Hypoxanthine deregulates genes involved in early neuronal development. Implications in Lesch-Nyhan disease pathogenesis.

Neurological manifestations in Lesch-Nyhan disease (LND) are attributed to the effect of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency on the nervous system development. HPRT deficiency causes the excretion of increased amounts of hypoxanthine into the extracellular medium and we hypothesized that HPRT deficiency related to hypoxanthine excess may then lead, directly or indirectly, to transcriptional aberrations in a variety of genes essential for the function and development of striatal progenitor cells. We have examined the effect of hypoxanthine excess on the differentiation of neurons in the well-established human NTERA-2 cl.D1 (NT2/D1) embryonic carcinoma neurogenesis model. NT2/D1 cells differentiate along neuroectodermal lineages after exposure to retinoic acid (RA). Hypoxanthine effects on RA-differentiation were examined by the changes on the expression of various transcription factor genes essential to neuronal differentiation and by the changes in tyrosine hydroxylase (TH), dopamine, adenosine and serotonin receptors (DRD, ADORA, HTR). We report that hypoxanthine excess deregulate WNT4, from Wnt/ß-catenin pathway, and engrailed homeobox 1 gene and increased TH and dopamine DRD1, adenosine ADORA2A and serotonin HTR7 receptors, whose over expression characterize early neuro-developmental processes.

Incidence of electrocardiographic alterations in the preoperative period of non-cardiac surgery. / Incidencia de alteraciones electrocardiográficas en el preoperatorio de cirugía no cardiaca.

BACKGROUND: The electrocardiogram is the most widely used test to assess cardiovascular risk during the preoperative period. The objective of the present study is to evaluate the incidence of electrocardiographic alterations in the general population scheduled for non-cardiac surgery and to determine if the age greater than or equal to 65 years or the revised cardiac risk index ≥ 1 represent a risk factor for presenting these alterations. MATERIAL AND METHODS: Over a period of one month, all preoperative electrocardiograms (ECG) from the anesthesia clinic were analyzed. Various epidemiological data were collected and the revised cardiac risk index was calculated. Major alterations were defined as those requiring Cardiology follow-up. RESULTS: 476 patients were recruited, of whom 40.8% were ≥ 65 years, 32.6% had HTN, 14.4% DM and 27.9% dyslipidemia. 16.16% of the patients had a Lee Index ≥ 1. Of the entire sample, 80.5% had a normal ECG, 6.5% minor alterations and 13.0% major alterations. In the multivariate analysis, age ≥ 65 years and the presence of HTN were shown as independent risk factors for presenting alterations in the total and major ECG. The Lee index ≥ 1 was not associated with an increased risk of electrocardiographic abnormalities. CONCLUSIONS: Patients ≥ 65 years old and those with HTN are at greater risk of presenting major electrocardiographic abnormalities, so we recommend including the ECG as a routine diagnostic test in the preoperative period of non-cardiac surgery.

Incidence of electrocardiographic alterations in the preoperative period of non-cardiac surgery.

BACKGROUND: The electrocardiogram is the most widely used test to assess cardiovascular risk during the preoperative period. The objective of the present study is to evaluate the incidence of electrocardiographic alterations in the general population scheduled for non-cardiac surgery and to determine if the age greater than or equal to 65 years or the revised cardiac risk index ≥1 represent a risk factor for presenting these alterations. MATERIAL AND METHODS: Over a period of one month, all preoperative electrocardiograms (ECG) from the anesthesia clinic were analyzed. Various epidemiological data were collected and the revised cardiac risk index was calculated. Major alterations were defined as those requiring Cardiology follow-up. RESULTS: 476 patients were recruited, of whom 40.8% were ≥65 years, 32.6% had HTN, 14.4% DM and 27.9% dyslipidemia. 16.16% of the patients had a Lee Index ≥1. Of the entire sample, 80.5% had a normal ECG, 6.5% minor alterations and 13.0% major alterations. In the multivariate analysis, age ≥65 years and the presence of HTN were shown as independent risk factors for presenting alterations in the total and major ECG. The Lee index ≥1 was not associated with an increased risk of electrocardiographic abnormalities. CONCLUSIONS: Patients ≥65 years old and those with HTN are at greater risk of presenting major electrocardiographic abnormalities, so we recommend including the ECG as a routine diagnostic test in the preoperative period of non-cardiac surgery.

