Proteasome activity and expression of mammalian target of rapamycin signaling factors in skeletal muscle of dairy cows supplemented with conjugated linoleic acids during early lactation.

The mammalian target of rapamycin (mTOR) is a major regulator of protein synthesis via its main downstream effectors, ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein (4EBP1). The ubiquitin-proteasome system (UPS) is the main proteolytic pathway in muscle, and the muscle-specific ligases tripartite motif containing 63 (TRIM63; also called muscle-specific ring-finger protein 1, MuRF-1) and F-box only protein 32 (FBXO32; also called atrogin-1) are important components of the UPS. We investigated 20S proteasome activity and mRNA expression of key components of mTOR signaling and UPS in skeletal muscle of dairy cows during late gestation and early lactation and tested the effects of dietary supplementation (from d 1 in milk) with conjugated linoleic acids (sCLA; 100 g/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). Blood and muscle tissue (semitendinosus) samples were collected on d -21, 1, 21, and 70 relative to parturition. Dry matter intake increased with time of lactation in both groups. It was lower in sCLA than in CTR on d 21, which resulted in a reduced calculated metabolizable protein balance. Most serum and muscle concentrations of AA followed time-related changes but were unaffected by CLA supplementation. In both groups, serum and muscle 3-methylhistidine (3-MH) concentrations and the ratio of 3-MH:creatinine increased from d -21 to d 1, followed by a decline on d 21. The mRNA abundance of MTOR on d 21 and 70 was greater in sCLA than in CTR. The abundance of 4EBP1 mRNA did not differ between groups but was upregulated in both on d 1. The mRNA abundance of S6K1 on d 70 was greater in CTR than in sCLA, but remained unchanged over time in both groups. The mRNA abundance of FBXO32 (encoding atrogin-1) on d 21 was greater in sCLA than in CTR. The mRNA abundance of TRIM63 (also known as MuRF1) showed a similar pattern as FBXO32 in both groups: an increase from d -21 to d 1, followed by a decline. The mRNA for the α (BCKDHA) and ß (BCKDHB) polypeptide of branched-chain α-keto acid dehydrogenase was elevated in sCLA and CTR cows on d 21, respectively, suggesting a role of CLA in determining the metabolic fate of branched-chain AA. For the mTOR protein, no group differences were observed. The abundance of S6K1 protein was greater across all time points in sCLA versus CTR. The antepartum 20S proteasome activity in muscle was elevated in both groups compared with postpartum, probably reflecting the start of protein mobilization before parturition. Plasma insulin concentrations decreased in both groups postpartum but to a greater extent in CTR than in sCLA, resulting in greater insulin concentrations in sCLA than in CTR. Thus, the greater abundance of MTOR mRNA and S6K1 protein in sCLA compared with CTR might be mediated by the greater plasma insulin postpartum. The upregulation of MTOR mRNA in sCLA cows on d 21, despite greater FBXO32 mRNA abundance, may reflect a simultaneous activation of both anabolic and catabolic signaling pathways, likely resulting in greater protein turnover.

Glucose supplementation stimulates peripheral branched-chain amino acid catabolism in lactating dairy cows during essential amino acid infusions.

To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5 d with saline, 844 or 1,126 g/d of a complete essential AA mix, with and without the inclusion of 1,000 g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1 kg/d, milk protein by 256 g/d, milk fat by 95 g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

γ-enolase (ENO2) is methylated at the Nτ position of His-190 among enolase isozymes.

Protein methylation is mainly observed in lysine, arginine and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18 and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between the brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.

Quantification of histidine, 1-methylhistidine and 3-methylhistidine in plasma and urine by capillary electrophoresis UV-detection.

