Chemoenzymatic Stereodivergent Synthesis of All the Possible Stereoisomers of the 2,3-Dimethylglyceric Acid Ethyl Ester

2,3-dihydroxy-2-methylbutyric acid, also known as 2,3-dimethylglyceric acid, constitutes the acyl and/or the alcoholic moiety of many bioactive natural esters. Herein, we describe a chemoenzymatic methodology which gives access to all the four possible stereoisomers of the 2,3-dimethylglyceric acid ethyl ester. The racemic ethyl α-acetolactate, produced by the N-heterocycle carbene (NHC)-catalyzed coupling of ethyl pyruvate and methylacetoin was employed as the starting material. The racemic mixture was resolved through (S)-selective reductions, promoted by the acetylacetoin reductase (AAR) affording the resulting ethyl (2R,3S)-2,3-dimethylglycerate; the isolated remaining (S)-ethyl α-acetolactate was successively treated with baker’s yeast to obtain the corresponding (2S,3S) stereoisomer. syn-2,3-Dimethylgliceric acid ethyl ester afforded by reducing the rac-α-acetolactate with NaBH4 in the presence of ZnCl2 was kinetically resolved through selective acetylation with lipase B from Candida antarctica (CAL-B) and vinyl acetate to access to (2S,3R) stereoisomer. Finally, the (2R,3R) stereoisomer, was prepared by C3 epimerization of the (2R,3S) stereoisomer recovered from the above kinetic resolution, achieved through the TEMPO-mediated oxidation, followed by the reduction of the produced ketone with NaBH4. The resulting 2,3-dimethylglycertate enriched in the (2R,3R) stereoisomer was submitted to stereospecicific acetylation with vinyl acetate and CAL-B in order to separate the major stereoisomer. The entire procedure enabled conversion of the racemic α-acetolactate into the four enantiopure stereoisomers of the ethyl 2,3-dihydroxy-2-methylbutyrate with the following overall yields: 42% for the (2R,3S), 40% for the (2S,3S), 42% for the (2S,3R) and 20% for the (2R,3R).

Prenatal programming of the small intestine in piglets: the effect of supplementation with 3-hydroxy-3-methylbutyric acid (HMB) in pregnant sows on the structure of jejunum of their offspring

When discussing the scale of the occurrence of diseases of the digestive system in farm animals, particularly pigs in the weaning period, it may be beneficial to study physiological and nutritional factors that could potentially affect the growth, development, and modelling of the structure and function of the digestive tract. Taking into account the reports on the beneficial effects of ß-hydroxy-ß-methylbutyrate (HMB) administration in the prenatal period on the development of various systems it was assumed that the HMB supplementation to pregnant sows can influence intestinal development in the offspring during weaning. Thus, the present experiment was conducted to evaluate the effect of HMB treatment of pregnant sows on jejunum development in offspring at weaning. From 70th day until the 90th day of gestation, sows received either a basal diet (n = 12) or the same diet supplemented with HMB (n = 12) at the dose of 0.2 g/kg of body weight/day. HMB given during prenatal time reduced the thickness of the longitudinal muscle; the apoptotic cell index in epithelium also significantly decreased after the HMB supplementation. Vasoactive intestinal (poly)peptide (VIP) expression in submucosal ganglia significantly increases in prenatally HMB treated piglets. The same strong reaction was observed with the expression of occludin, claudin-3, E-cadherin, and leptin in the jejunal epithelium. The obtained results indicate that the administration of HMB to pregnant sows significantly influenced the expression of VIP, leptin and some proteins of the intestinal barrier of their offspring less influencing the basal morphology.

Prenatal programming of the small intestine in piglets: the effect of supplementation with 3-hydroxy-3-methylbutyric acid (HMB) in pregnant sows on the structure of jejunum of their offspring

