Purine metabolism in sprint- vs endurance-trained athletes aged 20‒90 years.

Purine metabolism is crucial for efficient ATP resynthesis during exercise. The aim of this study was to assess the effect of lifelong exercise training on blood purine metabolites in ageing humans at rest and after exhausting exercise. Plasma concentrations of hypoxanthine (Hx), xanthine (X), uric acid (UA) and the activity of erythrocyte hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were measured in 55 sprinters (SP, 20‒90 years), 91 endurance runners (ER, 20‒81 years) and 61 untrained participants (UT, 21‒69 years). SP had significantly lower levels of plasma purine metabolites and higher erythrocyte HGPRT activity than ER and UT. In all three groups, plasma purine levels (except UA in UT) significantly increased with age (1.8‒44.0% per decade). HGPRT activity increased in SP and ER (0.5‒1.0%), while it remained unchanged in UT. Hx and X concentrations increased faster with age than UA and HGPRT levels. In summary, plasma purine concentration increases with age, representing the depletion of skeletal muscle adenine nucleotide (AdN) pool. In highly-trained athletes, this disadvantageous effect is compensated by an increase in HGPRT activity, supporting the salvage pathway of the AdN pool restoration. Such a mechanism is absent in untrained individuals. Lifelong exercise, especially speed-power training, limits the age-related purine metabolism deterioration.

Systems biology analysis of the lung cancer­related secretome.

Tumorigenesis is closely and highly associated with developmental biology. The present study aimed to discover and identify marker proteins strongly associated with the occurrence and development of non­small cell lung cancer (NSCLC) in humans and to provide new ideas for investigating lung cancer markers by combining biological analyses of embryonic development. We established primary cultures for samples of tumor and control tissues from 9 patients with NSCLC and collected conditioned medium (CM). Subsequently, we used liquid chromatography and linear ion trap (LTQ) mass spectrometry to isolate and identify proteins in CM samples. Data mining of free proteins was conducted using the analogous analysis strategy in systems biology to obtain important lung cancer­associated proteins (plasma markers). Proteins with significant plasma enrichment in lung cancer patients were detected via enzyme­linked immunosorbent assay (ELISA). We identified 987 high­confidence proteins and established a primary database of free proteins associated with lung cancer. Furthermore, 511 high­confidence proteins were present in CM from primary tissue cultures from at least 2 of the 9 examined cases of lung cancer. Analysis using Gene Set Enrichment Analysis (GSEA) software revealed significant enrichment for 197 proteins from the CM of lung cancer samples in maternal­placental interface expression profiles for a mid­term placenta with strong invasiveness relative to RNA expression profiles for a human full­term placenta after delivery. ELISA results demonstrated that hypoxanthine phosphoribosyltransferase 1 (HPRT1) was associated with worse rates of disease­free survival (DFS) and overall survival (OS). The biological behaviors of embryonic implantation are similar to those of tumor invasion and metastasis, and the information obtained regarding developmental biology could facilitate the interpretation of tumor invasion and metastasis. Therefore, similar biological behaviors combined with analyses at different molecular levels from the perspective of systems biology will provide new ideas for tumor research.

Hypoxanthine: A Universal Metabolic Indicator of Training Status in Competitive Sports.

Cardiorespiratory and biochemical indicators typically used by contemporary elite athletes seem to have limited applicability. According to some recent studies, purine metabolism better reflects exercise response and muscle adaptation in this group. We propose using purine derivatives, especially plasma hypoxanthine concentration, as indicators of training status in consecutive training phases in highly trained athletes.

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.

The pharmacokinetic effect of adalimumab on thiopurine metabolism in Crohn's disease patients.

