Erythrocyte Folate Analysis: Saponin Added During Lysis of Whole Blood Can Increase Apparent Folate Concentrations, Depending on Hemolysate pH

[摘要] Background: The analysis of red cell folate (RCF) depends on complete hemolysis of erythrocytes, and it is assumed that complete hemolysis is achieved by 10-fold dilution of whole blood with hypotonic solutions of 10 g/L ascorbic acid/ascorbate. This report challenges this assumption.Methods: The conventional method of erythrocyte lysis was modified to include saponin, a known effective hemolyzing agent. The influence of saponin was determined at various lysate pHs, using the microbiological ( Lactobacillus rhamnosus ) folate assay. The effect of saponin during lysate preparation was subsequently compared with either the effect of 30 s of sonication or a single 1-h freeze-thaw cycle.Results: Saponin addition was found to increase assayable RCF up to ninefold, depending on lysate pH. Sonication of lysates had no effect, and freezing-thawing lysates once did not always guarantee complete hemolysis. Lysates created with 10 g/L ascorbic acid (a historically widely used diluent) without pH adjustment produced assayable folate concentrations significantly lower than optimal.Conclusions: A lysing agent should be incorporated into RCF assays to guarantee complete hemolysis. Ten-fold dilution of blood with 10 g/L ascorbic acid, without pH adjustment, produces lysates with pHs (pH 4.0) below the point (pH 4.7) at which hemoglobin can denature irreversibly. The optimum pH for hemolysates is ∼5.0.

[学科分类] 过敏症与临床免疫学