Thiamine and its derivates in cattle blood measured by HPLC in healthy animals, in patients suffering from CCN and in their cohorts

The aim of this project was to develop a routine method to measure thiamine and its derivates in blood, to detect the need of vitamin B-1-supply in cattle faster and more precise than until now using the transketolase test. For that, thiamine and its derivates (thiaminediphosphate = TDP,-monophosphate = TMP) were analysed in cattle blood by HPLC technique with post-column derivatization. Moreover, the levels in healthy cattle were compared with those in herds with single CCN cases. EDTA-blood-stored up to 10 days by 6 or 20 degrees C- was the most appropriate substrate. Sensitivitiy and specifity were significantly better than those of other methods - particularly of the transketolase test. The VCs for the diagnostically important parameters were as follows:TDP and total thiamine (TT, sum of all fractions): 1.95 % and 2.10 %; thiamine and TMP:7.02 % and 9.17%. The lowest concentration, which could be measured, was 0.5 ng/ml. 72 samples could be analysed daily. In the blood of 280 healthy calves and cows (group A), 201 clinically unsuspicious animals out of stocks with single cases of CCN (group B) and 12 patients with clinical obvious CCN (group C) thiamine and its phosphates were measured and the means calculated (ng/ml). After this, the thiamine status is characterized by (ng/ml): (A) thiamine 13.5 (4.61-28.8), TDP 51.0 (33.1-82.2), TMP 8.79 (2.23-18.1), TT 73.3 (44.6-114); (B) thiamine 8.73 (0.00-20.0), TDP 43.5 (15.5-75.7), TMP 6.15 (0.00-16.6), TT 59.4 (18.7-96.5); (C) thiamine 3.85 (0.00-11.5), TDP 14.5 (0.00-28.3), TMP 1.27 (0.00-4.87), TTP 19.6 (4.92-35.9). So, HPLC with post-column derivatization offers a precise, automatically working method for a fast diagnose of thiamine deficiency. The lowest critical value of TT is 40 ng/ml for healthy animals in the blood. The sample (EDTA-blood) can be posted simply at temperatures up to 20 degrees C. In comparison to other measuring methods, this HPLC-technique enables an early recognition of the risk to come down with CCN in endangered cattle herds.