BACKGROUND AND AIMS: A drug interaction between infliximab and azathioprine has previously been reported in Crohn's disease patients: the concentration of the main active thiopurine metabolites, the 6-thioguanine nucleotides (6-TGN), increased 1-3 weeks after the first infliximab infusion by 50% compared to baseline. The aim of this prospective study was to determine the effect of adalimumab on thiopurine metabolism in Crohn's disease patients, evaluated by 6-TGN and 6-methylmercaptopurine ribonucleotides (6-MMPR) concentration measurement. METHODS: Crohn's disease patients on azathioprine or mercaptopurine maintenance therapy starting with concomitant adalimumab treatment were included. 6-TGN and 6-MMPR concentrations were determined before initiation of adalimumab and after 2, 4, 6 and 12 weeks of combination therapy. The activity of three essential enzymes involving thiopurine metabolism, thiopurine S-methyltransferase (TPMT), hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and inosine-triphosphate pyrophosphatase (ITPase), was evaluated at baseline and week 4. Clinical outcome was evaluated by the Crohn's disease activity index and C-reactive protein concentrations at baseline, week 4 and week 12. RESULTS: Twelve Crohn's disease patients were analyzed. During the follow-up period of 12 weeks the median 6-TGN and 6-MMPR concentrations did not significantly change compared to baseline. TPMT, ITPase and HGPRT enzyme activity did not change either after 4 weeks. In two patients (17%) myelotoxicity was observed within 2-4 weeks, in whom both low therapeutic 6-TGN and 6-MMPR concentrations were found. CONCLUSIONS: In this study in Crohn's disease patients no pharmacokinetic interaction was shown between adalimumab and the conventional thiopurines, azathioprine and mercaptopurine.

PfHPRT: a new biomarker candidate of acute Plasmodium falciparum infection.

Plasmodium falciparum is a protozoan parasite that causes human malaria. This parasitic infection accounts for approximately 655,000 deaths each year worldwide. Most deaths could be prevented by diagnosing and treating malaria promptly. To date, few parasite proteins have been developed into rapid diagnostic tools. We have combined a shotgun and a targeted proteomic strategy to characterize the plasma proteome of Gambian children with severe malaria (SM), mild malaria, and convalescent controls in search of new candidate biomarkers. Here we report four P. falciparum proteins with a high level of confidence in SM patients, namely, PF10_0121 (hypoxanthine phosphoribosyltransferase, pHPRT), PF11_0208 (phosphoglycerate mutase, pPGM), PF13_0141 (lactate dehydrogenase, pLDH), and PF14_0425 (fructose bisphosphate aldolase, pFBPA). We have optimized selected reaction monitoring (SRM) assays to quantify these proteins in individual patients. All P. falciparum proteins were higher in SM compared with mild cases or control subjects. SRM-based measurements correlated markedly with clinical anemia (low blood hemoglobin concentration), and pLDH and pFBPA were significantly correlated with higher P. falciparum parasitemia. These findings suggest that pHPRT is a promising biomarker to diagnose P. falciparum malaria infection. The diagnostic performance of this marker should be validated prospectively.

Prolonged fasting increases purine recycling in post-weaned northern elephant seals.

Northern elephant seals are naturally adapted to prolonged periods (1-2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, food deprivation stimulates ATP degradation and decreases ATP synthesis, resulting in the accumulation of purines (ATP degradation byproducts). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism, which also promotes oxidant production. The contributions of HGPRT to purine recycling during prolonged food deprivation in marine mammals are not well defined. In the present study we cloned and characterized the complete and partial cDNA sequences that encode for HGPRT and xanthine oxidoreductase (XOR) in northern elephant seals. We also measured XO protein expression and circulating activity, along with xanthine and hypoxanthine plasma content in fasting northern elephant seal pups. Blood, adipose and muscle tissue samples were collected from animals after 1, 3, 5 and 7 weeks of their natural post-weaning fast. The complete HGPRT and partial XOR cDNA sequences are 771 and 345 bp long and encode proteins of 218 and 115 amino acids, respectively, with conserved domains important for their function and regulation. XOR mRNA and XO protein expression increased 3-fold and 1.7-fold with fasting, respectively, whereas HGPRT mRNA (4-fold) and protein (2-fold) expression increased after 7 weeks in adipose tissue and muscle. Plasma xanthine (3-fold) and hypoxanthine (2.5-fold) levels, and XO (1.7- to 20-fold) and HGPRT (1.5- to 1.7-fold) activities increased during the last 2 weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to ameliorating oxidant production and enhancing the supply of ATP, both of which would be beneficial during prolonged food deprivation and appear to be adaptive in this species.

