Sadao Kashiwazaki

Optimal Range of Serum Urate Concentrations to Minimize Risk of Gouty Attacks During Anti-Hyperuricemic Treatment

To find an optimal range of urate concentrations wherein the risk of attacks during the initial 6 months of treatment is minimized, data from 350 gouty patients treated with anti-hyperuricemic drugs were retrospectively analyzed. We determined the optimal range of urate concentrations to be 4.6-6.6 mg/dl. If urate concentrations were within this range, the risk ratio of an attack as opposed to outside of the range was 0.705 (95% confidence interval, 0.629-0.791). The increase (or decrease) in urate concentration in one month associated with minimal risk of gouty attacks was also determined. The lowest risk ratio of attack (0.451) occurred at a range of -0.1 to 0.6 mg/dl/month increase in urate concentrations (95% confidence interval, 0.310-0.655). In conclusion, we propose that urate concentrations during the initial 6 months of anti-hyperuricemic therapy should be maintained within a range of 4.6-6.6 mg/dl, and reduction in the urate concentrations during treatment should be as slow as possible.

Accelerated purine nucleotide degradation by anaerobic but not by aerobic ergometer muscle exercise

The exact conditions under which exercise causes purine nucleotide degradation are not well understood. We determined plasma hypoxanthine and uric acid levels serially in eight individuals during ergometer muscle exercise. When the load was increased gradually by 15 W/min, plasma hypoxanthine was elevated only after the status exceeded the anaerobic threshold (AT), as determined by analysis of expired gas. Nonstrenuous ergometer exercise, which kept the status continuously below the AT, induced neither blood lactic acid nor plasma hypoxanthine elevation. These results suggest that the AT is also the threshold for the acceleration of purine nucleotide degradation. Muscle exercise to a degree that does not exceed the AT does not cause major purine nucleotide degradation, and, therefore, is expected to be beneficial for patients with gout and/or hyperuricemia.

Additive effects of IL-1 and TNF on induction of prostacyclin synthesis in human vascular endothelial cells.

The effect of interleukin 1 (IL-1) and tumor necrosis factor (TNF) on the induction of prostacyclin (PGI2) synthesis in human vascular endothelial cells (EC) was investigated. Both IL-1 and TNF increased PGI2 production by EC in both a time- and dose-dependent manner, and a combination of the two cytokines additively enhanced PGI2 production. Metabolic inhibitors including indomethacin and cycloheximide abolished cytokine induced PGI2 synthesis. Since, the effect of TNF was not inhibited by neutralization with antibody to IL-1 alpha and IL-1 beta, it seems that the mechanism is not mediated by endogenous IL-1 induced by TNF.

Selection against blood cells deficient in hypoxanthine phosphoribosyltransferase (HPRT) in Lesch-Nyhan heterozygotes occurs at the level of multipotent stem cells

Lesch-Nyhan syndrome is caused by a severe genetic deficiency of hypoxanthine phosphoribosyltransferase (HPRT) and is characterized by central nervous system disorders, gout, and in some cases, macrocytic anemia. Women heterozygous for HPRT deficiency are healthy but their somatic cells are mosaic for enzyme deficiency owing to random inactivation of the X chromosome. Frequencies of red blood cells and T cells deficient in HPRT are significantly lower than the expected 50% in heterozygotes, suggesting that HPRT-negative blood cells are selected against in heterozygotes. To determine at which stage of hematopoiesis such selection occurs, we determined the frequencies of HPRT-negative T, B and erythroid precursor cells in three heterozygotes. Since the cloning efficiencies of T and B cells and colony forming efficiency of burst-forming unit erythroid (BFU-E) for sample from Lesch-Nyhan patients were similar to those of normal cells, HPRT deficiency does not seem to render the differentiated cells less efficient for proliferation. However, the frequencies of HPRT-negative T and B cells, and BFU-E were all less than 10% in each of the three heterozygotes. Although the frequencies of HPRT-negative cells showed tenfold variations between the heterozygotes, each heterozygote had similar frequencies of HPRT-negative cells in the three cell types. These results suggest that HPRT is important at early stages of hematopoiesis, but less so after the cells have differentiated into T cells, B cells and erythroid precursor cells.

