Α-hydroxyisocaproic Acid Research Articles

α-Hydroxyisocaproic Acid Decreases Protein Synthesis but Attenuates TNFα/IFNγ Co-Exposure-Induced Protein Degradation and Myotube Atrophy via Suppression of iNOS and IL-6 in Murine C2C12 Myotube.

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.

Formation of 3-Methylbutanal and 3-Methylbutan-1-ol Recognized as Malty during Fermentation in Swiss Raclette-Type Cheese, Reconstituted Milk, and de Man, Rogosa, and Sharpe Broth.

This work aimed to determine the formation over time of 3-methylbutanal and 3-methylbutan-1-ol recognized as malty during the manufacture of Raclette-type cheese and the fermention of reconstituted skim milk, and filter-sterilized MRS broth. Using dynamic headspace-vacuum transfer in trap extraction followed by gas chromatography coupled with mass spectrometry-olfactometry (DHS-VTT-GC-MS-O) as a screening method for the malty compounds, five compounds (2-methylpropanal, 2- and 3-methylbutanal, and 2- and 3-methylbutan-1-ol) were identified as potential compounds causing the malty aroma in starter culture development and Raclette-type cheeses. Focus on compounds having a predominant sensorial effect (3-methylbutanal and 3-methylbutan-1-ol), spikings of leucine, 13C-labeled leucine, α-ketoisocaproic acid, and α-ketoglutaric acid provided a better understanding of their formation pathway. This study highlighted the discrepancies in the formation of 3-methylbutanal and 3-methylbutan-1-ol between the growth media; namely, despite the presence of free leucine available in MRS and the addition of an excess, no increase of the target compounds was observed. The concentration of these compounds in MRS increased only when α-ketoglutaric acid or α-ketoisocaproic acid was added, and a preference for the pathway to α-hydroxyisocaproic acid instead of 3-methylbutanal was shown. In addition, a formation of 3-methylbutanal when the bacteria were not yet active was observed when spiking α-ketoisocaproic acid, which potentially indicates that this part of the metabolism could take place extracellularly. These results could potentially unveil other, not-yet-identified reactants, directly influencing the production of compounds responsible for the malty aroma in Raclette cheese.

Leucine metabolites do not induce changes in phase angle, bioimpedance vector analysis patterns, and strength in resistance trained men.

We aimed to assess the effects of off-the-shelf leucine metabolite supplements on phase angle (PhA), bioimpedance vector analysis (BIVA) patterns and strength during an 8-week resistance training protocol. Fifty-three male participants were allocated into 4 groups: α-hydroxyisocaproic acid (n = 12, age = 30.9 ± 9.3 years), β-hydroxy-β-methylbutyrate free acid (n = 12, age = 31.0 ± 9.3 years), calcium β-hydroxy-β-methylbutyrate (n = 15, age = 32.1 ± 5.2 years) or placebo (n = 14, age = 28.9 ± 6.6 years). Bioimpedance parameters and 1-repetition maximum (1RM) for back squat and bench press were assessed at baseline and at the end of weeks 4 and 8. Additionally, fat-free mass and fat mass were evaluated by dual-energy X-ray absorptiometry. No statistically group by time interactions were found, even adjusting for age. PhA and vector did not change over the training period, while time-dependent increases were observed for 1RM back squat and 1RM bench press. A direct association was observed between PhA and 1RM bench press changes (whole sample), while PhA and strength were correlated throughout the study, even when adjusting for fat-free mass and percentage of fat mass. Leucine metabolites have no effect on PhA, BIVA patterns or strength during an 8-week resistance training program, in resistance trained subjects. The trial was registered at ClincicalTrials.gov: NCT03511092. Novelty: Supplementation with leucine metabolites is not a supplementation strategy that improves bioelectrical phase angle, cellular health, and strength after an 8-week resistance training program. When consuming a high protein diet, none of the α-hydroxyisocaproic acid, β-hydroxy-β-methylbutyrate free acid, and calcium β-hydroxy-β-methylbutyrate metabolites resulted in an ergogenic effect in resistance trained men.