Successful unrelated umbilical cord blood transplantation in Lesch-Nyhan syndrome.

Lesch-Nyhan syndrome (LNS) is a chronic, progressive neurodevelopmental disorder causing motor and behavioral dysfunction due to decreased synthesis of the enzyme hypoxantine-guanine phosphoribosyltransferase (HPRT). Affected boys have mental retardation, delayed development, extrapyramidal motor disturbances and self-injuring behavior. As hematopoietic stem cell transplantation (HSCT) has been shown to be effective in several neurodevelopmental inborn errors, we hypothesized that it could be favorable in LNS as well. Following a myeloablative conditioning regimen (busulphan 3.2 mg/kg/day for 4 days, cyclophosphamide 60 mg/kg/day for 2 days with ATG Thymoglobin 2.5 mg/kg/day for 4 days) an unrelated umbilical cord blood unit was transfused at the age of 2 years. The graft was a 6/6 HLA-matched at HLA-A, B loci by antigen level, and at DRB1 by allelic level typing. Infused total nucleated cell dose was 3.6 × 10e7 per kilogram body weight. Serum HPRT levels reached normal values by the end of the sixth month post transplant. Slow neurodevelopmental improvement seen during the three-year follow-up and the missing self-injuring behavior can be considered as a proof for the presence of enzyme-competent cells behind the blood-brain barrier.

Identification of a new single-nucleotide mutation on the hypoxanthine-guanine phosphoribosyltransferase gene from 983 cases with gout in Taiwan.

OBJECTIVE: The frequency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency within the gout-affected population in Taiwan was unclear. We evaluated its frequency and sought to identify a new genetic variation in a case with HPRT deficiency. METHODS: From 2004 to 2005, a total of 983 patients with gout were followed among outpatients attending the Department of Rheumatology. Among these, 12 cases were suspected to have HPRT deficiency, and HPRT activity was examined by HPLC. In the index case found to have HPRT deficiency, genetic variation was analyzed by RT-PCR, direct sequencing, and SSCP. RESULTS: Only a single case proved to have partial HPRT deficiency among 12 suspicious cases. Both cDNA and genomic DNA analysis identified a new mutation on exon 2 with T to G transition at cDNA base 93, resulting in a change from aspartic acid to glutamic acid at position 31. It was designated as HPRTChia-Yi, from our case's residence at Chia-Yi Hsein, Taiwan. CONCLUSION: According to this hospital-based survey, HPRT deficiency is a rare trait in the Taiwanese gouty population. However, our index case with HPRT deficiency provided the first proven HPRT mutation in non-aboriginal Taiwanese patients with gout, which was different from a mutation previously found in aboriginal Taiwanese. Hence, in non-aboriginal Taiwanese gouty patients with HPRT deficiency, exon 2, rather than just exon 3, should be analyzed.

Analysis of genetic alterations and clonal proliferation in children treated for acute lymphocytic leukemia.

The development of risk-directed treatment protocols over the last 25 years has resulted in an increase in the survival rates of children treated for cancer. As a consequence, there is a growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapy is unknown. We previously reported that children treated for acute lymphocytic leukemia have significantly elevated somatic mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in their peripheral T cells. To understand the molecular etiology of the increase in mutant frequencies following chemotherapy, we investigated the HPRT mutation spectra and the extent of clonal proliferation in 562 HPRT T cell mutant isolates of 87 blood samples from 47 subjects at diagnosis, during chemotherapy, and postchemotherapy. We observed a significant increase in the proportion of CpG transitions following treatment (13.6-23.3%) compared with healthy controls (4.0%) and a significant decrease in V(D)J-mediated deletions following treatment (0-6.8%) compared with healthy controls (17.0%). There was also a significant change in the class type percentage of V(D)J-mediated HPRT deletions following treatment. In addition, there was a >5-fold increase in T cell receptor gene usage-defined mean clonal proliferation from diagnosis compared with the completion of chemotherapeutic intervention. These data indicate that unique genetic alterations and extensive clonal proliferation are occurring in children following treatment for acute lymphocytic leukemia that may influence long-term risks for multifactorial diseases, including secondary cancers.

