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Methods in Cell Physiology

Protein synthesis rates of human PBMC and PMN can be determined simultaneously in vivo by using small blood samples

¹Unité du Métabolisme Protéino-Energétique, Unité Mixte de Recherche Université d'Auvergne/Institut National de la Recherche Agronomique, Centre de Recherche en Nutrition Humaine, Centre Hospitalier Universitaire de Clermont-Ferrand 63009 Clermont-Ferrand Cedex 1, and ²Laboratoire de Biochimie, Biologie Moléculaire et Nutrition, Faculté de Pharmacie, Centre de Recherche en Nutrition Humaine, 63001 Clermont-Ferrand Cedex 1, France

Submitted 3 December 2002 ; accepted in final form 22 January 2004

Immune cell functions can be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing simultaneous in vivo assessment of FSR in two leukocyte populations in healthy human subjects, using small blood samples. Peripheral blood mononuclear cell (PBMC) and polymorphonuclear neutrophil (PMN) FSR were measured during primed continuous intravenous infusion of L-[1-¹³C]leucine. Immune cells from 6 ml of whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR using plasma [¹³C]leucine or -[¹³C]ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes, using enrichments of either intracellular or plasma free [¹³C]leucine as immediate precursor pools. The present approach showed a steady-state enrichment of plasma and circulating immune cell free [¹³C]leucine precursor pools. The linearity of labeled amino acid incorporation rate within mixed PBMC and PMN proteins also was verified. Postabsorptive protein FSR was 4.09 ± 0.39%/day in PBMC and 1.44 ± 0.08%/day in PMN when plasma [¹³C]KIC was the precursor pool. The difference between PBMC and PMN FSR was statistically significant, whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. Use of the plasma [¹³C]KIC pool led to an underestimation of leukocyte FSR compared with the intracellular pool (PBMC: 6.04 ± 0.94%/day; PMN: 2.98 ± 0.30%/day). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans by using small blood samples to directly indicate their metabolic activity in various clinical situations and in response to regulating factors.

peripheral blood mononuclear cells; polymorphonuclear neutrophils; protein metabolism; stable isotopes; leucine