Plasma arginine concentration decreases rapidly in whole blood held at room temperature, and this decrease is greatly attenuated by placing the blood on ice. Ornithine, the metabolic product of arginine metabolism by arginase, rises exponentially at room temperature, and this rise does not occur on ice, suggesting that it is due to an enzymatic process. Thus, it is likely that arginase is the primary mechanism of arginine degradation in ex-vivo blood samples. This arginase could come from either lysed RBCs or lysed leucocytes, but we did not evaluate the source of arginase, and thus cannot determine which of these was more important. In-vitro hemolysis is difficult to measure, as the released cell-free haemoglobin is immediately bound by haptoglobin. While we have not proven this hypothesis, our observations strongly suggest it.
Most other amino acids increase at room temperature but not on ice, which also implies an enzymatic reaction. Tryptophan is very stable both at room temperature and on ice. Taurine and glutamine are unusual, in that they increase markedly both at room temperature and on ice; this may be due to cellular release rather than enzymatic catabolism.
The rate of decrease of plasma arginine which we found in blood held at room temperature is similar to that found by Nuttall and colleagues in the only published paper to have reported plasma arginine concentrations at room temperature at more than two time points [18
]. The lack of early time points in other papers makes it difficult to estimate the rate of decline and whether it is linear or exponential. Nuttall et al. reported data in graphical form, from a single subject up to 2.5 hours post venepuncture. They found a fall from 89 μmol/L to approximately 60 μmol/L at 2 hours (a 33% drop), similar to our reported decrease of 25% at 2 hours.
The large increases seen in taurine and glutamate in our study have not previously been reported. Sahai et al. measured amino acid levels in whole blood from twenty-two volunteers, stored on ice for 1 hour or 2 hours, and found a less than 10% decrease in plasma taurine and glutamate at 1 and 2 hours [21
]. Shaeffer et al. reported a < 10% decrease in plasma taurine and glutamate at 6 hours in blood held at room temperature from one healthy volunteer [17
]. The reason for this discrepancy is unclear. Both papers used different methods for amino acid quantification than we did. Sahai et al did not measure time points beyond 2 hours, and most of the increase in both taurine and glutamine in our study occurred beyond 2 hours. However, until this finding is reproduced by other investigators, it should be regarded with caution.
The primary limitations of this study are the relatively small number of subjects and the lack of subjects suffering from sepsis, trauma or other conditions of interest. A larger number of subjects would allow a more accurate estimate of the time profile of arginine degradation over time. Considering arginase activity is increased in severe sepsis [22
] and trauma [23
], it is unclear if blood from patients with these conditions would yield the same results as we observed. We did not directly measure arginase activity in blood or plasma, and thus our inference that plasma arginase is primarily responsible for the observed ex-vivo arginine degradation is based on indirect evidence. However, the only other significant mechanism for arginine degradation likely to occur ex-vivo is the breakdown of arginine to NO and citrulline by nitric oxide synthase, which accounts for less than 5% of arginine metabolism in healthy humans [24
One potential implication of these data is that whole blood stored for the purpose of transfusion is likely to contain non-physiological concentrations of amino acids, which may have unintended immunosuppressive effects. These data also reinforce the importance of accurate methodological descriptions in papers reporting plasma amino acid levels. In a hospital setting, it is not always possible to process samples within 30 minutes of collection. It is therefore essential to note the time between collection and freezing when reporting concentrations of plasma amino acids. This is particularly important if the sample cannot be kept on ice - for example, if the blood is to be used for both peripheral blood mononuclear cell (PBMC) collection and amino acid analysis. As PBMCs are damaged by freezing, these samples must be kept at room temperature and processed as soon as possible to allow accurate analysis of both PBMC function and amino acid concentrations. Furthermore, where plasma amino acids are being measured for clinical applications, our data emphasise the importance of timely separation and freezing of plasma to avoid potential diagnostic errors.