We describe a new CE method with UV-detection for the quantification of histidine (His) and its methylated forms 1-methylhistidine and 3-methylhistidine, both in plasma and urine. Analytes were basically resolved using a 60 mmol/L Tris-phosphate run buffer pH 2.2 in less than 12 min. The use of a mixture of ACN/ammonia (80:20) for protein precipitation allows the quantitative recovery of all His from plasma. The optimization of the sample volume injection permits to reach an LOD of 20 nmol/L, thus improving the sensitivity of about hundred times in comparison to the previous described assays. Moreover, the opportunity to also measure creatinine in the same run makes it possible to evaluate the renal function contemporarily, thus avoiding further dosages with significant time saving. The application method has been proved by measuring His, 1-methylhistidine and 3-methylhistidine in 44 healthy subjects. In conclusion, our new method seems to be an inexpensive, fast and specific tool to assess large numbers of patients for routine analysis both in clinical and research laboratories.

Myofibrillar proteolysis in response to voluntary or electrically stimulated muscle contractions in humans.

Knowledge about the effects of exercise on myofibrillar protein breakdown in human subjects is limited. Our purpose was to measure the changes in the degradation of myofibrillar proteins in response to different ways of eliciting muscle contractions using the local interstitial 3-methyl-histidine (3-MH) concentration as a marker for myofibrillar protein breakdown. Untrained males (n=8, 22-27 years, range) performed 210 maximal isokinetic eccentric contractions with each leg on an isokinetic dynamometer. One leg performed voluntary (VOL) and the other leg performed electrically induced contractions (ES). Microdialysis probes were placed in m. vastus lateralis in both the legs immediately after, and 1 and 3 days post-exercise. Interstitial 3-MH was higher in ES vs VOL immediately after exercise (P<0.05). One and 3 days post-exercise no difference between the two exercise types was observed. Only after ES did the histochemical stainings show significant disruption of cytoskeletal proteins. Furthermore, intracellular disruption and destroyed Z-lines were markedly more pronounced in ES vs VOL. In conclusion, the local level of interstitial 3-MH in the skeletal muscle was significantly enhanced after ES compared with VOL immediately after exercise, while the level of 3-MH did not change in the post-exercise period after VOL. These results indicate that the local myofibrillar breakdown is accelerated after ES associated with severe myofiber damage.

Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.

Validation of a Method for the Determination of Balenine/Ophidine in Whale.

The aim of this study was to develop and validate a method for the determination of balenine/ophidine (hereafter referred to as "balenine") in whale extracts and muscle samples from Balaenoptera acutorostrata. Further, the goal was to evaluate the method's applicability for the determination of other histidine-containing dipeptides (HCDs): anserine and carnosine and their amino acids π-methylhistidine, τ-methylhistidine, histidine, and ß-alanine. For balenine, the LOD and LOQ were found to be 0.03 and 0.1 mg/g, respectively, and the linear range was validated up to 160 mg/g. Trueness was evaluated by spiking experiments with balenine, and the recovery was found to be 88-90%. A comparison of the results showed that most of the other analytes were within 80-120% of the value found with the previously developed and validated method. Precision and internal reproducibility for balenine was around 0.9 and 2%, respectively, with measurement uncertainties of 2-4%. Therefore, the method was found to be fit for purpose for the determination of balenine and other HCDs and their constituent amino acids in whale meat and extracts.

The effect of four different feeding regimens from rearing period to sexual maturity on breast muscle protein turnover in broiler breeder parent stock.

A study was conducted to evaluate the effect of four different feeding regimens on breast muscle protein turnover in broiler breeder Cobb-500 parent stock (PS) pullets and breeder hens. The four feeding regimens based on BW curves utilized for the study were as follows: Everyday feeding (STD-ED) (Cobb Standard BW curve), skip-a-day feeding (STD-SKIP) (Cobb Standard BW curve), lighter BW (LBW-SKIP) (BW curve 20% under), and heavier BW (HBW-SKIP) (BW curve 20% over). Each feeding regimen was provided to pullets from 4 wk to 21 wk of age. Protein turnover was determined in PS pullets/breeders at 6, 10, 12, 16, 21, 25, 31, 37, 46, and 66 wk of age. A completely randomized design was used with a 4 × 10 factorial arrangement (four feeding regimens, 10 ages), each pullet represented a replicate. Five pullets/breeders at each age were given an intravenous flooding-dose of 15N-Phe (15N phenylalanine 150 mM, 40 APE (atom percent excess)) at a dose of 10 mL/kg BW for the determination of fractional synthesis rate (FSR). After 10 min, birds were euthanized and the breast muscle (pectoralis major) excised for protein turnover and gene expression analysis. Excreta was collected from each pullet or breeder for 3-methylhistidine (3-MH) analysis. No feeding regimen affected protein turnover. There was an age effect for breast muscle FSR. The FSR in breast muscle of pullets significantly increased from 6 wk to 12 wk and then decreased significantly for 31 wk-old breeders. FSR in breeder breast muscle increased significantly from 31 wk to 66 wk. There was an age effect for breast muscle fractional breakdown rate (FBR). FBR in breast muscle significantly increased from 21 wk to 25 wk and 31 wk (peak egg production), then significantly decreased at 66 wk. The expression of the genes related to protein degradation (Atrogin-1, MURF-1) in breast muscle was significantly higher at peak egg production. Protein turnover in skeletal muscle tissue is believed to be a source of nutrients for egg production.