Analysis of the genotypes for aldehyde dehydrogenase 2 in Japanese patients with primary gout.

Alcoholic ingestion is one of the major factors for increasing serum uric acid levels. Genotypes of aldehyde dehydrogenase 2 (ALDH2, E.C.1.2.1.3), which regulates the sensitivity of an individual to ethanol, were determined in Japanese patients with gout and control subjects by allele specific oligonucleotide hybridization using PCR amplified gene. The most common allele ALDH2*1 codes for normal ALDH2 activity, while the less common allele ALDH2*2 codes for a lower enzyme activity. The frequency of homozygotes of ALDH2*2 was significantly lower in patients with gout than those with rheumatoid arthritis or a normal population. Plasma and urinary hypoxanthine levels were strikingly increased after ethanol drinking in homozygotes for ALDH2*1 but not in heterozygotes for ALDH2*1/ALDH2*2, indicated extensive purine nucleotide degradation in homozygote for ALDH2*1. These data indicated that alcohol ingestion may not be the requisite factor but is deeply involved in the pathogenesis of gout and hyperuricemia.

1,1′-Ethylidenebis(tryptophan) (peak E) induces functional activation of human eosinophils and interleukin 5 production from T lymphocytes: Association of eosinophilia-myalgia syndrome with al-tryptophan contaminant

This study was designed to clarify the important association between eosinophilia-myalgia syndrome (EMS) and thel-tryptophan contaminant, “Peak E.” To determine the functional activation of eosinophils induced by Peak E, eosinophil cationic protein (ECP) release was examined. Peak E augmented the release of ECP from peripheral blood normodense eosinophils by degranulation. Proliferative analysis using the human eosinophilic leukemia cell line EoL-3 showed prominent cellular replication in the presence of Peak E. Moreover, Peak E upregulated interleukin 5 (IL-5) receptor levels on normodense eosinophils. Of particular interest, Peak E-stimulated human splenic T cells produced bipactive and immunoreactive IL-5. Marked induction of IL-5 mRNA in Peak E-stimulated T cells was also shown by reverse-transcriptase polymerase chain reaction (RT-PCR). In contrast,l-tryptophan without the contaminant showed none of these effects. Thus, these data suggest that Peak E might be involved in the pathogenesis of EMS through bimodal mechanism including IL-5 generation by T cells and potentiation of eosinophil functional activation.

A germline mutation abolishing the original stop codon of the human adenine phosphoribosyltransferase (APRT) gene leads to complete loss of the enzyme protein

Abstract Adenine phosphoribosyltransferase (APRT) is a purine metabolic enzyme and a homozygous deficiency in this enzyme causes 2,8-dihydroxyadenine urolithiasis. Various germline abnormalities have been described, but we report here a unique type of germline mutation in a homozygous individual (SY) who had excreted 2,8-dihydroxyadenine crystals. In SY, TCA was substituted for the physiological stop codon TGA. This base substitution generates a new HinfI restriction site, and, using the polymerase chain reaction and subsequent digestion by this enzyme, it was confirmed that SY is homozygous for the base substitution. This base change is unique in that it generates an open reading frame that extends to the poly(A) addition site. The amount of mRNA in transformed B cells from SY was approximately a quarter of that in control subjects and no APRT proteins were detected. In eukaryotes, unlike in prokaryotes, no rescue systems for defective polypeptide termination caused by a missing stop codon have been found. Therefore, the outcome of the defect of SY is unclear from present knowledge about termination of polypeptide synthesis. Investigations into the mechanisms of the absence of protein in the cells of SY may lead to a better understanding of the physiological and nonphysiological termination of polypeptide synthesis in eukaryotic cells.

Divergent effects of methotrexate on the clonal growth of T and B lymphocytes and synovial adherent cells from patients with rheumatoid arthritis.

