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1 From the Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston (SLB, JMS, RR, GED, and JBB); the Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Munich, Germany (IG); and the Saga Nutraceuticals Research Institute, Saga, Japan (KH)
2 The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. 3 Supported by the Saga Nutraceuticals Research Institute of Otsuka Pharmaceutical Co, Ltd, Kanzaki, Japan; Pharmavite, Inc, San Fernando, CA; HERMES Arzneimittel GmbH, Grosshesselohe/Munich, Germany; the US Department of Agriculture Agricultural Research Service under Cooperative Agreement no. 58-1950-001; the American College of Sports Medicine, Indianapolis; and Life Fitness, Franklin Park, IL. Vitamin E and placebo were generously donated by HERMES Arzneimittel GmbH (Grosshesselohe/Munich, Germany) and the Pharmavite Corporation (San Fernando, CA). 4 Reprints not available. Address correspondence to SL Booth, Vitamin K Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111. E-mail: sarah.booth{at}tufts.edu.
ABSTRACT
Background: Cases of enhanced anticoagulant effect in response to high-dose vitamin E supplementation have been reported among patients taking oral anticoagulants. Although a vitamin E–vitamin K interaction was proposed to underlie this effect, it has not been systematically investigated in adults with normal baseline coagulation status.
Objective: The objective was to study the effect of 12 wk of supplementation with 1000 IU RRR--tocopherol/d on biochemical measures of vitamin K status in men and women not taking oral anticoagulants.
Design: Vitamin K status, which was assessed with the use of plasma phylloquinone concentrations, the degree of under--carboxylation of prothrombin (proteins induced by vitamin K absence–factor II, PIVKA-II), and the percentage of undercarboxylated osteocalcin (ucOC), was determined in 38 men and women with rheumatoid arthritis (study A) and in 32 healthy men (study B) participating in 2 independent, 12-wk randomized clinical trials of vitamin E supplementation (1000 IU/d).
Results: Mean (± SD) PIVKA-II increased from 1.7 ± 1.7 to 11.9 ± 16.1 ng/mL (P < 0.001) in study A and from 1.8 ± 0.6 to 5.3 ± 3.9 ng/mL (P < 0.001) in study B in response to 12 wk of vitamin E supplementation. An increase in PIVKA-II is indicative of poor vitamin K status. In contrast, the other measures of vitamin K status (ie, plasma phylloquinone concentration and percentage of ucOC) did not change significantly in response to the supplementation.
Conclusions: High-dose vitamin E supplementation increased PIVKA-II in adults not receiving oral anticoagulant therapy. The clinical significance of these changes warrants further investigation, but high doses of vitamin E may antagonize vitamin K. Whether such an interaction is potentially beneficial or harmful remains to be determined.
Key Words: Vitamin K • vitamin E • proteins induced by vitamin K absence • PIVKA-II • coagulation
INTRODUCTION
An adverse vitamin E–vitamin K interaction was reported among patients taking coumarin-based oral anticoagulants, such as warfarin and phenprocoumon (1–3). The mechanism of the apparent enhancement by vitamin E of the action of coumarin is unknown, although Dowd and Zheng (4) proposed a competitive inhibition between tocopherol quinone and the phylloquinone hydroquinone for the vitamin K–dependent -carboxylase. The vitamin K–dependent carboxylase is required for conversion of specific glutamyl residues to -carboxyglutamyl residues in certain proteins, including factors II, VII, IX, and X, and proteins C and S, which are involved in normal hemostatic function (5).
Although vitamin E antagonism of vitamin K–dependent coagulation may increase the risk of hemorrhagic disorders in patients taking oral anticoagulants, the vitamin E antagonism of vitamin K in healthy adults may be one mechanism by which vitamin E exerts its putative mild anticoagulant effect and is associated with a reduced risk of coronary artery disease (4). However, results from animal studies and clinical trials are equivocal as to whether vitamin E possesses an anticoagulant action (6, 7), and the mechanisms by which vitamin E may have a role in vascular homeostasis is still in an exploratory phase (8).
