Vitamin K Testing: An Osteoporosis “Risk Factor”

By Jonathan V. Wright, MD

Heightened public awareness of osteoporosis has led practitioners to request laboratory aid in evaluation, treatment, and “risk factor” analysis. Serum vitamin K measurement appears to be a valuable new tool for both preventive and therapeutic efforts.

Vitamin K has a crucial role in normal bone formation.¹ It catalyzes the conversion of glutamic acid to gamma-carboxyglutamic acid², a major functional center of osteocalcin, which is the protein matrix for new bone formation. The gamma-carboxyglutamic acid in osteocalcin binds calcium ions, leading to normal bone calcification.2. Without sufficient vitamin K, gamma-carboxylation does not proceed and (presumably) normal bone calcification does not either.

In much better known fashion, vitamin K performs exactly the same function in blood clotting. The gammacarboxyglutamic acid residues of prothrombin bind calcium in its transformation to thrombin. Without sufficient vitamin K, the gamma-carboxyglutamic acid residues do not form, calcium is not bound to prothrombin, and the blood does not clot.

Gamma-carboxylation is mediated by vitamin K2 phylloquinone, which is oxidized to the epoxide and reduced to the quinone again. Kl epoxide reduction is inhibited by coumadin and other vitamin K antagonists.³ Coumadin not only inhibits blood clotting, but severely retards new bone formation following experimental fractures.”⁴ There are reports of a high incidence of fetal bone malformations in women taking coumadin or other vitamin K antagonists.^5,6

Vitamin K deficiency has been assumed to be uncommon. However, vitamin K status (when considered) was assumed from studies of clotting function and not directly measured.

In a series of 15 individuals with osteoporosis resulting in vertebral or femoral neck fractures, serum vitamin Kl levels were only one-third those of age-matched controls.^7,8 In another study,⁹ vitamin K supplementation of osteoporotic individuals reduced calcium loss by 18-50%. Vitamin K supplementation accelerated healing of experimental fractures in rabbits on a presumably “vitamin K-adequate” diet.’

Whom to Test

In my practice, the serum vitamin K test has been equally useful as a preventive and therapeutic tool. “Younger” women with a family history of osteoporosis and low vitamin Kl are obviously at greater risk. Extrapolating from the same data, women with existing osteoporosis would appear to have much less chance of recalcification if suffering from vitamin K deficiency.

Considerable vitamin K is presumably synthesized by a “normal” intestinal microflora. However, an increasing number of clinicians are aware that “normal” microflora are much less frequent today than in the pre-antibiotic era.

Vitamin K insufficiency is also more likely amount those who do not eat their green vegetables, and those whose digestion and assimilation of nutrients (especially fat soluble vitamins) is impaired. I request serum vitamin K testing for everyone with osteoporosis and a substantial proportion of those women who related elders have osteoporosis. I recommend appropriate dietary change and/or supplementation for those with low or low-normal levels (as personally I think the stated “normal range” is too broad). Repeat testing almost always shows substantial improvement in just a few weeks.

To date, all our information on plasma levels has come from recently developed assays based on high-performance liquid chromatography (h.p.l.c.). Blood obtained by venipuncture is collected into EDTA. Specimens are collected in subdued light, and are kept in the dark during processing. All K vitamins are rapidly destroyed by light and alkali (it is essential to rinse all traces of detergents from glassware) but are relatively stable towards room temperatures, oxygen and dilute acids.

Vitamin Kl circulates in human plasma bound to lipoproteins, from which it may be readily extracted into a non-polar solvent after disruption of the lipoprotein molecules. Extraction with hexane after flocculation of proteins with ethanol is a simple extraction method. It is safe to remove solvents at temperatures up to 60C either in a rotary evaporator or under a stream of nitrogen. Following additional cleanup (some methods use a separate chromatographic step) to remove coextracted interferences vitamin Kl is resolved from remaining impurities and internal standard by isocratic reverse-phase h.p.l.c.¹¹

Several approaches have been applied for the detection of vitamin Kl by h.p.l.c. U.V. detection at 248nm has been used reliably, and is particularly suitable for measuring raised plasma levels after the administration of pharmacological doses of the vitamin (“absorption tests”). The reversible reduction of the quinone moiety to the hydroquinone makes electrochemical detection an attractive alternative, for it is both more sensitive and more selective than U.V. detection. Finally, although K vitamins possess no native fluorescence, they are readily reduced or degraded to forms that do fluoresce.¹¹

The test may not be valid if anti-coagulant drugs which cause vitamin K epoxide to accumulate in plasma (those in the coumarin family) have been administered. In some methods, the epoxide would lead to falsely elevated measurements. Heparin does not cause this problem.¹²

Learn More About Vitamin K Testing

References:

1. Gallop PM, Lian JB, Hauschka PV. Carboxylated calcium-binding proteins and vitamin K. N.Eng. J. Med. 302, 460 (1980).
2. Anon. Osteocalcin: a vitamin-K dependent calcium-binding protein in bone. Nutr. Rcv. 27, 54 (1979).
3. Whiton DS, Sadowski JA, Suttie JW. Mechanism of coumarin action: significance of vitamin K reductase inhibition. Biochem 17, 1371 (1978).
4. Dodds RA, Caterall A, Bitensky L, Chayen J. Effects on fracture healing of an antogonist of the vitamin K cycle. Calcif Tissue Int 36, 223 (1948).
5. Pettifor JM, Benson R. Congenital malformations associated with the administration of oral anticoagulants during pregnancy. J Pediat 86, 459 (1975).
6. Warkany J. A Warfarin embryopathy. AM J Dis Child 129, 287 (1975).
7. Hart JP et al. Electrochemical detection of depressed circulating levels of vitamin K1 in osteoporosis. J Clin Endo Metab 60,1268 (1985).
8. Hart JP, Catterall A, Dodds RA, Kienerman L, Shearer MJ. Circulating vitamin K1 levels in fractured neck of femur. Lancet 2, 283 (1948).
9. Tomita A. Postmenopausal osteoporosis ca-47 study with vitamin K2. Cin Endocrinol (Jpn) 19, 731 (1971).
10. Bouchaert, JH. Said AH. Fracture healing by vitamin K. Nature 185, 849 (1960).
11. Lim CK, ed.H.p.l.c. of small molecules. IRL Press, Oxford, England (1986).
12. Procedure manual, Meridian Valley Lab, Kent, WA (1987).