Tomoko Fukuuchi

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.

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.

Analysis of Intra- and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography

To evaluate cellular uptake and purine transport, we developed a high-performance liquid chromatography method for intra- and extracellular purine quantification. Our aim was to develop an effective method for simultaneously quantifying the substrate and metabolites with high sensitivity. C18 columns from different manufacturers were tested for simultaneous quantification of 22 different purine bases, nucleosides, and nucleotides. We used a YMC-Triart C18 column. The analysis conditions, including extraction solutions for the cells and cell culture medium, were optimized to achieve good quantification. Linearity, accuracy, determination limits, and recovery were assessed and showed good performance. The developed HPLC method was successfully applied to the qualitative analysis of 22 different intra- and extracellular purines, demonstrating that it is useful for studying the overall pattern of purine metabolism. This method could also be useful for evaluating metabolic dynamics of purines under a variety of stimulatory conditions of culture cells.

Proteomic Analysis to Examine the Role of Matrix Proteins in a Gouty Tophus from a Patient with Recurrent Gout

To examine the role of matrix proteins in the formation of gouty tophus, we analyzed the crystalline components and matrix proteins in a gouty tophus from a patient with recurrent gout. Micro-area X-ray diffraction analysis and infrared spectroscopy indicated that the tophus was composed of monosodium urate monohydrate. Proteomic analysis identified 134 proteins from the tophus as matrix proteins. Many proteins relevant to inflammation and host defense were identified, and immunoglobulin was detected in all four extracted fractions (KCl, formic acid, guanidine-HCl, and ethylenediaminetetraacetic acid) and from many spots throughout a broad molecular weight range after electrophoresis. It is thought that the process of biological defense including the immunity has occurred in the gouty tophus.

Proteomic analysis of a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate: A case report

The objective of the present study was to investigate the matrix protein of a rare urinary stone that contained calcium carbonate. A urinary stone was extracted from a 34‐year‐old male patient with metabolic alkalosis. After X‐ray diffractometry and infrared analysis of the stone, proteomic analysis was carried out. The resulting mass spectra were evaluated with protein search software, and matrix proteins were identified. X‐ray diffraction and infrared analysis confirmed that the stone contained calcium carbonate and calcium oxalate dihydrate. Of the identified 53 proteins, 24 have not been previously reported from calcium oxalate‐ or calcium phosphate‐containing stones. The protease inhibitors and several proteins related to cell adhesion or the cytoskeleton were identified for the first time. We analyzed in detail a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate. Considering the formation of a calcium carbonate stone, the new identified proteins should play an important role on the urolithiasis process in alkaline condition.

Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines

ABSTRACT Using Caco-2 cells and our previously developed high-performance liquid chromatography method for quantification of purine bases, nucleosides, and nucleotides, we evaluated cellular purine transport and uptake. The analytes were separated using YMC-Triart C18 column with gradient elution. We used Caco-2 cells as intestinal model cells and monitored purine transport across a monolayer for 2 h. The degree of change of purine concentrations in the permeate was very slight; however, it was possible to simultaneously determine these parameters for all purines because of our methods high sensitivity. In the present study, the purine bases (adenine, guanine, hypoxanthine, and xanthine) showed a relatively high permeability as compared with the nucleosides (adenosine, guanosine, inosine, and xanthosine). Increased concentration of metabolites in the permeate was also observed following the addition of purines. In a cell uptake assay, both the cell culture medium (extracellular) and the cells extracted from Caco-2 with acetonitrile:water (7:3) (intracellular) were measured. The additional nucleoside did not increase significantly within the cells. On the other hand, we observed that nucleotide, such as ATP, increased in the cell in a time-dependent manner following the addition of nucleoside. The additional nucleosides were considered to be rather recycled via the salvage pathway than metabolized to purine bases and/or uric acid in the cell. Such differences might have affected the increase in the serum uric acid levels depending on purine form.

Determination and Profiling of Purines in Foods by Using HPLC AND LC-MS

Purines in food are known to raise serum uric acid levels. We determined the purine content of sweet potato and beef by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The purine content of the samples was 118–1,034 μmol/100 g. The total purine content was also divided into purine bases, nucleosides, nucleotides, and nucleic acids. Our results suggest that differences in total purine content and in the ratio of purine types between vegetables and beef cause a difference in elevation of serum uric acid levels.

Proteomic Analysis after Sequential Extraction of Matrix Proteins in Urinary Stones Composed of Calcium Oxalate Monohydrate and Calcium Oxalate Dihydrate.

In this study, we performed proteomic analysis following sequential protein extraction on calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) urinary stones to determine the specific matrix proteins according to the crystal components of the stones. After X-ray and IR analysis of 13 urinary stones, matrix proteins were sequentially extracted with KCl, formic acid, guanidine-HCl, and EDTA, before SDS-electrophoresis followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The electrophoretic patterns of the extracted proteins differed from that of COM and COD stones. LC-MS/MS identified 65 proteins, of which many were cellular plasma proteins, and were frequently detected regardless of the crystal components. However, 6 proteins (protein Z, protein S, prothrombin, osteopontin, fatty acid binding protein 5, and ubiquitin) were detected in the final EDTA fractions of COM stones. These proteins are involved in the coagulation process or osteometabolism, and thus the roles they play are of particular interest.

Characterizing substrate properties of purine-related compounds with purine metabolism enzymes for enzymatic peak-shift HPLC method.

We have extended peak-shift method for measuring purine bases to make it suitable for other purine-related compounds. We optimized the reactions of the purine metabolism enzymes 5′-nucleotidase (EC 3.1.3.5), purine nucleoside phosphorylase (PNP) (EC 2.4.2.1), xanthine oxidase (XO) (EC 1.17.3.2), urate hydroxylase (EC 1.7.3.3), adenosine deaminase (ADA) (EC 3.5.4.4), and guanine deaminase (EC 3.5.4.3) by determining their substrate specificity and reaction kinetics. These enzymes eliminate the five purine base peaks (adenine, guanine, hypoxanthine, xanthine, and uric acid) and four nucleosides (adenosine, guanosine, inosine, and xanthosine). The bases and nucleosides can be identified and accurately quantified by comparing the chromatograms before and after treatment with the enzymes. Elimination of the individual purine compound peaks was complete in a few minutes. However, when there were multiple substrates, such as for XO, and when the metabolites were purine compounds, such as for PNP and ADA, it took longer to eliminate the peaks. The optimum reaction conditions for the peak-shift assay methods were an assay mixture containing the substrate (10 μL, 0.1 mg/mL), the combined enzyme solution (10 μL each, optimum concentration), and 50 mM sodium phosphate (up to 120 μL, pH 7.4). The mixture was incubated for 60 minutes at 37°C. This method should be suitable for determining the purine content of a variety of samples, without interference from impurities.