William L. Nyhan

Sephadex G-10 adsorption chromatography of purines and related compounds

The adsorption chromatography of purines and related heterocyclic compounds on Sephadex G-10 was investigated under different conditions of pH, ionic strength and flow rate and found to give excellent separations. The elution volumes were highly reproducible and recovery quantitative. A relation between a log function of the elution volumes and substituents on the purine ring was shown and the additivity of the resulting Δ log V6° values used to predict elution volumes. Some analytical and preparative applications have been undertaken.

Assay of hypoxanthine-guanine and adenine phosphoribosyl transferases: A simple screening test for the Lesch-Nyhan syndrome and related disorders of purine metabolism☆

Abstract A novel procedure has been developed for the assay of hypoxanthineguanine phosphoribosyl transferase (EC2.4.2.8.) and adenine phosphoribosyl transferase (EC2.4.2.7.). The assay is based on the precipitation by lanthanum chloride of 14 C-labeled IMP, GMP, and AMP, the nucleotide products generated in the reaction. The activity of the enzyme can be determined through the assay of the 14 C content of either the residual substrate in the supernatant fluid or of the lanthanum nucleomonophosphate in the precipitate. The determination of enzyme activity can be carried out on whole blood or on blood dried on filter paper. The assay is suitable for the screening diagnosis of disorders of purine metabolism and also for quantitative determination of enzyme activity.

Systemic infection with Hemophilus influenzae in very young infants.

Experience is described with systemic infection due to H. influenzae in infants less than4 months old. Five patients with meningitis were observed in a 9 month period. Severe disease with widespread involvement of multiple systems was common. One patient had osteomyelitis, one had purulent pericarditis, and one had pneumonia with pneumatoceles. Two died. The rare occurrence of systemic infection with this organism in this age group has been correlated with the transfer of bactericidal activity across the placenta. This property was studied in 4 of the mothers of these infants and found to be very low.

Acquired Fanconi syndromefollowing degraded tetracycline

Three patients have been found to manifest a Fanconi syndrome followingingestion of outdated tetracycline. Marked hypokalemia, changes in sensorium, and photosensitivity were prominent additional features. The generalized aminoaciduria was quantitated, and the pattern of its return to normal documented. The disorder appears to be a toxic effect of a degradation product of tetracycline.

Mevalonate Kinase in Lysates of Cultured Human Fibroblasts and Lymphoblasts: Kinetic Properties, Assay Conditions, Carrier Detection and Measurement of Residual Activity in a Patient with Mevalonic Aciduria

An assay has been developed for the measurement of mevalonate kinase activity in extracts of cultured human fibroblasts and lymphoblasts. Individual elements of the assay were investigated in order to achieve optimum conditions. Apparent Michaelis constants (KMapp) for the substrates mevalonic acid and adenosine-5-triphosphate were 22 +/- 10 mumol/l and 0.42-0.53 mmol/l, respectively, in lysates of control fibroblast lines. The same values in lysates of a control lymphoblast line were 17 mumol/l and 0.23 mmol/l, respectively. Mevalonate kinase activity in extracts of cultured fibroblasts derived from 6 control individuals was 3.24 +/- (SD) 0.91 nmol/min/mg protein. The activity in extracts of fibroblasts derived from a patient with mevalonic aciduria was 0.15 +/- 0.10 nmol/min/mg protein, approximately 5% of the control mean. The parents and brother of the patient displayed mevalonate kinase activities in fibroblast extracts approximating 38-42% of the control mean. Substantially higher mevalonate kinase activity was documented in extracts of cultured lymphoblasts. When assayed on various occasions, the mean activity of mevalonate kinase in extracts of lymphoblasts derived from the parents, brother and maternal grandmother of the patient ranged from 27 to 32% of the mean activity of 9.8 +/- (SD) 3.4 nmol/min/mg protein measured in a parallel control lymphoblast line, while the mean activity in a maternal and paternal uncle approximated 65-89% of the same control mean. The mean activity in extracts of lymphoblasts derived from the patient approximated 2% of the control mean. The data suggest that the parents, brother and maternal grandmother are carriers of the defective gene responsible for mevalonate kinase deficiency, consistent with an autosomal recessive mode of inheritance.

Cerebral gigantism: Concentrations of amino acids in plasma and muscle

Patients with cerebral gigantism have advanced linear growth and skeletal maturation, as well as mental retardation and characteristic facies. Two patients with these features have been studied. Both had dermatoglyphic abnormalities. Growth hormone concentrations were normal. The concentrations of essential amino acids in the plasma of these patients were considerably higher than those of control subjects. The ratios of glycine to valine and the ratios of other nonessential to essential amino acids were particularly effective in distinguishing patient from control. The data suggest that these ratios may be a meaningful way to assess cellular growth.

Concentrations of uric acid in the serum of neonatal infants and their mothers.

Abstract Serum concentration of uric acid were measured in 45 normal mother-infant pairs. At the time of delivery concentrations in mother and baby were highly significantly correlated and the cord to maternal ratio was 1.08. In the mothers, the serum concentrations rose significantly by 12 to 24 hours post partum. In the infants they also rose significantly by 12 to 24 hours, falling thereafter to a steady state after 3 days of life. Serum concentrations of uric acid in 98 normal infants could best be described by a quadriatic relationship. The data obtained provide a base for the detection of disorders of purine metabolism in infants and indicate that testing should be done after 3 days of life. The maternal-infant interrelationships observed and the course of time-related changes are important for an understanding of purine metabolism in this period.

An improved technique for the separation of glucose 6-phosphate dehydrogenase isoenzymes by disc electrophoresis on polyacrylamide gel.

A new method has been developed for the separation of glucose 6-phosphate dehydrogenase (G6PD) isoenzymes by means of disc electrophoresis. The effects of gel concentration, method of preparation, and application of samples on the separation of types A and B enzymes and hemoglobin were examined. The method not only separated the isoenzymes but also subresolved each isoenzyme into subbands. The formation of subbands may be related to the existence of enzyme subunits.

Theophylline: Induction of self-biting in rabbits

A tentative animal model is presented for studying the self-mutilating behavior seen in children with an abnormality of purine metabolism. Each of four groups of rabbits was given a quarter-normal diet and daily injections of saline or 46.0, 61.5, or 92.0 mg/kg of anhydrous theophylline (1, 3-dimethylxanthine). The major findings were that (1) the greatest number of self-biters were found in the 61.5 mg/kg group, (2) mortality was directly related to drug dosage, and (3) the onset of biting occurred earliest with the highest dosage.

Quantitation of oxypurines and allopurinol metabolites in biological fluids by cation-exchange chromatography

Abstract Chromatography of urine, cerebrospinal fluid, and deproteinized serum on 30 × 0.9 cm columns of AG 50 permits the convenient separation of hypoxanthine, xanthine, allopurinol, and its metabolites. Quantitation by determination of absorbance at 250 mμ is accurate and can be automated. Column preparation is simple, and the columns can be regenerated and used daily for several months with no loss of resolution.