Acute effects of oral mesna administration on the full amino acid profile and 3-methylhistidine: secondary results from the CYLOB dose-finding study.

Plasma total cysteine (tCys) is strongly associated with fat mass in humans. Mesna lowers plasma tCys in a dose-dependent manner, but it is not known whether it interferes with metabolism of other amino acids or protein. In this Phase-1 study, we show that a single dose of mesna administered at 400, 800, 1200 or 1600 mg to 6-7 individuals per dose only slightly affects amino acid profiles, with increases in plasma valine across dose levels. There were no effects of mesna on 3-methylhistidine, a marker of protein breakdown.

Iridium(III) solvent complex-based electrogenerated chemiluminescence method for the detection of 3-methylhistidine in urine.

3-Methylhistidine (3-MeHis) is increasingly used as an indicator of muscle protein breakdown. The development of a sensitive, simple, and non-invasive method for 3-MeHis assay is important in clinical practice. Herein, a sensitive, simple, and non-invasive electrogenerated chemiluminescence (ECL) method was proposed for the quantitation of 3-MeHis in urine by using an iridium(III) solvent complex ([Ir(dfppy)2(DMSO)Cl], dfppy = 2-(2,4-difluorophenyl)pyridine, Ir-DMSO) as a signal reagent. The photoluminescence (PL) and ECL responses of Ir-DMSO to 3-MeHis were studied. The ECL intensity of Ir-DMSO was enhanced in the presence of 3-MeHis because of the coordination recognition between Ir-DMSO and the imidazole group of 3-MeHis. Based on the enhancement of ECL intensity, 3-MeHis can be sensitively detected in the range of 5 to 25 µM. The detection limit was 0.4 µM. This is the first report of an ECL method for the quantitation of 3-MeHis. Further, to investigate the feasibility of the Ir-DMSO-based ECL method in practical applications, the developed ECL method was applied for 3-MeHis assay in urine samples of 28 healthy volunteers and 2 patients. The urine samples from patients hospitalized with obesity and kidney disease and healthy individuals were distinguished by the ECL responses of Ir-DMSO. The proposed ECL method based on the coordination recognition between iridium(III) solvent complex and the imidazole group of 3-MeHis allows inexpensive, fast, non-invasive, and sensitive detection of 3-MeHis in urine, which is promising for assessing large volumes of patients for routine analysis in clinical practices.

Variation in protein metabolism biomarkers during the transition period and associations with health, colostrum quality, reproduction, and milk production traits in Holstein cows.

The aims of this study were to assess (1) the variation of protein metabolism biomarkers and factors affecting them during the transition period, (2) the association of each biomarker with skeletal muscle reserves and their changes, and (3) the association of these biomarkers with postpartum health, colostrum quality, reproduction, and milk production. For this purpose, 238 multiparous Holstein cows from 6 herds were used in a prospective cohort study. Plasma concentrations of 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH) and serum concentrations of total protein (TP), albumin (ALB), urea nitrogen (BUN), and creatinine (SCR) were determined for each cow at -21, -7, 7, 21, and 28 d relative to calving. Clinical diseases were recorded during the first 28 d postcalving, and presence of subclinical ketosis (scKET) was investigated at 7 and 21 d. Colostrum quality was estimated by Brix refractometry. Reproduction data by 150 d in milk (DIM) and milk production records were also available. Linear mixed models including the fixed effects of time point, herd, parity, body condition score (-21 d), duration of dry period and postparturient diseases were fitted to assess the variation in each biomarker's concentration. The association between the biomarkers' concentration during the prepartum period with the odds for each postparturient disease and for a combined trait (CD_1-28), defined as the presence of at least one clinical condition during the first 28 d after calving, were assessed with separate binary logistic models for time points -21 d and -7 d. The relationship of each biomarker's concentration with longissimus dorsi thickness (LDT) and the changes in LDT (ΔLDT) was assessed with pairwise correlations. Separate general linear models were used to assess the association of each biomarker with colostrum Brix values and milk production traits. Finally, the associated hazard for first artificial insemination (AI) and for pregnancy by 150 DIM (PREG_150DIM) was assessed with Cox proportional hazard models, whereas odds for pregnancy to the first AI (PREG_1stAI) were assessed with binary logistic models. The level of 3-MH was affected mainly by herd, time points, and their interaction. Higher 3-MH was associated with increased odds for metritis and CD_1-28, increased hazard for PREG_150 DIM and with increased milk production. 1-Methylhistidine was affected mainly by herd, scKET and occurrence of displaced abomasum. Higher 1-MH was associated with better colostrum quality, increased odds for scKET, increased hazard for first AI by 150 DIM and with decreased milk production. Both 3-MH and 1-MH were weakly to moderately negatively correlated with LDT and moderately to strongly negatively correlated with ΔLDT at the corresponding time periods. Additionally, higher TP was associated with increased odds for metritis and CD_1-28 and increased milk production, while higher ALB was associated with increased odds for scKET and increased milk production. Moreover, higher BUN was associated with decreased odds for scKET, increased odds for PREG_1stAI and increased milk production. Higher SCR was associated with decreased odds for retained fetal membranes, metritis, and CD_1-28. Periparturient protein metabolism is significantly associated with postpartum health, colostrum quality, reproduction, and milk production; mechanisms involved require further investigation.

