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Vitamin K status in healthy volunteers Cite this: Food Funct., 2014, 5, 229
E. Theuwissen,*a E. J. Magdeleyns,a L. A. J. L. M. Braam,a K. J. Teunissen,a M. H. Knapen,a I. A. G. Binnekamp,b M. J. H. van Summerenb and C. Vermeera Vitamin K's recommended dietary allowance (RDA) is based on the hepatic requirement for clotting factor synthesis, but substantial concentrations of undercarboxylated extra-hepatic Gla-proteins are found in the circulation of non-supplemented individuals. This suggests that vitamin K intake above the RDA is required for an optimal extra-hepatic vitamin K status. Circulating uncarboxylated osteocalcin (ucOC) and desphospho-uncarboxylated matrix Gla-protein (dp-ucMGP) are considered markers of the vitamin K status in bone and the vasculature, respectively. We measured these markers in 896 samples of healthy volunteers and defined target groups for vitamin K supplementation based on increased levels indicative of tissue-specific vitamin K deficiency. We studied the response to vitamin K supplements at different states of vitamin K deficiency by measuring the circulating dp-ucMGP level in samples from two shortterm trials on menaquinone-7 (MK-7, vitamin K2) supplementation in 42 children and 68 adults. Children had high ucOC levels (3.4–96.9 ng ml1); other age groups had values in the range of 1.5–5.0 ng ml1.
Received 2nd October 2013 Accepted 18th November 2013
From the age of 40 years, dp-ucMGP levels gradually increased. Children and adults with more pronounced vitamin K deficiency gave the highest responses to MK-7 supplementation. Children and
DOI: 10.1039/c3fo60464k www.rsc.org/foodfunction
adults above 40 years showed the largest tissue-specific vitamin deficiency and accordingly may benefit from MK-7 supplementation to improve their extra-hepatic vitamin K status.
Introduction Vitamin K functions as a cofactor for g-glutamate carboxylase catalysing the posttranslational carboxylation in Gla-proteins of certain glutamate (Glu) into g-carboxyglutamate (Gla) residues.1,2 Beyond their central role in blood coagulation, Gla-proteins are involved in a number of other metabolic processes, including regulation of so tissue calcication (matrix Gla-protein, MGP) and bone mineralization (osteocalcin, OC).3 The recommended daily allowance (RDA) of vitamin K (75 mg; Commission Directive CD2008/100/EC; Commission of the European Communities, 2008) is based on the hepatic requirement for clotting factor synthesis. Indeed, at intakes in the order of the RDA all clotting factors are fully carboxylated. At the same time, however, substantial concentrations of undercarboxylated extra-hepatic Gla-proteins are found in the circulation of non-supplemented individuals.4–7 This suggests that vitamin K intake greater than the RDA is required for the optimal extra-hepatic vitamin K status. Vitamin K intake and circulating phylloquinone levels are not dependable as measures of the status because of biases inherent to dietary intake questionnaires and to uctuations in intake. Instead, the undercarboxylated fractions of vitamin K-dependent proteins are considered as indicators of the vitamin K status of certain tissues. While uncarboxylated prothrombin a
VitaK, Maastricht University, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands. E-mail: [email protected]
b
Department of Pediatrics, University Medical Centre Utrecht, The Netherlands
This journal is © The Royal Society of Chemistry 2014
reects the hepatic vitamin K status, circulating uncarboxylated fractions of OC (uncarboxylated OC, ucOC) and MGP (desphospho-uncarboxylated MGP, dp-ucMGP) are recognized markers for the extra-hepatic vitamin K status and have been associated with osteoporotic fractures and arterial calcication, respectively.8–10 In line, long-term vitamin K deciency has been indicated as an independent, but modiable risk factor for the development of age-related diseases, including osteoporosis and cardiovascular disease (CVD).11 High circulating levels of ucOC are found in children and menopausal women, which are characterized by increased metabolic bone activity.5,12–18 Although elderly were previously described to have higher plasma dp-ucMGP levels,7 limited information exists on the extra-hepatic vitamin K