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Effects of chronic β‐carotene supplementation on vitamin K status in adults a

c

Louise M. Canfield , James J. Corrigan Jr. , Patricia M. Plezia b

c

b

, Monette Jeter , Susan Sayers & David S. Alberts

d

a

Departments of Biochemistry and Family and Community Medicine , University of Arizona , Tucson, AZ, 85724 b

Pharmacy Practice (College of Pharmacy) , University of Arizona , Tucson, AZ, 85724 c

Pediatrics (College of Medicine) , University of Arizona , Tucson, AZ, 85724 d

Arizona Cancer Center , Tucson, AZ, 85724 Published online: 04 Aug 2009.

To cite this article: Louise M. Canfield , James J. Corrigan Jr. , Patricia M. Plezia , Monette Jeter , Susan Sayers & David S. Alberts (1990) Effects of chronic β‐carotene supplementation on vitamin K status in adults, Nutrition and Cancer, 13:4, 263-269, DOI: 10.1080/01635589009514068 To link to this article: http://dx.doi.org/10.1080/01635589009514068

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Effects of Chronic β-Carotene Supplementation on Vitamin K Status in Adults Louise M. Canfield, James J. Corrigan, Jr., Patricia M. Plezia, Monette Jeter, Susan Sayers, and David S. Alberts

Abstract Plasma vitamin K concentrations andprothrombin coagulation activity were determined in 26 normal adults who had received daily (S-carotene supplementation (0,15,30, or 60 mg) for six months. Neither plasma vitamin K nor coagulation activity were significantly decreased at any supplementation level. Thus, chronic β-carotene supplementation, even at high daily doses, is not expected to result in clinical vitamin K deficiency. The data suggest separate mechanisms for intestinal absorption of β-carotene and vitamin K. (Nutr Cancer 13, 263-269, 1990)

Introduction

With the discovery that dietary |3-carotene is inversely correlated with incidence of a variety of cancers (1) has come widespread clinical interest in the use of /3-carotene as a possible cancer preventive agent. A number of large, randomized supplementation studies to test this possibility are now underway (2). In addition, many patients have begun supplementing their diets with /3-carotene, which is readily available over the counter in a number of dosage forms. It is therefore critical to establish the safety of such a compound. Both synergistic and antagonistic: interactions of fat-soluble vitamins have been documented (3) but are poorly understood. High amounts of vitamin A reportedly impair intestinal vitamin K utilization in rats (4). Vitamin K inhibits retinyl palmitate hydrolase (5). High dietary intakes of vitamin E in rats antagonize vitamin K activity (6), and vitamin E inhibits vitamin K-dependent carboxylase in vitro (7,8). Because the effects of /3-carotene on vitamin K absorption have not been studied, by analogy to effects of other fat-soluble vitamins on vitamin K absorption and metabolism, we were concerned about the effects of chronic /3-carotene supplementation at low to high daily L.M. Canfield is affiliated with the Departments of Biochemistry and Family and Community Medicine, P. Plezia and S. Sayers with the Pharmacy Practice (College of Pharmacy), and J. Corrigan and M. Jeter with Pediatrics (College of Medicine), University of Arizona, Tucson, AZ 85724. D. Alberts (as is L.M. Canfield) is affiliated with the Arizona Cancer Center, Tucson, AZ 85724.

Copyright © 1990, Lawrence Erlbaum Associates, Inc.

doses on vitamin K status. Thus, this study was performed to determine whether subjects on /3-carotene supplementation trials are at risk for development of secondary vitamin K deficiency. Materials and Methods

