Vitamin B-I status assessed by direct measurement of thiamin pyrophosphatein erythrocytes or whole blood by HPLC: Comparison with erythrocyte transketolase activation assay

Background: The concentration of thiamin diphosphate (TDP) in erythrocytes is a useful index of thiamin status. We describe an HPLC method for TDP and its results in patients at risk of thiamin deficiency. Methods: We used reversed-phase HPLC with postcolumn derivatization with al kaline potassium ferricyanide and fluorescence detection. Samples were depr oteinized and injected directly onto a C-18 column. TDF concentrations in e rythrocytes were compared with those in whole blood. Reference intervals fo r erythrocyte TDP (n = 147; 79 males and 68 females; mean age, 54 years) an d whole blood TDP (n = 124; 68 males and 56 females; mean age, 54 years) we re determined in an apparently healthy population. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 pat ients who were considered at risk of thiamin deficiency. Results: The method was linear to at least 200 mu g/L. The between-run CV w as <8%. The lower limit of quantification for both whole blood and packed e rythrocytes was 300 pg on column with a detection limit of 130 pg on column . Recovery of TDP from blood samples was >90%. TDF in erythrocytes correlat ed strongly with that in whole blood (r = 0.97). Reference intervals for er ythrocyte and whole blood TDP were 280-590 ng/g hemoglobin and 275-675 ng/g hemoglobin, respectively. Of the 63 patients suspected of thiamin deficien cy, 46 were normal by both TDP and activation tests, 13 were deficient by b oth tests, 1 was deficient by the activation test but had normal erythrocyt e TDF concentrations, and 4 were normal by the activation test but had low TDP. Conclusions: The HPLC method is precise and yields results similar to the e rythrocyte activation assay. (C) 2000 Amccican Association for Clinical Che mistry.