When discussing the scale of the occurrence of diseases of the digestive system in farm animals, particularly pigs in the weaning period, it may be beneficial to study physiological and nutritional factors that could potentially affect the growth, development, and modelling of the structure and function of the digestive tract. Taking into account the reports on the beneficial effects of ß-hydroxy-ß-methylbutyrate (HMB) administration in the prenatal period on the development of various systems it was assumed that the HMB supplementation to pregnant sows can influence intestinal development in the offspring during weaning. Thus, the present experiment was conducted to evaluate the effect of HMB treatment of pregnant sows on jejunum development in offspring at weaning. From 70th day until the 90th day of gestation, sows received either a basal diet (n = 12) or the same diet supplemented with HMB (n = 12) at the dose of 0.2 g/kg of body weight/day. HMB given during prenatal time reduced the thickness of the longitudinal muscle; the apoptotic cell index in epithelium also significantly decreased after the HMB supplementation. Vasoactive intestinal (poly)peptide (VIP) expression in submucosal ganglia significantly increases in prenatally HMB treated piglets. The same strong reaction was observed with the expression of occludin, claudin-3, E-cadherin, and leptin in the jejunal epithelium. The obtained results indicate that the administration of HMB to pregnant sows significantly influenced the expression of VIP, leptin and some proteins of the intestinal barrier of their offspring less influencing the basal morphology.

Novel biomarkers of habitual alcohol intake and associations with risk of pancreatic and liver cancers and liver disease mortality

BackgroundAlcohol is an established risk factor for several cancers, but modest alcohol-cancer associations may be missed due to measurement error in self-reported assessments. The identification of biomarkers of habitual alcohol intake may enhance evidence on the role of alcohol in cancer onset.MethodsUntargeted metabolomics was used to identify metabolites correlated with habitual alcohol intake in a discovery dataset from the European Prospective Investigation into Cancer and Nutrition (EPIC; n=454). Significant correlations were replicated in independent datasets of controls from case-control studies nested within EPIC (n=281) and the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC; n=438) study. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals for associations of alcohol-associated metabolites and self-reported alcohol intake with risk of pancreatic cancer, hepatocellular carcinoma (HCC), liver cancer, and liver disease mortality in the contributing studies.ResultsTwo metabolites displayed a dose-response association with alcohol intake: 2-hydroxy-3-methylbutyric acid and an unidentified compound (m/z(+):231.0839). A 1-SD increase in log2-transformed levels of 2-hydroxy-3-methylbutyric acid was associated with risk of HCC (OR=2.14; 1.39-3.31) and pancreatic cancer (OR=1.65; 1.17-2.32) in EPIC and liver cancer (OR=2.00; 1.44-2.77) and liver disease mortality (OR=2.16; 1.63-2.86) in ATBC. Conversely, a 1-SD increase in log2-transformed questionnaire-derived alcohol intake was not associated with HCC or pancreatic cancer in EPIC or liver cancer in ATBC but was associated with liver disease mortality (OR=2.19; 1.60-2.98) in ATBC.Conclusions2-Hydroxy-3-methylbutyric acid is a candidate biomarker of habitual alcohol intake that may advance the study of alcohol and cancer risk in population-based studies.

Metabolic flux analysis of Branched-chain amino and keto acids (BCAA, BCKA) and β-Hydroxy β-methylbutyric acid (HMB) across multiple organs in the pig.

Objective: Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and β-Hydroxy β-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. However, their (inter)organ kinetics remain unclear. Therefore, BCAA (leucine (LEU), valine (VAL), isoleucine (ILE)), BCKA (α-ketoisocaproic acid (KIC), 3-methyl-2-oxovaleric acid (KMV), 2-oxoisovalerate (KIV)) and HMB across organ net fluxes were measured. Methods: In multi-catheterized pigs (n=12, ±25 kg), net fluxes across liver, portal drained viscera (PDV), kidney and hindquarter (HQ, muscle compartment) were measured before and 4h after bolus feeding of a complete meal (30% daily intake) in conscious state. Arterial and venous plasma were collected and concentrations were measured by LC- or GC-MS/MS. Data are expressed as mean[95%CI] and significance (p<0.05) from zero by Wilcoxon Signed Rank Test. Results: In the postabsorptive state (in nmol/kg bw/min), the kidney takes up HMB (3.2[1.3,5.0] ). BCKA is taken up by PDV (144[13,216]) but no release by other organs. In the postprandial state, the total net fluxes over 4h (in µmol/kg bw/4h) showed a release of all BCKA by HQ (46.2[34.2,58.2]), KIC by the PDV(12.3[7.0,17.6]) and KIV by the kidney(10.0[2.3,178]). HMB was released by the liver (0.76[0.49,1.0]). All BCKA were taken up by the liver (200[133,268]). Conclusions: Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.