Harmonised framework for ecological risk assessment of sediments from ports and estuarine zones of North and South Atlantic.

This paper presents a harmonised framework of sediment quality assessment and dredging material characterisation for estuaries and port zones of North and South Atlantic. This framework, based on the weight-of-evidence approach, provides a structure and a process for conducting sediment/dredging material assessment that leads to a decision. The main structure consists of "step 1" (examination of available data); "step 2" (chemical characterisation and toxicity assessment); "decision 1" (any chemical level higher than reference values? are sediments toxic?); "step 3" (assessment of benthic community structure); "step 4" (integration of the results); "decision 2" (are sediments toxic or benthic community impaired?); "step 5" (construction of the decision matrix) and "decision 3" (is there environmental risk?). The sequence of assessments may be interrupted when the information obtained is judged to be sufficient for a correct characterisation of the risk posed by the sediments/dredging material. This framework brought novel features compared to other sediment/dredging material risk assessment frameworks: data integration through multivariate analysis allows the identification of which samples are toxic and/or related to impaired benthic communities; it also discriminates the chemicals responsible for negative biological effects; and the framework dispenses the use of a reference area. We demonstrated the successful application of this framework in different port and estuarine zones of the North (Gulf of Cádiz) and South Atlantic (Santos and Paranaguá Estuarine Systems).

Abnormal adenosine and dopamine receptor expression in lymphocytes of Lesch-Nyhan patients.

Self-injurious behavior is the most outstanding feature of Lesch-Nyhan syndrome and has recently been ascribed to an obsessive-compulsive behavior. Lesch-Nyhan syndrome results from the complete enzyme deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) but the link between abnormal purine metabolism and its neurological and behavioral manifestations remains largely unknown. Previous studies led us to hypothesize that adenosine and dopamine receptor expression could be altered in HPRT-deficient cells. To test this hypothesis, we examined mRNA expressions of adenosine (ADORA2A and ADORA2B) and dopamine receptors (DRD1 and DRD2 like), and dopamine transporter (DAT1) in peripheral blood lymphocytes (PBLs) from Lesch-Nyhan patients. We also examined the influence of hypoxanthine in these expressions. As compared to normal PBLs, both ADORA2A and DRD5 expression were abnormal in PBLs from Lesch-Nyhan patients. In contrast, DAT1 expression was similar to control values in HPRT deficient PBLs. These results indicate an abnormal adenosine and dopamine receptor expression in HPRT-deficient cells and suggest disrupted adenosine and dopamine neurotransmission may have a significant role in the pathogenesis of the neurological manifestations of Lesch-Nyhan syndrome.

Integrated sediment quality assessment in Paranaguá Estuarine System, Southern Brazil.

Sediment quality from Paranaguá Estuarine System (PES), a highly important port and ecological zone, was evaluated by assessing three lines of evidence: (1) sediment physical-chemical characteristics; (2) sediment toxicity (elutriates, sediment-water interface, and whole sediment); and (3) benthic community structure. Results revealed a gradient of increasing degradation of sediments (i.e. higher concentrations of trace metals, higher toxicity, and impoverishment of benthic community structure) towards inner PES. Data integration by principal component analysis (PCA) showed positive correlation between some contaminants (mainly As, Cr, Ni, and Pb) and toxicity in samples collected from stations located in upper estuary and one station placed away from contamination sources. Benthic community structure seems to be affected by both pollution and natural fine characteristics of the sediments, which reinforces the importance of a weight-of-evidence approach to evaluate sediments of PES.

Regulation of transcription factor Pdr1p function by an Hsp70 protein in Saccharomyces cerevisiae.

Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae requires the expression of several ATP binding cassette transporter-encoding genes under the control of the zinc finger-containing transcription factor Pdrlp. The ATP binding cassette transporter-encoding genes regulated by Pdrlp include PDR5 and YOR1, which are required for normal cycloheximide and oligomycin tolerances, respectively. We have isolated a new member of the PDR gene family that encodes a member of the Hsp70 family of proteins found in this organism. This gene has been designated PDR13 and is required for normal growth. Overexpression of Pdr13p leads to an increase in both the expression of PDR5 and YOR1 and a corresponding enhancement in drug resistance. Pdr13p requires the presence of both the PDR1 structural gene and the Pdr1p binding sites in target promoters to mediate its effect on drug resistance and gene expression. A dominant, gain-of-function mutant allele of PDR13 was isolated and shown to have the same phenotypic effects as when the gene is present on a 2microm plasmid. Genetic and Western blotting experiments indicated that Pdr13p exerts its effect on Pdr1p at a posttranslational step. These data support the view that Pdr13p influences pleiotropic drug resistance by enhancing the function of the transcriptional regulatory protein Pdr1p.

Unapparent hypoxanthine-guanine phosphoribosyltransferase deficiency.

Complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity causes Lesch Nyhan disease (LND), characterized by hyperuricemia, severe action dystonia, choreoathetosis, ballismus, cognitive and attention deficit and self-injurious behavior. Partial HPRT deficiency is present in patients with Lesch-Nyhan variant (LNV), who present with HPRT-related gout and a variable degree of neurological involvement. The diagnosis of HPRT deficiency relies on clinical, biochemical, enzymatic and molecular data. Patients with HPRT deficiency present low or undetectable HPRT activity in hemolysates, with increased adenine phosphoribosyltransferase (APRT) activity. We present a 9-year-old boy who experienced an episode of macroscopic hematuria with dysuria and left flank pain. He presented hyperuricemia and hyperuricosuria. HPRT and APRT activities were both normal in hemolysate; however, HPRT activity assayed in intact erythrocytes was 50% of control levels. A new missense point mutation c.424 A>G (T142A) was found in the HPRT1 gene. The apparent Michaelis constant (Km) for 5-phosphoribosyl-pyrophosphate assayed in patient hemolysate was 20-fold of control levels. In conclusion, we report a patient with HPRT deficiency who presented with both normal HPRT and APRT activity in hemolysate, in which the enzyme activity determined in intact erythrocytes was of diagnostic utility.

Colour vision defects in asymptomatic carriers of the Leber's hereditary optic neuropathy (LHON) mtDNA 11778 mutation from a large Brazilian LHON pedigree: a case-control study.

AIMS: To determine if asymptomatic carriers from a previously identified large pedigree of the Leber's hereditary optic neuropathy (LHON) 11778 mtDNA mutation have colour vision deficits. METHODS: As part of a comprehensive analysis of over 200 members of a large Brazilian LHON pedigree spanning seven generations, colour vision tests were obtained from 91 members. Colour vision was tested one eye at a time using the Farnsworth-Munsell 100 (FM-100) hue colour vision test. The test was administered under uniform conditions, taking into account: ambient light levels, daylight colour temperature of 6700 kelvin, and neutral uniform background. Tests were scored using the FM-100 MS-Excel computer scoring program. Defects were determined and categorised as tritan, deutan, or protan. Categorisation of each dyschromatopsia was based on review of demonstrated axis computer generated plots and age adjusted error scores which coincided with Verriest 95% confidence intervals. Only the axis with the greatest magnitude error score was used to classify the defect. 55 of the 91 test subjects were LHON mtDNA 11778 J haplotype mutation carriers, proved by mtDNA analysis. The remaining 36 subjects were age matched non-blood relatives (off pedigree), who served as controls. RESULTS: 27 of 55 carriers (49.10%) were shown to have colour vision defects in one or both eyes. 13 of the 27 (48%) abnormal tests in the carrier group were tritan defects and the remaining 14 (52%) were deutan defects. Nine of the 27 (33%) abnormals in the carrier group were identified as having bilateral defects. Six of these were deutan, and the remaining three were tritan dyschromatopsias. Only six of the 36 (16.66%) age matched controls were found to have any type of dyschromatopsia. Five (83.3%) of these were deutan defects. The remaining one was a tritan defect. The difference between the two groups using a chi(2) test with one degree of freedom was statistically significant with a p value less that 0.001. CONCLUSIONS: Until now, LHON has always been characterised by a sudden, devastating vision loss. Asymptomatic carriers, those without vision loss, were considered unaffected by the disease. It now appears that asymptomatic carriers of the LHON mutation are affected by colour vision defects and may manifest other subtle, yet chronic, changes.

