Inaccuracy in measuring glycated albumin concentration by thiobarbituric acid colorimetry and by boronate chromatography.

We compared thiobarbituric acid colorimetry and boronate chromatography for measuring glycation of serum proteins. With 14C-glycated human albumin as a test material, both methods were acceptably linear and precise. However, comparable estimates (mmol/L) of albumin glycation ranged from 0.22 for thiobarbituric acid to 0.05 for boronate, representing yields relative to [14C]glycosylprotein of 42% and 10%, respectively. The low yield with thiobarbituric acid was corroborated independently on the basis of kinetic differences between the reactions of fructose standards and of glycosylprotein, leading to underestimation of glycosylprotein concentration. The lower estimate of glycosylprotein by boronate chromatography was related to an apparent requirement for two [14C]glyco groups per albumin molecule to effect binding.

Assay of adenosine 5′-P1-tetraphospho-P4-5′′′-adenosine and adenosine 5′-P1-tetraphospho-P4-5′′′-guanosine in Physarum polycephalum and other eukaryotes. An isocratic high-pressure liquid-chromatography method

A5′pppp5′A has been proposed to serve as a molecular signal that triggers DNA replication. When published methods proved to be inadequate for the assay of A5′pppp5′A in Physarum polycephalum by h.p.l.c. (high-pressure liquid chromatography), a set of purification procedures was developed that allowed assay of as little as 2pmol of A5′pppp5′A. A5′pppp5′A was purified from cellular extract by covalent boronate chromatography, treated with alkaline phosphatase to hydrolyse residual mononucleotides and analysed by isocratic ion-exchange h.p.l.c. The analysis was facilitated by a pre-column switching procedure that allowed early-eluted species to be diverted from the analytical column. By using this procedure A5′pppp5′A has been detected in Physarum polycephalum (1.4 pmol/mg of protein), Saccharomyces cerevisiae (3.6 pmol/mg of protein) and rat liver (3.3 pmol/mg of protein). In each case a minor peak was also seen, which was identified as A5′pppp5′G. The identity of both peaks was confirmed by co-elution with standards on isocratic and gradient h.p.l.c. and treatment with enzymes, including a dinucleoside polyphosphate pyrophosphohydrolase from Physarum polycephalum.