Gas chromatographic/mass spectrometric analysis of stable isotopes of 3-methylhistidine in biological fluids: application to plasma kinetics in vivo.

A simple and rapid method for measuring 3-methylhistidine (3MH) in plasma and urine is described. Internal standard, 1-methylhistidine (1MH), was added to plasma, acidified and absorbed onto cation-exchange columns. It was then eluted from columns, dried, and derivatized for gas chromatography/mass spectrometry. A major fragment of 3MH was monitored at 238 u and 3-methyl-(methyl-2H3)histidine (d3-3MH) (used for in vivo kinetics) at 241 u, whereas 1MH was monitored at 340 u and eluted 0.5 min later than 3MH. Standard curves for plasma analysis were linear and nanamole amounts of 3MH in plasma were determined with a precision of 3.5%. 3MH was also quantitated in urine; however, because of substantial amounts of 1MH, (18O2)1MH was used as the internal standard. Nanamole amounts of 3MH were determined in urine with a precision of 2.7%. Application of the 3MH analytical method was used to develop a kinetic compartmental model by using the stable isotope of 3MH, d3-3MH. Cattle, like humans, quantitatively excrete 3MH in the urine. A young bovine was injected with d3-3MH and the enrichment curve in plasma was evaluated in order to obtain a steady-state production rate of 3MH. The decay curve was modeled through the use of NIH-SAAM modeling program. The kinetics of d3-3MH from plasma were adequately described by a three-pool compartmental model. The de novo production rate of 3MH estimated in the calf was 665 mumol per day. This corresponded to an estimated fractional turnover rate of 1.56% per day, which was similar to estimates obtained from urine collections.(ABSTRACT TRUNCATED AT 250 WORDS)

Technical note: the use of a compartmental model to estimate the de novo production rate of N tau-methylhistidine in cattle.

Urinary N tau-methylhistidine (NMH) excretion has been used as an index of muscle protein breakdown in cattle. An alternative means to estimate muscle proteolysis in cattle is to estimate the de novo production of NMH from plasma kinetics isotopically. Three crossbred steers (average 229 kg) were given a 5.0-mg bolus intravenous injection of [methyl-2H3-N tau-methylhistidine (d3-NMH), after which 16 serial blood samples and three consecutive 24-h urine samples were taken. The enrichment of NMH in plasma was determined by gas chromatography-mass spectrometry, and compartmental analysis of the kinetic data was performed using the SAAM modeling program. The NMH production rates per day (NMHPR, micromoles per day) were 732, 782, and 725, and the fractional breakdown rates (FBR, percentage per day) were 1.61, 1.72, and 1.58 as determined by urinary excretion of NMH, by a three-pool catenary model (plasma kinetics, Model A), and by a more descriptive, three-pool model with two response curves (both plasma and urine kinetics, Model B), respectively. Model A and B estimates of NMHPR and FBR were similar (P greater than .25) to those of estimates obtained from urinary NMH excretion. Kinetic modeling also allows calculation of compartment mass and flux of NMH between compartments and indicates that when NMH exists the muscle pool it is rapidly excreted via the urine. In conclusion, kinetic modeling offers an alternative approach to estimating the NMH production rate.

Comparison of insulin-like growth factor I serum binding proteins in sheep and cattle: species differences in size and endogenous binding capacities.

Binding proteins (BP) for insulin-like growth factor I (IGF-I) were characterized in sheep and beef cattle serum for molecular weight (Mr) and binding characteristics. Serum was incubated with [125I] IGF-I at 37 degrees C before chromatography over a 1.6-cm X 94.0-cm column of Sephacryl S-300 (pH 7.4, 4 degrees C). Beef serum exhibited a 145 k Mr (mol. wt X 1,000) and a 35 to 39 k Mr BP. Sheep serum possessed a 170 to 190 k and a 35 to 38 k Mr protein. Binding of [125I] IGF-I was inhibited in the presence of excess unlabeled ovine somatomedin, demonstrating specific binding for each BP of both species. The high Mr component was pituitary-dependent in sheep, as evidenced by binding patterns from serum of hypophysectomized sheep. Direct binding studies of the Sephacryl-separated BP demonstrated that the native BP of high molecular weight of both species bound only minor amounts of [125I] IGF-I in a manner unrelated to BP concentration. The BP of low molecular weight of beef cows displayed a bell-shaped dose-response binding curve with maximum binding at 250 micrograms/ml BP, whereas binding to sheep BP of low molecular weight was independent of BP concentration. After chromatography on Sephadex G50 at pH 2.8, both BP from both species exhibited concentration-dependent binding that plateaued at 250 to 500 micrograms/ml of BP of low molecular weight but was curvilinear for the BP of high molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)