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Materials Radiolabeled vitamin Kj(2-3H-methyl-3-phytyl-l,4-naphthoquinone,53 mCi/mmol) and MK7 (l-methyl-3-fl//-//w?s-farnesylgeranylgeranyl-l,4-naphthoquinone), /3-carotene capsules (water-miscible beadlets), and placebos were generous gifts of Hoffmann LaRoche (Nutley, NJ). Patients received either 15, 30, or 60 mg of /3-carotene per day or a placebo that consisted of a like amount of excipients. |3-Carotene was furnished in 15-mg capsules. The /3-carotene content of the capsules was analyzed in our laboratory by high-pressure liquid chromatography (HPLC) and was found to agree within 95% of the stated dose. Blister packs were prepared by pharmacists in the in-patient pharmacy at University Medical Center. Each blister pack contained a 32-day supply of 0-carotene doses (15, 30, or 60 mg) or placebo doses. All solvents used for extraction and chromatography were HPLC grade or equivalent from J. T. Baker Chemical (Phillipsburg, NJ). Silica was from BioRad Laboratories (Richmond, CA) (BioSil HA-325). Tetrabutyl ammonium perchlorate (TBAP) was from Eastman Kodak (Rochester, NY). Brain thromboplastin was from Ortho Diagnostic (Raritan, NJ), and Factor H-deficient human plasma was from George King Bio-Medical (Overland Park, KS). E. carinatus venom (0.03 mg/ml, 0.85% NaCl solution) and all other chemicals (analytical or HPLC grade) were from Sigma Chemical (St. Louis, MO). Nitrogen (99.5%) from a local supplier was further purified by flowing through six feet of ice-cooled copper tubing (id = 55 mm) immediately prior to use. Subject Selection Twenty-six healthy nonsmoking male and female individuals aged 50-65 years were recruited via advertisement in the local newspaper for this randomized double-blind clinical pharmacology trial. All subjects were screened for determination of eligibility. The screening consisted of a medical, drug, and diet history, physical exam, and blood chemistries, including SMA-20, CBC, and serum lipids. Participants were considered eligible if they had no acute or chronic illness, were not taking any medications, vitamins, or other nutritional supplements, were less than 50% over their ideal body weight, were nonsmokers, and had normal blood chemistries and hematologies. All women were postmenopausal. The average age of all subjects was 56 ± 4 years (male subjects, 56 ± 4 yrs; female subjects, 57 ± 4 yrs). All participants signed an informed consent approved by the University of Arizona's Human Subjects Committee before entrance into the study. The demographic data on the 26 evaluable subjects is presented in Table 1. Treatment Schedule All participants completed a three-month lead-in period in which they received treatment with the placebo. The lead-in period allowed for assessment of compliance and documentation of normal dietary intake for all subjects. At Month 4, participants were randomly assigned to receive either the placebo or 15, 30, or 60 mg of /3-carotene daily for six months between 7 and 9 AM with a meal including some food high in protein and fat (e.g., milk, cheese, or eggs).

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Table 1. Demographic Characteristics of Subjects by Dosage Group"'6 Dosage Groups Placebo Age, yrs Sex

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Height, inches Males Females Weight, lbs Males Females Body surface area Males Females Race Caucasian Asian Black Hispanic

15 mg

30 mg

60 mg

57 ± 4 4 Males 2 Females

55 ± 4 5 Males 1 Female

58 ± 3 2 Males 4 Females

56 ± 4 3 Males 5 Females

72 65

± 1 ± 0

72 65

± 2 ± 0

68 62

± 1 ± 2

69 66

± 2 ± 2

204 128

± 16 ± 13

194 190

± 20 ± 0

190 137

± 21 ± 9

165 151

± 9 ± 18

2.19 ± 1.64 ±

0.03 0.08

5 0 0 1

2.09 ± 0.12 1.93 ± 0.00 5 0 1 0

1.99 ± 0.08 1.62 ± 0.10 5 0 0 1

1.90 ± 0.68 1.77 ± 0.11 8 0 0 0

a: Subjects were healthy, nonsmoking individuals aged 50-60 yrs and were screened for eligibility as described in Subject Selection, b: Values are means ± SD.

Sample Collection Plasma samples were obtained after six months of daily dosing with 0-carotene. Subjects fasted overnight and blood was collected in the morning prior to dosing in evacuated collection tubes containing sodium citrate and placed on ice until processing. Plasma was separated and stored at — 20°C. Compliance and Toxicity Monitoring Compliance was monitored monthly by pill counts and medication consumption reports completed by each subject. Toxicitj' evaluations were performed monthly. Dietary Assessment Because of the high day-to-day variation in /3-carotene intake, between 7 and 21 days are required to estimate the usual intake of an individual. Therefore, seven days of dietary records were kept during the lead-in period. Assay of Coagulation Activity E. carinatus prothrombin activity was determined as previously described (9). Venom (0.2 ml) was added to a mixture of 0.1 ml Factor II-deficient plasma, and 0.1 ml plasma (normal or test) was serially diluted (1:2, 1:4, 1:8, and 1:16 in veronal buffer, pH 7.5). Clotting times were determined in a semiautomatic clot timer apparatus (Fibrometer, BBL, Cockeysville,

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MD). Clotting times for normal plasma were plotted against dilutions (1:2 = 100 units/dl) using log-log paper. Clotting times for the test samples were read from the standard curve. The normal range is 50-150 units/dl. Factor II activity was performed using brain thromboplastin by the method of Owren and Aas (10). The normal range is 60-150 units/dl.

