Erythrocyte folate analysis: Saponin added during lysis of whole blood canincrease apparent folate concentrations, depending on hemolysate pH

Background: The analysis of red cell folate (RCF) depends on complete hemol ysis 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 asco rbic acid/ascorbate. This report challenges this assumption. Methods: The conventional method of erythrocyte lysis was modified to inclu de saponin, a known effective hemolyzing agent. The influence of saponin wa s determined at various lysate pHs, using the microbiological (Lactobacillu s 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 ninefol d, depending on lysate pH. Sonication of lysates had no effect, and freezin g-thawing lysates once did not always guarantee complete hemolysis. Lysates created with 10 g/L ascorbic acid (a historically widely used diluent) wit hout pH adjustment produced assayable folate concentrations significantly l ower than optimal. Conclusions: A lysing agent should be incorporated into RCF assays to guara ntee complete hemolysis. Tenfold dilution of blood with 10 g/L ascorbic aci d, without pH adjustment, produces lysates with pHs (pH 4.0) below the poin t (pH 4.7) at which hemoglobin can denature irreversibly. The optimum pH fo r hemolysates is similar to5.0. (C) 2000 American Association for Clinical Chemistry.