Hypoxanthine effect on equilibrative and concentrative adenosine transport in human lymphocytes: implications in the phatogenesis of Lesch-Nyhan syndrome.

We postulated that increased levels of hypoxanthine, a main characteristic of hypoxanthine phosphoribosyltransferase (HPRT) deficiency, may influence adenosine function which could be related to some of the neurological features of the Lesch-Nyhan syndrome. We have examined the effect of hypoxanthine on different adenosine transporters in peripheral blood lymphocytes from control subjects. Increased hypoxanthine concentrations (25 microM) significantly decreased adenosine transport. The equilibrative adenosine transporters (79.6% of the adenosine transport), both NBTI sensitive and NBTI insensitive, were affected significantly. In contrast, the concentrative adenosine transporters were not influenced by hypoxanthine. These results supports the hypothesis that increased hypoxanthine levels influence equilibrative (predominantly NBTI-insensitive type) adenosine transporters.

Efficacy and safety of allopurinol in patients with the Lesch-Nyhan syndrome and partial hypoxanthine- phosphoribosyltransferase deficiency: a follow-up study of 18 Spanish patients.

Allopurinol is used widely for the treatment of purine disorders such as gout, but efficacy and safety of allopurinol has not been analyzed systematically in an extensive series of patients with HPRT deficiency. From 1984 to 2004 we have diagnosed 30 patients with HPRT deficiency. Eighteen patients (12 with Lesch-Nyhan syndrome or complete HPRT deficiency, and 6 with partial HPRT deficiency) were treated with allopurinol (mean dose, 6.44 mg/Kg of weight per day) and followed-up for at least 12 months (mean follow-up 7,6 years per patient). Mean age at diagnosis was 7 years (range, 5 months to 35 years). Treatment with allopurinol was associated to a mean reduction of serum urate concentration of 50%, and was normalized in all patients. Mean urinary uric acid excretion was reduced by 75% from baseline values, and uric acid to creatinine ratio was close or under 1.0 in all patients. In contrast, hypoxanthine and xanthine urinary excretion rates increased by a mean of 6 and 10 times, respectively, compared to baseline levels. These modifications were similar in patients with complete or partial HPRT deficiency. In 2 patients xanthine stones were documented despite allopurinol dose adjustments to prevent markedly increased oxypurine excretion rates. Neurological manifestations did not appear to be influenced by allopurinol therapy. Allopurinol is a very efficacy and fairly safety drug for the treatment of uric acid overproduction in patients with complete and partial HPRT deficiency. Allopurinol was associated with xanthine lithiasis.

Familial nephropathy associated with hyperuricemia in Spain: our experience with 3 families harbouring a UMOD mutation.

