Yosuke Nakano

Studies on serine hydroxymethylase isoenzymes from rat liver

Abstract Serine hydroxymethylase ( l -serine:tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1) was found to occur in the soluble and mitochondrial fractions of rat liver. The enzymes were purified from the two fractions and some of their properties were investigated. 1. 1. The soluble fraction serine hydroxymethylase had a rather sharp pH optimum at 8.0, whereas the mitochondrial enzyme showed a broad pH optimum centering at 7.4. 2. 2. There was no significant difference in the Michaelis constants for substrates between the soluble fraction and mitochondrial serine hydroxymethylases. 3. 3. The mitochondrial enzyme was more stable than the soluble fraction enzyme. 4. 4. The soluble fraction serine hydroxymethylase was more anionic than the mitochondrial enzyme as evidenced by the affinity for DEAE-cellulose and electrophoretic mobility.

Mass screening for colorectal cancer by testing fecal occult blood

The validity of screening for colorectal cancer by testing for occult blood on 2 successive days was evaluated over a 2‐year period beginning April 1980 by testing 9449 individuals without symptoms. The Shionogi slide (Shionogi Pharmaceutical Co., Osaka, Japan), a commercial guaiac‐impregnated slide with moderate sensitivity, was used for screening subjects under dietary restriction. Of the 1401 persons (14.8%) who had a positive reaction for occult blood, 858 (61.2%) received further diagnostic examinations, and 265 of them proved to have one or more abnormalities of the gastrointestinal tract. Colorectal cancer was detected in 11 persons and polyps in 91 persons. Eight of the cancers were in an early stage. This screening method was found to be suitable for large‐scale mass screening, and appears to have high diagnostic value.

Development of a liquid chromatography-tandem mass spectrometry method for quantitative analysis of trace d-amino acids.

d-Amino acids have recently attracted much attention in various research fields including medical, clinical and food industry due to their important biological functions that differ from l-amino acid. Most chiral amino acid separation techniques require complicated derivatization procedures in order to achieve the desirable chromatographic behavior and detectability. Thus, the aim of this research is to develop a highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By optimizing MS/MS parameters, we established a quantification method that allowed the simultaneous analysis of 18 d-amino acids with high sensitivity and reproducibility. Additionally, we applied the method to food sample (vinegar) for the validation, and successfully quantified trace levels of d-amino acids in samples. These results demonstrated the applicability and feasibility of the LC-MS/MS method as a novel, effective tool for d-amino acid measurement in various biological samples.

Mechanistic study on the high-selectivity enantioseparation of amino acids using a chiral crown ether-bonded stationary phase and acidic, highly organic mobile phase by liquid chromatography/time-of-flight mass spectrometry

The separation mechanism of amino acid enantiomers using a chiral crown ether-bonded stationary phase, CROWNPAK CR-I(+), and acetonitrile (ACN)-rich mobile phases (MPs) was studied. The retention factors of proteinogenic l-amino acids (except proline) formed U-shaped plots against the ACN content in the MP with a sharp increase at a high ACN content, while d-amino acids showed much smaller increases or monotonous decreases in retention within the same range. The use of an acidic, highly organic MP with trifluoroacetic acid (TFA) provided a high enantioselectivity with a short separation time from the contribution of the increased binding of the ammonium group of the analytes to the crown ether functionality of the stationary phase and electrostatic repulsion counteracting the hydrophilic partition mechanism. Optimizing the sample diluent and MP alleviated the peak distortion caused by a moving water band that accompanied the hydrophilic interaction liquid chromatography-like elution conditions. The liquid chromatography/time-of-flight mass spectrometry method with the optimized MP - ACN/ethanol/water/TFA = 80/15/5/0.5 (v/v/v/v) - enabled the determination of eighteen pairs of proteinogenic amino acid enantiomers within 10 min. The conditions also provided the following advantages: (i) fast and highly reproducible separations under isocratic conditions, (ii) high sensitivity and low backpressure using the MP with a high organic content, and (iii) highly reliable peak identification by combining two columns (CR-I(+) and CR-I(-)), reversing the elution orders of the enantiomers.

Investigation of storage time-dependent alterations of enantioselective amino acid profiles in kimchi using liquid chromatography-time of flight mass spectrometry

Although naturally abundant amino acids are represented mainly by l-enantiomers, fermented foods are known to contain various d-amino acids. Enantiospecific profiles of food products can vary due to fermentation by bacteria, and such alterations may contribute to changes in food properties that would not be dependent exclusively on l-amino acids. Therefore, more attention should be paid to the study of temporal alterations of d-amino acid profiles during fermentation process. However, there have been very few studies reporting time-dependent profiling of d-amino acids because enantioseparation of widely targeted d-amino acids is technically difficult. This study aimed to achieve high throughput profiling of amino acids enantiomers. Enantioselective profiling of amino acids using CROWNPAK CR-I(+) column, liquid chromatography, time of flight mass spectrometry, and principle component analysis was performed to investigate time-dependent alterations in concentrations of free d- and l-amino acids in kimchi stored at 4°C or 25°C. We demonstrated significant changes in d- and l-amino acid profiles in kimchi stored at 25°C. In particular, concentrations of the amino acids d-Ala, d-Ser, d-allo-Ile, d-Leu, d-Asp, d-Glu, and d-Met became higher in kimchi with storage time. This is the first report of time-dependent alterations of d- and l-amino acid contents in kimchi. This study showed that our analytical method of enantioselective detection of amino acids using liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) with CROWNPAK CR-I(+) enables high throughput food screening and can be recommended for advanced studies of the relationship between d-amino acid content and food properties.