Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.

Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT.

[Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase presenting seizure and psychomotor retardation: a case report].

An 18-year-old man was admitted to our hospital because of convulsive seizure. He had psychomotor retardation and intellectual disability from childhood, and had been diagnosed with attention deficit-hyperactivity disorder when he was 12 years old. He showed mental deficit (Wechsler Adult Intelligence Scale-Revised: IQ 52) and tendon hyperreflexia without pathological reflexes, but no involuntary movements or self-injurious behavior. As he had hyperuricemia, we measured the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and adenine phosphoribosyltransferase (APRT) in erythrocytes. While HPRT activity had decreased to 57.4% of normal, APRT activity had increased to 140.5% of normal. Genetic analysis revealed a single-base substitution (c.179A>G) in the third exon of the HPRT gene, which resulted in a missense mutation (p.H60R) of the 60th amino acid. His mother was a heterozygous carrier of this mutation and presented partial deficiency (73.3%) of HPRT activity. Lesch-Nyhan disease is a neurogenetic disorder caused by complete deficiency of the enzyme HPRT. Variant forms of the disease caused by partial deficiency of HPRT do not show the typical clinical features, or show only mild neurological manifestations; these diseases are jointly referred to as HPRT-related neurological disease (HRND). The present case was unique in that the patient diagnosed as having HRND showed relatively higher HPRT residual activity in erythrocytes.

Clinical, biochemical and molecular diagnosis of a compound homozygote for the 254 bp deletion-8 bp insertion of the APRT gene suffering from severe renal failure.

OBJECTIVE: To determine the type of mutation in a patient with clinical diagnosis of suspected APRT deficiency. DESIGN AND METHODS: A 51-year-old male patient, with a clinical history of two prior episodes of renal colic with urinary stone excretion (reported as uric acid stones in the first episode and as calcium oxalate stones in the second), was admitted to the hospital with severe non-oliguric renal failure (1.06 mmol/L serum creatinine), severe hyponatremia (114 mmol/L Na(+)), metabolic acidosis (14 mmol/L HCO(3)(-)) and uricemia in the normal range. Abnormalities at renal scan and persistency of severe renal failure required to start haemodialysis. Results of renal biopsy prompted us to undertake a biochemical and molecular biological evaluation of the patient for suspected adenine phosphoribosyltransferase (APRT) deficiency. RESULTS: HPLC analysis of serum and urine, for determining purine derivative profile, showed the pathological presence of adenine in both biological fluids (3.57 micromol/L and 7.11 micromol/mmol creatinine in serum and urine, respectively; not detectable in both fluids in healthy controls). APRT assay in a sample of patient hemolysate showed no detectable activity of the enzyme (25.56+/-9.55 U/L red blood cells in control healthy subjects). Molecular biological analysis of the amplified APRT gene revealed that the patient harboured in exon 3 a homozygous 254 bp deletion-8 bp insertion, previously described only once in a compound heterozygote. Analysis of the patient family showed that heterozygotes for this APRT gene mutation, in spite of a 69% lower APRT enzymatic activity than that of healthy subjects, had no detectable adenine concentrations in both serum and urine. CONCLUSIONS: Results of the first patient harbouring the homozygous 254 bp deletion-8 bp insertion of the APRT gene strongly indicated that definitive diagnosis of APRT deficiency (often under or misdiagnosed) would require a combined clinical, biochemical and molecular biological evaluation.

2,8-Dihydroxyadenine renal stones in a 41-year-old man.

A case is presented of a 41-year-old man with a history of recurrent renal stones over 10 years. Analysis of the stone showed that, although it gave a positive reaction with the non-specific phosphotungstic acid test, uricase failed to identify any urate present. Analysis in a reference laboratory confirmed its composition as dihydroxyadenine. Patients who are homozygous for the rare autosomal-recessive adenine phosphoribosyltransferase deficiency, excrete large amounts of 2,8-dihydroxyadenine, which has poor solubility at normal urinary pH. Treatment with the xanthine oxidase inhibitor allopurinol induces a total cessation of stone formation. Increased awareness of the condition and knowledge of the limitations of some methods of laboratory analysis for renal stones should help to identify this type of stone and prevent renal damage.

Purine metabolism in Echinococcus multilocularis.

The activities of the enzymes in Echinococcus multilocularis metacestodes involved in purine salvage were studied by HPLC. As in most parasites, this cestode relies entirely on salvage of preformed bases and nucleosides for its purine requirement. Therefore, these enzymes may be targets for drugs in the chemotherapeutic treatment of diseases caused by this parasite. The animals used in this study were gerbils (Meriones unguiculatus). Enzyme activities from sera and hepatic tissue in control and infected animals were similar with the exception of adenine phosphoribosyltransferase which showed an activity 4-fold greater in the serum from control than in serum from infected animals. In the parasite, adenine and hypoxanthine-guanine phosphoribosyltransferases and adenosine deaminase had the highest activities. Therefore, in E. multilocularis metacestodes, this pathway seems to be important for the parasite's metabolism.

Adenine phosphoribosyltransferase-deficient mice develop 2,8-dihydroxyadenine nephrolithiasis.

Adenine phosphoribosyltransferase (APRT) deficiency in humans is an autosomal recessive syndrome characterized by the urinary excretion of adenine and the highly insoluble compound 2,8-dihydroxyadenine (DHA) that can produce kidney stones or renal failure. Targeted homologous recombination in embryonic stem cells was used to produce mice that lack APRT. Mice homozygous for a null Aprt allele excrete adenine and DHA crystals in the urine. Renal histopathology showed extensive tubular dilation, inflammation, necrosis, and fibrosis that varied in severity between different mouse backgrounds. Thus, biochemical and histological changes in these mice mimic the human disease and provide a suitable model of human hereditary nephrolithiasis.