Concentrations of carnosine, anserine, L-histidine and 3-methyl histidine in boar spermatozoa and sheep milk by a modified HPLC method.

The present study deals with the application of high-performance-liquid-chromatography (HPLC) method for a quantitative detection of carnosine, anserine, L-histidine and 3-methyl-L-histidine in biological material with o-phthaldialdehyde (OPA) post column derivatisation at the constant temperature of 50 degrees C. For this purpose, some mobile-phases were prepared with scalar acetonitrile concentrations. A complete separation of all molecules, particularly for carnosine and 3-methyl-L-histidine, was obtained with a solution of acetonitrile and 6mM hydrochloric acid with 0.48 M sodium chloride (5%:95% v/v). Post column derivatisation reaction at temperature of 50'C permitted to obtain an increase in sensibility of all molecules. This method has been utilised for detection of histidine dipeptides in boar spermatozoa and in sheep milk. Concentrations (mean +/- S.E. nmol/10(9) spermatozoa) of carnosine (0.96 +/- 0.14) and anserine (0.83 +/- 0.18) in boar spermatozoa were significantly lower than those of L-histidine (52.85 +/- 4.86) and 3-methyl-L-histidine (83.07 +/- 7.1). Positive correlation was found between carnosine and anserine contents (r = 0.740; p < 0.01) and between L-histidine and 3-methyl-L-histidine (r = 0.657; p < 0.01). All histidine dipeptides studied were also present in 40 samples of sheep milk. In a case of samples without unit-forming colonies (UFC) of Staphylococcus coagulase-positive, carnosine concentrations (9.17 +/- 0.89 nmol/ml) were higher than anserine (0.51 +/- 0.02 nmol/ml) and both were significantly lower in respect to L-histidine (49.51 +/- 6.48 nmol/ml) and 3-metyl-L-histidine (81.21 +/- 6.82 nmol/ml). A negative correlation was observed between carnosine milk levels (r = -0.773; p < 0.01) and UFC/ml of Staphylococcus coagulase-positive. In conclusion this very simple and fast method can be used to detect histidine dipeptides in biological compartments where their concentrations are very low.

Ntau-methylhistidine content of mixed proteins in various rat tissues.

In order to use Ntau-methylhistidine (3-methylhistidine) excretion in the urine as a measure of muscle protein breakdown, it is necessary to demonstrate that other tissues are not important sources of this protein constituent. Accordingly, the concentration of Ntau-methylhistidine in blood serum and in the mixed proteins of heart, brain, lung, kidney, diaphragm, spleen, testis, stomach, liver and hind leg skeletal muscle was measured in male rats of approx. 400 g body weight. The free Ntau-methylhistidine concentration of rat serum was less than 2 nmol per ml. In contrast, measurable amounts of Ntau-methylhistidine were found in the mixed proteins of all tissues and organs examined. The highest concentration was found in skeletal muscle (658 nmol/g tissue). Assuming muscle mass to be 45% of body weight, it has been estimated that the muscle contains more than ten times the total amount of this amino acid present in all of the other organs analyzed, which together account for about 20% of total body weight. These findings indicate that skeletal muscle is likely to be the major source of urinary Ntau-methylhistidine and the latter is, in consequence, a reflection of myofibrillar protein breakdown in skeletal muscle.