Leucine metabolites do not attenuate training-induced inflammation in young resistance trained men

ABSTRACT Leucine metabolites may reduce training-induced inflammation; however, there is scant evidence for this assertion. We conducted a double-blind randomized controlled pragmatic trial where 40 male participants were allocated into 4 groups: α-hydroxyisocaproic acid group ([α-HICA], n = 10, Fat-free mass [FFM] = 62.0 ± 7.1 kg), β-hydroxy-β-methylbutyrate free acid group ([HMB-FA], n = 11, FFM = 62.7 ± 10.5 kg), calcium β-hydroxy-β-methylbutyrate group ([HMB-Ca], n = 9, FFM = 65.6 ± 10.1 kg) or placebo group ([PLA]; n = 10, FFM = 64.2 ± 5.7 kg). An 8-week whole-body resistance training routine (3 training sessions per week) was employed to induce gains in skeletal-muscle thickness. Skeletal muscle thickness (MT), one repetition maximum (1RM), interleukin-6 (IL-6), high-sensitivity C-reactive protein (hsCRP) and tumour necrosis factor alpha (TNF-α) were assessed at baseline and at the end of weeks 4 and 8. Time-dependent increases were detected from baseline to week 8 for MT (vastus lateralis: p = 0.009; rectus femoris: p = 0.018), 1RM (back squat: α-HICA, 18.5% ± 18.9%; HMB-FA, 23.2% ± 16%; HMB-Ca, 10.5% ± 13.8%; PLA, 19.7% ± 9% and bench press: α-HICA, 13.8% ± 19.1%; HMB-FA, 15.5% ± 9.3%; HMB-Ca, 10% ± 10.4%; PLA, 14.4 ± 11.3%, both p < 0.001), IL-6, hsCRP (both p < 0.001) and TNF-α (p = 0.045). No differences were found between groups at any time point. No leucine metabolite attenuated inflammation during training. Additionally, backwards elimination regressions showed that no circulating inflammatory marker consistently shared variance with the change in any outcome. Using leucine metabolites to modulate inflammation cannot be recommended from the results obtained herein. Furthermore, increases in inflammatory markers, from training, do not correlate with any outcome variable and are likely the result of training adaptations.

Leucine Metabolites Do Not Enhance Training-induced Performance or Muscle Thickness.

We aimed to conduct a double-blind randomized controlled pragmatic trial to evaluate the effects of off-the-shelf leucine metabolite supplements of α-HICA, HMB-FA, and HMB-Ca on resistance training-induced changes in muscle thickness and performance. Forty men were randomly assigned to receive α-HICA (n = 10, fat-free mass [FFM] = 62.0 ± 7.1 kg), HMB-FA (n = 11, FFM = 62.7 ± 10.5 kg), HMB-Ca (n = 9, FFM = 65.6 ± 10.1 kg), or placebo (PLA; n = 10, FFM = 64.2 ± 5.7 kg). The training program consisted of whole body thrice weekly resistance training for 8 wk (seven exercises per session, three to four sets per session, at 70%-80% one repetition maximum). Skeletal muscle thickness by ultrasound, performance measures, and blood measures (creatine kinase, insulin-like growth factor 1, growth hormone, cortisol, and total testosterone) were evaluated at baseline and at the end of weeks 4 and 8. Time-dependent changes were observed for muscle thickness (P < 0.001), one repetition maximum bench press and squat (P < 0.001), Wingate peak power (P = 0.02), countermovement jump height (P = 0.03), power (P = 0.006), creatine kinase, insulin-like growth factor-1, growth hormone, and cortisol (all P < 0.001). No significant between-group or time-group interactions were observed. No leucine metabolite resulted in any ergogenic effects on any outcome variable. Supplementation with leucine metabolites-α-HICA, HMB-FA, or HMB-Ca-is not a supplementation strategy that improves muscle growth and strength development in young adult men.

No effect of HMB or α-HICA supplementation on training-induced changes in body composition

β-hydroxy-β-methylbutyrate (calcium: HMB-Ca and free acid: HMB-FA) and α-hydroxyisocaproic acid (α-HICA) are leucine metabolites that have been proposed to improve body composition and strength when combined with resistance exercise training (RET). In this double-blind randomized controlled pragmatic trial, we evaluated the effects of off-the-shelf supplements: α-HICA, HMB-FA and HMB-Ca, on RET-induced changes in body composition and performance. Forty men were blocked randomized to receive α-HICA (n = 10, fat-free mass [FFM] = 62.0 ± 7.1 kg), HMB-FA (n = 11, FFM = 62.7 ± 10.5 kg), HMB-Ca (n = 9, FFM = 65.6 ± 10.1 kg) or placebo (PLA; n = 10, FFM = 64.2 ± 5.7 kg). The training protocol consisted of a whole-body resistance training routine, thrice weekly for 8 weeks. Body composition was assessed by dual-energy x-ray absorptiometry (DXA) and total body water (TBW) by whole-body bioimpedance spectroscopy (BIS), both at baseline and at the end of weeks 4 and 8. Time-dependent changes were observed for increase in trunk FFM (p < 0.05). No statistically significant between-group or group-by-time interactions were observed. Supplementation with HMB (FA and Ca) or α-HICA failed to enhance body composition to a greater extent than placebo. We do not recommend these leucine metabolites for improving body composition changes with RET in young adult resistance trained men.

Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani.

Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate knockout mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway for sansalvamide. Sansalvamide is structurally related to the cyclic hexadepsipeptide destruxin, which both contain an α-hydroxyisocaproic acid (HICA) unit. A gene cluster responsible for destruxin production has previously been identified in Metarhizium robertsii together with a hypothetical biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium chrysogenum and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread.

Investigation of the Presence of β-Hydroxy-β-methylbutyric Acid and α-Hydroxyisocaproic Acid in Bovine Whole Milk and Fermented Dairy Products by a Validated Liquid Chromatography–Mass Spectrometry Method

A simple, rugged, quantitative, and confirmatory method based on liquid chromatography-mass spectrometry was developed and comprehensively validated for the analysis of the leucine metabolites β-hydroxy-β-methylbutyric acid (HMB) and α-hydroxyisocaproic acid (HICA) in bovine whole milk and yogurt. Mean accuracy (90-110% for HMB and 85-115% for HICA) and total precision (<10% RSD in most cases, except for <20% RSD for HMB at the limit of quantitation) at four concentration levels across three validation runs have been determined. Limits of quantitation for HMB and HICA in whole milk were 20 and 5 μg/L, respectively. Measured concentrations of HMB and HICA were <20-29 and 32-37 μg/L, respectively, in bovine whole milk and <5 and 3.0-15.2 mg/L, respectively, in yogurt. These concentrations are insufficient by large margins to deliver any musculoskeletal benefits, and fortification of milk and dairy products with HMB and/or HICA appears to be justified.

Chronic α-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy

Muscle disuse atrophy is observed routinely in patients recovering from traumatic injury and can be either generalized resulting from extended bed rest or localized resulting from single-limb immobilization. The present study addressed the hypothesis that a diet containing 5% α-hydroxyisocaproic acid (α-HICA), a leucine (Leu) metabolite, will slow the loss and/or improve recovery of muscle mass in response to disuse. Adult 14-wk-old male Wistar rats were provided a control diet or an isonitrogenous isocaloric diet containing either 5% α-HICA or Leu. Disuse atrophy was produced by unilateral hindlimb immobilization ("casting") for 7 days and the contralateral muscle used as control. Rats were also casted for 7 days and permitted to recover for 7 or 14 days. Casting decreased gastrocnemius mass, which was associated with both a reduction in protein synthesis and S6K1 phosphorylation as well as enhanced proteasome activity and increased atrogin-1 and MuRF1 mRNA. Although neither α-HICA nor Leu prevented the casting-induced muscle atrophy, the decreased muscle protein synthesis was not observed in α-HICA-treated rats. Neither α-HICA nor Leu altered the increased proteasome activity and atrogene expression observed with immobilization. After 14 days of recovery, muscle mass had returned to control values only in the rats fed α-HICA, and this was associated with a sustained increase in protein synthesis and phosphorylation of S6K1 and 4E-BP1 of previously immobilized muscle. Proteasome activity and atrogene mRNA content were at control levels after 14 days and not affected by either treatment. These data suggest that whereas α-HICA does not slow the loss of muscle produced by disuse, it does speed recovery at least in part by maintaining an increased rate of protein synthesis.

Biosynthesis of Athmu, a α,γ-hydroxy-β-amino acid of pahayokolides A–B

Pahayokolides A–B are cyanobacteria derived non-ribosomal peptides which exhibit cytotoxicity against a number of cancer cell lines. The biosynthetic origin of the 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu) moiety has been investigated using stable isotope incorporation experiments. While α-ketoisocaproic acid (α-KIC), α-hydroxyisocaproic acid (α-HIC), and leucine all serve as precursors to Athmu, the feeding of [1-13C] α-KIC results in more than threefold greater 13C enrichment than the other precursors. This result suggests that α-KIC is the immediate precursor which is selected and activated by the adenylation domain of the loading NRPS module and subsequently reduced in a fashion similar to that of the recently identified pathways for cryptophycins A–B, cereulide, and valinomycin.

Intrahippocampal administration of the branched-chain α-hydroxy acids accumulating in maple syrup urine disease compromises rat performance in aversive and non-aversive behavioral tasks

