TMAVA, a Metabolite of Intestinal Microbes, Is Increased in Plasma From Patients With Liver Steatosis, Inhibits γ-Butyrobetaine Hydroxylase, and Exacerbates Fatty Liver in Mice.

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 3H2O from [3H] 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.

Metabolites of milk intake: a metabolomic approach in UK twins with findings replicated in two European cohorts.

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 (ß = 0.012, SE = 0.002, P = 2.98 × 10-12) and the nucleotide uridine (ß = 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 (ß = 0.050, SE = 0.015, P = 7.53 × 10-4) and validated in the urine of 236 UK twins (ß = 0.091, SE = 0.032, P = 0.004). Two metabolites from the targeted platform, hydroxysphingomyelin C14:1 (ß = 0.034, SE = 0.005, P = 9.75 × 10-14) and diacylphosphatidylcholine C28:1 (ß = 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.

The development and validation of a high-throughput LC-MS/MS method for the analysis of endogenous ß-hydroxy-ß-methylbutyrate in human plasma.

A high-throughput, sensitive, and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid quantitation of ß-hydroxy-ß-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.

A (14)C-leucine absorption, distribution, metabolism and excretion (ADME) study in adult Sprague-Dawley rat reveals ß-hydroxy-ß-methylbutyrate as a metabolite.

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 ß-hydroxy-ß-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.

Comparison of availability and plasma clearance rates of ß-hydroxy-ß-methylbutyrate delivery in the free acid and calcium salt forms.

ß-Hydroxy-ß-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.

Protein synthesis in skeletal muscle of neonatal pigs is enhanced by administration of ß-hydroxy-ß-methylbutyrate.

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 ß-hydroxy-ß-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.

The relative bioavailability of the calcium salt of ß-hydroxy-ß-methylbutyrate is greater than that of the free fatty acid form in rats.

BACKGROUND: ß-Hydroxy-ß-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.

Effects of ß-hydroxy-ß-methylbutyrate free acid and cold water immersion on post-exercise markers of muscle damage.

The aim of the current study was to examine the effects of cold water immersion (CWI) with and without the free acid form of ß-hydroxy-ß-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.

Relationship of endogenous plasma concentrations of ß-hydroxy ß-methyl butyrate (HMB) with frailty in community dwelling older adults with type-2 diabetes mellitus.

BACKGROUND: Supplementation with ß-hydroxy ß-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.

Development and validation of LC-MS/MS method for the estimation of ß-hydroxy-ß-methylbutyrate in rat plasma and its application to pharmacokinetic studies.

A simple, sensitive and specific high-performance liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of ß-hydroxy-ß-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.

Free acid gel form of ß-hydroxy-ß-methylbutyrate (HMB) improves HMB clearance from plasma in human subjects compared with the calcium HMB salt.

The leucine metabolite, ß-hydroxy-ß-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.

Effect of chronic exercise in healthy young male adults: a metabolomic analysis.

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.

Redefining COVID-19 Severity and Prognosis: The Role of Clinical and Immunobiotypes.

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.

Effects of Co-Ingestion of ß-Hydroxy-ß-Methylbutyrate and L-Arginine α-Ketoglutarate on Jump Performance in Young Track and Field Athletes.

The aim of the study was to determine the effect of simultaneous supplementation of ß-hydroxy-ß-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 ß-hydroxy-ß-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.

Net Absorption and Metabolism of ß-Hydroxy- ß-Methyl Butyrate during Late Gestation in a Pig Model.

The leucine metabolite, ß-hydroxy-ß-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.

A double-blind placebo controlled trial into the impacts of HMB supplementation and exercise on free-living muscle protein synthesis, muscle mass and function, in older adults.

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 ß-hydroxy-ß-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 (D2O) 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.

Dimethylguanidino Valerate: A Lifestyle-Related Metabolite Associated With Future Coronary Artery Disease and Cardiovascular Mortality.

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.

Effect of BCAA supplement timing on exercise-induced muscle soreness and damage: a pilot placebo-controlled double-blind study.

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 ß-hydroxy-ß-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.

L-carnitine therapy in isovaleric acidemia.

Isovaleric acidemia, resulting from isovaleryl-coenzyme A dehydrogenase deficiency, is associated with marked reduction of free carnitine in both plasma and urine. Fast atom bombardment-mass spectrometry, hydrolysis, and gas chromatography/mass spectrometry have unequivocally identified the existence of isovalerylcarnitine, a new metabolite specific for this disorder. Administration of equimolar amounts of glycine or L-carnitine separately with leucine demonstrated that isovaleryl-coenzyme A is removed by supplemental L-carnitine in the form of isovalerylcarnitine as effectively as it is by glycine, in the form of isovalerylglycine. When L-carnitine is given alone, excretion of isovalerylglycine decreases in preference to enhanced excretion of isovalerylcarnitine and hippurate. Treatment with L-carnitine alone has proven effective in preventing further hospitalizations in a patient with this genetic disorder.

Nucleotide sequence of messenger RNA encoding human isovaleryl-coenzyme A dehydrogenase and its expression in isovaleric acidemia fibroblasts.

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.