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Background: Supplementation with β-hydroxy β-methyl butyrate (HMB) appears to be effective in preserving muscle in older adults. However, the association between endogenously produced HMB with frailty has not been studied in people with chronic disease., Objectives: The purpose of this study is to explore whether an association exists between endogenous HMB levels and frailty status in older adults with type-2 diabetes mellitus (T2DM)., Methods: Data were taken from the Toledo Study of Healthy Ageing, a community-dwelling aged (65 years+) cohort. Frailty was assessed at baseline and at 2.99 median years according to the Frailty Phenotype (FP) standardized to our population and the Frailty Trait Scale 12 (FTS12). The associations between HMB levels and frailty were assessed using three nested multivariate logistic regressions and segmented by sex. Glucose, HMB and glucose interaction, age and body composition were used as covariables., Results: 255 participants (mean age 75.3 years, 52.94% men) were included. HMB levels showed an inverse cross-sectional association with frailty, which was modified when the interaction term HMB*glucose was included, remaining significant only for FTS12 [OR (95% CI): 0.436 (0.253, 0.751), p-value 0.003]. The association between HMB endogenous levels and FTS12 appears to be independent of sex, in which the association was maintained after adjusting for the covariates. However, there appears to be threshold points for glucose levels, above which the protective effect of HMB is lost: 145.4 mg/dl adjusted by gender for the whole sample and 149.6 mg/dl and 138.9 mg/dl for men and women, respectively. Endogenous HMB levels were not found to be associated with incident frailty., Conclusions: Cross-sectional analysis revealed that endogenous HMB levels were inversely associated with frailty as assessed by the FTS12 in older people with T2DM. This association was found to be dependent on circulating fasted glucose levels., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Background: Most of the explanatory and prognostic models of COVID-19 lack of a comprehensive assessment of the wide COVID-19 spectrum of abnormalities. The aim of this study was to unveil novel biological features to explain COVID-19 severity and prognosis (death and disease progression)., Methods: A predictive model for COVID-19 severity in 121 patients was constructed by ordinal logistic regression calculating odds ratio (OR) with 95% confidence intervals (95% CI) for a set of clinical, immunological, metabolomic, and other biological traits. The accuracy and calibration of the model was tested with the area under the curve (AUC), Somer's D, and calibration plot. Hazard ratios with 95% CI for adverse outcomes were calculated with a Cox proportional-hazards model., Results: The explanatory variables for COVID-19 severity were the body mass index (BMI), hemoglobin, albumin, 3-Hydroxyisovaleric acid, CD8+ effector memory T cells, Th1 cells, low-density granulocytes, monocyte chemoattractant protein-1, plasma TRIM63, and circulating neutrophil extracellular traps. The model showed an outstanding performance with an optimism-adjusted AUC of 0.999, and Somer's D of 0.999. The predictive variables for adverse outcomes in COVID-19 were severe and critical disease diagnosis, BMI, lactate dehydrogenase, Troponin I, neutrophil/lymphocyte ratio, serum levels of IP-10, malic acid, 3, 4 di-hydroxybutanoic acid, citric acid, myoinositol, and cystine., Conclusions: Herein, we unveil novel immunological and metabolomic features associated with COVID-19 severity and prognosis. Our models encompass the interplay among innate and adaptive immunity, inflammation-induced muscle atrophy and hypoxia as the main drivers of COVID-19 severity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Torres-Ruiz, Pérez-Fragoso, Maravillas-Montero, Llorente, Mejía-Domínguez, Páez-Franco, Romero-Ramírez, Sosa-Hernández, Cervantes-Díaz, Absalón-Aguilar, Nuñez-Aguirre, Juárez-Vega, Meza-Sánchez, Kleinberg-Bid, Hernández-Gilsoul, Ponce-de-León and Gómez-Martín.)

The aim of the study was to determine the effect of simultaneous supplementation of β-hydroxy-β-methylbutyrate and L-Arginine α-ketoglutarate on lower limb power and muscle damage in medium distance runners aged 15.3 (±0.9) years old., Methods: The study group consisted of 40 volunteers aged 14-17 years practicing medium distance running for at least two years. The study lasted 12 days and followed a randomized, double-blind, placebo-controlled, parallel design. All subjects attended a familiarization session on day 0 before the test. The subjects were randomly divided into two groups: supplements and placebo group. The same training cycle protocol was used in both groups during the 12-day training period. Morning warm-up involved 10 min jogging at 60-75% of maximal heart rate and countermovement jump height measurement. Main training units were carried out for both groups with the same volume. Training load assessment (the daily session Rating of Perceived Exertion (s-RPE) method) method takes into consideration the intensity and the duration of the training session to calculate the "training load" (TL)., Results: At the end of the training cycle, a significant ( p = 0.002) decrease in the countermovement jump (CMJ) height was found in the placebo group when compared to the baseline. In the supplement group, there was no decrease in the countermovement jump height. Creatine kinase and lactate dehydrogenase concentration increased during the training days similarly in both groups and decreased on rest days. There were no differences between groups in enzymes concentration. The research results indicate that the supplement combination used in the supplements group prevented a reduction in the CMJ values. In contrast to the supplements group, in the placebo group, the CMJ changes were statistically significant: a noticeable ( p = 0.002) decrease in CMJ was noted between the baseline measurement and the 6th measurement. The well-being of the subjects from both groups changed significantly during the training period, and the intergroup differences in the mood level were similar and not statistically significant., Conclusions: The results of this study indicate that the daily co-supplementation with calcium salt of β-hydroxy-β-methylbutyrate (7.5 g) and L-Arginine α-ketoglutarate (10 g) during training might help to prevent decline in jump performance. No influence on muscle damage markers or mood was shown.

