Hiroo Yoshida

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry

An automated method for high-throughput amino acid analysis, using precolumn derivatization high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home-built auto-sampler system. Amino acids were derivatized with 3-aminopyridyl-N-hydroxysuccinimidyl carbamate, and a 3 microm Wakosil-II 3C8-100HG column (100 x 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra- and inter-precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%.

Advantage of LC-MS Metabolomics Methodology Targeting Hydrophilic Compounds in the Studies of Fermented Food Samples

The utility of a liquid chromatography mass spectrometry (LC-MS) method, using a pentafluorophenylpropyl (PFPP) bonded silica, was demonstrated in a metabolomics study of fermented food samples. Our LC-MS method was applied to Japanese fermented food (miso) of different stages of ripeness. The data acquired were evaluated by principal component analysis (PCA). The score plots indicated that the miso samples could be approximately classified into three groups, based on the stage of miso ripeness. The loading plots indicated that the ions responsible for group separation included not only amino acids and citric acid but also Amadori compounds. On the other hand, the miso samples were also analyzed by a conventional LC-MS method using an octadecyl (C(18)) column for comparison. The group separation of score plots from the conventional method was less clear than that from our method. The advantage of our LC-MS method is due to the different retention properties of the PFPP column and the C(18) column with hydrophilic compounds. Our LC-MS method will be useful for the metabolic profiling of fermented food samples.

Validation of an analytical method for human plasma free amino acids by high-performance liquid chromatography ionization mass spectrometry using automated precolumn derivatization.

The analysis of human plasma free amino acids is important for diagnosing the health of individuals, because their concentrations are known to vary with various diseases. The development of valid, reliable, and high-throughput analytical methods for amino acids analysis is an essential requirement in clinical applications. In the present study, we have developed an automated precolumn derivatization amino acid analytical method based on high-performance liquid chromatography/electrospray ionization mass spectrometry (so-called UF-Amino Station). This method enabled the separation of at least 38 types of physiological amino acids within 8min, and the interval time between injections was 12min. We also validated this method for 21 major types of free amino acids in human plasma samples. The results of the specificity, linearity, accuracy, repeatability, intermediate precision, reproducibility, limits of detections, lower limits of quantification, carry over, and sample solution stability were sufficient to allow for the measurement of amino acids in human plasma samples. Our developed method should be suitable for use in clinical fields.

The effects of pre-analysis sample handling on human plasma amino acid concentrations.

BACKGROUND The accurate and reliable quantification of amino acid concentrations in human plasma is important for the investigation of a number of diseases. However, few systematic studies investigating the changes in amino acid concentrations related to blood collection and storage conditions have been completed. METHODS Blood samples were collected with EDTA-Na2 from 3 healthy volunteers and subjected to a number of different treatments; hemolysis, temperature after blood collection, time from blood collection to cooling, the influence of platelets, long term storage conditions, and repeated freeze-thaw cycles. Changes in the concentrations of 22 amino acids were determined using an Amino Acid Analyzer. RESULTS Of the conditions influencing sample stability between blood collection and amino acid analysis, hemolysis, temperature after blood collection, and long-term storage at -20°C affected the concentrations of 11 amino acids. Time from blood collection to cooling, platelet contamination and repeated freeze-thaw cycles altered the levels of 4 amino acids. CONCLUSIONS We observed changes in amino acid concentrations relating to blood collection and storage conditions. If attention is paid to 4 key factors (hemolysis, temperature immediately following blood collection, time from collection to cooling, and long-term storage temperature) 19 amino acids can be reliably quantified.

Reference intervals for plasma-free amino acid in a Japanese population

Background Plasma amino acid concentrations vary with various diseases. Although reference intervals are useful in daily clinical practice, no reference intervals have been reported for plasma amino acids in a large Japanese population. Methods Reference individuals were selected from 7685 subjects examined with the Japanese Ningen Dock in 2008. A total of 1890 individuals were selected based on exclusion criteria, and the reference samples were selected after the outlier samples for each amino acid concentration were excluded. The lower limit of the reference intervals for the plasma amino acid concentrations was set at the 2.5th percentile and the upper limit at the 97.5th percentile. Results By use of the nested analysis of variance, we analysed a large dataset of plasma samples and the effects of background factors (sex, age and body mass index [BMI]) on the plasma amino acid concentrations. Most amino acid concentrations were related to sex, especially those of branched-chained amino acid. The citrulline, glutamine, ornithine and lysine concentrations were related to age. The glutamate concentration was related to body mass index. Conclusions The concentrations of most amino acids are more strongly related to sex than to age or body mass index. Our results indicate that the reference intervals for plasma amino acid concentrations should be stratified by sex when the background factors of age and body mass index are considered.

Clinical Implementation of Metabolomics

Metabolomics, which is also referred to as metabonomics, metabolic profiling or metabolic fingerprinting, is the comprehensive quantitative measurement of endogenous metabolites within a biological system (Fiehn, 2002; Kaddurah-Daouk et al, 2008; Spratlin et al, 2009). Detection of metabolites is in general carried out in cell extracts, tissue specimens, or various biological fluids including serum, plasma, urine and cerebrospinal fluid (CSF) by liquid chromatography mass spectrometry (LC-MS), gas chromatography–mass spectrometry (GCMS), capillary electrophoresis–mass spectrometry (CE-MS) or nuclear magnetic resonance spectroscopy (NMR). Metabolomics captures the status of diverse biochemical pathways in a particular situation and can define the metabolic status of an organism (Aranibar et al, 2011; DeFeo et al, 2011; Lu et al, 2008; Roux et al, 2011; Soga, 2007; Yuan et al, 2007). In clinical settings, biomarkers generated from metabolomics have become one of the most essential diagnostic criteria that can be objectively measured and evaluated as indicators of normal or pathological states, as well as a tool to assess responses to therapeutic interventions (Hunter, 2009; Spratlin et al, 2009; van der Greef et al, 2006; Zeisel, 2007). As we describe in this chapter, novel metabolomic markers, for instance, for cancer therapy, glucose intolerance, hepatic steatosis, nephrotic and psychiatric disorders, and their incorporation into clinical decision-making may considerably change future health care.