Progress 12/01/08 to 11/30/12
Outputs
OUTPUTS: In 2012, we presented our findings concerning the interactions of vitamins E and K at one international meeting. At the INSDH2012 (6th International Niigata Symposium on Diet and Health, Niigata, Japan), which was held at Niigata Convention Center "Toki Messe" on October 15 to 17, 2012. Dr. Maret Traber, project director, was an invited speaker and presented a talk entitled "The Role of Vitamin E in the Brain, from Adults to Zebrafish". The talk highlighted our findings that vitamin E has a major role in changing the concentrations of brain menaquinone-4 (MK-4). A major accomplishment this year has been the successful completion of Sherry Farley's PhD graduate studies. She was a major player in the investigation into the vitamin E and K interactions. She successfully completed her thesis defense Nov 12, 2012. Her thesis covered the major topics that we investigated to elucidate the mechanisms by which vitamin E alters vitamin K status. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The purpose of our project was to evaluate three different mechanisms by which vitamin K status could be decreased by increases in whole body vitamin E concentrations in rats supplemented with vitamin E by subcutaneous injections (100 mg alpha-tocopherol/kg body weight per day), the vitamin E intoxication model system developed in the Traber lab. The tested mechanisms were: 1) increasing vitamin K metabolism, 2) decreasing menaquinone-4 synthesis from dietary phylloquinone and 3) potentiating vitamin K excretion through xenobiotic pathways. Our studies have shown vitamin K status is decreased in alpha-tocopherol-injected rats as documented by decreases of both phylloquinone and menaquinone-4 concentrations in most extra-hepatic tissues. 1) Metabolism of vitamin K at the first step of omega-hydroxylation by cytochrome P4504F2 was not stimulated in response to alpha-tocopherol, moreover urinary excretion of vitamin K metabolites was not increased by vitamin E injections in vivo. Although increased excretion of a vitamin K catabolite was measured in the bile, quantitatively this catabolite did not account for all of the vitamin K loss observed in tissues. 2) Vitamin E did not specifically interfere with menaquinone-4 synthesis from dietary phylloquinone because menadione conversion to menaquinone-4 was equally impacted, showing the lack of specificity of the interference of vitamin E on this conversion. Alternatively, transport of phylloquinone and menadione to extra-hepatic tissues or menaquinone-4 recycling may have been inhibited in response to vitamin E. Further studies are needed to distinguish between these mechanisms. 3) We did observe increases in various membrane transporters, suggesting that vitamin E alterations in xenobiotic pathways may have impact on vitamin K status. Measures of transport activity will need to be carried out to evaluate the role of transporters in determining vitamin K tissue concentrations.
Publications
- Farley, S.M., Leonard, S.W., Labut, E.M., Raines, H.F., Card, D.J., Harrington, D.J., Mustacich, D.J., Traber, M.G. Vitamin E decreases extra-hepatic menaquinone-4 concentrations in rats fed menadione or phylloquinone. Mol Nutr Food Res 56, 912-922, 2012.
- Farley, S.M., Leonard, S.W., Taylor, A., Birringer, M., Rettie, A.E., Traber, M.G., omega-Hydroxylation of phylloquinone by CYP4F2 is not decreased by alpha-tocopherol. Molecular Nutrition and Food Research, submitted Dec 2012.
- Farley, S.M., Leonard, S.W., Card, D.J., Harrington, D.J., Labut, E.M., Traber, M.G., Biliary excretion of vitamin E and K and their catabolites by rats during subcutaneous vitamin E administration. Molecular Nutrition and Food Research, submitted Dec 2012.
