Therapeutic response to leflunomide in combo therapy and monotherapy is associated to serum teriflunomide (A77 1726) levels.

There is a significant rate of therapeutic failure in rheumatoid arthritis (RA) patients treated with leflunomide (LEF). This study investigates the utility values of teriflunomide levels (A77 1726) in identifying RA patients who remained with moderate or severe disease activity after the treatment with LEF. In this cross-sectional study, we compared: (a) RA patients who achieved a DAS28-ESR ≤ 3.2, and (b) RA patients who maintained a DAS28-ESR > 3.2 after treatment. ROC curves determined the cut-off of A77 1726 with the better performance to identify patients achieving a DAS28-ESR ≤ 3.2. Of the 115 patients treated with LEF, 69 (60%) remained with moderate/severe disease activity and 46 (40%) achieved low disease activity/remission. Higher A77 1726 levels showed a negative correlation with DAS28-ESR (r = - 0.42, p < 0.001) and other parameters of disease activity. We obtained the following utility values with the cut-off of A77 1726 > 10 µg/mL to identify RA patients who achieved a DAS28-ESR ≤ 3.2: sensitivity of 91.31%; specificity of 73.91%; positive predictive value of 70.00%; and negative predictive value of 92.73%. Serum A77 1726 discriminated between RA patients who remained with moderate/severe disease activity despite the treatment with LEF both as monotherapy and LEF as combo therapy.

Effect of incremental proportions of Desmanthus spp. in isonitrogenous forage diets on growth performance, rumen fermentation and plasma metabolites of pen-fed growing Brahman, Charbray and Droughtmaster crossbred beef steers.

Desmanthus (Desmanthus spp.), a tropically adapted pasture legume, is highly productive and has the potential to reduce methane emissions in beef cattle. However, liveweight gain response to desmanthus supplementation has been inconclusive in ruminants. This study aimed to evaluate weight gain, rumen fermentation and plasma metabolites of Australian tropical beef cattle in response to supplementation with incremental levels of desmanthus forage legume in isonitrogenous diets. Forty-eight Brahman, Charbray and Droughtmaster crossbred beef steers were pen-housed and fed a basal diet of Rhodes grass (Chloris gayana) hay supplemented with 0, 15, 30 or 45% freshly chopped desmanthus forage on dry matter basis, for 140 days. Varying levels of lucerne (Medicago sativa) hay were added in the 0, 15 and 30% diets to ensure that all diets were isonitrogenous with the 45% desmanthus diet. Data were analyzed using the Mixed Model procedures of SAS software. Results showed that the proportion of desmanthus in the diet had no significant effect on steer liveweight, rumen volatile fatty acids molar proportions and plasma metabolites (P ≥ 0.067). Total bilirubin ranged between 3.0 and 3.6 µmol/L for all the diet treatments (P = 0.67). All plasma metabolites measured were within the expected normal range reported for beef cattle. Rumen ammonia nitrogen content was above the 10 mg/dl threshold required to maintain effective rumen microbial activity and maximize voluntary feed intake in cattle fed low-quality tropical forages. The average daily weight gains averaged 0.5 to 0.6 kg/day (P = 0.13) and were within the range required to meet the target slaughter weight for prime beef markets within 2.5 years of age. These results indicate that desmanthus alone or mixed with other high-quality legume forages can be used to supplement grass-based diets to improve tropical beef cattle production in northern Australia with no adverse effect on cattle health.

Detection of γ-hydroxybutyric acid-related acids in blood plasma and urine: Extending the detection window of an exogenous γ-hydroxybutyric acid intake?

In crimes facilitated by γ-hydroxybutyric acid (GHB) administration, the frequent occurrence of anterograde amnesia of the victims as well as the short detection window and variations of endogenous GHB concentrations complicate obtaining analytical proof of GHB administration. Because elevated endogenous organic acid concentrations have been found in the urine of patients with succinic semialdehyde deficiency (leading to accumulation of GHB in human specimens) and after GHB ingestion, we searched for an alternative way to prove GHB administration via detection of elevated organic acid concentrations in blood plasma and urine. We collected blood and urine samples from narcolepsy patients (n = 5) treated with pharmaceuticals containing GHB sodium salt (1.86-3.72 g GHB as free acid per dose). Although GHB was detectable only up to 4 h in concentrations greater than the commonly used cutoff levels in blood plasma, 3,4-dihydroxybutyric acid (3,4-DHB) could be detected up to 12 h in blood plasma in concentrations exceeding initial concentrations of the same patient before GHB ingestion. Furthermore, four of the five patients showed an increase above endogenous levels described in the scientific literature. In urine, GHB concentrations above commonly used cutoff levels could be observed 4.5-9.5 h after GHB intake. Creatinine standardized initial concentrations were reached again for glycolic acid (GA), 3,4-DHB, and 2,4-dihydroxybutyric (2,4-DHB) acid at 6.5-22, 11.5-22, and 8.5-70 h after GHB intake, respectively. Therefore, 2,4-DHB, 3,4-DHB, and GA are promising and should be further investigated as potential biomarkers to prolong the detection window of GHB intake.

