用阳离子交换树脂测定尿三甲基组氨含量

因3-甲基组氨酸(3MH)从肌肉蛋白降解后,不重新参加蛋白质的合成,而从尿中排出,所以把它作为肌肉蛋白质分解的可靠指标。本文以Radha和Bess-man在1982年建立,Fitch等在1986年改进的阳离子交换树脂法。以大部国产试剂,对柱长、内径、pH等作了适当的改进,得到了满意的测定3MH的结果。从0.15到1.0μMol的三点标准曲线与Fitch等的相似,此法重复性好,回收率高,测定范围广,是较为简单,可推行的方法。用此法测定了烧伤和正常鼠尿3MH的含量,烧伤组14.5%Cv和对照组13.0%Cv,显示大鼠每天尿中排出的3MH含量比较稳定,说明烧伤和正常鼠肌肉蛋白质的分解比较恒定。烧伤组鼠尿3MH的每天排出量高于正常组35%以上,表明烧伤鼠肌肉蛋白质的分解速率高于对照组。     

Measurement of urinary 3-methylhistidine with cationic-exchange resin

A simple method is presented for measurement of urinary 3-methylhistidine (3MH) using a cationic exchange resin treatment followed by colorimetric analysis. Equations are given to correct for the interference by histidine (4.3% by mole) in the colorimetric analysis. This correction is especially important for measurement of urinary 3MH in pregnant women or in other subjects with elevated histidine excretion. Good recovery of added standard and good reproducibility of results are documented. Preliminary data from a study of pregnant women are reported, suggesting an increased excretion of 3MH during pregnancy. Large day-to-day variability of 3MH excretion was observed within subjects. It is recommended that repeated measurements be done on each subject when determining 3MH excretion.     

Biological activity and the 3-methylhistidine content of actin and myosin

1. The 3-methylhistidine content of myosin varies according to muscle type. It is highest in myosin from white skeletal muscle and lower values are obtained from myosin of red skeletal and smooth muscle. 2. The 3-methylhistidine content of actin was similar in all of the types of muscle from which it was isolated. 3. The 3-methylhistidine of rabbit actin is localized in a single tryptic peptide that was readily modified during fractionation procedures. 4. Photo-oxidation studies indicated that the 3-methylhistidine residues are not essential for adeonsine triphosphatase and actin-combining activities of myosin. 5. During photooxidation G-actin lost completely the ability to polymerize to the F form before all the 3-methylhistidine was destroyed.     

A practical and reliable method for determination of urinary 3-methylhistidine

A practical and reliable semiautomated method for analysis of urinary 3-methylhistidine (3-MH) was designed combining the isolation of 3-MH by ion-exchange chromatography with the color reaction given by ninhydrin-orthopthalaldehyde (ninhydrin-OPT) reagent after alkalinization. 2 ml of urine were passed through disposable columns packed with an ion-exchange resin (Dowex 50-X8, 200–400 mesh) and the acidic and neutral amino acids were eluted with 10 ml of 0.2 M pyridine solution. Then, the 3-MH was quantitatively eluted and separated from histidine with a volume of 9 ml of a 1.5 M pyridine solution. Standard Autoanalyzer equipment was used for the automation of spectrophotometry. The method permits the analysis of 40 samples in duplicate per day. The 3-MH color reaction was linear for concentrations from 0.015 to 0.24 μ mol/ml. The mean recoveries of 3-MH from standards and urine were 98.6 ± 1.3 and 99.0 ± 1.3%, respectively. Duplicate determinations of urine samples showed a variation coefficient of 1.88%. An excellent agreement was obtained between urine samples analyzed by the present method and by an amino acid analyzer. The need for the elimanation of the interfering amino acids was clearly demonstrated.     

Analysis of the amino acid 3-methylhistidine by gas-liquid chromatography

A method for the quantitative separation of 3-methylhistidine from other amino acids, by gas-liquid chromatography, has been developed. This method gives complete resolution of the N-heptafluorobutyryl isobutyl esters of 20 amino acids with the use of a single column packed with 3% SE-30 on $\text{100}\text{120}$ mesh Gas Chrom Q. Using this method the 3-methylhistidine content of urine and meat has been determined.     

Quantitative gas chromatographic determination of 3-methylhistidine in biological fluids

The gas chromatographic procedure is suggested to determine 3-methylhistidine in biological fluids. The amino acid fraction containing 3-methylhistidine is separated by ion-exchange chromatography. Amino acids are transformed into N-trifluoroacetyl-O-isobutyl esters which are analyzed by the gas chromatography instrument with micropacked columns and ionization-resonance detector. The limit of the quantitative determination of 3-methylhistidine is 50 ng per a probe.     

High-performance liquid chromatographic determination of imidazole dipeptides,histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres

The combined solid-phase extraction (Isolute PRS columns) and reversed-phase gradient HPLC method presented provides a sensitive, reproducible and selective quantification of carnosine, anserine, balenine, homocarnosine, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres. Recoveries were 91–115%. Lower limits of detection were 0.005–0.010 mmol kg⁻ dry muscle. The compounds were isolated from other physiological amino acids and small peptides and resolved within a single chromatographic run of 55 min. Concentrations of these compounds in equine myocardium, diaphragm, skeletal muscle, camel muscle and individual muscle fibres of both species are presented for the first time.     