Aims: To examine the metabolic adaptation to an 80-day exercise intervention in healthy young male adults where lifestyle factors such as diet, sleep, and physical activities are controlled., Methods and Results: This study involved cross-sectional analysis before and after an 80-day aerobic and strength exercise intervention in 52 young, adult, male, newly enlisted soldiers in 2015. Plasma metabolomic analyses were performed using liquid chromatography, tandem mass spectrometry. Data analyses were performed between March and August 2019. We analysed changes in metabolomic profiles at the end of an 80-day exercise intervention compared to baseline, and the association of metabolite changes with changes in clinical parameters. Global metabolism was dramatically shifted after the exercise training programme. Fatty acids and ketone body substrates, key fuels used by exercising muscle, were dramatically decreased in plasma in response to increased aerobic fitness. There were highly significant changes across many classes of metabolic substrates including lipids, ketone bodies, arginine metabolites, endocannabinoids, nucleotides, markers of proteolysis, products of fatty acid oxidation, microbiome-derived metabolites, markers of redox stress, and substrates of coagulation. For statistical analyses, a paired t-test was used and Bonferroni-adjusted P-value of <0.0004 was considered to be statistically significant. The metabolite dimethylguanidino valeric acid (DMGV) (recently shown to predict lack of metabolic response to exercise) tracked maladaptive metabolic changes to exercise; those with increases in DMGV levels had increases in several cardiovascular risk factors; changes in DMGV levels were significantly positively correlated with increases in body fat (P = 0.049), total and LDL cholesterol (P = 0.003 and P = 0.007), and systolic blood pressure (P = 0.006). This study was approved by the Departments of Defence and Veterans' Affairs Human Research Ethics Committee and written informed consent was obtained from each subject., Conclusion: For the first time, the true magnitude and extent of metabolic adaptation to chronic exercise training are revealed in this carefully designed study, which can be leveraged for novel therapeutic strategies in cardiometabolic disease. Extending the recent report of DMGV's predictive utility in sedentary, overweight individuals, we found that it is also a useful marker of poor metabolic response to exercise in young, healthy, fit males., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Background & Aims: Nonalcoholic fatty liver disease is characterized by excessive hepatic accumulation of triglycerides. We aimed to identify metabolites that differ in plasma of patients with liver steatosis vs healthy individuals (controls) and investigate the mechanisms by which these might contribute to fatty liver in mice., Methods: We obtained blood samples from 15 patients with liver steatosis and 15 controls from a single center in China (discovery cohort). We performed untargeted liquid chromatography with mass spectrometry analysis of plasma to identify analytes associated with liver steatosis. We then performed targeted metabolomic analysis of blood samples from 2 independent cohorts of individuals who underwent annual health examinations in China (1157 subjects with or without diabetes and 767 subjects with or without liver steatosis; replication cohorts). We performed mass spectrometry analysis of plasma from C57BL/6J mice, germ-free, and mice given antibiotics. C57BL/6J mice were given 0.325% (m/v) N,N,N-trimethyl-5-aminovaleric acid (TMAVA) in their drinking water and placed on a 45% high-fat diet (HFD) for 2 months. Plasma, liver tissues, and fecal samples were collected; fecal samples were analyzed by 16S ribosomal RNA gene sequencing. C57BL/6J mice with CRISPR-mediated disruption of the gene encoding γ-butyrobetaine hydroxylase (BBOX-knockout mice) were also placed on a 45% HFD for 2 months. Hepatic fatty acid oxidation (FAO) in liver tissues was determined by measuring liberation of 3 H 2 O from [ 3 H] palmitic acid. Liver tissues were analyzed by electron microscopy, to view mitochondria, and proteomic analyses. We used surface plasmon resonance analysis to quantify the affinity of TMAVA for BBOX., Results: Levels of TMAVA, believed to be a metabolite of intestinal microbes, were increased in plasma from subjects with liver steatosis compared with controls, in the discovery and replication cohorts. In 1 replication cohort, the odds ratio for fatty liver in subjects with increased liver plasma levels of TMAVA was 1.82 (95% confidence interval [CI], 1.14-2.90; P = .012). Plasma from mice given antibiotics or germ-free mice had significant reductions in TMAVA compared with control mice. We found the intestinal bacteria Enterococcus faecalis and Pseudomonas aeruginosa to metabolize trimethyllysine to TMAVA; levels of trimethyllysine were significantly higher in plasma from patients with steatosis than controls. We found TMAVA to bind and inhibit BBOX, reducing synthesis of carnitine. Mice given TMAVA had alterations in their fecal microbiomes and reduced cold tolerance; their plasma and liver tissue had significant reductions in levels of carnitine and acyl-carnitine and their hepatocytes had reduced mitochondrial FAO compared with mice given only an HFD. Mice given TMAVA on an HFD developed liver steatosis, which was reduced by carnitine supplementation. BBOX-knockout mice had carnitine deficiency and decreased FAO, increasing uptake and liver accumulation of free fatty acids and exacerbating HFD-induced fatty liver., Conclusions: Levels of TMAVA are increased in plasma from subjects with liver steatosis. In mice, intestinal microbes metabolize trimethyllysine to TMAVA, which reduces carnitine synthesis and FAO to promote steatosis., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The leucine metabolite, β-hydroxy-β-methyl butyrate (HMB), is widely used in human nutrition and animal production as a nutritional supplement. Although the HMB usage during late gestation has been demonstrated to have a positive effect on fetal development, knowledge on net absorption and metabolism of HMB and impact of HMB on branched chain amino acids (BCAAs) metabolism is lacking. To address this, we conducted a study using pigs during the perinatal period as a model organism. Eight-second parity sows were fitted with indwelling catheters in the femoral artery and in the portal, hepatic, femoral, and mesenteric veins. Eight hourly sets of blood samples were taken starting 30 min before the morning meal on day -10 and day -3 relative to parturition. Four control (CON) sows were fed a standard lactation diet from day -15 and throughout the experiment, and 4 HMB sows were fed the control diet supplemented with 15 mg Ca(HMB) 2 /kg body weight mixed in one third of the morning meal from day -10 until parturition. Blood gases, plasma metabolites, milk compositions, and apparent total tract digestibility of nutrients were measured. Arterial plasma concentrations of HMB ( p < 0.001), Cys ( p < 0.001), and Lys ( p < 0.10) were increased in HMB supplemented sows, while arterial plasma triglycerides concentration was decreased ( p < 0.05). The net portal recovery of Ala and Asp were increased in HMB sows ( p < 0.05). Sows fed HMB had increased hepatic vein flow and net hepatic fluxes of Met, Asn, and Gln ( p < 0.05). In contrast, the femoral extraction rates of Ala and Ser were decreased by dietary HMB supplementation ( p < 0.05). Dietary HMB treatment and sampling time relative to feeding had an interaction on arterial concentrations, net portal fluxes, and femoral extraction rates of BCAAs. The net portal recovery of HMB was 88%, while 14% of supplemented HMB was excreted through urine and 4% through feces. Moreover, the gastrointestinal tract metabolized 8% while the liver metabolized 12%. Finally, 26% of the daily intake of HMB was secreted via colostrum at the day of farrowing. This study demonstrated that dietary HMB supplementation increased net uptake of amino acids and increased fatty acid oxidation through improving blood flow and insulin sensitivity during the late gestation. Most importantly, oral HMB administration could maintain a stable postprandial absorption and altered metabolism in BCAAs. Net portal flux of HMB at 5.5 to 6.5 h after feeding approached zero, indicating that HMB ideally should be administrated two or three times, daily.

Background Identification of lifestyle modifiable metabolic pathways related to cardiometabolic disease risk is essential for improvement of primary prevention in susceptible individuals. It was recently shown that plasma dimethylguanidino valerate (DMGV) levels are associated with incident type 2 diabetes mellitus. Our aims were to investigate whether plasma DMGV is related to risk of future coronary artery disease and with cardiovascular mortality and to replicate the association with type 2 diabetes mellitus and pinpoint candidate lifestyle interventions susceptible to modulate DMGV levels. Methods and Results Plasma DMGV levels were measured using liquid chromatography-mass spectrometry in a total of 5768 participants from the MDC (Malmö Diet and Cancer Study-Cardiovascular Cohort), MPP (Malmö Preventive Project), and MOS (Malmö Offspring Study). Dietary intake assessment was performed in the MOS. Baseline levels of DMGV associated with incident coronary artery disease in both the MDC (hazard ratio=1.29; CI=1.16-1.43; P <0.001) and MPP (odds ratio=1.25; CI=1.08-1.44; P =2.4e-3). In the MDC, DMGV was associated with cardiovascular mortality and incident coronary artery disease, independently of traditional risk factors. Furthermore, the association between DMGV and incident type 2 diabetes mellitus was replicated in both the MDC (hazard ratio=1.83; CI=1.63-2.05; P <0.001) and MPP (odds ratio=1.65; CI=1.38-1.98; P <0.001). Intake of sugar-sweetened beverages was associated with increased levels of DMGV, whereas intake of vegetables and level of physical activity was associated with lower DMGV. Conclusions We discovered novel independent associations between plasma DMGV and incident coronary artery disease and cardiovascular mortality, while replicating the previously reported association with incident type 2 diabetes mellitus. Additionally, strong associations with sugar-sweetened beverages, vegetable intake, and physical activity suggest the potential to modify DMGV levels using lifestyle interventions.