Purine enzymes in rheumatoid arthritis: possible association with response to azathioprine. A pilot study.

OBJECTIVE: To study the possible association of purine enzyme activities with response to azathioprine (AZA) treatment in rheumatoid arthritis (RA) and their correlation with parameters of disease activity. PATIENTS AND METHODS: Lymphocyte activities of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT), adenine phosphoribosyltransferase (APRT), purine nucleoside phosphorylase (PNP) and 5'-nucleotidase (5NT), and erythrocyte activities of thiopurine methyltransferase (TPMT) were measured in 14 healthy controls and 36 patients with RA. Eight patients had not previously been treated with AZA. Response to AZA therapy in 28 patients, determined in a prospective trial, was considered good in nine (group 1), insufficient in seven (group 2). In 12 patients AZA was withdrawn because of adverse reactions (group 3). Disease activity parameters were obtained simultaneously with purine enzyme measurements. Purine enzyme levels in the different groups were compared. RESULTS: Levels of 5NT activity were significantly lower in patients with RA than in healthy controls. PNP activity was higher in patients with RA not using prednisone compared with those who did and healthy controls. No clear correlation between purine enzyme levels and disease activity parameters was found. 5NT activities were significantly higher in group one than in group three (p = 0.012; alpha = 0.017), and almost significantly higher than in group two (p = 0.03; alpha = 0.017). CONCLUSIONS: The results indicate that purine enzyme activities in patients with RA differ from healthy controls, are associated with the outcome of AZA treatment and seem not to be associated with disease activity. Our findings may offer a clue to predict the response to AZA therapy in RA.

Scanning electron microscopy of 2,8-dihydroxyadenine crystals and stones.

The lack of purine salvage enzyme, adenine phosphoribosyltransferase (APRT), leads to 2,8-dihydroxyadenine stone formation and/or crystalluria because it is insoluble in urine. Urolithiasis composed of 2,8-dihydroxyadenine is not only formed in a complete defect of APRT, but also in a partial deficiency of this enzyme. The defect is inherited as an autosomal recessive trait, the homozygous state is associated with high urinary levels of 2,8-dihydroxyadenine and with crystalluria, calculus formation, and potential nephrotoxicity. Determination of the APRT activity will facilitate quantification of the enzyme deficiency and elucidation of the hereditary history. 2,8-dihydroxyadenine excretion in the 24-hour urine and its circadian rhythm were determined using a new method of high performance liquid chromatography determination. By means of a standard case presentation, we illustrate the analysis of urinary sediments and calculi as well as the scanning electron microscopic images of this kind of stone.

Contrast in adenine uptake by chicken and rabbit erythrocytes in vitro.

1. Relative to rabbit erythrocytes, chicken red blood cells exhibit a much greater capacity to utilize [3H]adenine for nucleotide synthesis in vitro, even at 5 degrees C and in the absence of added inorganic phosphate. 2. This difference is largely due to a higher concentration of phosphoribosylpyrophosphate and greater activity of adenine phosphoribosyltransferase in the avian cells. 3. The capacity of avian erythrocytes for utilization of guanine and hypoxanthine is several fold less than that of adenine. 4. The data are consistent with lower activity for hypoxanthine/guanine phosphoribosyltransferase than for adenine phosphoribosyltransferase in intact chicken erythrocytes. 5. The results indicate that reutilization of adenine by chicken erythrocytes may be physiologically significant.

The effect of ammonium, phosphate, potassium, and hypotonicity on stored red cells.

The use of an experimental solution that maintained high ATP levels and produced greater than 70 percent viability of stored red cells (RBCs) for up to 18 weeks has been described by other investigators. This solution differed markedly from conventional storage media in that it lacked sodium; contained high concentrations of potassium, ammonium, and phosphate; and was hypotonic. It was not clear which feature or features were responsible for the observed effects. The effects of ammonium, phosphate, potassium, and hypotonicity on stored RBCs have been examined. It was determined that ammonium and phosphate were the important factors in ATP maintenance. The biochemical mechanism by which ammonium acts was studied. In fresh human blood samples, ammonium was found to relieve phosphofructokinase from inhibition by increased ATP concentrations, to have no significant effect on adenine phosphoribosyl transferase activity, and, unexpectedly, to increase the activity of AMP deaminase. Despite prolonged ATP maintenance by ammonium and phosphate-containing storage media, satisfactory viability of RBCs stored up to 77 days was not demonstrated in the rabbit transfusion model.

A new method for the determination of adenine phosphoribosyltransferase activity in human erythrocytes by reversed phase high performance liquid chromatography.

A new method for the determination of adenine phosphoribosyltransferase (APRT) activity in human erythrocytes is described. APRT activity was assayed by a non-radiochemical method in which adenosine monophosphate (AMP) and AMP metabolites produced from a substrate adenine were converted to inosine by alkaline phosphatase and adenosine deaminase. The inosine thus produced was quantitated by reversed phase HPLC. This method was simple, precise, sensitive and free from interference with other co-existing erythrocyte enzymes. Four patients with 2,8-dihydroxyadenine urolithiasis and others with several disorders in purine metabolism have been studied, showing that the present method is clinically useful for the diagnosis and the evaluation of the severity of some human diseases.