Noriko Yamaoka

Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography-electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 micromol/L (UMP and AMP) to 1.3 micromol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.

Analysis of Purine in Purine-Rich Cauliflower

Purine is a general term for purine nucleotides, nucleosides, bases, and nucleic acid. The amount of purine nucleotides, nucleosides, and bases in purine-rich cauliflower was determined with the use of LC-MS and HPLC, and the ratio of these molecules were compared with in raw and in heated condition. Total purine content of raw and heated cauliflower was 42.6 and 43.2 mg/100 g, respectively. Nucleotide content was increased from 0.02 to 50.8 μmol/100 g, and nucleoside content was decreased from 12.4 to 7.7 μmol/100 g, by heating.

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid-liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30-1000ng for nicotine and 0.06-1000ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8-15ng/mL and 156-372ng/mL, respectively.

An asymptomatic germline missense base substitution in the hypoxanthine phosphoribosyltransferase (HPRT) gene that reduces the amount of enzyme in humans

Abstract A 40-year-old normouricemic (5.5 mg/dl) male showed 46% hemolysate and 37% lymphoblast hypoxanthine phosphoribosyltransferase (HPRT) activities but was otherwise completely free of symptoms. His genomic DNA and cDNA had a missense base substitution (CAT-to-CGT in codon 60) leading to the amino-acid substitution His-to-Arg. Western blot analysis revealed that the amount of HPRT protein in lymphoblasts from this individual was 25%–50% of normal cells, suggesting that the decrease in the amount of enzyme protein was responsible for the partial deficiency. This provides the first clear evidence that a genomic missense mutation at the HPRT locus leads to a decrease in the amount of the enzyme protein but that otherwise it has no evident adverse effects in the hemizygote (asymptomatic mutation).

Direct Evidence for a Hot Spot of Germline Mutation at HPRT Locus

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a well-studied purine metabolic enzyme which converts hypoxanthine or guanine into IMP or GMP in the presence of 5-phosphoribosyl-l-pyrophosphate. A genetic deficiency of this enzyme causes either Lesch-Nyhan syndrome characterized by neurological symptoms and self-mutilation behavior or severe overproduction-type gout, both of which show X-linked inheritance. The germline mutations causing this enzyme deficiency have been studied extensively at the molecular level (Sculley et al. 1992). Typically, germline mutations associated with HPRT deficiencies differ from family to family. These data imply that the defective mutant genes at this locus do not expand in human populations and that mutations in patients from different families are a reflection of different germline mutations. Although the mutations in a limited number of families with this disease have shown the same molecular alteration, conclusive evidence that these mutations reflected independent events has been lacking (Davidson et al.1991, Sculley et al.1991, Marcus et al. 1992, Peterson et al. 1993).

Urinary Stone Analysis in a Patient with Hyperuricemia to Determine the Mechanism of Stone Formation

In order to determine the mechanism of urinary stone formation in patients with hyperuricemia, we analyzed the crystal components and matrix proteins in a urinary stone from such a patient. Micro-area X-ray spectrometry and infrared (IR) spectroscopy suggested that the outside of the stone was composed of calcium oxalate monohydrate (COM) and the inside of uric acid (UA). Proteomic analysis identified 37 and 14 proteins from the inside and outside of the stone, respectively, as matrix proteins. The proteins that were identified in an ethylenediaminetetraacetic acid (EDTA) fraction were able to bind calcium ions. Thus, calcium-binding proteins may play a significant role in the formation of urinary stones in patients with hyperuricemia.

Simultaneous Determination of Purine and Pyrimidine Metabolites in Hprt-Deficient Cell Lines

Genetic mutations in the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT), are known to cause Lesch–Nyhan syndrome and Kelley–Seegmiller syndrome. In patients, purine metabolism is different from that of normal persons. We have previously developed a method for simultaneously determining the concentration of purine and pyrimidine nucleosides and nucleotides. This system was applied to determine the concentrations of nucleosides and nucleotides in HPRT-deficient cell lines. The amount of inosine 5′-monophosphate (IMP) was different in Lesch–Nyhan syndrome, Kelley–Seegmiller syndrome, and control cell lines. The difference in the amount of IMP confirmed the mutation of the enzyme.

Molecular Analysis of Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency in Japanese Patients

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a purine salvage enzyme that catalyzes the conversion of hypoxanthine and guanine to IMP and GMP, respectively. Complete deficiency of HPRT causes the Lesch-Nyhan syndrome, which is characterized by hyperuricemia, mental retardation, choreoathetosis, and compulsive self-mutilation(1). Partial deficiency of HPRT leads to severe form of gout and nephrolithiasis(2). Several molecular analyses of HPRT deficiency in Caucasian patients have elucidated that the molecular abnormalities in HPRT deficiency are strikingly heterogeneous(3). Although an epidemiological survey has found more than 50 cases with Lesch-Nyhan syndrome in Japan(4), sequence-level analyses have not been performed well(5). We have identified two previously undescribed single point mutations and two unique deletions in the amplified HPRT cDNA derived from four unrelated Japanese patients with Lesch-Nyhan syndrome.

Evaluation of purine utilization by Lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine

Abstract It is well accepted that frequent and heavy intake of purine-rich foods causes elevation of serum uric acid levels, which is a risk factor of hyperuricemia. Reducing intestinal absorption of dietary purines may attenuate the elevation of serum uric acid levels and exacerbation of hyperuricemia. This reduction may be achieved by the ingestion of lactic acid bacteria that take up purines in the intestine. In this study, we investigated the degree of uptake and utilization of purines of three lactobacilli strains. Among them, Lactobacillus gasseri PA-3 (PA-3) showed the greatest incorporation of 14C-adenine. PA-3 also incorporated 14C-adenosine and 14C-AMP. Additionally, using defined growth medium, PA-3 demonstrated greater proliferation in the presence of these purines than in their absence. Although further investigation is required, ingestion of PA-3 may lower serum uric acid levels by reducing intestinal absorption of purines in humans.

Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA.

N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions and is oxidized by an equimolar amount of H2O2 to yield its decarboxylated product 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid (ADOA). Highly sensitive analytical methods are required to detect ADOA in the human body. We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) to enable their fluorometric detection, and developed a method using HPLC with fluorometric detection (HPLC-FD) for the simultaneous determination of the derivatized NANA and ADOA. The derivatized NANA and ADOA were separated by a hydrophilic interaction liquid chromatography (HILIC) column using an H2O/CH3CN/HCOOH (10/90/0.35) mobile phase. Fluorescence was monitored at excitation and emission wavelengths of 450nm and 560nm, respectively. Both intra- and inter-day (n=6) repeat determinations of the DBD-ED-derivatized NANA and ADOA gave relative standard deviations of less than 5%. The calibration curves for standard solutions of DBD-ED-derivatized NANA and ADOA were linear over the ranges from 576fmol to 2.0nmol and 556fmol to 2.0nmol, respectively. The method developed was highly specific and sensitive for NANA and ADOA. The presence of ADOA in biological samples was revealed for the first time using this method.