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Extraction of Vitamin K From Plasma Vitamin K was extracted from plasma using the method previously described from extracting vitamin K from milk (11) with the exception that the Silicar CC-4 column was omitted. Briefly, plasma was extracted with isopropanol/hexane (3:2 vol/vol) and protein precipitated by centrifugation for 15 minutes at 600 g (25 °C). Water was removed from the sample by rotary evaporation three times from chloroform/methanol (2:1 vol/vol) using a Buchi model REIH Rotavapor (Brinkmann Instruments, Westbury, NY). The residue was resuspended in hexane, and the supernatant was decanted from the flocculent precipitate, dried under nitrogen, and redissolved with 5 ml isooctane. The extract was applied to a 30-ml fritted glass column (25 x 100 mm) containing 6 g Bio-Sil and eluted with 100 ml hexane/chloroform (75:25 vol/vol). To control for changes in humidity, chromatography was performed under nitrogen atmosphere in a custom-made gas-tight chamber. HPLC Analysis To purified fractions, 90 pmol menaquinone (MK7) were added as internal standard and the mixture was injected onto a 5-/t CjgHPLC column (Radial Pak, Waters, Milford, MA) and eluted with a convex gradient (Waters no. 3) of ethanol/water (90:10 vol/vol) to ethanol/hexane (90:10 vol/vol) as previously described (12). Vitamin K was detected at 254 nm using a Waters model 450 ultraviolet detector. The portion of the gradient in which vitamin Kj and MK7 elute was collected, concentrated, reinjected onto a 10-/* C,8HPLC column (Radial Pak, Waters) and eluted isocratically with ethanol/hexane/water (90:6.5:3.5 vol/vol/vol) containing 25 mM TBAP. Vitamin K was detected with an amperometric dual glassy carbon electrode (LC-4B/17A, BioAnalytical Systems, West LaFayette, IN) using an applied potential of —0.6 V at the reductive electrode and +0.2 V at the oxidative electrode (12). Quality Control Procedures Batches were composed of individual samples from six subjects plus samples from a control pool and a radiolabeled pool. Plasma pools were constructed from plasma from 7-10 normal volunteers and divided in half. One-half received 1 ng/ml 3H-vitamin K, whereas the other half was unaltered. The tritiated pool was used to validate the recovery procedure through the open chromatography step (Bio-Sil). Recovery on HPLC was validated by the internal standard MK7. The unaltered control was assayed with each batch to calculate variation in procedure. Each procedure (e.g., extraction, chromatography) was performed on a single batch on the same day. Results and Discussion

Compliance with dosing was greater than 90% in all study subjects. There was no evidence of coagulation-related toxicities based on monthly clinical assessments. Concentrations of vitamin K in plasma of controls and treated subjects were 1-2 ng/ml. Similar concentrations have been reported by others (13). We detected no differences in vitamin K levels in plasma of carotene-supplemented subjects compared with controls. As shown in Table 2, individual variability is high; thus, the effect would have to be large to be outside the normal range.

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Table 2. Vitamin K in Plasma of Carotene-Supplemented Subjects" Vitamin K, ng/ml ± SD (range)

/8-Carotene Administered, mg/day 0

1.9 ± 2.1 (0.4-5.3) 1.3 ± 0.7 (0.4-2.3) 2.3 ± 1.1 (0.7-3.2) 1.3 ± 1.4 (0.1-4.3) 2.0 ± 0.8 (1.3-3.4)

15 30 60 Pools

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N 6 6 6 8 7

a: Plasma was obtained 6 mo after daily dosing with 0-cartotene or placebo. Vitamin K was extracted from plasma and analyzed on HPLC as described in HPLC Analysis.

