A sensitive and specific method for the determination of phylloquinone (K₁) and the menaquinones family (MK-n), between MK-4 and MK-10, was developed. Vitamin K₁ and MK-n extracted from biological materials were separated by high perfromance liquid chromatography using a Cosmosil 5 C₁₈ column with ethanol-water as a mobile phase. The separated K₁ and MK-n were detected by a fluorometry after their reaction with ethanolic sodium borohydride in a reaction coil connected by one-line to a chromatographic column. Minimum detectable quantities of K₁, MK-4 and MK-7 were 10, 10 and 50pg, respectively. The recovery rates of the serum and feces assay were assessed by adding a known amount of vitamins K to them. More than 95% and more than 94% of added K₁ and MK-7 were recovered in the serum and feces.
With a wide variation of vitamins K concentration in each sample, K₁, MK-4, MK-5, MK-6, MK-7, MK-8, MK-9 and MK-10 were detected in feces from 6 normal adults, as shown in figure 4 and table 1. On the other hand, only K₁ and MK-7 were detected in meconium collected from 6 normal newborn infants before the start of initial feeding (Table 2). The contents of vitamins K in meconium were less than 1 percent of those in adult feces. Vitamin K₁ and MK-7 were detected in normal adult serums, as shown in figure 5 and table 3. The mean serum concentration of K₁ in 5 normal adults was 0.47ng/ml (range 0.21-0.89), and the range of MK-7 concentration was from less than 0.05 to 0.92ng/ml. In 6 pooled serums of umbilical cord blood from 56 normal newborn infants, only MK-7 was detected, ranging from less than 0.014 to 0.188ng/ml (Table 4).
These results show that the measurement of contents of K₁ and MK-n in human biological materials is an important subject in the elucidation of the cause of vitamin K deficiency.