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Progress 12/01/10 to 11/30/11
Outputs
OUTPUTS: In 2011, we presented our findings concerning the interactions of vitamins E and K at two national meetings and one international meeting. At the Experimental Biology meeting, held in Washington, DC, my graduate student Sherry Farley, presented our in vivo data on the interactions between vitamin E and K in a poster at a session entitled "Fat Soluble Vitamins" on Tuesday April 12, 2011. Unfortunately, due to the government shut-down, we were unable to present our findings at the annual Stakeholder input session for the Agriculture and Food Research Initiative, National Institute of Food and Agriculture, U.S. Dept. of Agriculture (USDA) at the USDA headquarters. We also participated in the FASEB Summer Conference (06/26/11-07/01/11) entitled "Molecular, Structural & Clinical Aspects of Vitamin K & Vitamin K-Dependent Proteins" in Carefree, AZ, where Dr. Traber was an Invited Speaker and presented a talk entitled "Interactions Between Vitamin K and Vitamin E". She also was an invited speaker at the 21st International Conference of the Korean Society for Gerontology: Intervention and Age-related Disease (07/08/11-07/09/11) Busan, South Korea, where she discussed this topic. The Diet and Optimum Health Conference, Sept 13-15, 2011, Corvallis, Oregon, is sponsored by the Linus Pauling Institute. This conference emphasizes dietary and lifestyle approaches to improving human health and preventing or treating disease, including dietary supplements, micronutrients, and antioxidants. We presented our work at this conference in the form of a poster demonstrating our new in vivo findings concerning the effects of vitamin E on vitamin K in rats (see abstract list in Publications). PARTICIPANTS: Sherry Farley, graduate student TARGET AUDIENCES: Participants at conferences and workshops. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The purpose of our project is to identify mechanisms by which vitamin E interferes with vitamin K status and thus decreases blood clotting and increases the tendency to bleed. We hypothesized that alpha-tocopherol interferes with vitamin K1 (phylloquinone, PK) conversion to vitamin K2 (menaquinone 4, MK-4), the most potent form of extrahepatic tissue vitamin K. PK is converted to MK-4 by truncation of its side chain to form the intermediate menadione (MN), followed by the addition of an unsaturated side chain to form MK-4. We also hypothesized that elevated hepatic alpha-tocopherol concentrations may stimulate vitamin K metabolism and excretion. In order to investigate these hypotheses, rats were fed PK or MN-containing diets (2 micromol/kg) for 2.5 weeks. From day 10, rats were given daily subcutaneous injections of either alpha-tocopherol (100 mg/kg) or vehicle and were sacrificed 24 hours after the 7th injection. Irrespective of diet, alpha-tocopherol injections decreased MK-4 concentrations in brain, lung, kidney, and heart; as well as decreasing lung PK concentrations. These decreases were not accompanied by increased excretion of urinary 5C- or 7C-aglycone vitamin K metabolites; however, the urinary alpha-tocopherol metabolite (alpha-CEHC) increased ≥100 fold suggesting that alpha-tocopherol metabolism was not impeded. Moreover, alpha-tocopherol increases were accompanied by down-regulation of hepatic cytochrome P450 expression and modified expression of tissue ATP binding cassette transporters. Thus, in rats, high tissue alpha-tocopherol depleted tissue MK-4 without significantly increasing urinary vitamin K metabolite excretion. Our data suggest that changes in tissue MK-4 and PK levels rather than being changed by alpha tocopherol may be a result of alpha tocopherol altering the regulation of transporters that control vitamin K uptake and disposal. Further experiments are underway to test this hypothesis.
Publications
- Farley SM, Leonard SW, Labut EM, Raines HF, Card DJ, Harrington DJ, Mustacich DJ, and Traber MG. Vitamin E Injections Decrease Tissue Vitamin K in Rats (abstract). Experimental Biology Conference. Washington, DC. April, 2011.
- Farley SM, Leonard SW, Labut EM, Raines HF, Card DJ, Harrington DJ, Mustacich DJ, and Traber MG. Vitamin K Status is Reduced in Vitamin E-Injected Rats (abstract). FASEB Summer Research Conference: Molecular, Structural & Clinical Aspects of Vitamin K & Vitamin K-Dependent Proteins. Carefree, Arizona. June, 2011.