Since 1993 we have studied 5 Spanish families with familial nephropathy associated with hyperuricemia (FJHN). Among these families, 24 patients have been identified. All patients had some combination of hyperuricemia, gout, renal insufficiency, arterial hypertension, and reduced kidney size. The clinical presentation in the different families and in the members of the same family was heterogeneous. Allopurinol treatment did not appear to influence renal disease. From a clinical perspective, this syndrome is a distinctive interstitial nephropathy, inherited as an autosomal dominant trait, that progresses to renal failure and is not halted nor prevented by allopurinol therapy. In 2003, genetic linkage analysis in 3 of the 5 families showed linkage of FJHN to 16p 11.2. One family was not analyzed and one family did not show linkage to this region confirming the genetic heterogeneity of this syndrome. A mutation in UMOD gene was found in these 3 families as the cause of the FJHN. The mutations cluster in exon 4 and exon 5 and were point mutation that results in an amino acid change in the uromodulin or Tamm Horsfall protein. This fact allowed in 2004, the presymptomatic genetic diagnosis of an 8-years-old boy belonging to one of these 3 Spanish families. We conclude that in families with a history of renal failure and/or gout in which FJHN is suspected, UMOD mutation screening may enable a definite diagnosis. When a mutation is found, family members can be tested for a UMOD mutation and pre-symptomatic diagnosis may allow counseling to prevent or halt the progression to renal insufficiency.

A review of the implication of hypoxanthine excess in the physiopathology of Lesch-Nyhan disease.

Lesch-Nyhan disease is caused by HGprt deficiency, however, the mechanism by which enzyme deficiency leads to the severe neurological manifestations is still unknown. We hypothesized that hypoxanthine excess leads, directly or indirectly, through its action in adenosine transport, to aberrations in neuronal development. We found that hypoxanthine diminishes adenosine transport and enhances stimulation of adenosine receptors. These effects cause an imbalance between adenosine, dopamine, and serotonin receptors in HGprt deficient cells, and cells differentiated with hypoxanthine showed an increase in dopamine, adenosine and serotonin receptors expression. Hypoxanthine deregulates early neuronal differentiation increasing WNT4 and EN1 gene expression.

Ultrasonography in the diagnosis of asymptomatic hyperuricemia and gout.

Sonography has detected urate deposits in 34%-42% of the patients with asymptomatic hyperuricemia. This may prompt reclassification of asymptomatic hyperuricemia into "asymptomatic gout" and consideration of urate lowering therapy (ULT) to resolve urate deposits. In patients with gout and no visible tophi, sonography has detected urate deposits in half of the patients. This may allow diagnosing "tophaceous gout" and influencing the serum urate target level, prophylaxis to avoid acute gout flares during ULT, and clinical follow-up. Current accessibility to sonography may better classify patients with hyperuricemia and gout and contribute to delineate therapeutic objectives and clinical guidance.

Cell swelling activates the K+ conductance and inhibits the Cl- conductance of the basolateral membrane of cells from a leaky epithelium.

Necturus gallbladder epithelial cells bathed in 10 mM HCO3/1% CO2 display sizable basolateral membrane conductances for Cl- (GClb) and K+ (GKb). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K+/Cl- selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca2+]i) increased transiently during hyposmotic swelling (peak at approximately 30 s, return to baseline within approximately 90 s), but chelation of cell Ca2+ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10-12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (Vcs) produced a significant increase in the change in Vcs elicited by lowering basolateral solution [Cl-], whereas hyperpolarization of Vcs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases GKb and decreases GClb. These two effects appear to be linked, i.e., the increase in GKb produces membrane hyperpolarization, which in turn reduces GClb. (b) Hyperosmotic shrinkage has the opposite effects on GKb and GClb. (c) Cell swelling causes a transient increase in [Ca2+]i, but this response may not be necessary for the increase in GKb during cell swelling.

Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in thirteen Spanish families.

We have determined the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT; HPRT1) deficiency in eight Lesch-Nyhan patients and in five partially HPRT deficient patients with mild to severe neurologic symptoms. Eight of these thirteen mutations have not been previously described. HPRT Zaragoza II (a GG insertion in exon 2), HPRT Murcia (an AG deletion in exon 4), HPRT Asturias (a A deletion in exon 4) and HPRT Cartagena (a A insertion in exon 6) cause a frame-shift resulting in a premature stop codon. HPRT Sevilla is a splice-site mutation resulting in exon 8 skipping in the HPRT mRNA. HPRT Huelva, Madrid II and Zaragoza I are point mutations that result in single amino-acid changes in the mutated HPRT protein (118G-->A, G40R; 143G-->A, R 48 H; 397G-->A, V133 M, respectively). Three mutations have been previously described in unrelated families, and two mutations have been already published. All mutations that resulted in truncated proteins corresponded to patients with the Lesch-Nyhan phenotype. Characterization of the HPRT mutation allowed us to make carrier detection in 33 women and prenatal diagnosis in two fetuses. Hum Mutat 15:383, 2000.