Choline Metabolites, Hydroxybutyrate and HDL after Dietary Fiber Supplementation in Overweight/Obese Hypertensive Women: A Metabolomic Study.

Metabolomics has been increasingly used to evaluate metabolic changes associated with morbidities. The objective of this study is to assess the metabolic profile before and after intervention with mixed dietary fiber in overweight and obese hypertensive women. This is an intervention study, and the sample consists of 14 women aged 28 to 58 years. An intervention with 12 g of mixed soluble and insoluble fiber is performed for a period of eight weeks. Serum metabolites are identified using a Bruker 1H NMR spectrometer at 400 MHz. Multivariate data analysis, including principal component analysis (PCA), is used to differentiate the two groups. After supplementation with dietary fiber, there is a significant increase in the peak intensity values of the metabolites HDL-C (0.0010*), choline (0.0012*) and hydroxybutyrate (0.0010*) as well as a decrease in systolic (0.0013*) and diastolic (0.0026*) blood pressure. The analysis of the metabolomic profile allows the identification of metabolites that have been associated in the literature with hypertension and excess weight (choline, hydroxybutyrate and amino acids) and with fiber intake (choline, hydroxybutyrate and amino acids) in addition to an increase in HDL-C. The increase in the detection of the described metabolites possibly occurs due to the presence of pathologies and the use of fiber in the intervention, which also contributes to elevated HDL-c and reduced blood pressure.

The serum metabolome of COVID-19 patients is distinctive and predictive.

BACKGROUND: Metabolism is critical for sustaining life, immunity and infection, but its role in COVID-19 is not fully understood. METHODS: Seventy-nine COVID-19 patients, 78 healthy controls (HCs) and 30 COVID-19-like patients were recruited in a prospective cohort study. Samples were collected from COVID-19 patients with mild or severe symptoms on admission, patients who progressed from mild to severe symptoms, and patients who were followed from hospital admission to discharge. The metabolome was assayed using gas chromatography-mass spectrometry. RESULTS: Serum butyric acid, 2-hydroxybutyric acid, l-glutamic acid, l-phenylalanine, l-serine, l-lactic acid, and cholesterol were enriched in COVID-19 and COVID-19-like patients versus HCs. Notably, d-fructose and succinic acid were enriched, and citric acid and 2-palmitoyl-glycerol were depleted in COVID-19 patients compared to COVID-19-like patients and HCs, and these four metabolites were not differentially distributed in non-COVID-19 groups. COVID-19 patients had enriched 4-deoxythreonic acid and depleted 1,5-anhydroglucitol compared to HCs and enriched oxalic acid and depleted phosphoric acid compared to COVID-19-like patients. A combination of d-fructose, citric acid and 2-palmitoyl-glycerol distinguished COVID-19 patients from HCs and COVID-19-like patients, with an area under the curve (AUC) > 0.92 after validation. The combination of 2-hydroxy-3-methylbutyric acid, 3-hydroxybutyric acid, cholesterol, succinic acid, L-ornithine, oleic acid and palmitelaidic acid predicted patients who progressed from mild to severe COVID-19, with an AUC of 0.969. After discharge, nearly one-third of metabolites were recovered in COVID-19 patients. CONCLUSIONS: The serum metabolome of COVID-19 patients is distinctive and has important value in investigating pathogenesis, determining a diagnosis, predicting severe cases, and improving treatment.

Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity.

Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, ß-hydroxy acylcarnitines, and ß-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.

Effect of Oral Administration with Lactobacillus plantarum CAM6 Strain on Sows during Gestation-Lactation and the Derived Impact on Their Progeny Performance.