OBJECTIVE: To define the mechanisms whereby methotrexate (MTX) manifests its effects in patients with rheumatoid arthritis. METHODS: T and B cells from peripheral blood and rheumatoid synovial tissues, synovial adherent cells, and the human fibrosarcoma cell line HT1080 and its mutant (defective in an enzyme in the nucleotide salvage pathway) were tested for clonal growth when cultured with MTX. Normal human fibroblasts and those with a deficiency in a salvage pathway were cultured with MTX in the presence or absence of purine and pyrimidine bases. RESULTS: Clonal growth of T and B cells, but not synovial cells, was inhibited by clinically relevant concentrations of MTX. Slowly proliferating fibroblast lines were resistant to MTX, whereas their rapidly proliferating counterparts were not. However, mutant fibroblast lines deficient in a salvage pathway were sensitive to MTX despite slow proliferation. Similarly, while skin fibroblasts were resistant to MTX, germline mutant fibroblasts deficient in a salvage pathway were sensitive to small concentrations of MTX. CONCLUSION: T and B lymphocytes, but not synovial cells, may be the target of MTX in vivo. Resistance to MTX may be associated with slow proliferation and the ability to synthesise nucleotides via salvage pathways. MTX can inhibit proliferation of even slowly growing cells by restricting the supply of nucleotides obtained via a salvage pathway, by removal of purine and pyrimidine bases, or by inducing a deficiency in a salvage pathway. It may be possible to manipulate the therapeutic effect of MTX by adjusting the amounts of purines and pyrimidines available to the cells in vivo.

Adenine phosphoribosyltransferase deficiency identified by urinary sediment analysis: cellular and molecular confirmation

Adenine phosphoribosyltransferase deficiency is an autosomal recessive purine enzyme defect that causes urolithiasis and, in severe cases, renal failure. Most homozygotes with this disorder were identified by analyses of excreted or surgically removed urinary stones, but some were identified only because they were family members of symptomatic individuals. We report here the detection of adenine phosphoribosyltransferase deficiency in two cases by routine analysis of urinary sediments. 2,8‐Dihydroxyadenine‐like spherical crystals were observed in the urinary sediment, and a diagnosis of homozygous adenine phosphoribosyltransferase deficiency was confirmed by cellular and molecular methods. A molecular diagnostic system using the polymerase‐chain reaction and single‐strand conformational polymorphism analysis proved to be a rapid and sensitive method to identify the APRT*J alleie, a common mutant allele among the Japanese people. These methods will facilitate identification of symptomatic and asymptomatic individuals with homozygous adenine phosphoribosyltransferase deficiency.

The origin of the most common mutation of adenine phosphoribosyltransferase among Japanese goes back to a prehistoric era.

Abstract The incidence of adenine phosphoribosyltransferase (APRT) deficiency is higher among Japanese nationals than among other ethnic groups, and the most common mutation (APRT*J, ATG to ACG mutation at codon 136) accounts for 68% of the disease-causing genes among Japanese. To investigate the origin of these mutations, we studied the geographical distribution of the mutant genes in Japan. The APRT*J mutation is distributed nearly uniformly in the four main islands of Japan and Okinawa, suggesting a very early origin. The products of PCR amplification between positions 2344 and 2750 of the genomic APRT sequence were examined by SSCP analysis in random blood samples from Japanese, Korean, and Taiwanese nationals. Among 955 random Japanese blood samples, 7 (0.73%) were heterozygous for the APRT*J mutation, giving a calculated heterozygote frequency of 1.1% among Japanese for the entire APRT deficiency. None of 231 Taiwanese samples contained heterozygotes for the APRT*J mutation, while 2 (0.53%) of 356 Korean samples were heterozygous. In addition to the APRT*J sequence, a total of five variant sequences was found. Sequencing one variant revealed a base substitution in intron 4, suggesting therefore that they are harmless mutations. Since the APRT*J mutation is present in Koreans and Okinawans who share ancestors only before the Yayoi era (third century bc to third century ad), the origin of the APRT*J mutation predates 300 bc.