However, given the precision of the assay, as determined by replicate assay of serum pools (relative standard deviation = 38%), over the study we would have reliably detected significantly altered vitamin K level:;. However, because serum levels of vitamin K are not a sensitive indicator of vitamin K status (14), coagulation activity was also determined. Factor II (prothrombin), one of the vitamin K-dependent coagulation factors, is synthesized by the liver as preprothrombin (PIVKA-II). Glutamic acid residues in preprothrombin are then carboxylai ed to 7-carboxyglutamic acid in a vitamin K-dependent posttranslational carboxylation. Preprothrombin has no prothrombin activity using the physiological activator thromboplastin (15). However, both preprothrombin and prothrombin can be activated to produce thrombin by the venom from E. carinatus. Conversely, only prothrombin is detected in the Factor II assay using thromboplastin as activator. Using these methods, preprothrombin (PIVKA II) is determined by subtracting the prothrombin activity measured with thromboplastin from the activity measured with E. carinatus venom. In Vitamin K deficiency the amount of preprothrombin is greater than 10 units/dl (9). Coagulant activity, as measured either by Factor II or E. carinatus assay in subjects who received placebos or 15, 30, or 60 mg /3-carotene daily for six months, is shown in Table 3. Table 3. Coagulation Activity in Plasma of Carotene-Supplemented Subjects" Prothrombin Coagulant Activity, units/dl ± SD 0-Carotene Administered, mg/day 0 15 30 60

Thromboplastin

E. carinatus

101 ± 19 (70-126) 104 ± 24 (80-148) 102 ± 14 (88-123) 116 ± 19 (88-138)

108 ± 14 (88-125) 102 ± 12 (87-122) 100 ± 9 (88-112) 122 ± 11 (105-138)

a: Plasma was obtained after 6 mo of diiily dosing with ^-carotene or placebo and analyzed for coagulation activity as described in Assay of Coagulation Activity.

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Although there is a trend to slightly higher activities in both assays at the 60-mg dosage level, given the assay variability, coagulation activity was not different in supplemented subjects compared with controls. In none of the samples was there an elevation in preprothrombin levels. In addition, prothrombin time and thromboplastin times were also determined by Central Diagnostics Laboratory (Tucson, AZ), using a one-step thromboplastin test (Koagulab-16S). All samples were within normal limits for both of these tests, suggesting no effect of /3-carotene on these generalized screening tests for clotting factors. /3-Carotene is typically present in serum at 25-50 /tg/dl, concentrations that are 200- to 500-fold greater than those for vitamin K (0.1-0.2 /tg/dl). Similarly, vitamin K requirements are estimated at 0.045 mg/day (16). Thus, /3-carotene was administered at 60 mg daily for six months, approximately 1,300 times the recommended dietary intake for vitamin K. As reported elsewhere, over the same six months of supplementation (17), the increase over baseline in plasma /3-carotene was minimal, ranging from 98 /tg/dl in subjects receiving 15 mg /3-carotene per day to 125 /tg/dl in subjects receiving 60 mg/day. The resistance of vitamin K status to this systemic excess of /3-carotene is remarkable and suggests a specific and stringent homeostatic regulation of vitamin K levels. Such a control would be expected as vitamin K, which is required to prevent hemorrhage, is present in trace quantities, and is not appreciably stored (14). At present, it is not known whether such control is limited to absorption or also applies to transport and metabolism of vitamin K. Despite the biologic importance of the fat-soluble vitamins, their mechanisms of absorption and transport have received little study (18). It is not known whether there are specific proteins that mediate their absorption or whether the specificity of absorption resides solely in the micellar vehicle in which they are presented to the intestine (19). However, the available data indicate that absorption of /3-carotene and vitamin K is regulated differently in humans. In the rat (20), intestinal absorption of vitamin K is via an energy-mediated, saturable process occurring primarily in the proximal bowel. Absorption is not enhanced by long-chain saturated fatty acids and is not inhibited by vitamin K, analogues (menaquinones). In contrast, /3-carotene is absorbed via passive diffusion in a nonsaturable process, and no appreciable difference in absorption between the proximal and distal segments of bowel was measured. Long-chain polyunsaturated fatty acids inhibit /3-carotene absorption (21). Taken in context, the present data suggest that separate and specific absorption mechanisms exist for /3-carotene and vitamin K in humans. Thus, we conclude that /3-carotene may be safely administered even in large doses over extended periods of time without causing secondary vitamin K deficiency. Acknowledgments and Notes The authors thank Anne Lima and Jeanne Burr for expert technical assistance and Paula Leece for assistance in preparation of the manuscript. The work was supported in part by Contract NO1-HD2992 and Grant No. CA-27502 from the National Institutes of Health (Bethesda, MD). Address reprint requests to Dr. L.M. Canfield, Dept. of Biochemistry, University of Arizona, Tucson, AZ 85721. Submitted 11 August 1989; accepted in final form 20 November 1989.