BACKGROUND: To evaluate the biological response of the sows and their offspring with oral administration of Lactobacillus plantarum CAM6 in breeding sows, a total of 20 Pietrain breeding sows with three farrowings and their descendants were used, randomly divided into two groups of 10 sows each. Treatments included a basal diet (T0) and basal diet +10 mL biological agent containing 109 CFU/mL L. plantarum CAM6 (T1). No antibiotics were used throughout the entire experimental process of this study. RESULTS: The L. Plantarum CAM6 supplementation in sows' feeding did not affect (P > 0.05) the reproductive performance of the sows; however, the number of deaths for their offspring before weaning (P ≤ 0.05) decreased. In addition, the oral administration of Lactobacillus plantarum CAM6 in sows increased (P ≤ 0.05) the content of lactose, nonfat solids, mineral salts, and the density of sows' milk, with a decrease in milk fat. Moreover, the probiotic feed orally to the sows improved the body weight (P ≤ 0.05) and reduced the diarrhea incidence of their offspring (P ≤ 0.05). Also, the probiotic administration of sows changed (P ≤ 0.05) the serum concentration of Na+, pCO2, and D-ß-hydroxybutyrate and increased (P ≤ 0.05) the leukocytes, lymphocytes, and platelets in their piglets. CONCLUSION: Oral administration of Lactobacillus plantarum CAM6 in breeding sows improved body weight, physiological status, and the health of their offspring. And preparing the neonatal piglets physiologically is of great importance to the pig farming industry which could decrease the operational cost and medication (especially antibiotics) consumption of the pig producers.

Plasma 3-hydroxyisobutyrate (3-HIB) and methylmalonic acid (MMA) are markers of hepatic mitochondrial fatty acid oxidation in male Wistar rats.

OBJECTIVE: Discovery of specific markers that reflect altered hepatic fatty acid oxidation could help to detect an individual's risk of fatty liver, type 2 diabetes and cardiovascular disease at an early stage. Lipid and protein metabolism are intimately linked, but our understanding of this crosstalk remains limited. METHODS: In male Wistar rats, we used synthetic fatty acid analogues (3-thia fatty acids) as a tool to induce hepatic fatty acid oxidation and mitochondrial biogenesis, to gain new insight into the link between fatty acid oxidation, amino acid metabolism and TCA cycle-related intermediate metabolites in liver and plasma. RESULTS: Rats treated with 3-thia fatty acids had 3-fold higher hepatic, but not adipose and skeletal muscle, expression of the thioesterase 3-hydroxyisobutyryl-CoA hydrolase (Hibch), which controls the formation of 3-hydroxyisobutyrate (3-HIB) in the valine degradation pathway. Consequently, 3-thia fatty acid-stimulated hepatic fatty acid oxidation and ketogenesis was accompanied by decreased plasma 3-HIB and increased methylmalonic acid (MMA) concentrations further downstream in BCAA catabolism. The higher plasma MMA corresponded to higher MMA-CoA hydrolase activity and hepatic expression of GTP-specific succinyl-CoA synthase (Suclg2) and succinate dehydrogenase (Sdhb), and lower MMA-CoA mutase activity. Plasma 3-HIB correlated positively to plasma and hepatic concentrations of TAG, plasma total fatty acids, plasma NEFA and insulin/glucose ratio, while the reverse correlations were seen for MMA. CONCLUSION: Our study provides new insight into TCA cycle-related metabolic changes associated with altered hepatic fatty acid flux, and identifies 3-HIB and MMA as novel circulating markers reflective of mitochondrial ß-oxidation in male Wistar rats.

The effect of circuit resistance training on plasma levels of amino acids, alpha-hydroxybutyrate, mannose, and urinary levels of glycine conjugated adducts in obese adolescent boys.

Few studies have examined the improving effects of exercise on the association between metabolites of impaired protein metabolism and insulin resistance in obese children. Therefore, this study aims to investigate the effect of circuit resistance training (CRT) on plasma levels of amino acids, alpha-hydroxybutyrate (α-HB), mannose, and urinary levels of glycine conjugated adducts in obese adolescent boys. Forty obese adolescent boys (body mass index above the 95th percentile) with an age range of 14-17 years were randomly divided into the CRT group (n = 20) and control group (n = 20). The CRT program (3 times/week, 70%-80% of 1-repetition maximum) was performed for 8 weeks. The results indicated that the body composition and plasma levels of glucose, insulin resistance, valine, mannose, lysine, and the sum of branched-chain amino acids (BCAA) were decreased because of CRT. The plasma levels of asparagine, glycine, serine, and urinary levels of glycine conjugated adduct also increased in the CRT group. Although α-HB level decreased during CRT, it had no significant difference from that of the control group. It can be concluded that the improvement in obesity complications including insulin resistance in obese adolescent boys after CRT may be due to decrease in plasma levels of mannose and BCAA and increase urinary metabolites. Novelty: CRT improves glucose metabolism and insulin resistance in obese adolescent boys. CRT decreases plasma levels of mannose and BCAA and normalizes other amino acids. CRT increases urinary levels of glycine conjugated adducts.

