HPLC method for plasma vitamin K1: effect of plasma triglyceride and acute-phase response on circulating concentrations.

BACKGROUND: The plasma concentration of vitamin K(1) (phylloquinone) is the most reliable index for assessing vitamin K status. Our aim was to analytically validate an HPLC method for quantifying phylloquinone in plasma and to examine the effect of plasma triglyceride concentration on the phylloquinone reference interval. We also examined the effect of acute-phase response on phylloquinone concentration in plasma. METHODS: Phylloquinone was extracted from fasting plasma samples by deproteinization and C18 solid-phase extraction, separated by reversed-phase HPLC, and detected fluorometrically after postcolumn reduction with a platinum catalyst. We synthesized a novel internal calibrator, docosyl naphthoate. RESULTS: The recovery of phylloquinone was >90%. Between-run imprecision was 8.7%-9.0%, and within-run imprecision was 3.8%-7.0%. The linearity was up to 44.8 nmol/L, limit of detection 0.08 nmol/L, and limit of quantification 0.14 nmol/L. The correlation between plasma phylloquinone and triglyceride concentrations was r = 0.7 in the reference population. The 95% reference interval for the phylloquinone:triglyceride ratio was 0.20 to 2.20 nmol/mmol. Plasma concentrations of C-reactive protein were significantly increased, whereas triglyceride and phylloquinone but not the phylloquinone:triglyceride ratio were transiently decreased >50% after surgery. CONCLUSION: Phylloquinone population reference intervals should be expressed as a ratio of the triglyceride concentration. Phylloquinone concentrations in plasma are decreased in acute-phase response and, unless corrected for plasma triglyceride concentration, are unlikely to be a reliable index of vitamin K status.

Biological variation of vitamins in blood of healthy individuals.

BACKGROUND: Components of biological variation can be used to define objective quality specifications (imprecision, bias, and total error), to assess the usefulness of reference values [index of individuality (II)], and to evaluate significance of changes in serial results from an individual [reference change value (RCV)]. However, biological variation data on vitamins in blood are limited. The aims of the present study were to determine the intra- and interindividual biological variation of vitamins A, E, B(1), B(2), B(6), C, and K and carotenoids in plasma, whole blood, or erythrocytes from apparently healthy persons and to define quality specifications for vitamin measurements based on their biology. METHODS: Fasting plasma, whole blood, and erythrocytes were collected from 14 healthy volunteers at regular weekly intervals over 22 weeks. Vitamins were measured by HPLC. From the data generated, the intra- (CV(I)) and interindividual (CV(G)) biological CVs were estimated for each vitamin. Derived quality specifications, II, and RCV were calculated from CV(I) and CV(G). RESULTS: CV(I) was 4.8%-38% and CV(G) was 10%-65% for the vitamins measured. The CV(I)s for vitamins A, E, B(1), and B(2) were lower (4.8%-7.6%) than for the other vitamins in blood. For all vitamins, CV(G) was higher than CV(I), with II <1.0 (range, 0.36-0.95). The RCVs for vitamins were high (15.8%-108%). Apart from vitamins A, B(1), and erythrocyte B(2), the imprecision of our methods for measurement of vitamins in blood was within the desirable goal. CONCLUSIONS: For most vitamin measurements in plasma, whole blood, or erythrocytes, the desirable imprecision goals based on biological variation are obtainable by current methodologies. Population reference intervals for vitamins are of limited value in demonstrating deficiency or excess.