Age-related sarcopenia and dynapenia are associated with frailty and metabolic diseases. Resistance exercise training (RET) adjuvant to evidence-based nutritional intervention(s) have been shown as mitigating strategies. Given that β-hydroxy-β-methyl-butyrate (HMB) supplementation during RET improves lean body mass in younger humans, and that we have shown that HMB acutely stimulates muscle protein synthesis (MPS) and inhibits breakdown; we hypothesized that chronic supplementation of HMB free acid (HMB-FA) would enhance MPS and muscle mass/function in response to RET in older people. We recruited 16 healthy older men (Placebo (PLA): 68.5 ± 1.0 y, HMB-FA: 67.8 ± 1.15 y) for a randomised double-blind-placebo controlled trial (HMB-FA 3 × 1 g/day vs. PLA) involving a 6-week unilateral progressive RET regime (6 × 8 repetitions, 75% 1-RM, 3 · wk -1 ). Deuterium oxide (D 2 O) dosing was performed over the first two weeks (0-2 wk) and last two weeks (4-6 wk) with bilateral vastus lateralis (VL) biopsies at 0-2 and 4-6 wk (each time 75 ± 2 min after a single bout of resistance exercise (RE)) for quantification of early and later MPS responses and post-RE myogenic gene expression. Thigh lean mass (TLM) was measured by DXA, VL thickness and architecture (fibre length and pennation angle) by ultrasound at 0/3/6 wk, and strength by knee extensor 1-RM testing and MVC by isokinetic dynamometry (approx. every 10 days). RET induced strength increases (1-RM) in the exercised leg of both groups (398 ± 22N to 499 ± 30N HMB-FA vs. 396 ± 29N to 510 ± 43N PLA (both P < 0.05)). In addition, maximal voluntary contraction (MVC) also increased (179 ± 12 Nm to 203 ± 12 Nm HMB-FA vs. 185 ± 10 Nm to 217 ± 11 Nm PLA (both P < 0.05); with no group differences. VL muscle thickness increased significantly in the exercised leg in both groups, with no group differences. TLM (by DXA) rose to significance only in the HMB-FA group (by 5.8%-5734 ± 245 g p = 0.015 vs. 3.0% to 5644 ± 323 g P = 0.06 in PLA). MPS remained unchanged in the untrained legs (UT) 0-2 weeks being 1.06 ± 0.08%.d -1 (HMB-FA) and 1.14 ± 0.09%.d -1 (PLA), the trained legs (T) exhibited increased MPS in the HMB-FA group only at 0-2-weeks (1.39 ± 0.10%.d -1 , P < 0.05) compared with UT: but was not different at 4-6-weeks: 1.26 ± 0.05%.d -1 . However, there were no significant differences in MPS between the HMB-FA and PLA groups at any given time point and no significant treatment interaction observed. We also observed significant inductions of c-Myc gene expression following each acute RE bout, with no group differences. Further, there were no changes in any other muscle atrophy/hypertrophy or myogenic transcription factor genes we measured. RET with adjuvant HMB-FA supplements in free-living healthy older men did not enhance muscle strength or mass greater than that of RET alone (PLA). That said, only HMB-FA increased TLM, supported by early increases in chronic MPS. As such, chronic HMB-FA supplementation may result in long term benefits in older males, however longer and larger studies may be needed to fully determine the potential effects of HMB-FA supplementation; translating to any functional benefit., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The aim of the present study was to compare the effects of branched-chain amino acid (BCAA) supplementation taken before or after exercise on delayed onset muscle soreness (DOMS) and exercise-induced muscle damage (EIMD)., Methods: Fifteen young men (aged 21.5±0.4 years) were given either BCAA (9.6 g·day-1) or placebo before and after exercise (and for 3 days prior to and following the exercise day) in three independent groups: the control group (placebo before and after exercise), the PRE group (BCAA before exercise and placebo after exercise), and the POST group (placebo before exercise and BCAA after exercise). Participants performed 30 repetitions of eccentric exercise with the non-dominant arm. DOMS, upper arm circumference (CIR), elbow range of motion (ROM), serum creatine kinase (CK), lactate dehydrogenase (LDH), and aldolase, BCAA, and β-hydroxy-β-methylbutyrate (3HMB) were measured immediately before and after the exercise and on the following 4 days., Results: Serum BCAA and 3HMB concentrations increased significantly in the PRE group immediately after the exercise, recovering to baseline over the following days. In the days following the exercise day, DOMS, CIR, and ROM were significantly improved in the PRE group compared to the control group, with weaker effects in the POST group. Serum activities of CK, LDH, and aldolase in the days following the exercise day were significantly suppressed in the PRE group compared to control group., Conclusions: The present study confirmed that repeated BCAA supplementation before exercise had a more beneficial effect in attenuating DOMS and EIMD induced by eccentric exercise than repeated supplementation after exercise.

Purpose: Milk provides a significant source of calcium, protein, vitamins and other minerals to Western populations throughout life. Due to its widespread use, the metabolic and health impact of milk consumption warrants further investigation and biomarkers would aid epidemiological studies., Methods: Milk intake assessed by a validated food frequency questionnaire was analyzed against fasting blood metabolomic profiles from two metabolomic platforms in females from the TwinsUK cohort (n = 3559). The top metabolites were then replicated in two independent populations (EGCUT, n = 1109 and KORA, n = 1593), and the results from all cohorts were meta-analyzed., Results: Four metabolites were significantly associated with milk intake in the TwinsUK cohort after adjustment for multiple testing (P < 8.08 × 10 -5 ) and covariates (BMI, age, batch effects, family relatedness and dietary covariates) and replicated in the independent cohorts. Among the metabolites identified, the carnitine metabolite trimethyl-N-aminovalerate (β = 0.012, SE = 0.002, P = 2.98 × 10 -12 ) and the nucleotide uridine (β = 0.004, SE = 0.001, P = 9.86 × 10 -6 ) were the strongest novel predictive biomarkers from the non-targeted platform. Notably, the association between trimethyl-N-aminovalerate and milk intake was significant in a group of MZ twins discordant for milk intake (β = 0.050, SE = 0.015, P = 7.53 × 10 -4 ) and validated in the urine of 236 UK twins (β = 0.091, SE = 0.032, P = 0.004). Two metabolites from the targeted platform, hydroxysphingomyelin C14:1 (β = 0.034, SE = 0.005, P = 9.75 × 10 -14 ) and diacylphosphatidylcholine C28:1 (β = 0.034, SE = 0.004, P = 4.53 × 10 -16 ), were also replicated., Conclusions: We identified and replicated in independent populations four novel biomarkers of milk intake: trimethyl-N-aminovalerate, uridine, hydroxysphingomyelin C14:1 and diacylphosphatidylcholine C28:1. Together, these metabolites have potential to objectively examine and refine milk-disease associations.

A high-throughput, sensitive, and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid quantitation of β-hydroxy-β-methylbutyrate (HMB) in human plasma has been developed and validated for routine use. The method uses 100 μL of plasma sample and employs protein precipitation with 0.1% formic acid in methanol for the extraction of HMB from plasma. Sample extracts were analyzed using LC-MS/MS technique under negative mode electrospray ionization conditions. A 13 C-labeled stable isotope internal standard was used to achieve accurate quantitation. Multiday validation was conducted for precision, accuracy, linearity, selectivity, matrix effect, dilution integrity (2×), extraction recovery, freeze-thaw sample stability (three cycles), benchtop sample stability (6 h and 50 min), autosampler stability (27 h) and frozen storage sample stability (146 days). Linearity was demonstrated between 10 and 500 ng/mL. Inter-day accuracies and coefficients of variation (CV) were 91.2-98.1 and 3.7-7.8%, respectively. The validated method was proven to be rugged for routine use to quantify endogenous levels of HMB in human plasma obtained from healthy volunteers., (Copyright © 2016 John Wiley & Sons, Ltd.)