References 1. Peto, R, Doll, R, Buckley, JD, and Sporn, MB: "Can Dietary Beta-Carotene Materially Reduce Human Cancer Rates?" Nature 290, 201-208, 1981. 2. Greenwald, P, Sondik, E, and Lynch, BS: "Diet and Chemoprevention in NCI's Research Strategy to Achieve National Cancer Control Objectives." Annu Rev Publ Health 1, 267-291, 1986. 3. Kuksis, A: "Absorption of Fat-Soluble Vitamins." In Fat Absorption, A Kuksis (ed). Boca Raton, FL: CRC, 1986, vol II. 4. Doisy, EA, Jr, and Matschiner, JT: "Biochemistry of Vitamin K." In Fat-Soluble Vitamins, RA Morton (ed). Oxford, UK: Pergamon, 1970, p 293.

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5. Napoli, JL, McCormick, AM, O'Meara, B, and Dratz, EA: "Vitamin A Metabolism: Alpha Tocopherol Modulates Tissue Retinol Levels In Vivo and Retinyl Palmitate Hydrolysis In Vitro." Arch Biochem Biophys 230, 194-202, 1984. 6. Olson, RE, and Jones, JP: "Inhibition of Vitamin K Action by Dietary Vitamin E." Fed Proc 41, 344, 1982. 7. Canfield, LM, Davy, LA, and Thomas, GL: "Anti-Oxidant/Pro-Oxidant Reactions of Vitamin K." Biochem Biophys Res Commun 128, 211-219, 19!;5. 8. Bettger, WJ, and Olson, RE: "Effect of α-Tocopherol and α-Tocopherolquinone on Vitamin K-Dependent Carboxylation in the Rat." Fed Proc 41, 442, 1982. 9. Corrigan, JJ, Jr, and Earnest, DL: "Factor II Antigen in Liver Disease and Warfarin-Induced Vitamin K Deficiency: Correlation With Coagulant Activity Using Echis Venom." Am J Hematol 8, 249-255, 1980. 10. Owren, PA, and Aas, K: "The Control of Dicumerol Therapy and the Quantitative Determination of Prothrombin and Proconvertin." Scand J Clin Lab Invest 3, 201-208, 1951. 11. Canfield, LM, and Hopkinson, JM: "State of the Art; Vitamin K in Human Milk." J Pediatr Gastroenterol Nutr 8, 430-441, 1989. 12. Canfield, LM, and Holzman, RB: "Reaction of Vitamin K and Dithiothreitol on Reversed-Phase C18 High-Performance Liquid Chromatographic Columns." J Chromatogr 299, 225-231, 1984. 13. Ueno, T, and Suttie, JW: "High Pressure Liquid Chromatographic Reductive Electrochemical Detection Analysis of Serum trans-Phylloquinone." Anal Biochem 133, 62-67, 1983. 14. Suttie, JW: "Vitamin K." In Handbook of Vitamins, Nutritional, Biochemical and Clinical Aspects, LJ Machlin (ed). New York: Dekker, 1984, pp 147-148. 15. Suttie, JW, and Jackson, CM: "Prothrombin Structure, Activation and Biosynthesis." Physiol Rev 57, 1-70, 1977. 16. Olson, JA: "Recommended Dietary Intakes (RDI) of Vitamin K in Humans." Am J Clin Nutr 45, 687-692, 1987. 17. Plezia, PM, Alberts, DS, Sayers, S, Peng, YM, Xu, MJ, and Ritenbaugh, C: "Evaluation of Plasma and Skin Concentrations of β-Carotene, Retinol and Retinyl Palmitate in Normal Subjects Receiving Daily Oral Doses of β-Carotene (abstr)." 2nd Int Meeting Assoc Vitamins Nutr Oncol, Charleston, SC, June 25-29, 1989. 18. Hollander, D: "Intestinal Absorption of Vitamins A, E, D and K." J Lab Clin Med 97, 449-462, 1981. 19. Ikeda, I, Tanaka, K, Sugano, M, Vahouny, GV, and Gallo, LL: "Inhibition of Cholesterol Absorption in Rats by Plant Sterols." J Lipid Res 29, 1573-1582, 1988. 20. Hollander, D: "Vitamin K1 Intestinal Absorption In Vivo: Influence of Luminal Contents on Transport." Am J Physiol 232, E69-74, 1977. 21. Hollander, D, and Ruble, PE: "β-Carotene Intestinal Absorption: Bile, Fatty Acid, pH and Flow Rate Effects on Transport." Am J Physiol 234, E686-691, 1978.

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Effects of chronic beta-carotene supplementation on vitamin K status in adults.

Plasma vitamin K concentrations and prothrombin coagulation activity were determined in 26 normal adults who had received daily beta-carotene suppleme...
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