A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP18e or Ascentis Express C18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.
- MeSH
- aminokyseliny analýza metabolismus MeSH
- embryo savčí chemie metabolismus MeSH
- fertilizace in vitro MeSH
- kultivace embrya * MeSH
- kultivační média chemie metabolismus MeSH
- lidé MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zygota chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Amino acids are the building blocks of proteins. In case of insulin resistance, which is typical for type 2 diabetes mellitus (T2DM), proteolysis is increased and protein synthesis is decreased; therefore, we can observe changes in the levels of amino acids in diabetics vs. non-diabetics. OBJECTIVES: The aim of this study was to find differences in the levels of selected amino acids between patients with diabetes (type 2) and a control group. MATERIAL AND METHODS: Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of potassium cyanide to form fluorescent 1-cyanobenz(f)isoindole product. Amino acids derivatives were measured using a high-performance liquid chromatography with fluorescence detection. The serum levels of glucose were determined using an automatic biochemistry analyzer, glycated hemoglobin HbA1c was measured by cation exchange chromatography. RESULTS: A total of 19 serum amino acids in T2DM patients and non-diabetics were measured. There were 9 amino acids, which were significantly different in these groups (p<0.05). Significantly decreased levels of arginine, asparagine, glycine, serine, threonine and significantly increased levels of alanine, isoleucine, leucine, valine in diabetics were found. CONCLUSIONS: Significant difference in metabolism of amino acids between diabetics and non-diabetics were observed. The altered levels of amino acids in diabetic patients could be a suitable predictor of diabetes.
- MeSH
- aminokyseliny krev MeSH
- biologické markery krev MeSH
- chromatografie iontoměničová MeSH
- diabetes mellitus 2. typu krev diagnóza MeSH
- fluorescenční spektrometrie MeSH
- glykovaný hemoglobin analýza MeSH
- krevní glukóza analýza MeSH
- laboratorní automatizace MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
