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Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111
3To whom correspondence should be addressed. E-mail: SBooth{at}HNRC.Tufts.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • vitamin K • absorption • phylloquinone • triglycerides • age
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). It has been estimated that green vegetables contribute 40–50% of total phylloquinone intake in the American diet, followed by certain phylloquinone-rich plant oils, such as soybean, canola and cottonseed (1
). Dietary phylloquinone in vegetables may be absorbed less than that in oils because it is a fat-soluble vitamin and is tightly associated with chloroplasts (2
). When defined as the area under the plasma phylloquinone curve, absorption of phylloquinone was greater from an oil-based supplement compared with spinach (3
,4
). Conversely, plasma phylloquinone concentrations after a 5-d broccoli diet were not different from those after a 5-d phylloquinone-fortified oil diet (5
). The equivocal results among studies may reflect the different types of vegetables used and/or differences in the approaches used to assess absorption.
To address differences in reported strategies used to study the absorption of phylloquinone, a 24-h absorption study was conducted as part of the aforementioned larger metabolic study with a 5-d phylloquinone supplementation period (5
). In this study, plasma phylloquinone concentrations were compared over a 24-h period in response to consuming phylloquinone from broccoli or a phylloquinone-fortified oil. Absorption was defined as the area under the plasma phylloquinone curve. To determine whether age-related differences exist, the absorption of phylloquinone was compared in younger and older adults, unadjusted and adjusted for plasma triglyceride concentrations.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Men and women (n = 36) were stratified into four groups of nine, according to age (20–40 y and 60–80 y) and sex (5
). The mean ± SEM age (y) and body mass index (kg/m2) were 31.2 ± 1.9 and 26.0 ± 0.8 for younger men, 30.7 ± 2.1 and 23.5 ± 1.1 for younger women, 70.0 ± 1.8 and 26.0 ± 1.4 for older men, and 70.9 ± 1.6 and 26.8 ± 1.5 for older women, respectively. All subjects were in good health, and fulfilled the following eligibility criteria: 1) no history of cardiovascular, hepatic, gastrointestinal or renal disease; 2) no alcoholism; 3) no antibiotic or supplemental vitamin and/or mineral use within 4 wk before the start of the study; and 4) no smoking. None of the women were pregnant, lactating or using exogenous hormones. The Human Investigation Review Committee of Tufts University and the New England Medical Center approved the study protocol, and written informed consent was obtained from each subject.
Study design.
Each subject resided in the Metabolic Research Unit at the Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University for three residency periods of 15 d each. There was a free-living period of 
6 wk between each residency period when each subject consumed a self-selected diet. As described in greater detail elsewhere, a weight-maintaining baseline diet (using 3-d rotating menus) was provided throughout the three residency periods (6
). For one residency period of 15 consecutive days, subjects consumed the baseline diet only. In the baseline diet, phylloquinone was present in foods consumed at lunch and dinner (27 and 68% of the total daily intake of phylloquinone, respectively). On d 6 to 10 of another residency period (broccoli diet), each subject consumed a 102.4-g serving of microwaved broccoli at both lunch and dinner, in addition to the baseline diet. On d 6 to 10 of a third residency period (oil diet), the corn oil in the baseline diet was fortified with exogenous phylloquinone at both lunch and dinner. As described in detail elsewhere (5
), the daily phylloquinone intakes from the baseline diet, the broccoli diet and the oil diet were (mean ± SD) 100 ± 12, 377 ± 46 and 417 ± 45 µg/d, respectively. The order of the residency periods was randomly assigned for each subject.
On d 6 of each residency period, an indwelling catheter was inserted before 0800 h, and hourly blood samples were drawn from 0800 (0 h) to 2400 h on d 7 (16 h). The catheter was removed, and two additional specimens were drawn at 0400 and 0800 h on d 7 (20 and 24 h, respectively). Meals on d 6 for each residency period were served subsequent to the blood draws at 0800 (0 h), 1200 (4 h) and 1700 h (9 h), and were standardized for all subjects. No other food or beverages were allowed during the study with the exception of deionized water. Ambulating was permitted when the catheter was inserted, although some subjects chose bed rest. Four subjects had insufficient or missing blood samples (total of 17 samples) for laboratory analysis because of inadequate venous access.
Methods.
Plasma phylloquinone was analyzed by reverse-phase HPLC using postcolumn reduction of phylloquinone to its hydroquinone, followed by fluorometric detection (7
). Plasma triglycerides were analyzed on a COBAS Mira (Roche Instruments, Belleville, NJ).
Statistical methods.