Our objective was to develop a quick and simplified method for the determination of β-Hydroxy-β-methylbutyrate (HMB) and ɑ-ketoisocaproic acid (KIC) concentrations and enrichments by GC/MS/MS to determine the turnover rate of HMB in humans. In experiment 1, we provided a pulse of L-[5,5,5- 2 H 3 ]leucine to younger adults in the postabsorptive state then collected blood samples over a 4h time period. In experiment 2, we provided a pulse of [3,4,methyl- 13 C 3 ]HMB to older adults in the postabsorptive state then collected blood samples over a 3h time period. Plasma concentrations of KIC and HMB and MPE of KIC and HMB were determined by GC/MS/MS. Plasma enrichment of leucine was determined by LC/MS/MS. To determine plasma enrichment of [5,5,5- 2 H 3 ]HMB and [3,4,methyl- 13 C 3 ]HMB, samples were derivatized using pentafluorobenzyl bromide and analyzed using chemical ionization mode. The final methods used included multiple reaction monitoring of transitions 117.3>59.3 for M+0 and 120.3>59.3 for M+3. In experiment 1, peak MPE of Leu peaked at 9.76% generating a peak MPE of KIC at 2.67% and a peak HMB MPE of 0.3%. In experiment 2, the rate of appearance for HMB was  0.66μmol/kg ffm/h. We calculated that production of HMB in humans accounts for 0.66% of total leucine turnover., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The maintenance of muscle mass and muscle strength is important for reducing the risk of chronic diseases. The age- related loss of muscle mass and strength is associated with adverse outcomes of physical disability, frailty and death. β-Hydroxy β-Methyl Butyrate (HMB), a metabolite of leucine, has beneficial effects on muscle mass and strength under various catabolic conditions. The objectives of the present study were to determine if age- related differences existed in endogenous plasma HMB levels, and to assess if HMB levels correlated to total appendicular lean mass and forearm grip strength. Anthropometry, dietary and physical activity assessment, and the estimation of fasting plasma HMB concentrations and handgrip strength were performed on the 305 subjects (children, young adults and older adults). Lean mass, which serves as a surrogate for muscle mass was measured using dual energy X-ray absorptiometry (DEXA). Mean plasma HMB concentrations were significantly lower with increasing age groups, with children having highest mean HMB concentration (p<0.01) followed by young adults and older adults. Female subjects (across all ages) had significantly lower plasma HMB concentrations. A significant positive correlation between HMB concentrations and appendicular lean mass normalized for body weight (%), appendicular lean mass (r=0.37; p<0.001) was observed in the young adults and older adults group. Handgrip strength was positively associated with plasma HMB concentrations in young adults (r=0.58; p<0.01) and the older adults group (r=0.28; p<0.01). The findings of the present study suggest that there is an age- related decline in endogenous HMB concentrations in humans and the HMB concentrations were positively correlated with appendicular lean mass and hand grip strength in young adults and older adults group., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The loss of muscle mass is considered to be a major factor contributing to strength decline during aging. β-Hydroxy-β-Methylbutyrate (HMB), a metabolite of leucine has been shown to enhance muscle protein synthesis and attenuate loss of muscle mass by multiple pathways. However, the production and regulation of endogenous levels of HMB over the lifespan have not been investigated., Objective: The objective of the present study was to do a cross-sectional analysis of the basal plasma levels of HMB in male Sprague-Dawley rats of different ages and to compare the efficiency of conversion of leucine to HMB in young versus older rats., Methods: Plasma levels of HMB and α-ketoisocaproate (KIC) were analyzed in rats of different age groups (3, 9, 12 and 24months old, n=10 per group). Levels of 4-HPPD, the enzyme involved in the conversion of KIC to HMB in the liver were determined by ELISA. The conversion efficiency of leucine to HMB was compared between 3 and 24month rats after an oral bolus dose of leucine., Results: Endogenous circulating levels of HMB were significantly reduced in older age rats compared to young rats (100±3.7 vs 156±10 (mean±SEM), ng/mL, p<0.001). A significant negative correlation was seen between HMB levels and age. The liver levels of 4-HPPD were found to be significantly lower in old versus young rats. Consistent with this, the conversion efficiency of leucine to HMB was significantly lower in the aged versus young cohorts., Conclusions: In summary, this study depicts for the first time that the basal levels of HMB, a metabolite of amino acid leucine, declines with age, and that this decline is due to perturbations in the key enzyme 4-HPPD which catalyzes the conversion of KIC to HMB. As a consequence, the efficiency of conversion of leucine to HMB is diminished in older rats compared to younger rats., (Copyright © 2016 Elsevier Inc. All rights reserved.)

β-Hydroxy-β-methylbutyrate (HMB), a leucine metabolite, has long been supplemented as a Ca salt (Ca-HMB) to increase strength and performance gains with exercise and to reduce recovery time. Recently, the free acid form of HMB (HMB-FA) has become commercially available in capsule form (gelcap). The current study was conducted to compare the bioavailability of HMB using the two commercially available capsule forms of HMB-FA and Ca-HMB. We also compared the pharmacokinetics of each form when administered mixed in water. Ten human subjects (five male and five female) were studied in a randomised crossover design. There was no significant sex by treatment interaction for any of the pharmacokinetic parameters measured. HMB-FA administered in capsules was more efficient than Ca-HMB capsule at HMB delivery with a 37 % increase in plasma clearance rate (74·8 (sem 4·0) v. 54·5 (sem 3·2) ml/min, P<0·0001) and a 76 % increase in peak plasma HMB concentration (270·2 (sem 17·8) v. 153·9 (sem 17·9) μmol/l, P<0·006), which was reached in one-third the time (P<0·009). When HMB-FA and Ca-HMB were administered in water, the differences still favoured HMB-FA, albeit to a lesser degree. Plasma HMB with HMB-FA administered in water was greater during the early phase of absorption (up to 45 min postadministration, P<0·05); this resulted in increased AUC during the first 60 min after administration, when compared with Ca-HMB mixed in water (P<0·03). In conclusion, HMB-FA in capsule form improves clearance rate and availability of HMB compared with Ca-HMB in capsule form.

Leucine is an essential branched-chain amino acid that acts as a substrate for protein synthesis and as a signaling molecule. Leucine not incorporated into muscle protein is ultimately oxidized through intermediates such as β-hydroxy-β-methylbutyrate (HMB) which itself is reported to enhance muscle mass and function in rats and humans. HMB has been reported in the plasma following oral leucine administration in sheep and pigs but not in Sprague-Dawley rats, the standard preclinical model. Therefore, we conducted radiolabeled absorption, distribution, metabolism and excretion (ADME) studies in rats using a low (3 mg/kg) or high dose (1,000 mg/kg) of (14)C-leucine. Blood, tissue, and urine samples were analyzed for (14)C-leucine and its metabolites by HPLC-MS. Our results show for the first time that (14)C-HMB appears in plasma and urine of rats following an oral dose of (14)C-leucine. (14)C-leucine appears in plasma as (14)C-α-ketoisocaproic acid (KIC) with a slower time course than (14)C-HMB, a putative product of KIC. Further, two novel metabolites of leucine were detected in urine, N-acetyl leucine and glycyl leucine. Mass balance studies demonstrate that excretory routes accounted for no more than 0.9 % of the radiolabel and approximately 61 % of the dose was recovered in the carcass. Approximately 65 % of the dose was recovered in total, suggesting that approximately one-third of the leucine dose is oxidized to CO2. In conclusion, this study demonstrates endogenous production of HMB from leucine in adult rats, a standard preclinical model used to guide design of clinical trials in nutrition.