Detection, characterisation, and quantification of carnosine and other histidyl derivatives in cardiac and skeletal muscle.

Isocratic reverse phase analytical high performance liquid chromatography (HPLC) has been used to examine naturally occurring imidazoles of cardiac and skeletal muscles. Elution of muscle extracts with a phosphate buffer mobile phase from columns packed with hypersil ODS (5 micron) resulted in good separation of the skeletal muscle imidazole-containing dipeptides carnosine and anserine. Measured concentrations corresponded to published values. N-Acetyl forms that were not commercially available were prepared from their parent compounds and their identities verified by NMR-spectroscopy. Examination of frog cardiac muscle confirmed the presence of N-acetylhistidine and also indicated the presence of its 1-methyl derivative. Extracts of mammalian cardiac muscle were examined by HPLC which indicated the presence of low concentrations of carnosine but substantial amounts of N-acetyl forms of histidine, 1-methylhistidine, carnosine and anserine. Fractions corresponding to the numerous peaks were examined using staining systems specific for certain chemical features and compared to results obtained for commercial or synthetic standards. Results of these tests supported the chromatographic data. The total concentrations in cardiac muscle of these imidazole-containing substances (approx. 10 mM) is sufficient to alter significantly the sensitivity of their contractile apparatus to calcium ions.

Myofibrillar protein breakdown in skeletal muscle is diminished in rats with chronic streptozocin-induced diabetes.

Previous reports have suggested that insulin may not regulate the breakdown of myofibrillar proteins in skeletal muscle. To further test the role of insulin, insulinopenia was produced by treating rats with streptozocin. After treatment, protein breakdown in skeletal muscle was evaluated with the isolated perfused rat hindquarter preparation. After the inhibition of protein synthesis with cycloheximide, total and myofibrillar protein breakdown were assessed by measuring the release of tyrosine and 3-methylhistidine, respectively, in the perfused hindquarters of diabetic and age-matched control rats. Streptozocin-induced (65 mg/kg) diabetes (3- to 28-day duration) resulted in hyperglycemia, hypoinsulinemia, hyperphagia, increased plasma lipid levels, arrested body and muscle growth, and increased urea and 3-methylhistidine excretion. Despite this, protein breakdown in skeletal muscle diminished. The release of 3-methylhistidine by the perfused hindquarters of diabetic rats decreased, whereas the release of tyrosine remained unchanged, suggesting that the breakdown of myofibrillar proteins was affected specifically. 3-Methylhistidine (unbound) levels in skeletal muscle of unperfused diabetic rats as well as in skin decreased, whereas they increased twofold in the gastrointestinal tract. More severe diabetes (125 mg/kg streptozocin), which resulted in ketoacidosis, augmented protein breakdown in muscle; however, this response was due to a marked fall in food consumption (it was also evident when control rats were pair fed). These data reinforce previous conclusions that insulin does not play a major role in the regulation of myofibrillar protein breakdown in skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence of Acanthamoeba actin.

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.

Effects of fasting and refeeding on expression of proteolytic-related genes in skeletal muscle of chicks.

This experiment was conducted to study the effects of fasting and refeeding on proteolytic-related gene expression in skeletal muscles of chicks. Chicks were fasted for 24 h, and refed for 2 h. Plasma Ntau-methylhistidine concentration, as an index of myofibrillar protein degradation, was increased by fasting, and that increment was reduced by refeeding. We also examined the expression of the protease mRNAs (calpain, proteasome, cathepsin and caspase-3) by real-time PCR of cDNA in skeletal muscles of fasting and refeeding chicks. Calpain (m-, mu-, and p94/calpain-3) mRNA expressions were also increased by fasting, and their increment was reduced by refeeding. Ubiquitin and 20S proteasome alpha subunit (alpha6 and alpha7) mRNA expressions as well as cathepsin B, and caspase-3 mRNA expression were likewise increased by fasting, with their increment also reduced by refeeding. These results indicate that fasting stimulates proteolytic-related gene expression, resulting in an increase in myofibrillar protein degradation, and that refeeding suppresses proteolytic-related gene expression, resulting in a decrease in myofibrillar protein degradation in chicks.

