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
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ABSTRACT |
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peripheral blood mononuclear cells; polymorphonuclear neutrophils; protein metabolism; stable isotopes; leucine
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MATERIALS AND METHODS |
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The purpose and the potential risks of the study were fully explained, and written informed consent was obtained from each participant. The experimental protocol was approved by the Ethics Committee of the Auvergne region.
Materials. L-[1-13C]leucine (99 molar percent excess) was obtained from Cambridge Isotope Laboratories (Andover, MA). The isotopic and chemical purities of leucine were checked by gas chromatography-mass spectrometry (GC-MS; Hewlett-Packard 5971A; Hewlett-Packard, Palo Alto, CA). Solutions of tracers were tested for sterility and pyrogenicity before use and were prepared in sterile apyrogenic water. Throughout each experiment, tracers were membrane-filtered through 0.22-µm-pore-size filters.
Experimental protocol. Data were collected in the postabsorptive state after a 10-h overnight fast. On the day of the experiment, two venous tracts were laid on the subject's arms. A catheter was retrogradely inserted into a dorsal vein of the left arm and used for blood sampling. The subject's hand was introduced into a ventilated box heated to 60°C to obtain arterialized blood. Another catheter was inserted into the contralateral arm and used for tracer. After a primed dose of [13C]leucine [8.4 µmol/kg fat-free mass (FFM)], continuous (0.14 µmol·kg FFM1·min1) infusion of [13C]leucine was started and lasted for 34 h.
In a first study performed in eight subjects, blood samples were taken before any infusion (20 and 0 min) and at 120 and 180 min after infusion. Blood was collected into EDTA-containing tubes for plasma isotopic enrichments and for measurement of PBMC and PMN protein synthesis rates after cell isolation.
In a second study, blood samples from five subjects were removed before any infusion (20 and 0 min) and at 120, 180, and 240 min after infusion. Blood was collected to evaluate the intracellular free [13C]leucine enrichment and to test the linearity of the incorporation rate of the tracer into PBMC and PMN proteins.
White blood cell isolation. Six milliliters of whole blood were collected in EDTA-treated tubes. Cycloheximide (0.5 mM; Sigma, Saint-Quentin-Fallavier, France) was rapidly added to the fresh blood to prevent further leucine incorporation during cell separation. Leukocytes were isolated by density gradient centrifugation, as previously described (29). Briefly, whole blood was carefully layered onto a double-discontinuous Ficoll-Hypaque density gradient (Histopaque 1077 and 1119; Sigma) with equal volumes and spun at 700 g for 30 min at room temperature. After centrifugation, the Ficoll-Hypaque layers were aspirated, and PBMC and PMN were collected on the corresponding layers at densities ranging from 1.024 to 1.077 g/ml and 1.077 to 1.119 g/ml, respectively. PBMC and PMN were washed twice with phosphate-buffered saline (PBS; pH 7.4, 150 mM), centrifuged, and resuspended in 1 ml of PBS. Cells were then tested for purity (>95%) and viability (>95%) by using May-Grunwald-Giemsa staining and the trypan blue dye exclusion test, respectively. PMN and lymphocyte suspensions were counted in a Malassez chamber, and their proteins were then precipitated in TCA stored at 80°C before analysis.
Analytical methods.
In the first study, plasma [13C]leucine and -[13C]ketoisocaproate (KIC) enrichments and concentrations were measured by GC-MS as previously described (1) and were used as precursor pool enrichments to calculate the fractional rate of protein synthesis (FSR). In the second study, we used isotopic enrichment of intracellular free L-[13C]leucine as precursor pool enrichment, which was determined from the 6-ml blood samples. After cell isolation and lysis by four freezing-defrosting cycles, intracellular amino acids were extracted by cation exchange chromatography (Dowex 50W 8X; Bio-Rad Laboratories, Hercules, CA), eluted in 4 M NH4OH, dried down in a SpeedVac (Savant Instruments), and analyzed with a gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) system (Isochrom; Micromass, Manchester, UK) as their N-acetyl-propyl derivatives. Intracellular protein enrichments were measured after intracellular protein hydrolysis by using GC-C-IRMS with the use of N-acetyl-propyl derivatization.