Animal data suggest that there is a critical ratio of vitamin E to vitamin K, with elevated doses of vitamin E having an anticoagulant effect when vitamin K intake is low (9, 10). Average vitamin K intakes among US adults (11) are below current recommended intakes (12), and the use of dietary supplements containing vitamin K is uncommon (13). Although dietary vitamin E intakes are low relative to recommended intakes, vitamin E is among the most frequently purchased single nutrient supplement in the United States, particularly among older adults (14, 15). Elevated vitamin E intakes associated with supplementation were not shown to alter coagulation times in healthy adults in short-term (30 d) (16) or longer-term (4 mo) trials (17, 18). However, coagulation times are not a sensitive measure of vitamin K status because plasma prothrombin concentrations must decrease by 50% before a change in coagulation time is observed (19). In previous metabolic studies of various age groups, dietary restriction of phylloquinone (vitamin K1) to <35 µg/d caused rapid decreases in plasma phylloquinone and increases in the under--carboxylated forms of the vitamin K–dependent proteins osteocalcin and prothrombin (20–22). In contrast, coagulation times consistently remained stable. The purpose of the present study was to examine the effect of high-dose supplementation with natural source -tocopherol on sensitive biochemical measures of vitamin K status in men and women participating in 2 independent, randomized clinical trials with vitamin E.
SUBJECTS AND METHODS
Subjects
Study A design
Men (n = 7) and women (n = 31) aged 18 y who had rheumatoid arthritis (RA) that was not in remission were recruited from rheumatology clinics at the Tufts University–New England Medical Center and from advertisements in local newspapers to participate in a study that examined the effectiveness of 1000 IU vitamin E/d as an adjunct to their current therapeutic regimens. All subjects met American College of Rheumatology criteria for RA (23, 24), were taking a nonsteroidal antiinflammatory drug on a daily basis, and had a stable antirheumatic medication regimen for 3 mo. Exclusion criteria included prednisone at doses 10 mg/d, severe anemia, thrombocytopenia, oral anticoagulant use, donation of blood (>450 mL) in the previous 2 mo, obesity, renal insufficiency, hepatic disease, or pregnancy. Subjects did not take supplements containing fish oil or daily supplements containing 400 IU vitamin E or 10 mg ß-carotene for 3 mo before enrollment and did not take daily supplements containing 500 mg vitamin C for 1 mo before enrollment.
Study B design
Physically active young (18–35 y; n = 16) and older (65–80 y; n = 16) healthy men were recruited from the general population to study the age-related effects of an acute bout of eccentric exercise (downhill running) and vitamin E supplementation on oxidative stress status (25). Exclusion criteria included coronary artery disease or angina, congestive heart failure, diabetes, cancer other than nonmelanoma skin cancer, thyroid problems, arthritis or other autoimmune conditions, severe anemia, liver or kidney disease, poorly accessible veins, and current use of addictive drugs, tobacco, or steroid hormone medications. Subjects did not take vitamin C supplements for 1 mo before or during the protocol or vitamin E supplements for 3 mo before the study period.
In both studies, fasting blood samples were drawn before and after 12 wk of daily administration of vitamin E or vehicle placebo supplement in a randomized design. In study A, vitamin E was provided on a daily basis in the form of 2 soft gel gelatin capsules, each containing 500 IU (335.5 mg) RRR--tocopherol in soybean oil. In study B, vitamin E was provided as 1000 IU (671 mg) RRR--tocopherol in soybean oil (25). The placebo was provided in an identical gelatin capsule containing soybean oil. For both studies, subjects were instructed to take vitamin E capsules daily with their largest meal. Vitamin E and placebo were generously donated by HERMES Arzneimittel GmbH (Grosshesselohe/Munich, Germany) (study A) and the Pharmavite Corporation (San Fernando, CA) (study B). Adherence to the regimen was confirmed by having subjects return to the laboratory 3 times during the study for pill counts and a blood draw for serum -tocopherol (at 4, 8, and 12 wk) (data not shown for 4- and 8-wk time points).
The Tufts University–New England Medical Center Investigation Review Board approved both study protocols. The study was fully explained to each subject, and his or her written informed consent was obtained.