3-Methylhistidine content and pH dependency of ATPase activity of adult and embryonic chicken cardiac ventricular myosins.

A distinct difference in the 3-methylhistidine (3-MeHis) content and the pH-dependency curve for calcium-activated adenosine triphosphatase (Ca-ATPase) activity was observed between chicken and mammalian cardiac ventricular myosins. The 3-MeHis content and pH dependency of the Ca-ATPase activity of myosins from adult and embryonic chicken cardiac ventricular muscles and chicken fast white and slow red muscles were almost the same.

Musle protein breakdown rates in humans based on Ntau-methylhistidine (3-methylhistidine) content of mixed proteins in skeletal muscle and urinary output of Ntau-methylhistidine.

Samples of psoas muscle from nine infants (aged 1 day to 14 mo) and of several skeletal muscles from seven adult males (age 19-74 yr) were analyzed for content of protein-bound Ntau-methylhistidine (3-methylhistidine; 3-Mehis). The mean content of 3-Mehis (expressed as mumoles/g mixed protein) was 3.2 (range 2.4-3.7) in infants and 4.2 (range 3.7-4.6) in adults. The daily urinary excretion of 3-Mehis was measured in four young adult males receiving an egg-protein, flesh-free diet. Mean excretion of 3-Mehis was 211 (range 167-252) mumoles/day. From these two sets of data the mean rate of muscle protein breakdown in adult males was estimated to be 50 g/day, or 0.7 +/- 0.1 g/kg body weight/day. These results are compared with reported values for the 3-Mehis content of mixed proteins in muscle of various species, and with published estimates, computed by other techniques, of the rate of muscle protein breakdown in human subjects.

Effects of cimaterol, a beta-adrenergic agonist, on protein metabolism in rats.

Fractional accretion rates of total body 3-methylhistidine containing proteins (actin and myosin) were elevated 40% to 120% in rats fed a high-carbohydrate diet containing 10 or 100 ppm cimaterol for 1 week. Fractional degradation and fractional synthesis rates of these proteins were examined by measuring total body 3-methylhistidine content and urinary excretion of 3-methylhistidine. Consumption of the diet containing 100 ppm cimaterol for 1 week caused a 25% reduction in fractional degradation rates and a concomitant 32% increase in fractional synthesis rates of 3-methylhistidine containing proteins. Effects of cimaterol on fractional accretion, degradation, and synthesis rates of 3-methylhistidine containing proteins diminished after 1 week. Cimaterol failed to influence plasma insulin, triiodothyronine, or corticosterone concentrations. The dramatic increase in accretion of 3-methylhistidine containing proteins observed during the first week rats are fed diets containing cimaterol is caused by reciprocal action on protein degradation and synthesis.

Contractile protein breakdown in human leg skeletal muscle as estimated by [2H3]-3-methylhistidine: a new method.

3-Methylhistidine urinary excretion and net balances across the leg or forearm have been used as markers of contractile protein breakdown in muscle tissue. Here we investigate whether infusion of labeled 3-methylhistidine and the measurement of the arteriovenous dilution of the tracer with unlabeled 3-methylhistidine will result in more consistent and precise measurements of 3-methylhistidine rates of appearance and consequently muscle contractile protein breakdown rates in comparison with conventional arteriovenous concentration difference measurements. Six healthy volunteers were studied in the postabsorptive state and received a primed continuous infusion of 3-[2H3-methyl]- methylhistidine and L-[ring-2H5]-phenylalanine for 4 hours. 2H3-3-methylhistidine reached an isotopic steady state after 210 minutes in all subjects. Arteriovenous differences of 3-methylhistidine, measured by high-performance liquid chromatography (HPLC), showed both uptake and release from skeletal muscle, which is theoretically not likely to occur. The enrichment of 2H3-3-methylhistidine was consistently lower in the femoral vein than in the artery, and therefore a constant net release of 3-methylhistidine from the leg was observed. The mean rates of appearance for 3-methylhistidine and phenylalanine were 0.44 +/- 0.30 nmol x min(-1) x 100 mL(-1) and 11.2 +/- 5.7 nmol x min(-1) x 100 mL(-1), respectively. In summary, arteriovenous difference measurement of 2H3-3-methylhistidine enrichment is more reliable than measurement of arteriovenous difference of unlabeled 3-methylhistidine. Consequently, measuring rates of appearance from leg muscle using labeled 3-methylhistidine resulted in more consistent and accurate values of contractile protein degradation rates in human skeletal muscle.