UROMODULIN mutations cause familial juvenile hyperuricemic nephropathy.

Gout, which is commonly associated with hyperuricemia, affects 0.2% of the population. Hyperuricemia has a heterogeneous etiology that may be due to either over production and/or reduced renal clearance, of urate. In order to identify the mechanisms underlying reduced excretion of urate, we undertook positional cloning studies of familial juvenile hyperuricaemic nephropathy (FJHN), which is an autosomal dominant disorder characterized by hyperuricaemia, a low fractional renal excretion of urate, and chronic renal failure that is associated with interstitial fibrosis. The FJHN locus has been previously localized to a 22 centiMorgan interval flanked centromerically by D16S401 and telomerically by D16S3069, on chromosome 16p11-p13. This interval contains over 120 genes and we selected 13 renal expressed sequences to search for mutations in 5 unrelated FJHN families that contained 21 affected and 24 unaffected members. This revealed 5 heterozygous missense mutations (Cys77Tyr, Cys126Arg, Asn128Ser, Cys255Tyr and Cys300Gly) that altered evolutionary conserved residues in the gene encoding UROMODULIN. UROMODULIN, which is an 85 Kda glycoprotein, has roles in renal stone formation, the modulation of immune responses, and urothelial cytoprotection. The results of our studies, which have identified the gene causing FJHN, now indicate a further, novel role for UROMODULIN in urate metabolism.

The spectrum of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency. Clinical experience based on 22 patients from 18 Spanish families.

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the reutilization of hypoxanthine and guanine to the purine nucleotides IMP and GMP, respectively. HPRT deficiency is an X-linked disorder characterized by uric acid overproduction and variable neurologic impairment. The complete deficiency of HPRT is diagnostic of Lesch-Nyhan syndrome manifested by choreoathetosis, spasticity, mental retardation, and self-injurious behavior. In some HPRT-deficient patients the enzyme defect appeared to be "partial" and the neurologic symptoms mild to severe (Kelley-Seegmiller syndrome). This has prompted the classification of HPRT deficiency in 2 distinct groups: Lesch-Nyhan syndrome and Kelley-Seegmiller syndrome, which has created much confusion. A spectrum of clinical consequences of HPRT deficiency has been recognized in small series of patients, but the complete spectrum of the neurologic disorder has not been described in a single series of patients examined by the same observers. We analyzed our experience with 22 patients belonging to 18 different families with HPRT deficiency diagnosed at "La Paz" University Hospital in Madrid over the past 16 years. The clinical spectrum of these HPRT-deficient Spanish patients was similar to the different phenotypes occasionally reported in the literature, in some cases diagnosed as Lesch-Nyhan "variants." The clinical, biochemical, enzymatic, and molecular genetic studies on these 22 patients allowed us to delineate a new classification of HPRT deficiency. Based on the neurologic symptoms, dependency for personal care, HPRT activity in hemolysate and in intact erythrocytes, and predicted protein size, patients were classified into 4 groups: Group 1 (2 patients), normal development with no neurologic symptoms, HPRT activity was detectable in hemolysates and in intact erythrocytes, and the mutation did not affect the predicted protein size. Group 2 (3 patients) mild neurologic symptoms that did not prevent independent lives, HPRT activity was detectable in intact erythrocytes, and the protein size was normal. Group 3 (2 patients), severe neurologic impairment that precluded an independent life, no residual HPRT activity, and normal protein size. Group 4 (15 patients), clinical characteristics of Lesch-Nyhan syndrome (some may not show self-injurious behavior), no residual HPRT activity, and in most (7 of 8 patients in whom the mutation could be detected) the mutation affected the predicted protein size. This classification of HPRT deficiency into 4 groups may be more useful in terms of accuracy, reproducibility, assessment for treatment trials and prognosis. The study of this Spanish series allows us to conclude that HPRT deficiency may be manifested by a wide spectrum of neurologic symptoms; the overall severity of the disease is associated with mutations permitting some degree of residual enzyme activity; and mutation analysis provides a valuable tool for prognosis, carrier identification, and prenatal diagnosis.