Rationale: Sepsis therapeutics have a poor history of success in clinical trials, due in part to the heterogeneity of enrolled patients. Pharmacometabolomics could differentiate drug response phenotypes and permit a precision medicine approach to sepsis., Objectives: To use existing serum samples from the phase 1 clinical trial of l-carnitine treatment for severe sepsis to metabolically phenotype l-carnitine responders and nonresponders., Methods: Serum samples collected before (T0) and after completion of the infusion (T24, T48) from patients randomized to either l-carnitine (12 g) or placebo for the treatment of vasopressor-dependent septic shock were assayed by untargeted (1)H-nuclear magnetic resonance metabolomics. The normalized, quantified metabolite data sets of l-carnitine- and placebo-treated patients at each time point were compared by analysis of variance with post-hoc testing for multiple comparisons. Pathway analysis was performed to statistically rank metabolic networks., Measurements and Main Results: Thirty-eight metabolites were identified in all samples. Concentrations of 3-hydroxybutyrate, acetoacetate, and 3-hydroxyisovalerate were different at T0 and over time in l-carnitine-treated survivors versus nonsurvivors. Pathway analysis of pretreatment metabolites revealed that synthesis and degradation of ketone bodies had the greatest impact in differentiating l-carnitine treatment response. Analysis of all patients based on pretreatment 3-hydroxybutyrate concentration yielded distinct phenotypes. Using the T0 median 3-hydroxybutyrate level (153 μM), patients were categorized as either high or low ketone. l-Carnitine-treated low-ketone patients had greater use of carnitine as evidenced by lower post-treatment l-carnitine levels. The l-carnitine responders also had faster resolution of vasopressor requirement and a trend toward a greater improvement in mortality at 1 year (P = 0.038) compared with patients with higher 3-hydroxybutyrate., Conclusions: The results of this preliminary study, which were not readily apparent from the parent clinical trial, show a unique metabolite profile of l-carnitine responders and introduce pharmacometabolomics as a viable strategy for informing l-carnitine responsiveness. The approach taken in this study represents a concrete example for the application of precision medicine to sepsis therapeutics that warrants further study.

Background: β-Hydroxy-β-methylbutyrate (HMB) supplementation has been demonstrated to enhance muscle protein synthesis and attenuate loss of muscle mass by multiple pathways. The beneficial effects of HMB have been studied by using either the calcium salt, monohydrate, of HMB (CaHMB) or the free acid form (FAHMB)., Objective: The present study was designed to compare the pharmacokinetics and relative bioavailability of the 2 forms of HMB administered as a liquid suspension in male Sprague-Dawley rats., Methods: CaHMB at 30, 100, and 300 mg/kg and equivalent doses of FAHMB at 24.2, 80.8, and 242 mg/kg were administered orally as a liquid suspension to male Sprague-Dawley rats. A single i.v. dose of 5 mg/kg CaHMB, corresponding to an equivalent dose of 4.04 mg/kg FAHMB, was also administered. Plasma concentrations of HMB were analyzed by liquid chromatography tandem mass spectrometry, and pharmacokinetic variables and relative bioavailability of the 2 forms of HMB were determined., Results: After oral administration, the area under the plasma concentration time curve (AUC) from time 0 to time t (0-t) and from time 0 to infinity (0-∞) and the maximum (peak) plasma concentration (Cmax) for CaHMB were significantly greater than for FAHMB, whereas the time to reach Cmax did not differ from that of FAHMB. The relative bioavailability of CaHMB was 49%, 54%, and 27% greater than that of FAHMB for the 3 respective oral doses tested. After i.v. administration, the AUCs 0-t and 0-∞ of the calcium salt were significantly greater than those of FAHMB. The relative bioavailability of CaHMB was 80% greater than that of FAHMB. The higher relative bioavailability of CaHMB may be attributable to its low systemic clearance compared with FAHMB., Conclusions: This study demonstrates the enhanced relative bioavailability of CaHMB compared with FAHMB. Further studies are warranted to understand the physiologic mechanisms contributing to the differences in systemic clearance., (© 2014 American Society for Nutrition.)

The aim of the current study was to examine the effects of cold water immersion (CWI) with and without the free acid form of β-hydroxy-β-methylbutyrate (HMB-FA) on markers of muscle damage following acute lower body resistance exercise. Forty recreationally resistance-trained men (22.3 ± 2.4 years) were randomly divided into one of the four groups: (1) Placebo (PL); (2) HMB-FA; (3) HMB-FA-CWI; (4) PL-CWI. HMB-FA groups ingested 3 g day(-1) and CWI groups submersed their lower body into 10-12 °C water for 10-min post-exercise. No differences between groups were observed for CK; however, PL-CWI had significantly greater elevations in myoglobin 30-min post-exercise compared to HMB-FA (p = 0.009) and PL (p = 0.005), and HMB-FA-CWI was significantly greater than HMB-FA (p = 0.046) and PL (p = 0.028). No differences between groups were observed for IL-6 and IL-10, although CRP was significantly greater 24-h post-exercise for PL-CWI compared to HMB-FA-CWI (p = 0.02) and HMB-FA (p = 0.046). Only HMB-FA-CWI showed significantly (p = 0.02) greater improvements in average power per repetition. CWI appeared to elevate myoglobin compared to other groups, while HMB-FA may have attenuated the increase in CRP when combined with CWI. Nevertheless, HMB-FA or CWI treatments did not appear to provide benefit over PL for recovery. Instead, the combination of CWI and HMB-FA improved performance recovery compared to other groups.

Many low-birth-weight infants experience failure to thrive. The amino acid leucine stimulates protein synthesis in skeletal muscle of the neonate, but less is known about the effects of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB). To determine the effects of HMB on protein synthesis and the regulation of translation initiation and degradation pathways, overnight-fasted neonatal pigs were infused with HMB at 0, 20, 100, or 400 μmol·kg body wt(-1)·h(-1) for 1 h (HMB 0, HMB 20, HMB 100, or HMB 400). Plasma HMB concentrations increased with infusion and were 10, 98, 316, and 1,400 nmol/ml in the HMB 0, HMB 20, HMB 100, and HMB 400 pigs. Protein synthesis rates in the longissimus dorsi (LD), gastrocnemius, soleus, and diaphragm muscles, lung, and spleen were greater in HMB 20 than in HMB 0, and in the LD were greater in HMB 100 than in HMB 0. HMB 400 had no effect on protein synthesis. Eukaryotic initiation factor (eIF)4E·eIF4G complex formation and ribosomal protein S6 kinase-1 and 4E-binding protein-1 phosphorylation increased in LD, gastrocnemius, and soleus muscles with HMB 20 and HMB 100 and in diaphragm with HMB 20. Phosphorylation of eIF2α and elongation factor 2 and expression of system A transporter (SNAT2), system L transporter (LAT1), muscle RING finger 1 protein (MuRF1), muscle atrophy F-box (atrogin-1), and microtubule-associated protein light chain 3 (LC3-II) were unchanged. Results suggest that supplemental HMB enhances protein synthesis in skeletal muscle of neonates by stimulating translation initiation.

Objectives: For the quantification of β-hydroxybutyrate (BHB) and β-hydroxy-β-methylbutyrate (HMB) in human whole blood, a method using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was developed, which does not require chemical modification of the analytes., Design and Methods: Samples were deproteinised by a mixture of methanol and acetonitrile, and the extracts were cleaned-up using both polymeric strong cation exchange and strong anion exchange sorbents. The analytes and their structural isomers were separated using a column with a zwitterionic stationary phase. Isotope dilution of both analytes was used for quantitative analysis., Results: Separation of BHB from isobaric interferences was achieved through chromatography. The relative intra-laboratory reproducibility standard deviations were better than 10% for blood samples at concentration levels of 10-20μM BHB and 1μM HMB and better than 5% at concentration levels 10 times higher. The mean true extraction recoveries were close to 100%. The trueness expressed as the relative bias of test results was within ±5% at concentration levels of 10-1000μM BHB and 1-20μM HMB. The lower limits of quantification were estimated to be 3μM for BHB and 0.4μM for HMB., Conclusions: A simple and highly sensitive and selective HILIC-MS/MS method was developed that is suitable for the quantification of BHB and HMB in whole blood., (© 2013.)

Gamma-valerolactone (GVL) is reported to be a substance that can be used as a legal substitute for gamma-hydroxybutyric acid (GHB), which is currently a controlled substance in several countries. Unlike gamma-butyrolactone and 1,4-butanediol, GVL is not metabolized to GHB, which causes the effects after uptake of these two chemicals. In the case of GVL, the lactone ring is split to gamma-hydroxyvaleric acid (GHV or 4-methyl-GHB) by a lactonase. Because of its affinity for the GHB receptor, GHV reveals similar effects to GHB, although it is less potent. Intoxications with GVL, or its use as a date rape drug, are conceivable. Despite these facts, there are no publications in the literature regarding detections of GHV in human samples. This study reports three cases, including five urine samples, in which GHV could be detected in concentrations between 3 and 5.8 mg/L. In one of these cases, a drug-facilitated sexual assault (DFSA) was assumed; four of these samples were from two people suspected of abusing GHB. The results indicate that GVL is used as an alternative to GHB and its precursors and should be taken seriously. GVL or GHV should be included in toxicological analysis, particularly in DFSA cases. More information is needed regarding the pharmacokinetics of GVL/GHV for the meaningful interpretation of positive or negative results.

