinolic acid, fukiic acid, caffeic acid, and cimiracemate B. Additional quinoid metabolites were formed from hydroxytyrosol and dihydroxyphenyl lactic acid, neither of which had been isolated previously from black cohosh. However, mercapturate conjugates of these black cohosh constituents were not detected in urine samples from women who consumed single oral doses of up to 256 mg of a standardized black cohosh extract. Therefore, for moderate doses of a dietary supplement containing black cohosh, this study found no cause for safety concerns over the formation of quinoid metabolites in women.

Selenium status in an iodine deficient population of the West Ivory Coast.

Selenium is an essential trace element which is part of the active site of seleno-dependent glutathione peroxidase and type 1 deiodinase. Therefore, it plays a key role in thyroid hormone metabolism. The present work was undertaken in order to evaluate selenium status in two Ivory Coast populations: the first with high (Glanlé) and the second with low (Abidjan) prevalence of iodine deficiency. Selenium, glutathione peroxidase, glutathione reductase, glutathione and diglutathione were determined in blood and/or urine. In plasma and erythrocytes, selenium and glutathione peroxidase were dramatically low in Glanlé. Compared to Abidjan, selenium, glutathione peroxidase, vitamin E and riboflavin status were decreased whereas diglutathione was increased in Glanlé. The results clearly demonstrate a selenium deficiency and suggest an oxidant stress in Glanlé. Causes and consequences of this selenium deficiency and oxidant stress remain to be determined.

Effects of a low selenium state in patients with phenylketonuria.

Eighty-seven participants of the German Collaboratory Study for Children with Phenylketonuria (PKU) presented low plasma, whole blood and hair selenium (Se) values, reduced urinary selenium excretion, and decreased plasma and erythrocyte glutathione peroxidase activity in comparison with a healthy reference group (all figures p < 0.001). Aspartate amino transferase and thyroxine (T4) concentrations in plasma were inversely correlated with the selenium blood values of the PKU children. Somatic measurements showed a negative standard deviation score of body height in the PKU children compared with reference values. Despite the different Se supply, the infants did not present any specific Se deficiency symptoms.

A therapeutic trial with N-acetylcysteine in subjects with hereditary glutathione synthetase deficiency (5-oxoprolinuria).

In a therapeutic trial, the effect of short-term low-dosage N-acetylcysteine supplementation on glutathione metabolism was investigated in two patients with hereditary glutathione deficiency (5-oxoprolinuria). Clinical and neurophysiological examinations of the patients indicated progressive neurological damage. The pretreatment concentrations of total and free glutathione in leukocytes were 15-20% of normal, whereas the corresponding gamma-glutamylcysteine levels were increased. In plasma, the glutathione concentrations were similarly decreased, but no gamma-glutamylcysteine was detected. Total glutathione in erythrocytes was markedly decreased. Low urinary excretion of cysteinylglycine, cyst(e)ine, taurine, N-acetylcysteine, mercaptolactate and mercaptoacetate and reduced leukocyte taurine levels constituted additional evidence of decreased intracellular availability of cysteine, i.e. glutathione. Oral supplementation with N-acetylcysteine (5 mg/kg x 3/day) had no effect on acid-base balance, erythrocyte glutathione levels or 5-oxoproline concentrations in plasma and urine. In leukocytes, the glutathione concentrations were increased by 20-30%, whereas the gamma-glutamylcysteine levels were essentially unaltered. In parallel, the urinary excretion of cysteinylglycine was increased and the leukocyte levels and urinary outputs of sulphur amino acids were restored. No side-effects of the treatment were noted. The results indicate that N-acetylcysteine may be of value in increasing the low intracellular glutathione concentrations and cysteine availability in patients with hereditary glutathione synthetase deficiency.