HPRT deficiency as the cause of ESRD in a 24-year-old patient: a very rare presentation of the disorder.

A 24-year-old male with end-stage renal disease (ESRD) and disproportionately high uric acid plasma concentration was admitted to our unit. After studying the patient's medical history, as well as that of the entire family, hyperuricemia was discovered in his brother, while microscopic examination of his brother's and mother's urine revealed abundant uric acid crystals. After performing purine metabolic studies, it was determined that the two siblings suffered from partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency (Kelley-Seegmiller syndrome). This report highlights the importance of clinical awareness and a thorough examination of the patient's medical history for establishing an early diagnosis and commencing treatment for such rare inherited metabolic disorders to prevent renal failure.

Lesch-Nyhan disease in a female with a clinically normal monozygotic twin.

Lesch-Nyhan disease (LND) is an inborn error of purine metabolism caused by defective activity of the enzyme hypoxanthine guanine phosphoribosyl transferase (HPRT, EC 2.4.2.8), resulting from mutation in the corresponding gene on the long arm of the X chromosome (Xq26). The classic phenotype occurs almost exclusively in males and is characterized by hyperuricemia, mental retardation, severe dystonia, and self-injurious behavior. Heterozygous carrier females are usually clinically normal. However, a small number of clinically affected females have been described. In all previous cases there was a mutation in one HPRT allele and non-random inactivation of the X chromosome carrying the normal HPRT gene. We have analyzed a female MZ twin pair discordant for Lesch-Nyhan disease. The mother and both twins are heterozygous carriers of a HPRT splicing mutation (IVS8 + 4A > G; c.609 + 4A > G) and all three express the mutant allele at similar frequencies in peripheral blood T cells. The mother and one sister are clinically normal. In the affected twin, the clinical phenotype is classical for Lesch-Nyhan disease, despite the fact that HPRT activity in the blood was also normal. X inactivation analysis showed a skewed pattern in the fibroblasts of the affected twin sister, with the X chromosome carrying the normal HPRT allele preferentially inactivated. As in many other reported cases of X-linked diseases, the discordant phenotype of the two monozygous twin sisters suggests that the process responsible for monozygotic twinning can trigger skewed X inactivation.

Hereditary hyperuricemia and renal disease.

Hyperuricemia and gout have long been known to run in families. As well as an apparently multifactorial genetic component to classic gout itself, 2 rather unusual sex-linked single-gene disorders of purine biosynthesis or recycling have been defined: deficiency of the enzyme hypoxanthine-guaninephosphoribosyl transferase (HPRT), and overactivity of PPriboseP synthase. Both result in overproduction of urate, hyperuricemia, and secondary overexcretion that may lead to acute or chronic renal damage. Familial juvenile hyperuricemic nephropathy (FJHN) and autosomal-dominant medullary cystic kidney disease (ADMCKD) are more common but less well-defined hyperuricemic conditions resulting from a decrease in the fractional excretion of filtered urate, with normal urate production. Although having features in common, ADMCKD is distinguished in particular by the presence of medullary cysts. One major group of both disorders is associated with mutations in the gene for uromodulin, but this accounts for only about one third of cases, and genetic heterogeneity is present. Whether the genes involved in these latter disorders contribute to the polygenic hyperuricemia and urate underexcretion of classic gout remains unexplored.

Somatic mutant frequency at the HPRT locus in children associated with a pediatric cancer cluster linked to exposure to two superfund sites.