A simple, sensitive and specific high-performance liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of β-hydroxy-β-methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water-acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C(8) (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30-4600 ng/mL (r > 0.998) for HMB. The intra- and inter-day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench-top, autosampler freeze-thaw cycles and long-term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats., (Copyright © 2012 John Wiley & Sons, Ltd.)

Objective: To evaluate the use of metabolomics for the first-trimester detection of maternal metabolic dysfunction and prediction of subsequent development of early-onset preeclampsia (PE)., Study Design: This was a case-control study of maternal plasma samples collected at 11-13 weeks' gestation from 30 women who had subsequently developed PE requiring delivery before 34 weeks and 60 unaffected controls. Nuclear magnetic Resonance (NMR) spectroscopy was used to identify and quantify metabolomic changes in cases versus controls. Both genetic computing and standard statistical analyses were performed to predict the development of PE from the metabolite concentrations alone as well as the combination of metabolite concentrations with maternal characteristics and first-trimester uterine artery Doppler pulsatility index (PI)., Results: Significant differences between cases and controls were found for 20 metabolites. A combination of four of these metabolites (citrate, glycerol, hydroxyisovalerate, and methionine) appeared highly predictive of PE with an estimated detection rate of 75.9%, at a false-positive rate (FPR) of 4.9%. The predictive performance was improved by the addition of uterine artery Doppler PI and fetal crown-rump length (CRL) and with an estimated detection rate of 82.6%, at a FPR of 1.6%., Conclusion: A profound change in the first-trimester metabolite profile was noted in women who had subsequently developed early-onset PE. Preliminary algorithms appeared highly sensitive for first trimester prediction of early onset PE.

Pancreatic cancer is a malignant tumor with the worst prognosis among all cancers. At the time of diagnosis, surgical cure is no longer a feasible option for most patients, thus early detection of pancreatic cancer is crucial for its treatment. Metabolomics is a powerful new analytical approach to detect the metabolome of cells, tissue, or biofluids. Here, we report the application of (1)H nuclear magnetic resonance (NMR) combined with principal components analysis to discriminate pancreatic cancer patients from healthy controls based on metabolomic profiling of the serum. The metabolic analysis revealed significant lower of 3-hydroxybutyrate, 3-hydroxyisovalerate, lactate, and trimethylamine-N-oxide as well as significant higher level of isoleucine, triglyceride, leucine, and creatinine in the serum from pancreatic cancer patients compared to that of healthy controls. Our data demonstrate that the subtle differences in metabolite profiles in serum of pancreatic cancer patients and that of healthy subjects as a result of physiological and pathological variations could be identified by NMR-based metabolomics and exploited as metabolic markers for the early detection of pancreatic cancer.

The leucine metabolite, β-hydroxy-β-methylbutyrate (HMB), is a nutritional supplement that increases lean muscle and strength with exercise and in disease states. HMB is presently available as the Ca salt (CaHMB). The present study was designed to examine whether HMB in free acid gel form will improve HMB availability to tissues. Two studies were conducted and in each study four males and four females were given three treatments in a randomised, cross-over design. Treatments were CaHMB (gelatin capsule, 1 g), equivalent HMB free acid gel swallowed (FASW) and free acid gel held sublingual for 15 s then swallowed (FASL). Plasma HMB was measured for 3 h following treatment in study 1 and 24 h with urine collection in study 2. In both the studies, the times to peak plasma HMB were 128 (sem 11), 38 (sem 4) and 38 (sem 1) min (P < 0·0001) for CaHMB, FASW and FASL, respectively. The peak concentrations were 131 (sem 6), 249 (sem 14) and 239 (sem 14) μmol/l (P < 0·0001) for CaHMB, FASW and FASL, respectively. The areas under the curve were almost double for FASW and FASL (P < 0·0001). Daily urinary HMB excretion was not significantly increased resulting in more HMB retained (P < 0·003) with FASW and FASL. Half-lives were 3·17 (sem 0·22), 2·50 (sem 0·13) and 2·51 (sem 0·14) h for CaHMB, FASW and FASL, respectively (P < 0·004). Free acid gel resulted in quicker and greater plasma concentrations (+185%) and improved clearance (+25%) of HMB from plasma. In conclusion, HMB free acid gel could improve HMB availability and efficacy to tissues in health and disease.

A rapid dried-filter paper plasma-spot analytical method was developed to quantify organic acids, amino acids, and glycines simultaneously in a two-step derivatization procedure with good sensitivity and specificity. The new method involves a two-step trimethylsilyl (TMS) - trifluoroacyl (TFA) derivatization procedure using GC-MS/ selective ion monitoring (GC-MS/SIM). The dried-filter paper plasma was fortified with an internal standard (tropate) as well as a standard mixture of distilled water and methanol. Methyl orange was added to the residue as an indicator. N-methyl-N-(trimethylsilyl-trifluoroacetamide) and N-methyl-bis-trifluoroacetamide were then added and heated to 60 degrees C for 10 and 15 min to produce the TMS and TFA derivatives, respectively. Using this method, the silylation of carboxylic functional groups was carried out, which was followed by the trifluoroacyl derivatization of the amino functional group. The derivatives were analyzed by GC-MS/SIM. A calibration cure showed a linear relationship for the target compounds between concentrations of 10-500 ng/mL. The limit of detection and quantification on a plasma spot were 10-90 ng/mL (S/N=9) and 80-500 ng/ mL, respectively. The correlation coefficient ranged from 0.938 and 0.999. When applied to the samples from positive patients, the method clearly differentiated normal subjects from the patients with various metabolic disorders such as PKU, MSUD, OTC and a Propionic Aciduria. The new developed method might be useful for making a rapid, sensitive and simultaneous diagnosis of inherited organic and amino acid disorders. In addition, this method is expected to be an alternative method for screening newborns for metabolic disorders in laboratories where expensive MS/MS is unavailable.

The purpose of this study was to determine the effect of beta-hydroxy beta-methylbutyrate (HMB) supplementation on maximal oxygen consumption (.V(O)(2)peak) and the onset of blood lactate accumulation (OBLA) in endurance-trained cyclists. Eight cyclists randomly (double blind) completed 3 2-week supplementation periods (HMB, 3g.day(-1); leucine [LEU], 3g.day(-1); placebo [CON], 3g.day(-1)) followed by a 2-week washout period. Testing consisted of a graded cycle ergometry test to measure .V(O)(2)peak and OBLA, the .V(O)(2) at 2 mM blood lactate. .V(O)(2)peak was unaffected by HMB (4.0 +/- 1.4%), LEU (-1.9 +/- 1.3%), and CON (-2.6 +/- 2.6%). HMB resulted in a greater time to reach .V(O)(2)peak, whereas LEU and CON did not affect this time (HMB, 3.6 +/- 1.5 min, LEU, -1.2 +/- 1.5 min; CON, -3.6 +/- 3.5 min). Lactate accumulation peak was unaffected by supplementation (HMB, 8.1 +/- 1.1 mM; LEU, 6.2 +/- 0.8 mM; CON, 7.5 +/- 1.3 mM). OBLA increased with HMB (9.1 +/- 2.4%) and LEU (2.1 +/- 1.5%), but not in the CON trial (0.75 +/- 2.1%). Blood glucose was significantly greater during the HMB trial compared with the LEU trial. It is concluded that HMB supplementation may have positive affects on performance by increasing the onset of blood lactate accumulation; however, the mechanism is unknown.

