Whole-body nitrogen and splanchnic amino acid metabolism differ in rats fed mixed diets containing casein or its corresponding amino acid mixture.

Whole-body and splanchnic metabolism of dietary amino acids derived from casein (CAS) or the corresponding crystalline L-amino acid mixture (AA) were compared. Male adult rats were adapted for 9 d to two isoenergetic, isonitrogenous diets (15 g/100 g protein, 5 g/100 g fat) containing either CAS or AA. On d 10, the rats were fed a single mixed meal (3 g dry mass) containing either intrinsically (13)C-labeled goat casein or the amino acid mixture containing [U-(13)C(6)] leucine and [alpha-(15)N] lysine. Rats were killed before and 1, 3, 5 and 7 h after meal ingestion and samples of plasma, stomach wall and contents, small intestine and liver were collected. (13)C and (15)N enrichments of free and protein-bound amino acids in plasma and tissues were analyzed by gas chromatography-combustion isotope ratio mass spectrometry. Urinary nitrogen excretion was higher (P < 0.05) and weight gain lower (P < 0.05) in rats given the AA diet, indicating a lower whole-body net protein synthesis. Free (13)C-leucine from the AA diet appeared in the intestinal mucosa free pool more rapidly (P < 0.05) than the CAS-(13)C-leucine, probably due to the faster transit through the stomach of the AA group. However, the incorporation of dietary leucine into plasma and liver proteins was higher in the CAS group 7 h after the meal (P < 0.05), whereas lysine incorporation into liver protein was higher in the AA group (P < 0.05). We conclude that whole-body protein homeostasis is better supported by dietary casein-bound than crystalline free amino acids, and that protein-bound leucine, but not lysine, is used more efficiently for liver protein synthesis than dietary free leucine.

13C gas chromatography-combustion isotope ratio mass spectrometry analysis of N-pivaloyl amino acid esters of tissue and plasma samples.

We present the analysis of the stable carbon isotope compositions of 14 individual N-pivaloyl-isopropyl (NPP) amino acid esters by gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS). The mean reproducibility of derivatization procedure and GC-C-IRMS analysis was 0.45 per thousand (range, 0.12-0.68), whereas the mean analytical error was 0.26 per thousand delta(13)C (range, 0.13-0.42). Furthermore, the delta(13)C values of N-pivaloyl-isopropyl and N-acetyl-n-propyl (NAP) amino acid esters were compared. Due to a reproducible isotopic fractionation introduced by the derivatization process an empirical correction factor for each individual amino acid was derived separately for both derivatives (NPP, -1.13 to -2.52 (lysine, +2.09) per thousand delta(13)C; NAP, -2.36 to -3.97 (lysine, +1.91) per thousand delta(13)C), and the original delta(13)C value of the underivatized amino acid was calculated. Further, we performed an animal study where rats (n = 5) ingested a mixed meal containing uniformly (13)C-labeled casein (indispensable amino acids 1.3 to 1.7 at.%). One hour after the meal delta(13)C values of protein-bound amino acids from small intestinal mucosa and liver and of free amino acids from mucosa and plasma were determined. Significant (13)C enrichments of indispensable amino acids of the free pools of mucosa and plasma (range, 0.0518 to 0.1700 at.% excess) and in mucosa and liver proteins (range, 0.0021 and 0.0161 at.% excess) were observed. The feasibility of various derivatives for the measurement of carbon isotopic composition is discussed.

Improved in vitro stability of serum osteocalcin by using a new commercially available antiproteolytic compound.

The effect of a commercially available antiproteolytic compound (OSCAstabil) on the degradation in vitro of serum osteocalcin (Oc) was investigated in serum samples stored at 22, 4 or -30 degrees C (n = 20) or subjected to repeated freeze-thaw cycles (n = 8). The addition of the stabilizing agent immediately after serum preparation increased the Oc stability from < 3 to 6 h at 22 degrees C, from < 3 h to 3 days at 4 degrees C and from 1 day to more than 3 days at -30 degrees C. Up to three freeze-thaw cycles had no influence on the Oc stability, either with or without the stabilizer. The obviously prolonged Oc stability at 22 and 4 degrees C offers a more convenient and reliable Oc estimation procedure for clinical practice. The use of this antiproteolytic agent can be recommended in order to retard the in vitro degradation of serum Oc, not only for clinical routine, but also for studies on metabolic bone diseases.