Results are expressed as means ± SEM, unless otherwise specified. Differences were considered significant if the observed, two-sided significance level (P-value) was < 0.05. Because the within-group variance was heterogeneous, a logarithmic transformation was applied to the plasma phylloquinone and triglyceride data before all analyses.
The plasma phylloquinone responses (both adjusted and unadjusted for triglyceride concentrations) were expressed as the area under the curve (AUC).4 The AUC was calculated using the trapezoidal method, after subtraction of the 0800-h fasting plasma sample on d 6 (0 h). Diet (oil/broccoli/baseline) was analyzed as a within-subject factor, and age and sex were analyzed as between-subject factors. Data were analyzed using the MIXED procedure in SAS, version 6.12 (SAS Institute, Cary, NC), which adjusts for missing data points in a repeated-measures analysis, without excluding a subject entirely. Tukey’s honestly significant difference test was used to compare the three diets within each age group. For comparison of fasting plasma phylloquinone concentrations at 0 h with those at 24 h for each diet, full factorial models were fitted that included fixed effects of age, sex, diet and time.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Older adults had significantly higher mean unadjusted plasma phylloquinone concentrations at 0 h and 24 h compared with younger adults after consumption of all three diets (P < 0.001; Table 1
). This age difference persisted when the unadjusted plasma phylloquinone concentrations were expressed as AUC (P < 0.001).
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. After adjustment for triglycerides, the plasma phylloquinone AUC did not differ between the two age groups (P = 0.13) (Fig. 1A and B
). Adjustment of plasma phylloquinone for triglycerides reduced the CV of the AUC from 43 to 27%.
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The mean AUC for plasma phylloquinone concentrations (both unadjusted and adjusted for triglycerides) were greater after the oil diet than after the broccoli diet in both younger and older adults (P < 0.001) (Table 1)
. The AUC for plasma phylloquinone concentrations (both unadjusted and adjusted for triglycerides) in response to the baseline diet was less than for either the oil or broccoli diet (P < 0.001 for both) (Fig. 1
A and B, Table 1
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,4
). The implications of a greater area under the plasma phylloquinone curve for the phylloquinone-fortified oil diet compared with the broccoli diet observed in this study is not well understood when considered in the context of the findings of the larger metabolic study (5
). The degree of carboxylation of the vitamin K–dependent proteins, such as osteocalcin, is considered to be a sensitive indicator of vitamin K nutritional status (8
). When changes in the percentage of undercarboxylated osteocalcin (%ucOC) were measured in the larger metabolic study, there were no significant differences between the effects of the phylloquinone-fortified oil and the broccoli diet. Differences in absorption, as indicated by the 24-h area under the plasma phylloquinone curve, may not be critical if there are no concomitant differences in the %ucOC and plasma phylloquinone after five consecutive days of supplementation. Conversely, the %ucOC may not be as sensitive an indicator of vitamin K status as previously considered (8
).
The amount of phylloquinone consumed from the broccoli or the phylloquinone-fortified oil in addition to the baseline diet, was in excess of 370 µg/d, which is four times the current adequate intake (AI) for vitamin K (90–120 µg/d) (9
). The supplemental phylloquinone was introduced after each subject had consumed a diet approximating the AI for vitamin K for five consecutive days. Therefore, study subjects were not vitamin K–deficient preceding supplementation. It is possible that once a certain dietary phylloquinone intake is attained, additional phylloquinone does not increase the plasma concentration or reduce the %ucOC, which would have limited our ability to detect differences in biological activity from broccoli compared with phylloquinone-fortified oil (5
). Furthermore, broccoli was used as the vegetable source of phylloquinone. Absorption of phylloquinone from broccoli has been reported to be greater than from spinach (3
). Others have reported differences in relative bioavailability of phylloquinone from a supplement when the fat content of the diet was manipulated (4
), whereas a mean intake of 25.6% energy from dietary fat was maintained throughout this study. Collectively, these observations highlight the importance of consistency when interpreting studies using different strategies for measuring absorption and bioavailability of a nutrient, particularly because potential differences in bioavailability have important implications when making dietary recommendations.
