ABSTRACT
Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography-tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1-250 pg per 50 microL. The recoveries of fat-soluble vitamins were 91-105%. Inter-assay CV values of each vitamin were 1.9-11.9%. The mean concentrations of retinol, vitamin D3, 25-hydroxyvitamin D3, alpha-tocopherol, phylloquinone and menaquinone-4 were 0.455 microg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 microg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n=82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.
Subject(s)
Chromatography, Liquid/methods , Milk, Human/chemistry , Tandem Mass Spectrometry/methods , Vitamin A/analysis , Vitamin D/analysis , Vitamin E/analysis , Female , Humans , Reproducibility of Results , Sensitivity and Specificity , Vitamin K/analysisABSTRACT
Thioredoxin is crucial for the maintenance of the redox status of cells of all types. Mammalian thioredoxin is secreted from various types of cells, although the mechanism underlying has not yet been clarified. Previously, we demonstrated that thioredoxin was released from Saccharomyces cerevisiae after treatment with ethanol. In this paper, we show that as well as ethanol, low-pH shock and hypoosmotic shock release thioredoxin. Low-molecular-weight proteins in yeast cells were preferentially released by treatment with ethanol and low-pH shock. A cell wall integrity pathway seems partially involved in the hypoosmotic shock-induced release of thioredoxin. Considerable amounts of thioredoxin were present in the insoluble fractions of the cells, a portion of which was associated with lipid microdomains that are resistant to nonionic detergent at 4 degrees C. The intracellular localization of thioredoxin may influence the efficiency of its release from yeast cells with ethanol.
Subject(s)
Ethanol/metabolism , Saccharomyces cerevisiae/metabolism , Thioredoxins/metabolism , Osmotic Pressure , SolubilityABSTRACT
Thioredoxin, an antioxidant protein, is a promising molecule for development of functional foods because it protects the gastric mucosa and reduces the allergenicity of allergens. To establish a method for obtaining an ample amount of yeast thioredoxin, we found here that thioredoxin is released from Saccharomyces cerevisiae by treatment with 20% ethanol. We also found that Japanese sake contains a considerable amount of thioredoxin.