Calculations.
In the first study, leucine kinetics were calculated according to the reciprocal pool model by using plasma leucine or KIC concentrations as an indicator of intracellular leucine enrichment. Plasma [13C]leucine and [13C]KIC enrichments were used as precursor pool enrichments to calculate the FSR of protein in PBMC and PMN. The FSR of PBMC and PMN was expressed as percent per day and was calculated as the ratio between the time difference of [13C]leucine enrichments in PBMC and PMN at 180 (E180) and 120 min (E120) and the enrichment of plasma [13C]KIC as the precursor pool enrichment (EKIC)
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Statistical analysis. Statistical comparison of rates of protein synthesis between PBMC and PMN was made using the unpaired Student's t-test. The achievement of steady state was verified using the repeated-measures ANOVA test. The data are expressed as means ± SE, and differences were taken to be statistically significant if the P value was <0.05.
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RESULTS |
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DISCUSSION |
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Park et al. (25) previously measured the in vivo protein turnover of lymphocytes from healthy male subjects by using the flooding dose procedure. The advantage of this method is that the assessment of precursor enrichment is facilitated (14, 15). Ideally, the measurement of precursor enrichment should be made on the aminoacyl-tRNA of the tissue. Nevertheless, because of the extremely small size, rapid turnover, and instability of this pool, this would be impracticably difficult and quite infeasible.
To the best of our knowledge, the present report represents the first documented use of the continuous intravenous infusion method of labeled amino acid to determine the protein synthesis of immune cell preparation. In this technique, the tracer is given by constant infusion at a rate sufficient to achieve, at steady state, a labeling in the plasma pool of 510% of the total free amino acid pool. Intracellular amino acid enrichments also did not change over time, ranging from 3 to 5% of the total free intracellular amino acid pool. These observations demonstrated that a steady state was actually reached in the intracellular pool. There is evidence in tissues such as muscle (26) that the labeling of KIC, a metabolite generated intracellularly from leucine, after tracer infusion is an accurate reflection of the intracellular tRNA pool (22, 26, 31). However, as demonstrated in this study, enrichment of the free amino acid pool in other tissues may be lower than in the plasma by an amount that varies with the metabolic activity of the tissue considered. This observation led, in our experiment, to an underestimation of protein FSR with the use of the plasma labeled amino acid enrichment. Protein synthesis rate as calculated from intracellular enrichment was 50% higher with a large variation among subjects corresponding to an increase from 35 to 80% compared with that calculated from plasma enrichment. Still, should the technique, in the future, be applied in pathological situations, amino acid concentration will change, as will enrichments in intracellular amino acids pools. This observation will require more rigorous validation in such situations.