This study examined the effects of supplemental beta-hydroxy-beta-methylbutyrate (HMB) on muscle damage as a result of intense endurance exercise. Subjects (n = 13) were paired according to their 2-mile run times and past running experience. Each pair was randomly assigned a treatment of either HMB (3 g/day) or a placebo. After 6 wk of daily training and supplementation, all subjects participated in a prolonged run (20-km course). Creatine phosphokinase and lactate dehydrogenase (LDH) activities were measured before and after a prolonged run to assess muscle damage. The placebo-supplemented group exhibited a significantly greater (treatment main effect, P = 0.05) increase in creatine phosphokinase activity after a prolonged run than did the HMB-supplemented group. In addition, LDH activity was significantly lower (treatment main effect, P = 0.003) with HMB supplementation compared with the placebo-supplemented group. In conclusion, supplementation with 3.0 g of HMB results in a decreased creatine phosphokinase and LDH response after a prolonged run. These findings support the hypothesis that HMB supplementation helps prevent exercise-induced muscle damage.

The present experiment was conducted to study the impact of portal-drained visceral (PDV) metabolism of arterial 3-OH-butyrate on estimates of the portal recovery of intraruminally infused butyrate. Three multicatheterized and rumen-fistulated Leicester ewes were subjected to three intraruminal infusion protocols in a Latin square design: control (C; water), butyrate (B; 20 mmol x h(-1)), and butyrate (20 mmol x h(-1)) + propionate (40 mmol x h(-1)) (BP). During the experiments, the sheep were infused with 1,2,3,4-13C4-D-3-OH-butyrate in a mesenteric vein. Portal recoveries of intraruminally infused butyrate and propionate were obtained by comparing Treatments B and BP, respectively, with Treatment C. The portal net appearance of butyrate and the portal net appearance of butyrate + 3-OH-butyrate accounted for 20 +/- 2% and 48 +/- 14% of intraruminally infused butyrate, respectively. Metabolism by the PDV tissues accounted for 32 to 44% of the whole-body irreversible loss rate of 3-OH-butyrate (12.0 to 24.7 +/- 0.5 mmol x h(-1)). The portal net appearance of butyrate plus the unidirectional PDV output of 3-OH-butyrate accounted for 62 +/- 5% of the intraruminally infused butyrate, and this estimate was comparable to the portal recovery of intraruminally infused propionate (62 +/- 7%). The results from the present study show that the extent of epithelial butyrate oxidation is overestimated and the portal recovery of butyrate carbon underestimated if only portal net appearance rates of butyrate and 3-OH-butyrate are considered.

In nephrotic syndrome, significant amounts of plasma proteins, mostly of hepatic origin, are lost in urine. Total hepatic protein synthesis increases, suggesting that other protein pools must be conserved to maintain steady state. This can be accomplished either by decreased amino acid oxidation or decreased protein synthesis in other organs to replace lost liver-derived proteins. To determine the effect of nephrotic syndrome on total-body protein metabolism, we compared whole-body valine use in seven nephrotic patients and five controls using a primed continuous infusion of [1-(13)C]-valine, with additional priming of NaH(13)CO(3). Plasma [(13)C]-valine, (13)C alpha ketoisovaleric acid, and the expired (13)CO(2) enrichments were used to assess whole-body valine flux, valine oxidation, and nonoxidative valine disposal (NOVD). The valine flux into the blood compartment (97.7 +/- 3.0 versus 95.3 +/- 3.3 micromol/kg/h), oxidation of valine (19.4 +/- 1.9 versus 21.2 +/- 2. 8 micromol/kg/h), and NOVD (78.3 +/- 2.5 versus 74.2 +/- 2.7 micromol/kg/h) were not statistically different in patients compared with controls. Valine oxidation correlated positively with urinary urea excretion (r = 0.70; P = 0.01) in all subjects. Compared with control subjects who have similar urinary urea excretion, nephrotic subjects do not compensate for urinary loss of protein by decreased amino acid oxidation or decreased nonoxidative valine disposal. Previous studies have shown that synthesis of several hepatic proteins increases when subjects are fed the same dietary regime, whereas the present study shows that total-body protein synthesis does not increase. This would imply reduced synthesis of nonhepatic protein pools.

Using serum biotin concentration as the indicator, a previous study reported biotin deficiency resulting from long-term anticonvulsant therapy. However, serum biotin may not be a good indicator of tissue biotin status. Using better indicators of biotin status in anticonvulsant-treated subjects, we found increased urinary excretion of biotin catabolites and 3-hydroxyisovaleric acid, an organic acid produced in greater quantities secondary to reduced activity of a biotin-dependent carboxylase. We conclude that anticonvulsant treatment led to increased biotin catabolism and probably to reduced biotin status.

Clinical and metabolic changes in the central nervous system are described in a patient with biotinidase deficiency before and after biotin treatment. Lactate, pyruvate and 3-hydroxyisovaleric acid as metabolic disease markers were measured in blood, cerebrospinal fluid and brain tissue by biochemical analyses or localized magnetic resonance proton spectroscopy. The patient improved markedly with biotin treatment. Nevertheless, neurological sequelae and abnormal intracerebral lactate concentrations persisted despite normalized metabolic disease markers in extracerebral fluids. Therefore, localized in vivo measurements of intracerebral metabolites may be a valuable tool for elucidating the pathogenesis of biotinidase deficiency.

Beta-hydroxy-beta-methyl butyrate (HMB), a compound formed during catabolism of leucine, was fed to 256 crossbred steers as 0 or .03% of diet dry matter. Effects on performance, carcass characteristics, and tissue composition were measured. Groups of 32 steers per diet were slaughtered after 105, 119, 133, and 147 d on feed. The HMB was fed to each group only during the final 82 d they were fed. Averaged across slaughter date, animal performance was not altered by HMB; however, an interaction between HMB and time on feed was detected. Feeding HMB increased (P < .01) daily gain of steers slaughtered at 105 d but decreased (P < .01) daily gain of steers slaughtered at 147 d. Steers fed HMB had numerically higher marbling scores that resulted in a trend toward higher carcass quality grades. Steers receiving HMB tended to have less (P < .08) s.c. fat and fewer steers with yield grades of 4 or greater (1.6 vs 4.7%). Supplementation of HMB to feedlot steers tended to increase (P < .07) the ratio of intramuscular fat to subcutaneous fat. Steers fed HMB had higher (P < .001) blood plasma concentrations of HMB (3.06 vs 1.70 mg/L) and lower (P < .03) blood plasma concentrations of cholesterol (108.4 vs 118.7 mg/dL). Feeding HMB tended to increase (P < .10) lipid content of the longissimus muscles of those steers slaughtered at 105 d.

Plasma concentrations of the branched-chain amino acids leucine, isoleucine and valine, and those of leucine's and isoleucine's transamination products alpha-ketoisocaproic acid (KICA) and alpha-keto-beta-methylvaleric acid (KMVA), respectively, are known to increase after a protein meal or during extended fasting, but little or no increase in the concentration of valine's transamination product, alpha-ketoisovaleric acid (KIVA), has been observed under these conditions. To determine whether this could be explained by the conversion of KIVA to its alpha-hydroxy analogue, we measured the plasma concentrations of KICA, KMVA and KIVA, as well as their alpha-hydroxy analogues [alpha-hydroxyisocaproic acid (HICA), alpha-hydroxy-beta-methylvaleric acid (HMVA) and alpha-hydroxyisovaleric acid (HIVA)], in normal volunteers immediately after a protein meal or during a 60-h fast. We also determined the oxidoreduction equilibrium constants for HIVA/KIVA and HICA/KICA and their extent of plasma protein binding. In subjects in the postabsorptive state, the plasma concentrations of KICA and KMVA were 100 times those of HICA and HMVA, whereas that of KIVA was only twice that of HIVA. Shortly after a protein meal, KICA and KMVA concentrations increased significantly by 30 and 60%, respectively, whereas that of KIVA decreased by 25% (P < 0.05). HICA, HMVA and HIVA concentrations did not change. During prolonged fasting the plasma concentrations of all six metabolites increased gradually. The high plasma keto/hydroxy acid ratios were not related to their K(eq), which favored alpha-hydroxy analogue formation. The reduction of the branched-chain alpha-keto acids to their alpha-hydroxy analogues seems to take place too slowly to attain thermodynamic equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The plasma 4,5-dioxovaleric acid (DOVA) levels of two different breeds of chicken were determined and found to be higher in the group with a higher porphyrin eggshell content. 2. The erythrocyte and uterus glyoxalase II activity, investigated by means of a new spectrophotometric method, was found to be significantly higher in the group with a low porphyrin eggshell content. 3. A comparative genetic study of two chicken populations, one with white and one with dark eggshells, showed different gene frequencies for the glyoxalase I polymorphism. 4. An interpretation of these data suggests that the glyoxalase pathway may be involved in the metabolism of early porphyrin precursors.