Assessing the Impact of Factors that Influence the Ketogenic Response to Varying Doses of Medium Chain Triglyceride (MCT) Oil.

Objectives, Design, Setting: The ketogenic effect of medium chain triglyceride (MCT) oil offers potential for Alzheimer's disease prevention and treatment. Limited literature suggests a linear B-hyroxybutyrate (BHB) response to increasing MCT doses. This pharmacokinetic study evaluates factors affecting BHB response in three subject groups. PARTICIPANTS: Healthy subjects without cognitive deficits <65years, similarly healthy subjects >=65years, and those with Alzheimer's Disease were assessed. INTERVENTION: Different doses (0g,14g, 28g, 42g) of MCT oil (99.3% C8:0) were administered, followed by fasting during the study period. MEASUREMENTS: BHB measured by finger prick sampling hourly for 5 hours after ingestion. Each subject attended four different days for each ascending dose. Data was also collected on body composition, BMI, waist/hip ratio, grip strength, gait speed, nutrient content of pre-study breakfast and side effects. RESULTS: Twenty-five participants: eight healthy; average age of 44yr (25-61), nine healthy; 79yr (65-90) and eight with AD; 78.6yr (57-86) respectively. Compiled data showed the expected linear dose response relationship. No group differences, with baseline corrected area under the blood vs. time curve (r2=0.98) and maximum concentrations (r2=0.97). However, there was notable individual variability in maximum BHB response (42g dose: 0.4 -2.1mM), and time to reach maximum BHB response both, within and between individuals. Variability was unrelated to age, sex, sarcopenic or AD status. Visceral fat, BMI, waist/hip ratio and pretest meal CHO and protein content all affected the BHB response (p<0.001). CONCLUSION: There was a large inter-individual variability, with phenotype effects identified. This highlights challenges in interpreting clinical responses to MCT intake.

Precision Medicine With Leflunomide: Consideration of the DHODH Haplotype and Plasma Teriflunomide Concentration and Modification of Outcomes in Patients With Rheumatoid Arthritis.

OBJECTIVE: Leflunomide is a commonly used disease-modifying drug in the treatment of rheumatoid arthritis (RA). Its effects are mediated via inhibition of dihydroorotate dehydrogenase (DHODH) by its active metabolite teriflunomide, and the pharmacokinetics of teriflunomide are highly variable. Our objective was to examine the association between the DHODH haplotype and plasma teriflunomide concentration with response to leflunomide in patients with RA where leflunomide was added to an existing disease-modifying drug regimen after failure to achieve an adequate response with conventional triple therapy. METHODS: Patients with RA who were taking, or were about to initiate, leflunomide were included. Participant characteristics, including the DHODH haplotype, were determined. Up to 5 plasma samples were collected after leflunomide was initiated for assays of total and free teriflunomide concentration. Disease activity was determined via the 28-joint Disease Activity Score (DAS28). The association between DAS28 scores and patient covariates was determined by linear mixed-effects modeling. RESULTS: A total of 67 patients were included in the study. The DAS28 score after initiation of leflunomide was associated with the baseline DAS28 score (ß = 0.70, P < 0.001) and was higher in those who carried the DHODH haplotype 2 (ß = 0.56. P = 0.01) and did not carry the shared epitope (ß = 0.56, P = 0.013). As total and free plasma teriflunomide concentration increased, the DAS28 score was significantly lower (P < 0.001 and P = 0.001, respectively). When considering threshold concentrations, teriflunomide concentrations >16 mg/liter were associated with a DAS28 score that was 0.33 lower, and when free teriflunomide concentration was >35 µg/liter, the DAS28 score was 0.32 lower. CONCLUSION: Teriflunomide concentration and carriage of the DHODH haplotype 2 are associated with response to leflunomide in patients with RA, and a total plasma teriflunomide concentration of at least 16 mg/liter is needed to maximize the likelihood of response.