status across age groups in terms of MGP carboxylation. In fact, circulating dp-ucMGP values in children are lacking. Several randomized controlled trials (RCTs) have shown that the extra-hepatic carboxylation of OC and MGP can be improved by increased vitamin K intake.4,6,19–21 Maximizing carboxylation of these markers by optimal vitamin K intake may reduce the risk of osteoporosis and CVD. Vitamin K occurs naturally in food as phylloquinone (vitamin K1) and menaquinones (MK-n, vitamin K2). Phylloquinone is the primary dietary form mainly found in green leafy vegetables.1 The short-chain menaquinone-4 (MK-4) is endogenously produced from menadione (synthetic form present in the animal feed) or converted from phylloquinone, and occurs in animal products, such as meat and eggs. The long-chain menaquinones (MK-7 to MK-10) generally are of microbial
Food Funct., 2014, 5, 229–234 | 229
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origin and are found in cheese and the Japanese fermented soybeans known as natto. Supplemental vitamin K comes in only three forms, i.e. phylloquinone, MK-4, and MK-7. Synthetic phylloquinone and MK-4 are given therapeutically to treat haemorrhagic disease of the new-born and osteoporosis, respectively. Phylloquinone is also used as an antidote to vitamin K antagonists (VKA) and in nutritional supplements, including multivitamins. MK-7 is, on the other hand, a natural fermentation product found in dietary supplements used almost invariably for maintaining bone health. The described forms of vitamin K have different chemical structures and pharmacokinetics probably accounting for differences in bioavailability. Two RCTs have been published showing higher bioavailability of MK-7 compared to phylloquinone and MK4.22,23 Sato et al. reported poor bioavailability of MK-4 at a nutritional level dose, whereas MK-7 was well absorbed in healthy women.22 Likewise, MK-7 was shown to be better absorbed and to be more potent in increasing carboxylation of both hepatic and extra-hepatic Gla-proteins than phylloquinone.23 The favourable effects of MK-7 supplementation on extra-hepatic carboxylation were conrmed in two independent short-term RCTs in healthy adults.4,6 Signicant associations were seen between plasma MK-7 concentrations and MK-7induced changes in circulating Gla-proteins; the higher the end MK-7 values, the larger the decrease in circulating ucOC and dpucMGP levels.4 Next to improved carboxylation, the long-term use of MK-7 supplements was recently shown to benecially affect bone strength24 and arterial stiffness (Braam et al., 2013, unpublished results). The rst aim of this paper was to measure circulating ucOC and dp-ucMGP levels across age groups in order to establish the tissue-specic vitamin K status during human life. Accordingly, target groups for vitamin K supplementation were appointed based on increased levels of these markers indicative of tissuespecic vitamin K deciency. Therefore, we measured circulating ucOC and dp-ucMGP in 896 samples of healthy volunteers. The second aim was to study the response to vitamin K supplements at different states of vitamin K deciency. Therefore, we examined the association between baseline dp-ucMGP levels and the vitamin K-induced effects on dp-ucMGP levels in healthy children and adults who participated in two independent randomized trials with MK-7 supplements.
Paper
Fig. 1
Circulating ucOC levels in healthy volunteers.
seen at 30 years but levels increased again at 40 years. Similar to men, levels were stable between 40 years and 70 years. Statistics showed that only the children had signicantly higher values as compared to the other study groups (including young adults). Accordingly, children were appointed as a target group for supplementation to improve their vitamin K status of bone. In contrast to circulating ucOC, low dp-ucMGP levels were found in children (mean 200 pmol l1, Fig. 2). A trend towards higher levels with increasing age was seen, with a statistical signicant increase from the age of 40 years (as compared to the young adults). A second signicant increase (as compared to 40 years) was observed aer the age of 70 years. Hence, adults >40 years were appointed as a target group for supplementation to improve their vitamin K status of the vasculature.
