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A two-step non-radioactive method that uses reverse-phase high-performance liquid chromatography (RP-HPLC) is described for the determination of phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) activity in human erythrocytes. The method is accurate and easily reproducible in different chromatographic systems; it is based on the quantification of phosphoribosylpyrophosphate by conversion into orotidine monophosphate and uridine monophosphate. Phosphoribosylpyrophosphate synthetase activity was determined in the erythrocytes of healthy adults and children, the latter showing significantly higher activity than the former. The enzyme activity assayed in children with different neurological disorders was significantly lower in patients with Rett syndrome than in control children or in autistic or mentally retarded patients.

The nucleotide concentrations in acid and alkaline erythrocyte extracts have been measured by RP-HPLC in healthy controls and in patients bearing different inherited disorders, with altered erythrocyte NAD(P) levels. The objective was the simultaneous determination of the nucleotide profile and of the oxidative state of pyridine coenzymes by the most suitable extraction method. Both alkaline and acid extractions were necessary to obtain the complete pattern, due to defective recovery of the oxidized or reduced coenzymes, respectively, during the extraction procedures. Purine nucleotide quantification seemed to be reliable by all methods. High NADP+ levels were confirmed in two glucose-6-phosphate dehydrogenase deficient patients, coupled with raised NAD levels, lowered NADPH/NADP+ ratio and increased NADH/NAD+ ratio. Higher NAD+ and normal or lower NADH/NAD+ ratios were found in two hypoxanthine-phosphoribosyltransferase deficient patients, while a patient with superactive phosphoribosylpyrophosphate synthetase showed a decreased NADH level in addition to the low NAD+ level previously found.

A spectrophotometric method for the determination of 5-phosphoribosyl 1-pyrophosphate (PRPP) is presented which shows several advantages in comparison to the radiochemical techniques, such as a relatively simple, rapid and less expensive procedure. This technique has been used to evaluate PRPP content in erythrocytes, leukocytes and lymphocytes of normal subjects and individuals with partial hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) deficiency. The results obtained proved to be completely reliable in both groups of subjects examined, with values of PRPP similar to those observed by radiochemical techniques.