Total and myofibrillar protein breakdown in different types of rat skeletal muscle: effects of sepsis and regulation by insulin.

Proteolysis is increased in sepsis, but it is not known whether myofibrillar and non-myofibrillar proteins are broken down in the same fashion, or respond to the same regulatory forces as in non-septic muscle. In this study, therefore, the effect of sepsis on total and myofibrillar protein breakdown in incubated rat extensor digitorum longus (EDL) and soleus (SOL) muscles was determined, and the response in vitro to different concentrations of insulin (10 to 10(5) microU/mL) of protein degradation was studied in incubated EDL muscles from control and septic rats. Sepsis was induced in rats weighing 40 to 60 g by cecal ligation and puncture (CLP). Control animals were sham operated. Sixteen hours after CLP or sham operation, intact EDL and SOL muscles were incubated for two hours in oxygenated Krebs-Henseleit bicarbonate buffer containing glucose (10 mmol/L) and cycloheximide (0.5 mmol/L), and total and myofibrillar protein breakdown was assessed from release into incubation medium of tyrosine and 3-methylhistidine (3-MH), respectively. Tyrosine and 3-MH were determined fluorometrically by high performance liquid chromatography (HPLC). Tissue levels of tyrosine and 3-MH remained stable both in control and septic muscles during incubation for two hours. The rate of tyrosine release was increased during sepsis by 58% (P less than .001) and 15% (NS) in EDL and SOL muscle, respectively. The corresponding figures for 3-MH were 103% (P less than .001) and 21% (NS). Tyrosine release was reduced by insulin at a concentration of 10(3) microU/mL in control muscle and at a concentration of 10(4) microU/mL in septic muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of amniotic fluid 3-methyl histidine to creatinine molar ratio for the diagnosis of intrauterine growth retardation.

To determine if the amniotic fluid 3-methyl histidine to creatinine molar ratio (3MH:CR) could prove useful for the antepartum detection of intrauterine growth retardation (IUGR), the 3MH:CR was determined retrospectively in 3 groups of human amniotic fluids. Group A consisted of amniotic fluids from pregnancies yielding IUGR fetuses whose birth weight was less than or equal to the tenth percentile for gestational age; group B consisted of amniotic fluid from pregnancies yielding infants whose birth weight was greater than the tenth but less than or equal to the 25th percentile for gestational age; group C consisted of amniotic fluids from pregnancies yielding infants whose birth weight was greater than the 25th but less than or equal to the 75th percentile for gestational age. The mean 3MH:CR x 10(-3) for groups A, B, and C were 15.9 +/- 1.9, 5.4 +/- 0.8, and 6.2 +/- 0.5, respectively. The mean 3MH:CR x 10(-3) was statistically different between groups A and B (P less than or equal to .001) and between groups A and C (P less than or equal to .001), but not statistically different between the 2 control groups. Employing an upper limit of normal of 8 for the 3MH:CR x 10(-3), 13 of 15 IUGR neonates were correctly identified as IUGR, and 23 of 27 neonates were correctly identified as being of normal birth weight for gestational age (sensitivity 86.7%, specificity 85.2%, incidence of correct diagnosis 85.7%). No consistent relationship was shown to exist between maternal serum and amniotic fluid 3-methyl histidine level. There was no statistically significant relationship between 3MH:CR x 10(-3) and gestational age. The comparison of the data generated in this study to that obtained with previously reported ultrasonic and biochemical techniques suggests that the amniotic fluid 3MH:CR ratio may prove helpful in establishing the antenatal diagnosis of IUGR, particularly in cases where the gestational age is uncertain.