Purine metabolism in women with primary gout.

PURPOSE: Uncontrolled studies have shown that women with gout have higher serum urate concentrations and similar or lower urinary uric acid excretion rates than do men with gout. These observations suggest a more defective tubular transport of uric acid in women than in men with gout. In this prospective study we assessed purine metabolism in women with primary gout under controlled conditions. We also examined whether there are sex-related differences in plasma and urinary purine concentrations among patients with primary gout. SUBJECTS AND METHODS: Ten women with crystal-proved primary gout and normal serum creatinine levels (below 116 mmol/L) were studied while they were on a purine-restricted diet and taking no medications known to influence uric acid metabolism. For comparison, 20 men with primary gout and 10 women without gout, matched for age, race, and body mass index, were studied under the same conditions. In each subject, plasma and 24-hour urinary uric acid, hypoxanthine, and xanthine concentrations were measured. The mean of three consecutive determinations for plasma purines and five for urinary purines was used. Standard formulas were used to calculate the renal clearances and the fractional excretion of purines. RESULTS: Mean plasma urate, hypoxanthine, and xanthine levels were significantly higher in women patients with primary gout compared with normal women (P < 0.05). Mean 24-hour urinary uric acid excretion was similar in both groups. Daily urinary hypoxanthine and xanthine excretion rates were significantly lower in gouty women patients than in control women (P < 0.05). The renal clearances and the fractional excretion of uric acid, hypoxanthine, and xanthine were markedly lower in women with primary gout than in control women (P < 0.05). Plasma and urinary purine concentrations were similarly increased and diminished, respectively, in women and men patients with primary gout. Plasma urate, hypoxanthine, and xanthine levels were inversely and significantly associated with the fractional excretion of uric acid (r = -0.520; P = 0.003), hypoxanthine (r = -0.555; P = 0.002), and xanthine (r = -0.384; P = 0.040), respectively. CONCLUSION: Women with primary gout have markedly diminished uric acid, hypoxanthine, and xanthine excretion rates. The disturbance of purine metabolism appears to be of a similar magnitude to that observed in gouty men. The absence of significant sex-related differences in plasma and urinary purine concentrations suggests a similar tubular dysfunction for purine excretion in women and men with primary gout.

A simplified method for the determination of phosphoribosylpyrophosphate synthetase activity in hemolysates.

Methods currently employed for measurement of phosphoribosylpyrophosphate synthetase (ribophosphate pyrophosphokinase; PRPPs) activity are cumbersome and expensive, requiring an auxiliary enzyme reaction, a radioactive nucleobase and multiple incubations, or do not allow a complete kinetic study. The aim of the present study is to describe a simplified, single step, non-isotopic method for determination of PRPPs in hemolysate, appropriate for a complete screening of PRPPs activity disorders. Briefly, the charcoal-treated hemolysate is incubated with saturating amounts of substrates and P1 P5 di(adenosine 5') pentaphosphate (Ap5A), an inhibitor of human adenylate kinase, to prevent conversion of AMP to ADP. AMP generated in this reaction is then measured by HPLC. Adenylate kinase activity was fully inhibited by Ap5A, allowing the accurate determination of AMP. The method was sensitive and reproducible and mean and variance values compared closely with those reported using other, more complicated, assay procedures. The hyperbolic curve relating Pi concentration to initial reaction velocity was shifted to sigmoidal by addition of 0.02 mmol/l GDP which inhibited PRPPs activities only at inorganic phosphate concentrations < 2 mmol/l. This suggests that this method should provide sensitive and accurate screening for regulatory, as well as catalytic, defects underlying PRPPs superactivity.