Effect of swimming on protein degradation: 3-methylhistidine and creatinine excretion

The effect of 5-km noncompetitive swimming (moderate exercise) and 2-km competitive speed swimming (intensive exercise) on protein breakdown was studied in a group of young male volunteers (16-20 years old) who followed a 3-MH-free diet throughout the study. Urinary 3-MH and creatinine were determined over a period of 24 and 48 hr as an index of protein degradation. Basal 3-MH levels in the two groups of swimmers were 2.85 and 3.07 mumole X kg-1 X day-1. Mean rates of 3-MH excretion were, respectively, 1.54 and 1.94 mumole X kg-1 X day-1 for the 48 hr after moderate exercise and the 24 hr after intensive exercise. The decrease in 3-MH urinary excretion was still evident when calculated as the urinary 3-MH-to-creatinine ratio.     

Eimeria acervulina infection elevates plasma and muscle 3-methylhistidine levels in chickens

To assess muscle breakdown during avian coccidiosis, the level of the nonmetabolizable amino acid 3-methylhistidine (3MH) was determined in muscle, plasma and excreta from chickens infected with Eimeria acervulina. The changes in 3MH levels during infection were assessed at 1-29 days postinoculation (DPI) in animals given 5 x 10(5) oocysts per bird. The effect of levels of parasitism were evaluated at 8 DPI in birds receiving 5 x 10(3), 5 x 10(4), 5 x 10(5) or 1 x 10(6) oocysts each. The 3MH levels of plasma, muscle, and excreta samples were determined by high-pressure liquid chromatography after derivatization with fluorescamine. Weight gains, breast muscle weight, eviscerated weight, plasma carotenoid levels, dry weight of muscle, and gross lesion scores were also determined. Infected birds had significantly elevated plasma and muscle 3MH at 4 and 8 DPI following a single dose of E. acervulina. The increase in 3MH levels had an inverse relationship with the time course of weight gain and plasma carotenoid levels. Plasma and muscle 3MH levels returned to control values by 15 DPI and remained unchanged from control values through the remainder of the experiment (29 DPI). Breast weight was decreased in infected birds, but the ratio of breast weight to eviscerated body weight was unchanged. Excretion of 3MH decreased relative to controls at 4 and 8 DPI and returned to control levels on 15 DPI. The plasma and muscle levels of 3MH were related to severity of infection; however, levels of excreted 3MH were not. The results suggested that muscle breakdown, as assessed by plasma and muscle levels of 3MH, increased during the acute stage of E. acervulina infection. The underlying causes for this muscle breakdown was unclear but could involve a physiological response to anorexia and decreased food intake during the acute phase of infection. Levels of excreted 3MH did not increase during infection and this may be the result of decreased excreta output during infection. Plasma and muscle levels of 3MH were correlated with severity of E. acervulina infections but may not be as sensitive an indicator of infection as plasma carotenoid levels or other physiological parameters.     

Simple and rapid high-performance liquid chromatographic method for the quantification of 3-methylhistidine

A procedure for the high-performance liquid chromatographic determination of tobramycin in serum is described using pre-column derivatisation with 1-fluoro-2,4-dinitrobenzene and subsequent chromatographic analysis on a reversed-phase column with ultraviolet detection. Gentamicin is used as the internal standard. The sensitivity is 0.5 mg/l with 50-μl samples. Precision, expressed as the coefficient of variation, is 3% or better in the concentration range 0.5–16 mg/l. The absolute recovery of tobramycin is 41%.The analyses of serum samples obtained in an in vivo experiment correlated well with the results from a microbiological assay. The influence of variation of derivatisation conditions and the implications for the reliability of the internal standardisation were studied. The 2,4-dinitrophenyl tobramycin derivative was synthesized and its structure was proved to be the fully derivatized tobramycin. Side-products of the derivatisation reaction were isolated.     

Urinary 3-methylhistidine derivative as indicator of nutrients intake in low-birth-weight infants

Urinary excretion levels of N-methylhistidine derivatives and N-methylhistidine/creatinine ratios were studied in a group of 20 small for date newborns, 10 premature infants and 8 normal infants, at birth and at one week of life. All infants were fed with an adapted milk formula supplying 2.8 g protein/kg body weight. 1-methyl and 3-methylhistidine urinary excretion were increased in all groups of infants from birth to the 7th day of life. Creatinine and N-methyl derivatives/creatinine ratios were also significantly increased at one week of life. The two ratios showed a higher level in small for date and premature infants than in normal infants at birth which continued relatively increased at one week of life. 3-methyl-histidine/creatinine ratio appears as a useful indicator of the turnover rate of muscular proteins in low-birth-weight infants.     

Ntau-methylhistidine (3-methylhistidine) and muscle protein turnover: an overview.

Actin and myosin, the contractile proteins of skeletal muscle, are methylated following peptide bond synthesis, with production of Ntau-methylhistidine (3-methylhistidine, 3-MeHis). During intracellular breakdown of these proteins, the 3-MeHis is released and excreted in the urine. Studies on tissue distribution of 3-MeHis and on its qunatitative excretion following administration to rats and to man show that urinary output of this amino acid provides a reliable index of the rate of myofibrillar protein breakdown in the musculature of intact rats and human subjects. Estimates of the fractional rate of muscle protein breakdown based on 3-MeHis data are consistent with rates computed by other techniques. By this technique, it has been shown that the fractional rate of muscle protein breakdown is not significantly different in the elderly as compared with young adults. However, since muscle mass is less in the elderly, it makes a smaller contribution to whole body protein breakdown with aging in humans. Output of 3-MeHis diminishes in growing rats and obese human subjects with protein or energy restriction, though the initial response of myofibrillar protein breakdown in growing rats to protein and protein-energy restriction differs. Measurement of 3-MeHis excretion has also proved useful in exploring the effects of physical and thermal trauma on the rate of muscle useful in exploring the effects of physical and thermal trauma on the rate of muscle protein breakdown.