<Originals>Studies on the measurement of blood vitamin K

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As a measurement for blood vitamin K (VK, VK_1 and VK_2) by high-performance liquid chromatography (HPLC), the fluorescence technique using a reduced form of VK emitting fluorescence is appropriate for routine examination because of its simplicity and high sensitivity. A nitrogen laser-excited fluorescence detector that is connected to HPLC and also a specific pretreatment method for serum samples were experimentally devised, and evaluated in the usefulness to this new VK measurement for clinical examination. After VK is separated by delivering a eluent of 96 percent ethanol into a silica ODS C_<18> column, a sodium borohydride ethanol solution is delivered by the post column method. The reduction product is measured by using HPLC nitrogen laser-excited fluorescence detector at an excitation wavelength of 337 nm and a fluorescent wavelength of 450 nm. For the pretreatment of serum samples, distilled water is added to the serum and after removing proteins with isopropanol, the remaining solution is treated with n-hexane. The extract solution is applied to a minicolumn containing silica. After washing with n-hexane, the VK fraction is eluted with a 6.0 percent ether n-hexane solution. The eluent is dried, redissolved in isopropanol and measured by HPLC. This method produced a linear measurement curve and good results in terms of simultaneous reproducibility, infer-day variation and the recovery rate and did not affect the measurement of other serum vitamins and elements. Measurement of the blood VK concentration in healthy adults and patients with various liver diseases showed that the VK level was significantly lower (p<0.05) in patients with hepatocellular carcinoma and compensated liver cirrhosis than in healthy individuals. The blood VK concentration was also determined before and after the administration of VK_1 in patients with liver diseases, and changes in the level of protein induced by vitamin K absense or antagonists-II (PIVKA-II) and the results of various coagulation tests were examined. The VK concentration showed a moderate correlation with the positivity for PIVKA-II but no marked correlations with the results of coagulation tests or biochemical liver function tests. To examine the antagonizing effect of warfarin on VK, the blood VK concentration was serially monitored before and after warfarin administration in patients who underwent valve replacement. The nitrogen laser-excited fluorescence detector used in this method is more sensitive (limit of detection : 0.02 ng) than the conventional fluorometer and highly specific measurement is possible. In addition, its size is far smaller than the other conventional laser detectors, and laser amplification occurs with a low pressure (1.0 kg/cm^2). This method may be useful as a routine examination for diagnosis and treatment of various diseases.

<Originals>Studies on the measurement of blood vitamin K

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