One important observation in this study was the absence of differences between the younger and older adults when plasma phylloquinone concentrations were adjusted for triglycerides in the calculation of the AUC. There is a strong correlation between plasma phylloquinone and triglyceride concentrations because phylloquinone is transported in triglyceride-rich lipoproteins (10
). However, there is currently a lack of consensus regarding the use of total lipids to adjust plasma phylloquinone concentrations (11
). Older adults have significantly higher circulating phylloquinone concentrations than younger adults (1
), an observation supported by data in this study and consistent with previous reports that older adults may be more resistant to dietary vitamin K deficiency than younger adults (12
). The current study does not support previous data suggesting that older subjects have a lower plasma phylloquinone to triglyceride ratio than younger adults (13
). Use of the phylloquinone to triglyceride ratio in the current study minimized age-related differences in the response to two different diets, consistent with the results of the larger metabolic study in which there were no significant age effects in the responses of %ucOC and plasma phylloquinone to five consecutive days of supplementation (5
). However, in both studies, older adults had consistently higher absolute circulating phylloquinone concentrations than the younger adults at 0 and 24 h, even after adjustment for triglycerides. Furthermore, the older adults had lower %ucOC compared with the younger adults in the larger metabolic study, which is consistent with a more favorable vitamin K status.
In summary, when absorption is defined as a 24-h area under the plasma phylloquinone curve, phylloquinone is absorbed better from a phylloquinone-fortified oil than from a green vegetable. These conclusions are inconsistent with those of the larger metabolic study, which used a 5-d change in vitamin K biochemical markers to define bioavailability. The disparities among studies emphasize the need for standardized approaches to study absorption and bioavailability. The use of triglycerides to adjust plasma phylloquinone concentrations removed age-related differences in the AUC, but not in fasting plasma phylloquinone concentrations. The implications of these differences remain unclear.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Presented in part at Experimental Biology 1999, April 1999, Washington, DC [Booth, S. L., Conlin, R. S., Kaszynski, B. B. & Davidson, K. W. (1999) Comparison of younger and older adults in absorption of phylloquinone (vitamin K1) from vegetable and oil sources. FASEB J. 13: A239 (abs.)].
4 Abbreviations used: AI, adequate intake; AUC, area under the curve; %ucOC, percentage of undercarboxylated osteocalcin.
Manuscript received 26 April 2002. Initial review completed 5 June 2002. Revision accepted 14 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Booth, S. L. & Suttie, J. W. (1998) Dietary intake and adequacy of vitamin K. J. Nutr. 128:785-788.
2. Shearer, M. J. (1992) Vitamin K metabolism and nutriture. Blood Rev 6:92-104.[Medline]
3. Garber, A. K., Binkley, N. C., Krueger, D. C. & Suttie, J. W. (1999) Comparison of phylloquinone bioavailability from food sources or a supplement in human subjects. J. Nutr. 129:1201-1203.
4. Gijsbers, B. L., Jie, K. S. & Vermeer, C. (1996) Effect of food composition on vitamin K absorption in human volunteers. Br. J. Nutr. 76:223-229.[Medline]
5. Booth, S. L., O’Brien-Morse, M. E., Dallal, G. E., Davidson, K. W. & Gundberg, C. M. (1999) Response of vitamin K status to different intakes and sources of phylloquinone-rich foods: comparison of younger and older adults. Am. J. Clin. Nutr. 70:368-377.
6. Booth, S. L., Charnley, J. M., Sadowski, J. A., Saltzman, E., Bovill, E. G. & Cushman, M. (1997) Dietary vitamin K1 and stability of oral anticoagulation: proposal of a diet with constant vitamin K1 content. Thromb. Haemost. 77:504-509.[Medline]
7. Davidson, K. W. & Sadowski, J. A. (1997) Determination of vitamin K compounds in plasma or serum by high-performance liquid chromatography using postcolumn chemical reduction and fluorometric detection. Methods Enzymol 282:408-421.[Medline]
8. Gundberg, C. M., Nieman, S. D., Abrams, S. & Rosen, H. (1998) Vitamin K status and bone health: an analysis of methods for determination of undercarboxylated osteocalcin. J. Clin. Endocrinol. Metab. 83:3258-3266.
9. Institute of Medicine (2001) Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc 2001 National Academy Press Washington, DC. .
10. Cham, B. E., Smith, J. L. & Colquhoun, D. M. (1999) Interdependence of serum concentrations of vitamin K1, vitamin E, lipids, apolipoprotein A1, and apolipoprotein B: importance in assessing vitamin status. Clin. Chim. Acta 287:45-57.[Medline]
11. Traber, M. G. & Jialal, I. (2000) Measurement of lipid-soluble vitamins—further adjustment needed?. Lancet 355:2013-2014.[Medline]
12. Ferland, G., Sadowski, J. A. & O’Brien, M. E. (1993) Dietary induced subclinical vitamin K deficiency in normal human subjects. J. Clin. Invest. 91:1761-1768.
13. Sadowski, J. A., Hood, S. J., Dallal, G. E. & Garry, P. J. (1989) Phylloquinone in plasma from elderly and young adults: factors influencing its concentration. Am. J. Clin. Nutr. 50:100-108.
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