Atorvastatin Decreases Menaquinone‐4 Formation in C57Bl6 Male Mice

Abstract

Vitamin K is a fat‐soluble vitamin found in two forms, phylloquinone (PK) and menaquinones. The two forms differ in saturation and length of their side chain. Menaquinone‐4 (MK4) is a unique menaquinone because it is: (1) formed from dietary PK by means of tissue‐specific conversion; and (2) has unique functions beyond the traditional coagulation role of vitamin K such as providing protection against oxidative damage. Geranylgeranyl pyrophosphate, an intermediate in the endogenous cholesterol synthesis pathway, has been indirectly implicated in the synthesis of MK4. Statins (HMG‐CoA reductase inhibitors) are effective suppressants of endogenous cholesterol synthesis, hence can be used to elucidate the role of geranylgeranyl pyrophosphate in the conversion of PK to MK4.The purpose of this study was to determine the effect of statin treatment on endogenous production of MK4 in both young and old male mice given a diet supplemented with PK.Four month (n=32) and 20‐month old (n=32) C57BL6 male mice were randomly assigned to a diet containing 300mg atorvastatin/kg diet and 2.67±0.33 mg PK/kg or a control diet containing 2.77±0.02 mg PK/kg for 8 weeks. During week 8, all mice received deuterium‐labeled PK in the diet. Labeled and unlabeled PK and MK4 in tissues were measured by HPLC‐MS. HMG‐CoA reductase expression gene expression in liver, kidney, brain, and intestine was quantified by reverse transcriptase‐PCR. Tissue MK4 and PK concentrations were compared between statin treatment groups using general linear models. We also examined the effect of statins in young and old mice.Mice given statins had less total MK4 and deuterium‐labeled MK4 tissue concentrations compared to mice not given statins (p=0.02 and 0.04, respectively, age‐adjusted). In statin‐treated mice, kidney MK4 concentration was reduced by 41% (p≤0.05), and percent deuterium‐labeled MK4 was reduced by 46.5% compared to mice not given statins (p=0.001). However, total and deuterium‐labeled MK4 were not affected by statin treatment in brain and intestine (p>0.31). The effect of statins on MK4 did not differ by age. Liver PK was not affected by statins (p=0.43). In liver, HMG‐CoA reductase expression increased in response to statin treatment by 1.5‐fold and 4‐fold in both young and old mice respectively (all p‐values<0.05). There were no differences in HMG‐CoA expression in kidney, brain, or intestine.In male mice, statin treatment effectively reduced endogenous MK4 formation in the kidney, but not other organs. Our results support previous studies demonstrating that liver is not a site of PK to MK4 conversion. In the brain MK4 is the sole form but the specific statin used for the intervention does not cross the blood brain barrier. Therefore, brain MK4 concentrations are not expected to be effected. These observations are consistent with our hypothesis that geranylgeranyl pyrophosphate is a key factor in the generation of MK4. Further research is needed to understand potential regulatory mechanisms and the unique functions of MK4.Support or Funding InformationSupported by the USDA agricultural Research Service under Cooperative Agreement No. 58‐1950‐7‐707, and the NIH/NIDDK grant T32 DK062032