Exogenous Ketosis Impairs 30-min Time-Trial Performance Independent of Bicarbonate Supplementation.

PURPOSE: We recently demonstrated that coingestion of NaHCO3 to counteract ketoacidosis resulting from oral ketone ester (KE) intake improves mean power output during a 15-min time trial (TT) at the end of a 3-h cycling race by ~5%. This ergogenic effect occurred at a time when blood ketone levels were low, as ketosis was only induced during the initial ~2 h of the race. Therefore, in the current study, we investigated whether performance also increases if blood ketone levels are increased in the absence of ketoacidosis during high-intensity exercise. METHODS: In a double-blind crossover design, 14 well-trained male cyclists completed a 30-min TT (TT30') followed by an all-out sprint at 175% of lactate threshold (SPRINT). Subjects were randomized to receive (i) 50 g KE, (ii) 180 mg·kg-1 body weight NaHCO3 (BIC), (iii) KE + BIC, or (iv) a control drink (CON). RESULTS: KE ingestion increased blood d-ß-hydroxybutyrate to ~3-4 mM during the TT30' and SPRINT (P < 0.001 vs CON). In KE, blood pH and bicarbonate concomitantly dropped, causing 0.05 units lower pH and 2.6 mM lower bicarbonate in KE compared with CON during the TT30' and SPRINT (P < 0.001 vs CON). BIC coingestion resulted in 0.9 mM higher blood d-ß-hydroxybutyrate (P < 0.001 vs KE) and completely counteracted ketoacidosis during exercise (P > 0.05 vs CON). Mean power output during TT30' was similar between CON and BIC at 281 W, but was 1.5% lower in the KE conditions (main effect of KE: P = 0.03). Time to exhaustion in the SPRINT was ~64 s in CON and KE and increased by ~8% in the BIC conditions (main effect of BIC: P < 0.01). DISCUSSION: Neutralization of acid-base disturbance by BIC coingestion is insufficient to counteract the slightly negative effect of KE intake during high-intensity exercise.

Quercetin Suppresses AOM/DSS-Induced Colon Carcinogenesis through Its Anti-Inflammation Effects in Mice.

Colorectal cancer (CRC) is the fourth leading cause of tumor-related deaths worldwide. In this study, we explored the in vivo effects of quercetin, a plant flavonol from the flavonoid group of polyphenols with antioxidant effects, on colon carcinogenesis induced by azoxymethane/dextran sodium sulfate (AOM/DSS). Thirty mice were randomly assigned into three groups: the control group, the AOM/DSS group, and the quercetin+AOM/DSS group. CRC was induced by AOM injection and a solution of 2% DSS in the drinking water. In the AOM/DSS-induced colon cancer mice model, quercetin treatment dramatically reduced the number and size of colon tumors. In addition, quercetin significantly restored the leukocyte counts by decreasing the inflammation caused by AOM/DSS. We also observed that the expression of oxidative stress markers, such as lipid peroxide (LPO), nitric oxide (NO), superoxide dismutase (SOD), glucose-6-phosphate (G6PD), and glutathione (GSH), could be reduced by quercetin, suggesting that the anti-inflammatory function of quercetin comes from its antioxidant effect. Moreover, potential biomarkers were identified with serum metabolite profiling. Increased levels of 2-hydroxybutyrate, 2-aminobutyrate, and 2-oxobutyrate and decreased levels of gentian violet, indole-3-methyl acetate, N-acetyl-5-hydroxytryptamine, indoxyl sulfate, and indoxyl were also found in the AOM/DSS-treated mice. However, quercetin treatment successfully decreased the levels of 2-hydroxybutyrate, 2-aminobutyrate, 2-oxobutyrate, endocannabinoids, and sphinganine and increased the levels of gentian violet, N-acetyl-5-hydroxytryptamine, indoxyl sulfate, and indoxyl. Together, our data demonstrated that quercetin could maintain relatively potent antitumor activities against colorectal cancer in vivo through its anti-inflammation effect.

3-Hydroxyisobutyrate, A Strong Marker of Insulin Resistance in Type 2 Diabetes and Obesity That Modulates White and Brown Adipocyte Metabolism.

