Genetic and Environmental Factors Affecting Hemostasis in Yup'ik People
Au, Nicholas T.
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Fish oil has been widely studied for its protective effects against heart disease. These effects are attributed to omega-3 PUFA content, which are also found in sea mammals in addition to fish. The mechanism by which omega-3 PUFAs provide cardiovascular benefits remain unclear but experimental data have alluded to effects on hemostasis. Omega-3 PUFAs may also modify bleeding risk based on anecdotal reports in the literature. The studies presented in this dissertation examine the effect of omega-3 PUFA consumption on measures of hemostasis among Yup'ik study participants, for whom fish and marine mammals are a dietary staple. Measures of clotting potential included clotting factors II and V, PT/INR, PTT, and sP-selectin. These data were assessed for an association with d15N, a validated biomarker of omega-3 PUFA intake. We found that the platelet biomarker, sP-selectin, correlated strongly and inversely with d15N. This supports earlier findings of diminished platelet activation in Greenland Inuits and suggests that platelet activity varies with omega-3 PUFA content in blood. There was also an unexpected bimodal distribution in plasma clotting factor V activity where approximately 11% of samples had values below 30%. While we validated the factor V activity data by measuring factor V protein concentrations by ELISA, the low values were not produced during repeat visits by some of the same individuals. This observation warrants investigation. We then explored whether low vitamin K status is evident in Yup'ik people. Given that the main dietary source of vitamin K is green leafy vegetables, which are only intermittently available in the remote sub-Arctic regions where the Yup'ik live, and that vitamin K is necessary for the production of vitamin K-dependent clotting factors, it is plausible that this population may be more prone to a hypocoagulable state because of limited vitamin K intake. We further hypothesized that Yup'ik people may also harbor unique polymorphisms in the genes of vitamin K cycle-associated enzymes that modify vitamin K status. Acute vitamin K status was determined by directly quantifying vitamin K concentrations in plasma using a new stable-label, LC-MS assay, while chronic vitamin K status was assessed by measuring concentrations of PIVKA-II in plasma. We then analyzed these data for statistically significant associations with genetic variants of the vitamin K hydroxylases, CYP4F2 and CYP4F11, as well as VKORC1, which catalyzes the recycling of the vitamin in the liver. Additionally, we measured the vitamin K content of tundra greens that are part of the traditional Yup'ik diet to determine if these foods are a significant, dietary source of vitamin K. While no significant associations with CYP4F11 and VKORC1 were found, we observed that the allele frequency of CYP4F2*3 was more than twice that of other world populations. Because CYP4F2*3 codes for an enzyme with low catalytic activity, this SNP may have a role in conserving vitamin K levels in the body. This hypothesis was supported by the observation that carriers of CYP4F2*3 possessed significantly higher plasma phylloquinone levels compared to wild-type participants. This SNP also associated with a reduced likelihood of having chronically low vitamin K status (PIVKA-II levels above 2 ng/mL). Furthermore, the vitamin K content of tundra greens were very similar to that of market vegetables measured in this study, suggesting that tundra greens are an excellent seasonal source of vitamin K. Finally, based on the association between CYP4F2*3 and vitamin K status in Yup'ik people, we explored the extent to which the genetic profile of CYP4F enzymes plays a role in altering hepatic stores of vitamin K. First, we quantified the vitamin K content of liver tissue obtained from the UW School of Pharmacy Human Liver Bank. Despite measuring a wide range of phylloquinone levels, we were not able find any association of CYP4F2*3 genotype with hepatic vitamin K content due to likely to a lack of dietary control in samples from these liver donors, as well as low numbers of the variant homozygote genotype in this largely Caucasian population. Therefore, we next measured hepatic vitamin K levels in livers of cyp4f14 knockout mice in comparison to cyp4f14 wild-type control mice. Cyp4f14 is a reported vitamin E omega-hydroxylase in mice, and is a candidate for the murine ortholog of CYP4F2. Although this study did not reveal differences in hepatic vitamin K content between cyp4f14 knockouts and controls, there was a substantial increase in hepatic phylloquinone content in female mice compared to males. This difference in hepatic vitamin K content between sexes has also been previously reported for vitamin E, but more studies are needed to understand these findings and implications for human health. Overall, the data from this thesis shed new light on the genetic and environmental factors that contribute to variability in coagulation and vitamin K status of Yup'ik people. These factors may be particularly important considerations when prescribing anticoagulant and antiplatelet medications or treating medical conditions involving hemostasis in this population. Continued research in this area is warranted to validate and extend our observations.
- Medicinal chemistry