The somatic mutant frequency (Mf) of the hypoxanthine phosphoribosyl transferase (HPRT) gene has been widely used as a biomarker for the genotoxic effects of exposure but few studies have found an association with environmental exposures. We measured background Mfs in 49 current and former residents of Dover Township, New Jersey, who were exposed during childhood to industrially contaminated drinking water. The exposed subjects were the siblings of children who developed cancer after residing in Dover Township, where the incidence of childhood cancer has been elevated since 1979. Mfs from this exposed group were compared to Mfs in 43 age-matched, presumably unexposed residents of neighboring communities with no known water contamination and no increased cancer incidence. Statistical comparisons were based on the natural logarithm of Mf (lnMF). The mean Mf for the exposed group did not differ significantly from the unexposed group (3.90 x 10(-6) vs. 5.06 x 10(-6); P = 0.135), but unselected cloning efficiencies were higher in the exposed group (0.55 vs. 0.45; P = 0.005). After adjustment for cloning efficiency, lnMf values were very similar in both groups and age-related increases were comparable to those previously observed in healthy children. The results suggest that HPRT Mf may not be a sensitive biomarker for the genotoxic effects of environmental exposures in children, particularly when substantial time has elapsed since exposure.

Are allopurinol and metabolites found in HPRT deficient erythrocytes responsible for increased NAD synthesis?

Aim of this study was to ascertain whether allopurinol, usually administered to hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficient patients, or metabolites abnormally increased in HPRT deficient erythrocytes (NAD, PPribP) could be directly responsible for the reported increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NADsynthetase (NADs) in these patients. No direct effect of the mentioned metabolites was demonstrated.

Acquired DNA mutations associated with in vivo hydroxyurea exposure.

Hydroxyurea (HU) is an effective therapeutic agent for patients with myeloproliferative disorders (MPDs) or sickle cell disease (SCD). Short-term HU toxicities primarily include transient myelosuppression, but long-term HU risks have not been defined. The mutagenic and carcinogenic potential of HU is not established, although HU has been associated with an increased risk of leukemia in some patients with MPD. In this study, 2 assays were used to quantitate acquired somatic DNA mutations in peripheral blood mononuclear cells (PBMCs) after in vivo HU exposure. The HPRT assay measures hypoxanthine phosphoribosyl transferase (hprt) mutations, while the VDJ assay identifies "illegitimate" T-cell receptor Vgamma-Jbeta interlocus recombination events. PBMCs were analyzed from patients with MPD, adults and children with SCD, and normal controls. MPD patients with prolonged HU exposure had numbers of DNA mutations equivalent to patients with low HU exposure or controls. Similarly, adults with SCD had equivalent numbers of DNA mutations regardless of HU exposure. Children with SCD and 30-month HU exposure had equivalent hprt(-) mutations but significantly more VDJ mutations (1.82 +/- 1.20 events per microg DNA) than children with 7-month HU exposure (1.58 +/- 0.87 events) or no HU exposure (1.06 +/- 0.45 events), P =.04 by analysis of variance. Taken together, these data suggest that the mutagenic and carcinogenic potential of in vivo HU therapy is low. Although increased numbers of illegitimate VDJ recombination events do not directly portend leukemia, young patients with SCD and HU exposure should be monitored serially for increases in DNA mutations.

Kelley-Seegmiller syndrome due to a unique variant of hypoxanthine-guanine phosphoribosyltransferase: reduced affinity for 5-phosphoribosyl-1-pyrophosphate manifested only at low, physiological substrate concentrations.

A male child, who presented at the age of 3.5 years with acute renal failure, was diagnosed as having partial deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8). The underlying HPRT mutation was unique in that the specific activity of HPRT in erythrocyte and in fibroblast lysates was normal, but the rate of uptake of hypoxanthine into nucleotides of intact cultured fibroblasts was markedly reduced (23% of normal). The low functioning of HPRT in the intact fibroblasts was associated with decreased utilization of endogenously generated hypoxanthine and with decreased utilization of the cosubstrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The non-utilized hypoxanthine was excreted into the incubation medium. The accumulation of PRPP was indicated by the 2.3-fold increase in the rate of uptake of adenine into intact cell nucleotides and by the 7. 5-fold enhancement of the rate of de novo purine synthesis. Kinetic studies of HPRT activity in fibroblast lysates revealed reduced affinity of the enzyme for PRPP (apparent K(m) 500 microM in comparison to 25 microM in control lysates), manifested in low activity at low (physiological), but not at high PRPP concentrations. The apparent K(m) for hypoxanthine was normal (23 microM in comparison to 14.2 microM in control lysates). With allopurinol treatment, our patient has had no problems since presentation, and is developing normally at 5 years of age.