Maple syrup urine disease (MSUD) is an inherited metabolic disease predominantly characterized by neurological dysfunction. Although a variable degree of psychomotor/delay/mental retardation is found in a considerable number of MSUD patients, the mechanisms underlying the neuropathology of this disorder are yet not defined. The present study investigated the effect of acute intrahippocampal administration of the branched-chain α-hydroxy acids (BCHA) accumulating in MSUD on rat behavior in non-aversive (open field) and aversive (inhibitory avoidance) tasks. Cannulated 60-day-old male Wistar rats received bilateral intrahippocampal injection of α-hydroxyisocaproic acid (HIC, 1.5 μmol), α-hydroxyisovaleric acid (HIV, 2.5 μmol), α-hydroxy-β-methyl-n-valeric acid (HMV, 1.5 μmol), or NaCl (2.5 μmol)(controls) immediately after or 10 min before training. Testing session was performed 24 h later. Administration of the hydroxy acids immediately after training caused no effect on the cognitive performance of the rats. In contrast, HIV and HMV administered 10 min before training provoked a habituation deficit in the open field task. Motor activity, assessed by crossing responses, was the same for the groups infused with BCHA and NaCl. The effect of MK-801, succinate, creatine, and the antioxidants ascorbic acid plus α-tocopherol on the behavioral alterations provoked by HIV in the open field task revealed that only the energetic substrates (succinate and creatine) prevented these effects, reflecting a possible compromise of brain energy production by HIV. We also observed that rats pretreated with HIC, HIV, or HMV did not increase their latency in the testing session in the step-down inhibitory avoidance task, revealing an impairment of retrieval (memory retention or acquisition) in this task. Furthermore, no differences between controls and rats receiving BCHA were detected in the latency to leave the platform in the training test, suggesting similar motor activity of all groups. The data indicate that the α-hydroxy acids accumulating in MSUD impair cognition and may be implicated in the neuropathology and psychomotor delay/mental retardation observed in the affected patients.

Enantioselective multidimensional gas chromatography–mass spectrometry in the analysis of urinary organic acids

Enantioselective multidimensional gas chromatography–mass spectrometry is a valuable tool for the enantioselective analysis of compounds from complex matrices. Samples are separated initially on a precolumn and selected substances then transferred on-line to a main-column coated with suitable chiral stationary phase for enantioselective analysis with subsequent mass selective detection. In this paper the method is used as an adjunct to urinary organic acid analysis to provide information in patients with suspected inborn errors of metabolism. Lactic acid, α-hydroxyisocaproic acid, 3-phenyllactic acid and 3-(4-hydroxyphenyl)-lactic acid are separated in a single run. In addition, the enantioselective analysis of pyroglutamic acid is presented. d-Enantiomers of amino acids and α-hydroxycarboxylic acids derived from amino acids, point to a bacterial origin whereas the l-enantiomer is predominantly of endogenous origin. Therefore the enantiomeric ratio of chiral metabolites is an important parameter for the understanding of metabolic processes.

Effects of stereoisomeric isoleucines on sporidesmolide biosynthesis by Pithomyces chartarum.

SUMMARY As well as the known sporidesmolides I, II and III, Pithomyces chartarum produces sporidesmolides or similar compounds which contain residues of erythro-isoleucine and α-hydroxyisocaproic acid. When erythro-L-isoleucine was added to the growth medium, synthesis of sporidesmolides I and III was inhibited and synthesis of isoleucine-containing sporidesmolides, mainly Sporidesmolide II, was promoted. Threo-L-isoleucine in the medium was poorly utilized, but its presence resulted in production of a very complex mixture of sporidesmolides containing a higher proportion of isoleucine residues than the control. Epimerization of erythro-D-isoleucine occurred in the medium. Neither epimer was well utilized, and this isomer had little effect on sporidesmolide production or composition; it promoted some increase in the synthesis of isoleucine-containing sporidesmolides. Threo-D-isoleucine had effects qualitatively similar to but less pronounced than those of erythro-L-isoleucine, probably because of extracellular epimerization. The use of epimeric pairs of amino acids as tools for investigating biochemical processes involving inversion of configuration at an amino acid α-carbon atom is suggested.

Production of α-hydroxy fatty acids by two strains of Lactobacillus casei

The principal α-hydroxy acids other than lactic acid produced by L. casei strains 7469 and 280-16A were isolated by countercurrent extraction and identified as α-hydroxyisocaproic acid and α-hydroxyisovaleric acid. The amount of these acids and of lactic acid were estimated from counter-current distribution data, and the degrees of racemization were estimated from specific rotation values and from the results of optically specific microbiological assays. The maximal concentrations of α-hydroxyisocaproic and α-hydroxyisovaleric acids produced by L. casei were approximately 0.6 and 0.2 μeq./ml., respectively. The lactic acid, α-hydroxyisovaleric acid, and α-hydroxyisocaproic acid produced by L. casei 7469 were approximately 16%, 62%, and 97% racemic (the rest l-), respectively, whereas the same acids produced by L. casei 280-16A were much less racemic. This was particularly true of the L. casei 280-16A lactic acid, which appeared to be 99.3% l-lactic acid. Racemization of the α-hydroxy acids appears to be of importance in L. casei since strain 280-16A, which produces only slightly racemized acids, fails to grow in the usual media unless provided with a d-α-hydroxy acid source.