- Farley SM, Leonard SW, Labut EM, Raines HF, Card DJ, Harrington DJ, Mustacich DJ, and Traber MG.Vitamin K Status is Reduced in Vitamin E-Injected Rats (abstract). Diet and Optimum Health Conference. Corvallis, Oregon. Sept, 2011.
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Progress 12/01/09 to 11/30/10
Outputs
OUTPUTS: In 2010, we presented our findings concerning the interactions of vitamins E and K at two national meetings. In April 2010, Traber lab members attended Experimental Biology 2010. Scott Leonard presented a poster describing his efforts at measuring vitamin K. Sherry Farley, a Nutrition PhD graduate student, presented her poster at two different venues of the meeting; the Vitamins & Minerals Research Interest Group held on Saturday afternoon, as well as at the vitamin E session. Dr. Debbie Mustacich, Co-PI, attended the Stakeholder input session for the Agriculture and Food Research Initiative, National Institute of Food and Agriculture, U.S. Dept. of Agriculture at the IFT meeting in Chicago. She presented a poster outlining our progress in this project. We plan in 2011 to again attend the Experimental Biology meeting to be held in Washington, DC. We now have in vivo data on the interaction and plan to present this information in poster form at a session entitled Fat Soluble Vitamins on Tuesday April 12, 2011. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Vitamin E can antagonize vitamin K-dependent coagulation processes resulting in increased bleeding tendencies, a phenomenon that can be reversed with vitamin K supplementation. The mechanisms of vitamin E and K interactions remain unknown. Vitamin E and K are structurally similar compounds and there is strong evidence that they share similar metabolic pathways involving xenobiotic enzymes and transporters. Our in vitro cytochrome P450 (CYP) assays investigating the initial step in the metabolism of vitamins E and K have confirmed identification of human CYP4F2 as the primary enzyme responsible for omega-hydroxylation of both vitamins. Following the initial omega-hydroxylation of their side chains by CYP4F2, they then undergo several rounds of beta-oxidation and conjugation to glucuronide or sulfate to yield their respective urinary metabolites, carboxy ethyl hydroxy chroman (CEHC) for vitamin E and 5C- and 7C-aglycone for vitamin K. We investigated the hypothesis that vitamin E supplementation may be up-regulating these xenobiotic pathways to enhance metabolism and excretion of vitamin K in the urine. We also proposed that alpha-tocopherol may be interfering with the mechanism for side chain removal of phylloquinone (PK) to form menadione (MN) for synthesis of the tissue-specific vitamin K form, menaquinone-4 (MK-4). Both of these mechanisms would result in reduced vitamin K status, thereby disrupting proper functioning of vitamin K-dependent proteins. We investigated these hypotheses in vitro using insect microsomes expressing human CYP4F2 and in vivo using rats. The in vitro CYP4F2 experiments and in vivo investigations with rats do not support the hypothesis that vitamin E is up-regulating vitamin K metabolism. Results from the insect microsomal experiments indicate that vitamin K, at least in vitro, is preferred over vitamin E as a substrate for CYP4F2. The in vivo investigations in rats demonstrated that elevated alpha-tocopherol concentrations had pronounced consequences on vitamin K status by depleting both PK and MK-4 concentrations in extra-hepatic tissues; and that the reduction in vitamin K concentrations occurred irrespective of dietary vitamin K form. Thus, it is unlikely that vitamin E interferes with the synthesis of MK-4, by inhibiting the conversion of PK to MN. Moreover, loss of vitamin K in tissues was not accounted for in the urine of these rats since the excretion of vitamin K metabolites, 5C- and 7C-aglycone, remained unchanged. This suggests that the reduction in vitamin K concentrations in tissues is likely not a result of increased vitamin K metabolism. However, the up-regulated expression of xenobiotic transporters may be facilitating the removal of vitamin K from tissues and potentiating its excretion into the bile. Further investigations are in progress to test this latter mechanism.
Publications
- Scott William Leonard, Tyler Barker, Debbie J Mustacich, and Maret G Traber. Measurement of vitamin K homologues in biological fluids and tissues by APCI LC/MS FASEB J April 6, 2010 24:533.1.
