served not only decreases in FA concentrations (assumingly attributable to FA peroxidation), but also increases. A second process may be ongoing during storage: the formation of erythrocyte microparticles. In vitro, glycolysis will lower the glucose concentration in erythrocytes, causing depletion of ATP. Consequently, the erythrocytes cannot maintain membrane integrity, and microparticles will be released (15 ). Over time, the concentration of plasma phospholipid-associated FAs may thus become enriched with erythrocyte membrane phospholipids containing relatively high amounts of polyunsaturated FAs.Our study has some limitations. One limitation is that we did not measure in duplicate to adjust for intraassay variation. In addition, serum samples were not analyzed within one run to minimize interassay variability. However, standardized procedures were used for analysis, with CVs Յ7.9% for serum analytes and Յ5.6% for plasma phospholipid-associated FAs. In addition, the reliability analyses showed high agreement between follow-up and baseline values. Another limitation is that samples were not randomized before analysis. Possible bias from order of draw, although unlikely, can therefore not be ruled out. A third limitation is that we were not able to test the reliability and validity of CRP over the 28-h storage period. However, changes in concentration were small (Յ5.5%), which, as mentioned before, is in line with results from other studies (3, 5 ).In conclusion, this study shows that in the context of epidemiologic studies investigating (nutritional) status during routine care, a pragmatic approach to blood collection may validly be applied to determine CRP, retinol, ferritin, folic acid, or FA status. Although storage will diminish the precision of estimates, standard (correlational) epidemiologic analyses will not be compromised in samples stored for a maximum of 28 h.

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Although most studies of protein phosphorylation have focused on intracellular protein kinases, evidence for protein kinase activity on the surface of several types of cells has been described. Evidence was recently provided for the existence of ecto-protein kinase activity on the surface of human neutrophils. Evidence for three distinct ecto-protein kinase activities was detected, one that phosphorylates endogenous surface proteins, one that phosphorylates exogenous substrates in a cAMP-independent manner and is released in the presence of substrate, and a low level of activity of one that phosphorylates exogenous Kemptide in a cAMP-dependent manner. To begin to elucidate its role in neutrophil function, we have characterized several properties of the releasable ecto-protein kinase activity on human neutrophils. This enzyme activity was inhibited by impermeant stilbene disulfonic acids, which are known to alter neutrophil function, as well as by impermeant sulfhydryl reactive agents. Enzyme activity was detectable at physiologic concentrations of Mg2+, but was higher in the presence of Mn2+. Protein kinase activity was strongly inhibited by heparin, whereas trifluoperazine, cAMP, and cGMP had little effect on kinase activity. Protein kinase activity was selectively removed from the cell surface by incubation with the ecto-kinase substrates casein and phosvitin, but the enzyme was not released by phosphatidylinositol-specific phospholipase C. Repeated exposure of neutrophils to substrate depleted ecto-protein kinase activity from the cell surface, but activity was rapidly restored by incubation in buffer lacking substrate. The released protein kinase had a Km for ATP of approximately 0.5 microM and a pH maximum between 7.0 and 7.5. At least four ecto-protein kinase substrates were detected in serum; vitronectin was identified as one of these substrates by immunoprecipitation studies. Although the exact role of ecto-protein kinase activity in neutrophil function remains undefined, the identification of vitronectin as a serum substrate suggests that it interacts with a physiologically important substrate.

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