Analytic procedures
Serum -tocopherol was measured with the use of reverse-phase HPLC (Waters Associates HPLC, Model 840 Data Station; Milford, MA) according to Bieri et al (26). In study B, serum total cholesterol and triacylglycerols were analyzed with the use of a Cobas Mira analyzer (Roche Diagnostic Systems Inc, Indianapolis). Lipid data were not available for study A. Phylloquinone was analyzed by reverse-phase HPLC as described by Davidson and Sadowski (27). In study A, prothrombin time was determined by photometric detection with an MLA Electra automated clot timer (Medical Laboratory Automations Inc, Pleasantville, NY). No coagulation measures were available for study B. PIVKA-II (proteins induced by vitamin K absence–factor II), a functional measure of the biological activity of vitamin K in a hepatic vitamin K–dependent protein, was analyzed in citrated plasma with an enzyme-linked immunosorbent assay from American Bioproducts Company (Parsippany, NJ). Undercarboxylated osteocalcin (ucOC), a marker of vitamin K status in extrahepatic tissues, was determined by radioimmunoassay, as described by Gundberg et al (28, 29). The radioimmunoassay for osteocalcin uses human osteocalcin for standard and tracer and a polyclonal antibody directed to intact human osteocalcin (29). The antibody recognizes intact osteocalcin and the large N-terminal midmolecule fragment. UcOC is determined in this assay as plasma osteocalcin that does not bind in vitro to hydroxyapatite.
Statistical analysis
Data were analyzed with the use of SYSTAT, version 10 (SPSS Inc, Chicago). The effects of vitamins E and K were assessed by applying analysis of covariance models to each of the biochemical measures (plasma phylloquinone, PIVKA-II, percentage of ucOC, and prothrombin time). A separate analysis was performed for each of the 12-wk measures with treatment (vitamin E or placebo), study (A or B), and their interaction as study factors, and baseline measurement was used as a covariate. Because the distributions of PIVKA-II and vitamin E were highly positively skewed, a logarithmic transformation was applied before formal analysis. All tabular summaries are expressed as means ± SDs in the original units, unless indicated otherwise. Results were considered statistically significant if the observed, two-sided significance level (P value) was <0.05.
An interaction between treatment and baseline measurements was evaluated for each response, but because none was statistically significant, these measurements were not included in the final models. In addition, there were no significant treatment-by-study interactions.
RESULTS
A description of the study participants is presented in Table 1. There were no differences in age, body mass index, or baseline biochemical measures between the vitamin E–supplemented and placebo groups within each study.
View this table:
TABLE 1. Subject characteristics1
In the vitamin E–supplemented groups, plasma -tocopherol concentrations significantly increased (P < 0.001) from baseline (Table 1) to 12 wk (69.8 ± 23.9 and 55.8 ± 18.7 µmol/L in studies A and B, respectively), which was consistent with compliance (Figure 1). Similar increases were observed when vitamin E concentrations were adjusted for serum total cholesterol and triacylglycerols in study B, for which lipid data were available (data not shown). Plasma -tocopherol concentrations did not change significantly in either placebo group (12-wk concentrations: 30.4 ± 8.3 and 22.5 ± 5.9 µmol/L in studies A and B, respectively) over the 12-wk study period.
FIGURE 1.. Mean (± SEM) plasma -tocopherol and phylloquinone concentrations before and after 12 wk of supplementation with 1000 IU RRR--tocopherol/d in men and women (n = 38 in study A; n = 32 in study B). *Significantly different from 0 wk for same treatment and study group, P < 0.001 (analysis of covariance). There were no significant interactions between treatment and baseline measurements for each response.
In studies A and B, mean baseline measures of vitamin K status (plasma phylloquinone concentrations, PIVKA-II, and serum percentages of ucOC) were within the normal range for healthy adults in the United States (21, 30, 31). Within each study, plasma phylloquinone concentrations did not change significantly over the study period in either treatment group (P = 0.78; Figure 1). In study A, for which coagulation times were available, there was no change in prothrombin time in either treatment group (P = 0.51).
PIVKA-II significantly increased from baseline (P < 0.001) in both vitamin E–supplemented groups [12-wk concentrations: 11.9 ± 16.1 (range: 1.9–71.3) and 5.3 ± 3.9 (range: 1.3–14.5) ng/mL in studies A and B, respectively] but remained unchanged in the placebo groups (12-wk concentrations: 2.3 ± 3.3 and 2.0 ± 1.0 ng/mL in studies A and B, respectively) (Figure 2). Concentrations of PIVKA-II 2.4 ng/mL are considered abnormal (32). In contrast, serum percentages of ucOC did not significantly change over the study period (P = 0.45) in either treatment group within each study (Figure 2).