Circulating branched-chain amino acids (BCAAs) associate with insulin resistance and type 2 diabetes. 3-Hydroxyisobutyrate (3-HIB) is a catabolic intermediate of the BCAA valine. In this study, we show that in a cohort of 4,942 men and women, circulating 3-HIB is elevated according to levels of hyperglycemia and established type 2 diabetes. In complementary cohorts with measures of insulin resistance, we found positive correlates for circulating 3-HIB concentrations with HOMA2 of insulin resistance, as well as a transient increase in 3-HIB followed by a marked decrease after bariatric surgery and weight loss. During differentiation, both white and brown adipocytes upregulate BCAA utilization and release increasing amounts of 3-HIB. Knockdown of the 3-HIB-forming enzyme 3-hydroxyisobutyryl-CoA hydrolase decreases release of 3-HIB and lipid accumulation in both cell types. Conversely, addition of 3-HIB to white and brown adipocyte cultures increases fatty acid uptake and modulated insulin-stimulated glucose uptake in a time-dependent manner. Finally, 3-HIB treatment decreases mitochondrial oxygen consumption and generation of reactive oxygen species in white adipocytes, while increasing these measures in brown adipocytes. Our data establish 3-HIB as a novel adipocyte-derived regulator of adipocyte subtype-specific functions strongly linked to obesity, insulin resistance, and type 2 diabetes.

Eight Days of Water-Only Fasting Promotes Favorable Changes in the Functioning of the Urogenital System of Middle-Aged Healthy Men.

The aim of this study was to determine whether, after 8 days of water-only fasting, there are changes in the efficiency of the lower urinary tract, the concentration of sex hormones, and the symptoms of prostate diseases in a group of middle-aged men (n = 14). For this purpose, before and after 8 days of water-only fasting (subjects drank ad libitum moderately mineralized water), and the following somatic and blood concentration measurements were made: total prostate specific antigen (PSA-T), free prostate specific antigen (PSA-F), follicle stimulating hormone (FSH), luteotropic hormone (LH), prolactin (Pr), total testosterone (T-T), free testosterone (T-F), dehydroepiandrosterone (DHEA), sex hormone globulin binding (SHGB), total cholesterol (Ch-T), ß-hydroxybutyrate (ß-HB). In addition, prostate volume (PV), volume of each testis (TV), total volume of both testes (TTV), maximal urinary flow rate (Qmax), and International Prostate Symptom Score (IPSS) values were determined. The results showed that after 8 days of water-only fasting, Qmax and IPSS improved but PV and TTV decreased significantly. There was also a decrease in blood levels of PSA-T, FSH, P, T-T, T-F, and DHEA, but SHGB concentration increased significantly. These results indicate that 8 days of water-only fasting improved lower urinary tract functions without negative health effects.

Development and validation of volumetric absorptive microsampling coupled with UHPLC-MS/MS for the analysis of gamma-hydroxybutyric acid in human blood.

A volumetric microsampling (VAMS) device (20 µl) was evaluated and validated for the analysis of γ-hydroxybutyric acid (GHB) in venous blood using a simple ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. GHB was extracted from VAMS device by acetonitrile, after a re-hydration step in a temperature-controlled ultrasonic bath at 60°C for 10 min. Chromatographic analysis was carried out on a Kinetex C18 column using 0.1% formic acid in water and acetonitrile as binary gradient mobile phase (from 5 to 95% of acetonitrile from 1 to 2.5 min) at a flow rate of 0.3 ml/min. The VAMS method was fully validated according to current guidelines with satisfactory results in terms of linearity, selectivity, precision, absolute recovery, matrix effect and stability. The linearity was determined from 0.5 to 200 µg/ml and the lower limit of quantitation was 0.5 µg/ml. The novel VAMS-UHPLC-MS/MS method was successfully compared with plasma-based method in a GHB-treated patient as a proof of concept.

Treatment of γ-Hydroxybutyric Acid and γ-Butyrolactone Overdose with Two Potent Monocarboxylate Transporter 1 Inhibitors, AZD3965 and AR-C155858.

