Jeanne B. Li

A rapid, sensitive method for the determination of 3-methylhistidine levels in urine and plasma using high-pressure liquid chromatography

Abstract A method is presented for the precolumn derivatization and subsequent high-pressure liquid chromatographic separation of 3-methylhistidine from urine and plasma. The solvent system is 10 m m sodium phosphate (pH 7.5) and acetonitrile. The elution can be performed isocratically and requires less than 10 min. Both fluorescent and ultraviolet detection may be utilized. This method is at least 10 3 times more sensitive than conventional ion-exchange chromatography using ninhydrin. 3-Methylhistidine determinations performed on plasma and urine samples from normal volunteers correlated well with published literature values.

Vitamin D Deficiency, hypocalcemia, and increased skeletal muscle degradation in rats.

The myopathy associated with vitamin D deficiency was examined in vitamin D-deficient and vitamin D-supplemented rats. When compared with either vitamin D-supplemented ad lib. or pair-fed rats, weight gain and muscle mass were decreased in vitamin D-deficient hypocalcemic animals. With the exception of a modest decrease in muscle creatine phosphate levels, muscle composition was unchanged by vitamin D deficiency. Muscle protein turnover rates were determined in both in vivo and in vitro studies and demonstrated that myofibrillar protein degradation was increased in vitamin D deficiency. Normal growth rates could be maintained be feeding the rats vitamin D-deficient diets containing 1.6% calcium, which maintained plasma calcium within the normal range. In addition to its role in maintaining plasma calcium, vitamin D-supplemented rats had significantly higher levels of the anabolic hormone insulin. Vitamin D supplementation may affect muscle protein turnover by preventing hypocalcemia, as well as directly stimulating insulin secretion, rather than by a direct effect within skeletal muscle.

1274 MUSCLE PROTEIN TURNOVER AND GLUTAMINE (GLN) RELEASE IN ACUTE METABOLIC ACIDOSIS (MA)

In MA the major nitrogen source for ammoniagenesis is GLN and plasma levels rise within minutes after initiation of MA. GLN may be provided by increased catabolism (C) of muscle at the expense of anabolism and growth. We studied MA in an in vitro perfused muscle preparation obtained from normal, fed rats. The control perfusate consisted of Krebs-Henseleit buffer (NaCl 118 mM, NaHCO3 25 mM) with erythrocytes, glucose, albumin and amino acids. The MA buffer was identical except that 15 mM NaHCO3 was replaced by NaCl. Both perfusates were continually gassed with 95% O2/ 5% CO2. Measured pH values were 7.38 vs 6.99 at the start and 7.33 vs. 7.13 at 3 hours. Protein synthesis (S) and (C) were assessed using [14C] phenylalanine and GLN was determined spectro-photometrically. During the first hour, perfusate GIN rose higher in the MA preparations (0.303 vs. 0.188 mmol/g HC p = 0.04), but by 3h, there were no significant differences. Muscle GLN levels decreased between 1 and 3 h (-1.304 vs. -1.125 umol/g HC, p NS) so that net GLN production was no different between the the two groups. Individual S and C rates were not different but MA resulted in a 15% decrease in the rate of net muscle degradation (P 0.05). Thus, MA leads to transient redistribution of GLN between intra and extracellular spaces and to decreased and not increased muscle degradation. These results cast doubt on the importance of muscle as the source of nitrogen for ammoniagenesis in acute MA.

EVIDENCE SUGGESTING ABNORMAL MUSCLE PROTEIN SYNTHESIS AND DEGRADATION IN UREMIA

Children with chronic renal disease have decreased growth and diminished muscle mass suggesting that altered protein metabolism occurs in these children. We studied the effect of moderate, stable uremia in young, growing rats made uremic by 5/6 nephrectomy. Two weeks postop the uremic animals weighed less than the sham-operated controls (191 vs. 251 g) and plasma urea nitrogen levels were elevated (57.4 vs. 16.1 mg/dl). Muscle composition, as reflected in protein, water, total RNA and ATP content was unaltered when measured per gram wet weight. While total RNA concentration was unchanged in uremia, sucrose density centrifugation revealed an increase in the percentage of RNA sedimenting as 60s + 40s ribosomal subunits, suggesting a relative block in peptide chain initiation. Subunit levels were approximately 30% higher both in the post-fed state and after a 48 hour fast. 3-methylhistidine production, a measure of myofibrillar protein breakdown was no different under normal feeding conditions but was significantly elevated after 48 hours of fasting. Taken together, these findings suggest that the decreased anabolism seen in uremia may be related to increased polysome disaggregation and consequent decreased “efficiency” of muscle protein synthesis. Protein degradation may be effected as well after the imposition of additional metabolic stress (PHS grant AM 24061 and MDA).