- Sherry M. Farley, Scott W. Leonard, Debbie J. Mustacich, Edwin M. Labut, and Maret G. Traber Investigation of {omega}-hydroxylation of tocopherols by CYP4F2 and by rat liver slices FASEB J April 6, 2010 24:534.2
- Sherry M Farley, Scott W Leornard, Edwin M Labut, Hannah F Raines, David J Card, Dominic J Harrington, Debbie J Mustacich, Maret G Traber. Vitamin E Injections Decrease Tissue Vitamin K in Rats FASEB J April 12, 2011 25: 996.16
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Progress 12/01/08 to 11/30/09
Outputs
OUTPUTS: The purpose of our project is identify mechanisms by which vitamin E interferes with vitamin K status and thus decreases blood clotting and increases the tendency to bleed. To evaluate our outcomes, we have been developing new methodologies to measure vitamin K and its omega-hydroxylated side chain. We have identified that cytochrome P450 F2 (CYP4F2) is the CYP(s) responsible for omega-hydroxylation of phylloquinone. These experiments have provided us the materials for assessment of vitamin K metabolism by LC/MS. A sensitive and specific liquid chromatography mass spectrometry (LC/MS) method to measure K1, K2 (MK-4 & MK-7), and K3 in biological fluids and tissues has been developed. A Micromass single quadrupole ZQ 2000 MS detector coupled to a Waters 2695 HPLC was used. For K1 and K2 analysis an isocratic method consisting of 100% methanol delivered at 1.0 mL/min for 30 min was used. For K3, a a gradient of 50-90% methanol delivered over 30 min at 0.2 mL/min was used. The calibration curves for the K forms generated a linear response from 1 to 50 nM. K1 was detectable in non-supplemented human plasma, but MK-4 was not. In livers from male Sprague-Dawley rats on a K3-containing diet, MK-4 was detectable but K1 was not; large amounts of K3 were measured in the urine. LC/MS with an APCI source is sensitive and specific enough to measure vitamin K homologues in biological fluids and tissues. A manuscript describing these studies is currently in preparation. Interaction of metabolism of vitamins E and K studied in vitro The interaction of metabolism of vitamins E and K have been studied in vitro using cytochrome P450 (CYP4F2). Insect microsomes (50 pmol) expressing recombinant human CYP4F2 (Gentest BD Biosciences) were pre-incubated for up to 20 min at 37C with 50 microM deuterated (d)6-RRR- alpha-tocopherol (d6- alpha-T), d2-gamma-tocopherol (d2-gamma-T), d4-phylloquinone (d4-K1), unlabeled phylloquinone (K1), or vehicle (ethanol) in 100 mM KH2PO4 (pH 7.4). The reaction was initiated by adding 1 mM NADPH and incubated for 30 min at 37C. The reaction was halted, and analyzed using a Shimadzu HPLC system coupled to a triple quadrupole mass spectrometer operated in negative mode (Applied Biosystems/MDS Sciex API 3000). Analytes were detected using selected reaction monitoring (SRM).Omega-Hydroxylation is the first step in metabolism of vitamins E and K. Our preliminary findings suggest that vitamin E strongly inhibits vitamin K hydroxylation by CYP4F2. Events,Diet and Optimum Health 2009, Portland Oregon, The program featured a session, which was proposed by and chaired by Dr. Traber, entitled "Vitamin K: New Functions and Biological Mechanisms". We presented a poster demonstrating our new techniques for measuring vitamin K in biological samples. Dissemination, Dr. Debbie Mustacich discussed "Mitochondrial beta-Oxidation of alpha-Tocopherol Challenges the Currently Accepted Paradigm" emphasizing the similarities of vitamin E and K metabolism at the College of Veterinary Medicine, February 2010 (audience is Veterinarians, Veterinary students and Biomedical Sciences faculty, staff and graduate students). PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Development of an HPLC/mass spectrometric method for vitamin K analyses We wrote