Interestingly, the labeled amino acid can be infused as a tracer with little or no effect on the overall metabolism of the cells. With the flooding dose, the exposure of the subjects to a novel stimulus, i.e., a loading dose of amino acids, could induce an activation of immune cells and cause an artifactually greater availability of tracer amino acid to the tRNA synthetase than that which occurred before the application of the flood (8, 26). The potential disadvantage is the possibility that the change in amino acid concentration might alter directly or indirectly the rate of protein synthesis in the tissue under study. This latter phenomenon may explain the higher values that were observed by others (25) concerning the rate of protein synthesis measured in immune cells by the leucine flooding dose technique (9%/day for lymphocytes) compared with the infusion procedure (6.04 and 2.98%/day for PBMC and PMN, respectively). More recently, the users of the flooding dose method have turned to the use of [2H5]phenylalanine as the flooding amino acid (14). However, this research group subsequently produced the same data for protein FSR in healthy immune cells as previously described with leucine (5, 12, 13, 1719, 23). Other authors working with different tissues also reported that the values obtained with the flooding dose protocol appear to be higher than those obtained with a constant infusion technique. The differences described in the literature may be partly explained by the stimulating effect of a flooding dose of amino acids, such as leucine, on protein synthesis but also may be related to the different precursor pool used to calculate protein turnover in these two procedures. Another explanation is that the immune cells export a large part of protein synthesized. Thus the flooding dose is performed during the period when the newly synthesized export proteins remain in the cell, where they contribute appropriately to the estimation of synthesis rate. This later observation means that the flooding dose procedure may reflect the synthesis rate of immune exported proteins, whereas the continuous infusion method may evaluate the constitutive protein pool turnover of this tissue. Ideally, the contribution of exported proteins should have been estimated during the continuous tracer infusion procedure by measuring the synthesis rate of specific proteins, such as immunoglobulins, from plasma samples. It is interesting to note that the FSR of immunoglobulins is higher than the FSR of constitutive proteins (9).
Blood requirements for isolating PBMC and PMN by density gradient centrifugation and measuring [13C]leucine enrichment by mass spectrometry can be reduced to 6 ml per sample with our study method (instead of 2060 ml in flooding dose studies), an acceptable blood removal for kinetic studies even in frail populations such as patients, the elderly, or neonates. This new method should be useful to assess in vivo the immune modifications during long-term immunomodulating treatments or pathological situations. In addition, the technique of cell isolation used in this study allows assessment of protein synthesis rate in all white blood cell subsets, i.e., PBMC and PMN, from the same blood sample. From these determinations, it appears that protein synthesis rate is higher in the PBMC subpopulation compared with the neutrophil fraction in healthy young adult subjects. A recent study (19) measured the in vivo protein turnover in lymphocytes and total leukocytes (i.e., 29% lymphocytes, 8% monocytes, 61% PMN, and a negligible count of eosinophils and basophils) from the same healthy volunteers. In this latter work, the in vivo protein synthesis was 67% higher in lymphocytes (9%/day) than in total white blood cells (3.2%/day), confirming the lower FSR in the PMN population, which represents >60% of the total leukocytes (19). Taken together, these observations suggest large differences in synthetic activities and functions of immune cells of healthy subjects, with a lower metabolic rate in PMN. This probably reflects the fact that, first, PMN are not active in the absence of stimulation, and second, all activated PMN left the blood compartment to synthesize immunoregulatory factors in tissues. Papet et al. (24) previously presented, in the rat model, the first quantification of protein synthesis in circulating lymphocytes and primary lymphoid tissues. In healthy, well-nourished rats, the protein synthesis rate decreased progressively in bone marrow, which is composed of stem cells, then in thymus, which is the site of maturation, followed by the circulating mature cells. This is consistent with their replicative and metabolic activities. Finally, the protein synthesis rate that we measured in human PBMC was lower than in rat as already described for muscle and intestine proteins and for albumin (1, 2, 3, 6, 21, 27, 28).
In conclusion, we have set up a new method allowing the determination of the rate of labeled amino acid incorporation into immune cell proteins by using a simple continuous infusion technique. The determination of the protein FSR in mononuclear and neutrophil subpopulations is possible at the same time and from the same sample with this procedure. However, the choice of the free labeled amino acid precursor pool seems to be mandatory because the absolute FSR values obtained by using the plasma tracer enrichment underestimated the FSR of PBMC and PMN. In addition, intracellular amino acid concentration may change depending on the pathological situation with or without any modification of the plasma amino acid profile. Therefore, intracellular pools have to be taken into consideration to calculate white blood cell FSR. Additional effort directed at purification of individual immune proteins for synthesis rate analysis is also warranted. Focusing on the specific proteins within immune system is likely to reveal additional differences in physiopathological situations.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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