Response to vitamin K supplements at different states of vitamin K deciency While circulating dp-ucMGP levels increased (non-signicantly, P ¼ 0.053) in the control group of children (placebo capsules), a signicant decrease was found in the children that received the MK-7 supplements (P ¼ 0.013, Table 1, Fig. 3). The change in the placebo group differed signicantly from the change in the MK-
Results Vitamin K status during life time In children, extremely high serum ucOC levels were found (35 ng ml1, Fig. 1). These values were >6 times higher as compared to the other age groups (range 1.5–5.5 ng ml1). In the total group, a trend towards lower ucOC levels with increasing age was seen from 20 years to 80 years. In the male adults, the highest levels were seen in the young adults with a rst decline in serum ucOC at 30 years (Fig. 1, upper right box, black bars). Between 40 years and 70 years, stable levels were seen with a second decrease in circulating ucOC levels at 70 years. In the female adults, the highest levels were also seen in the young adults (Fig. 1, upper right box, grey bars). Further, a decline was
230 | Food Funct., 2014, 5, 229–234
Fig. 2
Circulating dp-ucMGP levels in healthy volunteers.
This journal is © The Royal Society of Chemistry 2014
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7 group (P ¼ 0.002). A large variability was seen in the baseline values and MK-7 responses in our population of healthy children. Both in children and adults, MK-7-induced changes in dpucMGP levels were dependent on the baseline dp-ucMGP values. Higher baseline values gave larger decreases in circulating dp-ucMGP levels (Fig. 4).
Discussion In this paper, we investigated the vitamin K status during life time by measuring circulating levels of two extra-hepatic Glaproteins, namely circulating ucOC and dp-ucMGP, across several age groups. High ucOC levels were seen in the group of children, indicating an unusually poor vitamin K status of growing bone, which may increase osteoporosis risk in later life. Previous studies consistently showed elevated ucOC values in healthy children,12,14–16,25 and the higher metabolic activity of growing bone probably accounts for the observed vitamin K deciency in children. Thus, maximizing the accretion of bone during growth by optimal nutrition (including vitamin K intake) may reduce the risk of developing osteoporosis.26
Table 1 Circulating dp-ucMGP values in healthy children (before and after treatment)a
Placebo (n 21)
dp-ucMGP (pmol l1, week 0) dp-ucMGP (pmol l1, week 8) D dp-ucMGP (pmol l1) D dp-ucMGP (%)
MK-7 (n 21)
Median
Range (min; max)
156a
(55; 370)
172a
(32; 546)
178
(97; 340)
138
(45; 401)
24a
(111; 97)
21b
(145; 44)
18a
(30; 76)
18b
(46; 45)
Median
Range (min; max)
a
Between-group differences were assessed using the Mann–Whitney U test. Median values within a row with unlike superscript letters were signicantly different (P < 0.05).
Changes in circulating dp-ucMGP levels after treatment in 42 healthy children.
Fig. 3
This journal is © The Royal Society of Chemistry 2014
MK-7-induced changes in circulating dp-ucMGP levels in relation to baseline values in children and young adults.
Fig. 4
Unfortunately, the importance of vitamin K during growth has only been studied to a limited extent. Kalkwarf et al. found that a better vitamin K status (high plasma phylloquinone and low %ucOC) was associated with lower bone turnover (BAP, NTX) in healthy girls (3–16 years).27 The vitamin K status was however not consistently associated with the bone mineral content (BMC). O'Connor et al. showed in healthy Danish girls (11–12 years) that a better vitamin K status (low %ucOC) did relate to increased BMC, but not to bone turnover [OC, pyridinoline (PYD) and deoxypyridinoline DPD].28 van Summeren et al. conrmed the association between vitamin K status (the ratio of ucOC to carboxylated OC) and (total-body) BMC in healthy Dutch children (8–14 years).14 Consistently, reduced bone density was seen in vitamin K-decient (high ucOC) children as caused by (long-term) VKA therapy.29,30 Children using VKA presented higher levels of bone resorption markers [DPD/ creatinine, cross-linked telopeptide of type I collagen (ICTP)] and lower levels of bone formation markers [carboxylated OC, type I procollagen N-terminal propeptide (PINP)].30 We earlier reported that MK-7 (45 mg per day, i.e. RDA for children) supplementation improved the vitamin K status (28% reduction in ucOC levels) in healthy children, but data on bone mass are lacking.13 Altogether, these ndings suggest potentially adverse effects of low vitamin K status on growing bone, but the question remains whether higher vitamin K intake improves bone health in healthy