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Thiamine (vitamin B1) deficiency is associated with severe diseases such as beriberi and Wernicke encephalopathy. Although most Americans have sufficient dietary intake, thiamine deficiency is observed in the alcohol-dependent and elderly populations. Measurement of thiamine concentration in whole blood provides an assessment of vitamin B1 status in at-risk individuals. We used TCA to precipitate proteins in whole blood. Thiamine and its phosphate esters were derivatized using potassium ferricyanide to thiochromes, which were separated by gradient elution on a reversed-phase HPLC column and detected by fluorescence. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Results obtained with this method were compared with those produced by the method currently used in our clinical laboratory. Reference values of thiamine and its phosphate esters were determined in samples obtained from self-reported healthy adults who were not taking vitamin supplements.

To shorten analysis time, our method used whole blood rather than washed erythrocytes, did not require lengthy enzymatic dephosphorylation, and had a simple mobile phase. The method was linear to 4000 nmol/L. The lower limit of quantification was 3 nmol/L. The within-run CV was <3.5% and total CV was <9.4%. This method correlated with our current method (r = 0.97). Approximately 90% of the total thiamine content in whole blood was present as thiamine diphosphate (TDP).

The means (ranges) for an apparently healthy population were 114 (70-179) nmol/L for TDP and 125 (75-194) nmol/L for total thiamine. Results for separation and measurement of free thiamine and thiamine phosphate esters in whole blood were obtained within 5.5 min. We developed an HPLC method that allows separation and measurement of free thiamine and thiamine phosphate esters in whole blood and provides more rapid results than other methods.

Discussion

Although we receive approximately equal numbers of whole blood and plasma specimens for thiamine assessment, whole blood is the superior sample type for analysis of thiamine concentration. Losa et al. showed that plasma contains mainly thiamine and TMP, whereas TDP predominates in erythrocytes (9). Our data also demonstrate that TDP accounts for 90% of the thiamine content in whole blood. As has been reported previously (3, 9,11), thiamine triphosphate was not detected in whole blood specimens. For monitoring TDP, the specific index of thiamine nutrition, whole blood is the ideal specimen type. No significant difference was noted in results from blood samples anticoagulated with EDTA or heparin. This finding is consistent with results reported by Ihara et al. (14).

Talwar et al. (3) reported significant discrepancies between TDP concentrations in whole blood stored at -20 °C and -70 °C. We found that incompletely frozen specimens yielded low thiamine concentrations. Some samples kept at - 20 °C for 24 h did not produce good hemolysates. These samples yielded higher concentrations following overnight storage at -70 °C. Specimens must be frozen completely to ensure lysis of the erythrocytes.

Because approximately 90% of all thiamine in whole blood is TDP, measurement of the diphosphate ester should be representative of thiamine status. In fact, we believe that monitoring of this physiologically active form of thiamine is preferable to the analysis of total thiamine. For clinical assessment, our laboratory reports TDP concentration in whole blood.

The reference interval for TDP concentration has been reported as 90-140 nmol/L for whole blood (15). Our population reference range for TDP is comparable with this published interval. Vitamin concentrations depend solely on dietary intake. Diets are influenced by many factors and vary geographically. Therefore a local reference interval is essential for recommendations for optimal intake of vitamins to maintain health (3).

In conclusion, we developed an HPLC method that allows measurement of free thiamine and its phosphate esters in whole blood. Well-resolved peaks elute in 5.5 min using a simple gradient, and consumption of mobile phase and other reagents is much less for this method than for other methods.