A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.

Effects of pre-slaughter fasting on antemortem skeletal muscle protein degradation levels and postmortem muscle free amino acid concentrations in broiler chickens.

This study investigated the effects of pre-slaughter fasting time on the relationship between skeletal muscle protein degradation levels at slaughter and chicken meat quality after 48 h of postmortem aging. Twenty-four broiler chicks at 0 d of age were used in this study until 28 d of age. At 27 d of age, the chickens were assigned to 4 treatment groups: 0 h of fasting (0H), 8 h of fasting (8H), 16 h of fasting (16H), or 24 h of fasting (24H). They were slaughtered at 28 d of age. Blood samples were collected before fasting and immediately before slaughter. Plasma Nτ-methylhistidine concentration, an index of skeletal muscle protein degradation level, and muscle free amino acid concentration were analyzed. Antemortem changes in individual plasma Nτ-methylhistidine concentrations were significantly increased in 8H, 16H, and 24H compared to that in 0H (P < 0.05). After 48 h of postmortem storage, the glutamic acid content in the pectoralis major muscles increased with fasting time (P < 0.05), and the umami taste of chicken soup in the fasting groups (8H, 16H, 24H) was higher than that in the 0H group (P < 0.05). The antemortem changes in plasma Nτ-methylhistidine concentrations were correlated with glutamic acid content in the pectoralis major muscles (r = 0.57, P < 0.05) and umami taste (r = 0.66, P < 0.05). These results suggest that skeletal muscle protein degradation levels at slaughter are related to postmortem chicken meat quality, especially glutamic acid content and umami taste.

Effects of branched-chain amino acids on muscles under hyperammonemic conditions

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1-14C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and alfa-ketoglutarate (alfa -KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in alfa-KG found in the NH3 group. We conclude that (I) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and alfa-KG levels and (II) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in alfa-KG

Growth hormone improves graft mucosal structure and recipient protein metabolism in rat small bowel transplantation / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To observe the effects of recombinant human growth hormone (rhGH) on graft structure and recipient protein metabolism in rat small bowel transplantation (SBT) and total parenteral nutrition (TPN) models.</p><p><b>METHODS</b>Twenty recipients of rat allogeneic heterotopic small bowel transplants (SD-->Wistar) were divided into two groups (GH group and control group). Both groups were supported by standard TPN. Acute rejection was suppressed with CsA 10 mg x kg(-1) x d(-1) intramuscularly. All rats in the experimental group received subcutaneous rhGH 1 U x kg(-1) x d(-1) after transplantation. Morphological mucosal indices of transplanted gut and metabolic parameters such as body weight, nitrogen balance, urinary 3-methyl histidine excretion and serum albumin of the recipients were compared between two groups.</p><p><b>RESULTS</b>The application of rhGH promoted graft recovery significantly compared with standard TPN support alone. On postoperative day 14, all morphological indexes of transplanted gut recovered to the preoperative state. Protein metabolism in the recipient was also significantly improved. rhGH decreased the catabolism of protein, accelerated regaining of positive nitrogen balance and corrected hypoalbuminemia.</p><p><b>CONCLUSION</b>GH is an effective metabolic intervention in SBT. It may promote the structural repair of the graft and correct the metabolic disturbance. It is useful in improving the outcome of clinical SBT.</p>

Whole-body protein turnover and nitrogen balance in young children at intakes of protein and energy in the region of maintenance

Nitrogen balance and whole-body protein turnover were measured in children aged about one year taking diets which provided 1.7 or 0.7 g milk protein/kg/d at three levels of metabolizable energy, 80, 90 and 100 kcal/kg/d. All the children were in positive nitrogen balance at all levels of energy intake on 1.7 g protein/ kg/d. Nitrogen equilibrium was maintained on 0.7 g protein/kg/d when the energy intake exceeded 90 kcal/kg/d, but on 80 kcal/kg/d nitrogen balance was negative. Whole-body protein turnover was measured from the enrichment in urinary ammonia following a continuous infusion of15N-glycine. The variation between individuals on the same diet was significantly greater than the variation within individuals at different levels of energy intake. For the group as a whole protein synthesis on 1.7 g protein/kg/d was 0.74, 0.75 and 0.87 g N/kg/d on 100, 90 and 80 kcal/kg/d respectively;whereas on 0.7 g protein/kg/d it was 0.37, 0.38 and 0.40 g N/kg/d. These results show that over this range of intakes protein synthesis decreased as dietary protein fell, but tended to increase as energy intake fell. (AU)