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We developed a method for measurement of skin DNA synthesis, reflecting cell division, in conscious rabbits by infusing e-[U-[sup.13][C.sub.6]]glucose and L-[[sup.15]N] glycine. Cutaneous protein synthesis was simultaneously measured by infusion of L-[ring-[sup.2][H.sub.5]] phenylalanine. Rabbits were fitted with jugular venous and carotid arterial catheters, and were studied during the infusion of an amino acid solution (10% Travasol). The fractional synthetic rate (FSR) of DNA from the de novo nucleotide synthesis pathway, a reflection of total cell division, was 3.26 [+ or -] 0.59%/d in whole skin and 3.08 [+ or -] 1.86%/d in dermis (P = 0.38). The de novo base synthesis pathway accounted for 76 and 60% of the total DNA FSR in whole skin and dermis, respectively; the contribution from the base salvage pathway was 24% in whole skin and 40% in dermis. The FSR of protein in whole skin was 5.35 [+ or -] 4.42%/d, which was greater (P < 0.05) than that in dermis (2.91 [+ or -] 2.52%/d). The FSRs of DNA and protein were not correlated (P = 0.33), indicating that cell division and protein synthesis are likely regulated by different mechanisms. This new approach enables investigations of metabolic disorders of skin diseases and regulation of skin wound healing by distinguishing the 2 principal components of skin metabolism, which are cell division and protein synthesis. KEY WORDS: * stable isotopes * mass spectrometer * fractional synthetic rate * rabbits

We assessed the approach of using intracellular free amino acid enrichment as precursor enrichment for calculating the fractional synthetic rate of using the stable isotope tracer incorporation technique. We assumed that the true rate of protein synthesis was reflected by the rate of tracer incorporation over time divided by the plateau enrichment in protein. Isolated human fibroblasts and myocytes were cultured in medium supplemented with [[sup.15]N]glycine, [[sup.15]N]proline, and [[d.sub.5]]phenylalanine. Culture medium and cells were collected daily from d 1 to 5. A portion of cells harvested on d 5 was subcultured for an additional 3 passages to d 20. Protein enrichments in both cell types reached a plateau after 20 d of cell culture. In fibroblasts, the true protein synthesis rates were 0.74, 0.85, and 0.86%/h, using protein plateau enrichments of [[sup.15]N]]glycine, [[sup.15]N]]proline, and [[d.sub.5]phenylalanine as precursor enrichments, respectively. When the corresponding intracellular free amino acid enrichments were used, protein synthesis rates were 0.76, 0.79, and 0.76%/h, respectively. Similarly, in myocytes, the true protein synthesis rates were 0.98 and 1.14%/h by protein plateau enrichments of [[sup.15]N]glycine and [[d.sub.5]]phenylalanine, respectively. The synthesis rates were 0.94 and 1.01 %/h by the corresponding intracellular enrichments, respectively. Extracellular amino acid enrichments resulted in underestimation of protein synthesis by a variable amount. We conclude that the intracellular free amino acid enrichment is an optimal surrogate for precursor enrichment to quantify protein synthesis. KEY WORDS: * protein synthesis * stable isotopes * precursor enrichment * GC-MS * plateau enrichment