24-25-dihydroxyvitamin-d-3 and Liver-Cirrhosis

Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the 'FDA Guidance for Industry Bioanalytical Method Validation'. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.

Topics: 24,25-Dihydroxyvitamin D 3; Calcifediol; Calcitriol; Chromatography, High Pressure Liquid; Dietary Supplements; Humans; Liver; Liver Cirrhosis; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Vitamin D Deficiency

To investigate the relationship between serum vitamin D3 levels and liver fibrosis or inflammation in treatment-naive Chinese patients with chronic hepatitis C (CHC).. From July 2010 to June 2011, we enrolled 122 CHC patients and 11 healthy controls from Dingxi city, Gansu Province, China. The patients were infected with Hepatitis C virus (HCV) during blood cell re-transfusion following plasma donation in 1992-1995, and had never received antiviral treatment. At present, all the patients except two underwent liver biopsy with ultrasound guidance. The Scheuer Scoring System was used to evaluate hepatic inflammation and the Metavir Scoring System was used to evaluate hepatic fibrosis. Twelve-hour overnight fasting blood samples were collected in the morning of the day of biopsy. Serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, cholinesterase, prothrombin activity, albumin, γ-glutamyl transpeptidase, hemoglobin, calcium and phosphorus were determined. Serum HCV RNA levels were measured by real-time PCR. Serum levels of 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were measured by high-performance liquid chromatography tandem mass spectrometry.. Serum levels of 25(OH)D3 but not 24,25(OH)2D3 were significantly lower in CHC patients than in control subjects. Serum 25(OH)D3 levels did not correlate with liver fibrosis, inflammation, patient age, or levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, prothrombin activity, cholinesterase or HCV RNA. However, serum 25(OH)D3 levels did correlate with serum 24,25(OH)2D3 levels. Serum 25(OH)D3 and 24,25(OH)2D3 levels, and the 25(OH)D3/24,25(OH)2D3 ratio, have no difference among the fibrosis stages or inflammation grades.. We found that serum levels of 25(OH)D3 and its degradation metabolite 24,25(OH)2D3 did not correlate with liver fibrosis in treatment-naive Chinese patient with CHC.

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Biomarkers; Biopsy; Case-Control Studies; China; Chromatography, High Pressure Liquid; Female; Hepatitis C, Chronic; Humans; Liver Cirrhosis; Liver Function Tests; Male; Middle Aged; Severity of Illness Index; Tandem Mass Spectrometry

A new, highly sensitive HPLC assay method using an electrochemical detector (ECD) for multiple assay of vitamin D metabolites is reported. The assay involves extracting lipids from plasma with methylene chloride and methanol, purification on Zorbax SIL column with 5.5% (v/v) iso-propanol in hexane and quantification by HPLC-ECD. A coulometric system, composed of the dual electrode analytical cell and a guard cell, was used for ECD of the eluting compounds. The potentials applied to detectors 1 and 2 in a dual electrode analytical cell were adjusted to +0.20 V and +0.60 V, respectively. This method is sensitive to 20 pg of 25-hydroxyvitamin D3 [25(OH)D3] and of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3]. Calibration curves gave linearity from 20-1000 pg for 25(OH)D3 and 24,25(OH)2D3. The detection limit was approximately 50 pg ml-1 for 25(OH)D3 and 24,25(OH)2D3 in plasma. This sensitivity combined with an overall recovery of 25(OH)D3 (81.5 +/- 2.6%, mean +/- S.E.) allows the measurement of trace amount of 25(OH)D3 with only 20 microliters of plasma. Intra- and interassay RSD values were 5.3 and 9.7% for 25(OH)D3 and 6.3 and 9.7% for 24,25(OH)2D3, respectively. Plasma levels of 25(OH)D3 and 24,25(OH)2D3 in normal adults were 15.9 +/- 2.8 ng ml-1 (n = 10) and 1.4 +/- 0.5 ng ml-1 (n = 10), respectively. This method allows the determination of 25(OH)D2 and 25(OH)D3 for evaluating their nutritional and clinical status. From these results, it is concluded that the proposed HPLC-ECD assay system is useful for the determination of vitamin D metabolites in biological fluids as a highly sensitive physicochemical method.

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Case-Control Studies; Chromatography, High Pressure Liquid; Electrochemistry; Humans; Leukemia; Liver Cirrhosis; Osteoporosis; Reference Values; Reproducibility of Results; Sensitivity and Specificity; Vitamin D