children. In adults, on the other hand, the association between vitamin K status and bone health has been extensively studied.31,32 Observational studies have indicated that vitamin K deciency (increased circulating ucOC) leads to age-related bone loss.33 Data from RCTs in adults, however, gave contradictory results for effects on bone mass, although the vitamin K status improved (decreased circulating ucOC) consistently in all studies.31,32 It could be that improving vitamin K status may have more pronounced effects on bone quality, i.e. microarchitecture of bone, than on bone mass. Moreover, carboxylation-independent mechanisms have been suggested for vitamin K's role in bone health.33 Largely based on the observational ndings, the European Food Safety Authorities (EFSA) approved the claim: “a cause and effect relationship has been established between vitamin K and maintenance of normal bone”.34 Our recent ndings that long-term MK-7 supplementation decreases Food Funct., 2014, 5, 229–234 | 231
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the age-related decline in bone strength in healthy postmenopausal women are consistent with the EFSA's opinion.24 Women in menopause are, like children, characterized by high bone metabolism with increased levels of ucOC.5,17,18,35 Women at their 20s and 50s were determined by Kim et al. as having elevated serum ucOC levels suggesting that vitamin K supplements may improve their bone metabolism.18 Aer a decline in circulating ucOC levels at their thirties, we found higher ucOC levels from 40 years in our population of healthy women. Unfortunately, levels were not signicantly different from those of other adult age groups. It should be noted that we excluded osteoporotic women, which may explain the relatively small augmentation in circulating ucOC levels from 40 years and the lack of an extra elevation with menopause. In healthy men, a trend towards lower levels with increasing age was seen, including a plateau phase between 40 years and 70 years. Previous studies also showed a decreasing trend in circulating ucOC levels with increasing age in men.5,17 This is the rst study in which circulating dp-ucMGP levels were measured in children. In contrast to high ucOC levels, children had relatively low levels of dp-ucMGP, but in all children levels were well above the detection limit. MGP is mainly found in cartilage and the vascular wall, where it is produced by chondrocytes and vascular smooth muscle cells, respectively. MGP is an important inhibitor of vascular calcication and its uncarboxylated form has been associated with arterial calcication in healthy adults and in patients at increased cardiovascular risk.7 The consequences of dysfunctional or lack of MGP are shown by two human syndromes. The rare autosomal recessive Keutel syndrome is caused by mutations in the MGP gene leading to excessive cartilage calcication.36 Administration of VKA to women during pregnancy can cause embryopathy and skeletal defects, known as foetal warfarin syndrome.37 MGP is therefore thought to play a role in promoting growth and skeletal modelling in children. As indicated, no studies on dpucMGP values in children have been published thus far. We found a broad range of plasma dp-ucMGP values (32–546 pmol l1) in our population of healthy children. In order to explain the discrepancy in ucOC and dp-ucMGP levels, we could hypothesize that in children the vasculature is less vitamin Kdecient than bone. We have shown in this study that shortterm MK-7 supplementation signicantly decreased circulating levels of dp-ucMGP in the group of children. Despite the different degrees of vitamin K deciency in bone (high levels of ucOC) and the vasculature (low levels of dp-ucMGP) in children, the effective level of MK-7 administration was similar for ucOC13 and dp-ucMGP, i.e. net changes (as compared to the placebo group) of 38% and 36%, respectively. We further found larger responses to supplementation in children with higher baseline dp-ucMGP values. This was conrmed in a cohort of healthy adults, indicating that more pronounced vitamin K deciency (higher baseline dp-ucMGP values) gives larger responses to MK-7 supplements. We found that in healthy adults circulating dp-ucMGP levels gradually increased with age; a signicant rise was seen above 40 years of age. In line, Cranenburg et al. showed a trend of increasing values with age with signicant higher values for
232 | Food Funct., 2014, 5, 229–234
Paper