Biochemical Effects of Routine Gonadectomy on Blood of Domestic Ferrets (Mustela putorius furo)

We studied domestic ferrets (Mustela putorius furo) to evaluate the physiologic effects of routine surgery. Standard plasma biochemistry panels and 1H-NMR spectroscopy of heparinized whole blood were performed on samples taken 24 h prior to and immediately after surgery from female and male ferrets undergoing routine gonadectomy. Increases in plasma glucose, phosphorus, potassium, and creatine kinase concentrations associated with the duration of surgery were identified on plasma biochemistry panels. Whole-blood NMR spectra allowed us to identify 42 metabolites and one drug residue. Variations between pre- and postoperative metabolite concentrations were most pronounced for female ferrets, which underwent more prolonged surgery than males. Affected metabolites included organic acids and osmolytes (betaine, methylmalonate, <small>D</small>-lactate), fatty acids and lipids (2-hydroxy-3-methylbutyric acid), and amino acid groups (acetylglycine, alloisoleucine, leucine, and isoleucine). These findings indicate that 1H-NMR spectroscopy of whole blood provides insight into metabolic perturbations in domestic ferrets undergoing surgery that are not detected in routine clinical chemistry panels.

Structural Elucidation of Enantiopure and Racemic 2-Bromo-3-Methylbutyric Acid

Halogenated carboxylic acids have been important compounds in chemical synthesis and indispensable research tools in biochemical studies for decades. Nevertheless, the number of structurally characterized simple α-brominated monocarboxylic acids is still limited. We herein report the crystallization and structural elucidation of (R)- and rac-2-bromo-3-methylbutyric acid (2-bromo-3-methylbutanoic acid, 1) to shed light on intermolecular interactions, in particular hydrogen bonding motifs, packing modes and preferred conformations in the solid-state. The crystal structures of (R)- and rac-1 are revealed by X-ray crystallography. Both compounds crystallize in the triclinic crystal system with Z = 2; (R)-1 exhibits two crystallographically distinct molecules. In the crystal, (R)-1 forms homochiral O–H···O hydrogen-bonded carboxylic acid dimers with approximate non-crystallographic C2 symmetry. In contrast, rac-1 features centrosymmetric heterochiral dimers with the same carboxy syn···syn homosynthon. The crystal packing of centrosymmetric rac-1 is denser than that of its enantiopure counterpart (R)-1. The molecules in both crystal structures adopt a virtually identical staggered conformation, despite different crystal environments, which indicates a preferred molecular structure of 1. Intermolecular interactions apart from classical O–H···O hydrogen bonds do not appear to have a crucial bearing on the solid-state structures of (R)- and rac-1.

Exploring the Benefit of 2-Methylbutyric Acid in Patients Undergoing Hemodialysis Using a Cardiovascular Proteomics Approach

Short-chain fatty acids (SCFAs) can reduce pro-inflammatory parameters and oxidative stress, providing potential cardiovascular (CV) benefits. Although some evidence links SCFAs with host metabolic health via several biological mechanisms, the role of SCFA on CV disease in patients with kidney disease remains unclear. Herein, we investigate the association between a SCFA, 2-methylbutyric acid, and target CV proteomics to explore the potential pathophysiology of SCFA-related CV benefit in patients with kidney disease. Circulating 2-methylbutyric acid was quantified by high-performance liquid chromatography and 181 CV proteins by a proximity extension assay in 163 patients undergoing hemodialysis (HD). The associations between 2-methylbutyric acid and CV proteins were evaluated using linear regression analysis with age and gender, and multiple testing adjustment. The selected CV protein in the discovery phase was further confirmed in multivariable-adjusted models and evaluated by continuous scale association. The mean value of circulating 2-methylbutyric acid was 0.22 ± 0.02 µM, which was negatively associated with bone morphogenetic protein 6 (BMP-6) according to the false discovery rate (FDR) multiple testing adjustment method. The 2-methylbutyric acid level remained negatively associated with BMP-6 (β coefficient −1.00, 95% confidence interval −1.45 to −0.55, p < 0.001) after controlling for other CV risk factors in multivariable models. The cubic spline curve demonstrated a linear relationship. In conclusion, circulating 2-methylbutyric acid level was negatively associated with BMP-6, suggesting that this pathway maybe involved in vascular health in patients undergoing HD. However, further in vitro work is still needed to validate the translation of the mechanistic pathways.