The illicit use of γ-hydroxybutyric acid (GHB), and its prodrug, γ-butyrolactone (GBL), results in severe adverse effects including sedation, coma, respiratory depression, and death. Current treatment of GHB/GBL overdose is limited to supportive care. Recent reports indicate that GHB-related deaths are on the rise; a specific treatment may reduce lethality associated with GHB/GBL. Pretreatment with inhibitors of monocarboxylate transporter 1 (MCT1), a transporter that mediates many of the processes involved in the absorption, distribution (including brain uptake), and elimination of GHB/GBL, has been shown to prevent GHB-induced respiratory depression by increasing the renal clearance of GHB. To identify whether MCT1 inhibition is an effective treatment of GHB overdose, the impact of two MCT1 inhibitors, (S)-5-(4-hydroxy-4-methylisoxazolidine-2-carbonyl)-1-isopropyl-3-methyl-6-((3-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)methyl)thieno[2,3-day]pyrimidine-2,4(1H,3H)-dione (AZD3965) and 6-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-5-[[(4S)-4-hydroxy-2-isoxazolidinyl]carbonyl]-3-methyl-1-(2-methylpropyl)thieno[2,3-day]pyrimidine2,4(1H,3H)-dione (AR-C155858), on the toxicokinetics and toxicodynamics of GHB/GBL was assessed when the administration of the inhibitor was delayed 60 and 120 minutes (post-treatment) after administration of GHB/GBL. AR-C155858 and AZD3965 reduced the toxicodynamic effects of GHB when GHB was administered intravenously, orally, or orally as the prodrug GBL. The impact of these inhibitors on GHB toxicokinetics was dependent on the route of GHB administration and the delay between GHB/GBL administration and administration of the MCT1 inhibitor. The reduction in GHB plasma exposure did not explain the observed effect of MCT1 inhibition on GHB-induced respiratory depression. The efficacy of MCT1 inhibition on GHB toxicodynamics is likely driven by the pronounced reduction in GHB brain concentrations. Overall, this study indicates that inhibition of MCT1 is an effective treatment of GHB/GBL overdose.

Capillary versus Serum b-hydroxybutyrate in Pediatric Diabetic Ketoacidosis.

OBJECTIVE: To find the strength of agreement between point-of-care and serum b-hydroxybutyrate. METHODS: 236 paired samples (capillary b-hydroxybutyrate by a point of care device and serum b-hydroxybutyrate by colorimetric enzymatic estimation) samples were collected from 26 children aged <13 years admitted with diabetic ketoacidosis. Inborn errors of metabolism and septic shock were excluded. RESULTS: Capillary b-hydroxybutyrate showed excellent agreement with serum â-hydroxybutyrate with mean (SD) bias of 0.027 (0.78); 95% limit of agreement -1.51, 1.56 and intraclass correlation 96.1% (95%CI 95%-97%, P<0.001). An increase in the bias noted for value above 5 mmol/L (P<0.001) (serum measurements were higher than capillary point-of-care measure-ments). Capillary â-hydroxybutyrate correlated significantly with blood pH, anion gap,bicarbonate and carbon dioxide levels on blood gas analysis (P<0.05). CONCLUSIONS: Capillary b-hydroxybutyrate estimation is a valid method for monitoring of ketonemia in pediatric diabetic ketoacidosis.

Quantification of GHB and GHB-GLUC in an 1,4-butanediol intoxication: A case report.

Gamma-hydroxybutyric acid (GHB) is an endogenous compound with known action at the neural level. Its psychoactive effects led to an illicit use context including recreational purposes, muscle building effects in bodybuilders and drug-facilitated crimes, specifically in sexual assaults. Besides the misuse of the main compound, there are precursors like Gammabutyrolactone (GBL) and 1,4-butanediol (1,4-BD), usually non controlled substances, becoming a much easier way to obtain the target-compound. The authors present the first reported intoxication case in Portugal with 1,4-Butanediol, including the quantification of GHB and GHB-GLUC in serum, by GC-MS/MS TQD. A suspicious liquid and a serum sample were sent by an hospital ER and analysed by GC-MS-single quadrupole and GC-MS/MS TQD, respectively. A methodology including protein precipitation and GC-MS/MS TQD analysis was used to detect and quantify GHB and GHB-GLUC in serum. Toxicological analysis revealed the presence of 1,4-Butanediol in the liquid and GHB [171 mg/L] and GHB-GLUC [13,7 mg/L] in serum. The victim reverted the coma with no neurological sequelae. This was the first detected case, in Portugal, with 1,4-Butanediol, suggesting that it is important to be aware that consumers have different options to obtain illicit compounds, such as GHB. On the other hand, GHB-GLUC was identified and quantified for the first time in a real case, due to intoxication. This case highlights the importance of analysing all samples for active compounds, precursors and metabolites that can lead to the main intoxication origin.