FIGURE 2.. Mean (± SEM) undercarboxylated prothrombin (proteins induced by vitamin K absence–factor II, PIVKA-II) and mean (± SEM) percentage of undercarboxylated osteocalcin (ucOC) before and after 12 wk of supplementation with 1000 IU RRR--tocopherol/d in men and women (n = 38 in study A; n = 32 in study B). *Significantly different from 0 wk for same treatment and study group, P < 0.001 (analysis of covariance). There were no significant interactions between treatment and baseline measurements for each response.
DISCUSSION
To the best of our knowledge, this is the first report of a direct effect of vitamin E supplementation on a biochemical measurement of vitamin K among adults not taking coumarin-based oral anticoagulants. High-dose vitamin E supplementation increased under--carboxylated prothrombin (PIVKA-II) concentrations to concentrations that are indicative of poor vitamin K status. These findings in adults not taking oral anticoagulants are consistent with observations in patients with cystic fibrosis and coronary artery disease taking anticoagulants (33). One caveat to the current studies was the involvement of patients with RA (study A). However, there are no reports of RA being associated with abnormal vitamin K metabolism, and vitamin K status measured at baseline among subjects in study A were similar to healthy subjects enrolled in study B.
Coagulation times require a decrease of >50% plasma prothrombin concentrations to become abnormal (19) and have not been shown to change in response to vitamin K dietary deficiency or supplementation (21, 34). Therefore, it was not surprising that coagulation times did not change in response to vitamin E supplementation in the current study. In contrast, PIVKA-II is a measure of the biologically inactive, undercarboxylated form of prothrombin and can detect abnormalities in prothrombin before coagulation times are prolonged. With use of a low-sensitivity assay for this protein (PIVKA-II concentrations for all time points reported as below the detection threshold of 0.5 µg/mL), Kitagawa and Mino (35) found 900 IU RRR--tocopherol did not change PIVKA-II concentrations. In the current studies that used a different PIVKA-II assay [minimum detection threshold of 1 ng/mL (32)], high-dose vitamin E supplementation increased mean PIVKA-II concentrations from within the normal range at baseline to abnormal concentrations (2.4 ng/mL) at 12 wk. Given the differences in detection thresholds between the 2 PIVKA-II assays, the vitamin E dose at which PIVKA-II increases cannot be determined by direct comparison of these studies. It is important to note the vitamin E–associated changes attained in the current studies are modest in comparison to PIVKA-II concentrations measured among patients stabilized with oral anticoagulants (750 ng/mL) (32). However, this effect of vitamin E is consistent with reported increases to abnormal concentrations in response to mild vitamin K deficiency (20, 21, 36) or antagonism (37). Although elevated PIVKA-II concentrations are strongly predictive of neonatal vitamin K–deficient bleeding syndrome (38), the long-term consequences of modestly elevated concentrations of PIVKA-II in an adult population are not known. It is plausible that there can be a mild anticoagulant effect associated with a minor inhibition by vitamin E of the -carboxylation of prothrombin.
In the current studies, high-dose vitamin E supplementation had no effect on vitamin K absorption as measured by fasting plasma phylloquinone concentrations. Thus, any inhibitory effect of vitamin E supplementation on -carboxylation of vitamin K–dependent proteins likely occurred within the tissue at the site of the reaction. In some animal studies, vitamin E supplementation decreased phylloquinone concentrations in plasma, liver, heart, and spleen (10, 39). Plasma phylloquinone concentrations respond within 48 h of dietary alterations of vitamin K (40) and have a higher intra-individual variance ratio compared with other fat-soluble vitamins (41). As the specific aims of studies A and B did not originally include the examination of potential interactions between vitamin E and vitamin K, dietary and supplemental vitamin K intakes were not standardized throughout the treatment period or were usual dietary vitamin K intakes assessed. Therefore, it is plausible that dietary and nondietary factors influencing plasma phylloquinone concentrations (42) attenuated our ability to detect any vitamin K absorption changes in response to vitamin E supplementation.