The oxidation of alpha-ketoisocaproate (KIC) to beta-hydroxy-beta-methylbutyrate (HMB) by the enzyme KIC dioxygenase has been previously described in rat and human liver; however, the importance of this pathway in normal leucine metabolism has not yet been assessed. A series of experiments was conducted in young lambs and pigs to determine whether HMB is produced from KIC in vivo and to estimate the importance of this pathway in leucine metabolism. In the first study, lambs were fed a bolus of KIC, and the change in plasma HMB concentration was monitored over a 24-h period. Administration of KIC increased plasma HMB from basal concentrations of 2 to approximately 7 microM 4 h after the supplementation. In the second experiment, lambs were infused with [6,6,6-2H3]KIC into the duodenum, and the appearance of labeled [2H3]HMB was measured. Under basal conditions, a minimum of 18% of the HMB was derived from KIC, but when unlabeled KIC was infused into the duodenum at a rate of 1.6 mumol.kg-1.min-1, plasma HMB concentration doubled, and essentially 100% of the HMB present was derived from KIC. In a third experiment, young pigs were infused with [6,6,6-2H3]leucine. At steady state, [2H3]-leucine and HMB enrichments were nearly identical, indicating that plasma HMB is derived solely from leucine. In a fourth experiment, both lambs and pigs were injected intravenously with 600 mg of HMB daily, and urinary HMB excretion was quantitated.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for measuring the branched chain hydroxy acid beta-hydroxy-beta-methyl butyrate (HMB, a product of leucine catabolism) has been described. A [2H6]HMB internal standard was added to plasma and standards, and samples were extracted with diethyl ether, backextracted into neutral phosphate, dried, and derivatized for gas chromatography and mass spectrometry. The natural HMB was monitored at 175 amu and the deuterated HMB was monitored at 181 amu. Standard curves were linear to at least 25 microns and were quantitatively recovered from plasma. Basal concentrations of plasma HMB were from 1 to 2 microM in sheep and increased three- to fourfold when leucine's alpha-ketoacid (alpha-ketoisocaproate, KIC) was fed to lambs. This method can also be adapted to quantitate KIC and other branched chain ketoacids in plasma during the same run.

A case of isovaleric acidemia appearing as diabetic ketoacidosis with acute encephalopathy and pancytopenia was reported. A three-year-old male patient, with mild psychomotor retardation, had recurrent bouts of acute encephalopathy and pancytopenia after episodes of upper respiratory infection. At admission, he had vomiting associated with dehydration, acidosis, ketonuria, coma and a pungent, rather unpleasant odor. Laboratory features included hyperglycemia, hyperammonemia, hyperamylasemia, hypocalcemia, neutropenia, thrombocytopenia and subsequent anemia. Urine organic acid profiles showed profuse amount of 3-beta-hydroxyisovaleric acid (295 mg/ml) and isovalerylglycine (616 mg/ml) by gas chromatography-mass spectrometry. Levels of amino acids in the serum and urine were normal. The patient received treatment with rehydration and insulin, with rapid improvement. After the acute illness, blood glucose levels returned to normal. The patient was doing well on a low-protein diet in recent 3 months.

Isovaleric acidemia (IVA) is caused by a genetic deficiency of isovaleryl-CoA dehydrogenase (IVD). At least five distinct variant IVD alleles are known. We isolated five overlapping IVD cDNA clones from a human placenta cDNA library. They covered the entire coding region, except the initiation codon, and 587 bp in the 3'-noncoding region plus the poly(A) tail. The structure of the initiation site was identified by the study of genomic DNA and by the sequence comparison with rat IVD. Human IVD shared 89.6, 35.8, and 31.6% identical amino acid residues with rat IVD and human short and medium chain acyl-CoA dehydrogenases, respectively. In the Northern blot analysis of normal human liver and fibroblast poly(A)+ RNA, three mRNA species of different sizes (4.6, 3.8, and 2.1 kb) hybridized to IVD cDNA. Three mRNA species with similar sizes were also detected in five IVA fibroblast lines of different genotypes (variants 1, 1 X 2, 2, 3, and 5), suggesting that these variants are each due to a point mutation or small deletion.

The purpose of this study was to evaluate the possible modifications of the plasma and urine short-chain-fatty acid (SCFA) patterns indiced by treatment with valproic acid (VPA). Increased amounts of SCFAs in patients under VPA treatment may explain the presence of VPA-induced hyperammonemia, toxic encephalopathies and rarer Rey-like syndromes recently observed. For this reason we assayed SCFA levels in the plasma and looked for propionic acid in the urine of 10 epileptic patients to whom it was decided to add VPA to their previously unsatisfactory anti-epileptic treatment. This was carried out prior to and during therapy with VPA. 5 of these patients developed toxic encephalopathy with hyperammonemia induced by VPA. Our data show that plasma and urine SCFAs are not modified by VPA treatment. This is so even in patients who have toxic encephalopathy with hyperammonemia indiced by this drug.

Organic acids in plasma and urine of a patient with isovaleric acidemia were measured serially during a severe ketoacidotic attack. Urinary ketone bodies, lactic acid and 2-hydroxy-n-butyric acid changed in parallel with the plasma isovaleric acid concentration, which was correlated closely with the severity of the clinical symptoms. A 2-day lag was observed between the time of the peak plasma isovaleric acid level and that of the highest urinary excretion of 3-hydroxyisovaleric acid, methylsuccinic acid, mesaconic acid and 2-hydroxyisobutyric acid, the total quantity of which amounted to one-third of that of isovalerylglycine on the 4th day of the attack. Urinary isovalerylglycine excretion remained at a plateau for 4 days after the highest level of plasma isovaleric acid was observed. The decrease of urinary hippuric acid excretion during the attack continued until the patient had recovered enough to be able to eat. Benzoic acid administration concomitant with leucine and glycine canceled the effect of glycine in lessening the rise of plasma isovaleric acid concentration after a leucine load.

Two metabolites, 4-hydroxyisovaleric acid and mesaconic acid, have been identified and quantified in the urine of a patient with isovaleric acidemia. These compounds do not appear to have been reported previously as being components of human metabolism. In addition, large quantities of 3-methylbutyrolactone, the lactone of 4-hydroxyisovaleric acid, were observed in the volatile profile obtained by headspace chromatography. The demonstration of 4-hydroxyisovaleric acid supports the contention that urinary methylsuccinic acid seen in patients with isovaleric acidemia has arisen by omega-oxidation of isovaleric acid. The identification of mesaconic acid may indicate that the methylsuccinic acid formed in these patients is subject to further metabolism.

Severe pancytopenia developed in two infants with isovaleric acidemia. Previous reports indicate these hematologic abnormalities are a leading cause of death in affected infants. Our findings suggest that the pancytopenia may be due to arrested maturation of hematopoietic precursors. Prompt transfusion of appropriate blood components prevented complications due to the hematologic abnormalities.