a manuscript describing a liquid-liquid extraction method followed by atmospheric pressure chemical ionization LC-MS to quantitate phylloquinone (K1) and menaquinone (K2; MK-4). The analytes were separated by a 20 minute reverse-phase isocratic method, using ubiquinone-4 as the internal standard. Calibration curves for K1 (m/z 450) and MK-4 (m/z 444) were linear from 1 to 100 nM, with similar ionization efficiencies. The lower limit of detection was 0.2 nM (S/N = 3/1). Plasma was collected from patients (n=24) undergoing anterior cruciate ligament (ACL) surgery at pre-surgery and 90 minutes, 72 hours, 7 days, and 3 months post-surgery. Plasma K1 (0.2 - 7.1 nM) was detectable in all samples, while MK-4 was not detectable. Following ACL surgery, plasma K1 decreased from 2.1 +/- 0.3 to 1.4 +/- 0.3 nM at 7 days (p<0.05), and returned to baseline concentrations after 3 months (2.1 +/- 0.2, p<0.05). This novel and sensitive method documents that depletion of plasma vitamin K occurs post surgery in otherwise normal healthy subjects. The manuscript was submitted, but the journal found that the information was focussed too clinically. We are currently developing the method further to allow investigation of biological samples, such as rodent tissues. Development of an HPLC/mass spectrometric method for hydroxylated vitamin K analyses In order to carry out our objectives, we have developed an advanced method for assessing hydroxylation of either vitamin E or vitamin K. Building upon our methods described above, we moved our methodologies to a more advanced system. Chromatography was carried out using a Shimadzu HPLC system with a C-18 column (Phenomenex) with methanol as the mobile phase. The HPLC system was coupled through a TurboIon Spray source to a triple quadrupole mass spectrometer operated in negative mode (Applied Biosystems/MDS Sciex API 3000). Analytes were detected using selected reaction monitoring (SRM): d6-alpha-T, m/z 435 to 169; d2-gamma-T, m/z 417 to 150; d4-K1, m/z 454 to 189; K1 m/z 450 to 185; d6-alpha-T-OH, m/z 451 to 169; d2-gamma-T-OH, m/z 433 to 150; d4-K1-OH, m/z 470 to 189; K1-OH m/z 466 to 185. Standard curves were constructed using 5 levels of the analytes: d6-alpha-T, d2-gamma-T, d4-K1 and K1. Menaquinone-4 (m/z 444 to 185) was used as an internal standard; quantitation was performed using Analyst software. Using this methodology we have been able to quantitate vitamin E and K hydroxylation and are now poised to measure their interactions. The interaction of metabolism of vitamins E and K have been studied in vitro using cytochrome P450 (CYP4F2). Although CYP3A was tested, it was not found to metabolize vitamin K (not shown).
Publications
- Farley, S. M.; Leonard, S. W.; Taylor, A.; Traber, M. G. 2010. Investigation of omega-hydroxylation of tocopherols and phylloquinone by CYP 4F2. IFT 10; USDA Project Director's Meeting, Chicago.
- Leonard, S. W.; Mustacich, D. J.; Traber, M. G. 2009. Measurement of vitamin K homologues in biological fluids and tissues by APCI LC/MS. Linus Pauling Institute Conference on Diet and Optimum Health, Portland, OR.
- Traber, M. G.; Farley, S.; Mustacich, D.; Leonard, S. W. 2009. Vitamin E and K Interactions. IFT09; USDA Project Director's Meeting, Anaheim.
- Publications Traber, M. G. 2008. Vitamin E and K interactions--a 50-year-old problem. Nutr Rev. 66: 624-629.
- Leonard, S. W.; Barker, T.; Taylor, A. W.; Traber, M. G. 2010. Quantitation of vitamin K homologues in biological samples using liquid chromatography mass spectrometry. J Lipid Res. in preparation.
- Abstracts Leonard, S. W.; Barker, T.; Mustacich, D. J.; Traber, M. G. 2010. Measurement of vitamin K homologues in biological fluids and tissues by APCI LC/MS. Experimental Biology 2010, Anaheim, CA.
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