Because percentage of ucOC is considered a sensitive marker of vitamin K status in extrahepatic tissues (31), the lack of response of percentage of ucOC to vitamin E supplementation in the current studies was surprising given the significant increase of PIVKA-II in response to the vitamin E supplementation. If vitamin E supplementation decreased absorption of vitamin K, one would predict that undercarboxylation of both hepatic and extrahepatic vitamin K–dependent proteins would have increased, as previously observed in response to vitamin K depletion (21) or antagonism (37). The lack of a response of percentage of ucOC to the vitamin E supplementation in the current studies could reflect a tissue-specific vitamin E antagonism of the vitamin K–dependent carboxylase in liver but not bone. Alternatively, fluctuations in vitamin K intake can influence percentage of ucOC to a greater extent than that of PIVKA-II, which would have attenuated our ability to detect a response of percentage of ucOC to vitamin E supplementation because dietary vitamin K intakes were not controlled in these studies.
Dowd and Zheng (4) proposed extensive inhibition of the vitamin K–dependent carboxylase with the addition of micromolar concentrations of tocopherol quinone. Alternatively, tocopherol quinone can oxidize vitamin K hydroquinone, thus depleting the vitamin K cycle of a cofactor, a mechanism previously proposed for the in vitro inhibition of carboxylase by numerous synthetic and natural benzoquinones and naphthoquinones (43). On the basis of animal models, the inhibitory effects of vitamin E appear to be modulated by the amount of dietary vitamin K consumed, with supplements of vitamin K being able to prevent inhibition (10). This relation suggests the inhibitory effects of vitamin E on the carboxylation of prothrombin would be most pronounced among individuals with a poor vitamin K status. In the current studies, there was no significant interaction between baseline plasma phylloquinone concentrations and response of PIVKA-II to vitamin E supplementation. However, subjects participating in the current studies had mean plasma phylloquinone concentrations within the normal range for healthy adults, so this situation precluded an examination of a dose-response effect.
The hemorrhagic toxicity of high doses of vitamin E (>500 mg · kg–1 · –1d), reversible with supplemental vitamin K, was observed in chicks and rats (44, 45). The first report of a hemorrhagic syndrome in a patient receiving warfarin therapy after the supplemental intake of 800–1200 IU vitamin E/d (2) indicated the potential for an adverse interaction between high–vitamin E and low–vitamin K status in humans. However, at doses of 100–400 IU vitamin E/d, no changes were reported in coagulant activity among patients with coronary artery disease receiving warfarin (46). With one exception (47), no randomized clinical trial of vitamin E supplementation has reported signs of hemorrhagic toxicity, such as altered coagulation time or increased risk of stroke. The minimum dose, form, and duration of vitamin E intake required to induce a clinically significant, untoward effect on the maintenance of coagulation pathways is not known, but our observation of an inhibition by vitamin E of the -carboxylation of prothrombin underscores the caution already proffered about vitamin E supplementation by patients receiving stable oral anticoagulant therapy. Among healthy adults, it is plausible that the minor affects of vitamin E on prothrombin or its putative inhibition of platelet aggregation and adhesion and thrombin generation could contribute to a reduction in atherogenesis and the risk of coronary artery disease (48, 49).
In summary, supplementation with 1000 IU RRR--tocopherol decreased the -carboxylation and functionality of prothrombin, a vitamin K–dependent protein, among adults not receiving oral anticoagulant therapy. Further research is required to elucidate the mechanism by which vitamin E has an inhibitory effect on vitamin K and the potential beneficial or harmful outcomes of this action.
ACKNOWLEDGMENTS
We thank Marion (Molly) Damon and Giana Angelo for their technical assistance, the staff of the Jean Mayer USDA HNRCA Metabolic Research Unit and Tufts University–NEMC Rheumatology Clinic, and the volunteers for their participation in these studies.
SLB, JBB, IG, JMS, and RR contributed to the study design; SLB, JBB, JMS, KH, and RR contributed to the data collection; GD contributed to the data analysis; and all authors contributed to the writing of the manuscript. The authors had no financial or other interests in any company or organization sponsoring the research, except for IG, who was affiliated with HERMES Arzneimittel GmbH during the conduct of study A, KH, who is an employee of the Saga Nutraceuticals Research Institute, one of the sponsors of study B, and JBB, who serves as a member of the Scientific Advisory Board of Pharmavite, Inc. RR became an employee of Millennium Pharmaceuticals Inc after these data were collected and analyzed.
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