elderly (66–80 years) as compared to young adults (25–40 years).7 Four different randomized trials are published, all showing improvements in circulating dp-ucMGP levels aer phylloquinone20 or menaquinone4,6,38 (Braam et al., 2013, unpublished results) supplementation. Effective daily doses ranged from 90 mg to 500 mg with effective levels of 30% to 80%, respectively. The question remains whether improved carboxylation of MGP benecially affects cardiovascular health. Only three published RCTs investigated the health effects of vitamin K supplementation on vascular parameters (CAC and arterial stiffness)20,39 (Braam et al., 2013, unpublished results). All showed positive results, but individual changes in circulating dp-ucMGP levels could not be linked to changes in vascular measures. These ndings are in contrast with the results of case-control studies that have reported higher plasma dpucMGP levels among patients at increased cardiovascular risk, including patients with aortic valve disease, aortic stenosis, and chronic kidney disease.10 So, the association between MGP and vascular measures may be stronger in less healthy populations. Furthermore, it is plausible that vitamin K inuences cardiovascular health through carboxylation-independent mechanisms. In vitro studies suggested that vitamin K may decrease gene expression of proinammatory cytokines.40
Experimental Vitamin K status during life time Study population. Blood samples from 896 apparently healthy children (age range, 6–19 years) and adults (age range, 20–80 years) were analysed retrospectively. Only healthy volunteers were selected as determined by interviews and questionnaires. Exclusion criteria were: abnormal weight or height (children: weight or height outside p3–p97, adults: BMI > 30 kg m2), use of vitamin K supplements, use of oral anticoagulants (VKA), use of drugs that inuence bone metabolism, corticoid treatment, pregnancy (when applicable), gastrointestinal diseases, osteoporosis, chronic diseases, anaemia, and/or recent blood donation (
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Vitamin K status in healthy volunteers Cite this: Food Funct., 2014, 5, 229
E. Theuwissen,*a E. J. Magdeleyns,a L. A. J. L. M. Braam,a K. J. Teunissen,a M. H. Knapen,a I. A. G. Binnekamp,b M. J. H. van Summerenb and C. Vermeera Vitamin K's recommended dietary allowance (RDA) is based on the hepatic requirement for clotting factor synthesis, but substantial concentrations of undercarboxylated extra-hepatic Gla-proteins are found in the circulation of non-supplemented individuals. This suggests that vitamin K intake above the RDA is required for an optimal extra-hepatic vitamin K status. Circulating uncarboxylated osteocalcin (ucOC) and desphospho-uncarboxylated matrix Gla-protein (dp-ucMGP) are considered markers of the vitamin K status in bone and the vasculature, respectively. We measured these markers in 896 samples of healthy volunteers and defined target groups for vitamin K supplementation based on increased levels indicative of tissue-specific vitamin K deficiency. We studied the response to vitamin K supplements at different states of vitamin K deficiency by measuring the circulating dp-ucMGP level in samples from two shortterm trials on menaquinone-7 (MK-7, vitamin K2) supplementation in 42 children and 68 adults. Children had high ucOC levels (3.4–96.9 ng ml1); other age groups had values in the range of 1.5–5.0 ng ml1.
Received 2nd October 2013 Accepted 18th November 2013
From the age of 40 years, dp-ucMGP levels gradually increased. Children and adults with more pronounced vitamin K deficiency gave the highest responses to MK-7 supplementation. Children and
DOI: 10.1039/c3fo60464k www.rsc.org/foodfunction
adults above 40 years showed the largest tissue-specific vitamin deficiency and accordingly may benefit from MK-7 supplementation to improve their extra-hepatic vitamin K status.
Introduction Vitamin K functions as a cofactor for g-glutamate carboxylase catalysing the posttranslational carboxylation in Gla-proteins of certain glutamate (Glu) into g-carboxyglutamate (Gla) residues.1,2 Beyond their central role in blood coagulation, Gla-proteins are involved in a number of other metabolic processes, including regulation of so tissue calcication (matrix Gla-protein, MGP) and bone mineralization (osteocalcin, OC).3 The recommended daily allowance (RDA) of vitamin K (75 mg; Commission Directive CD2008/100/EC; Commission of the European Communities, 2008) is based on the hepatic requirement for clotting factor synthesis. Indeed, at intakes in the order of the RDA all clotting factors are fully carboxylated. At the same time, however, substantial concentrations of undercarboxylated extra-hepatic Gla-proteins are found in the circulation of non-supplemented individuals.4–7 This suggests that vitamin K intake greater than the RDA is required for the optimal extra-hepatic vitamin K status. Vitamin K intake and circulating phylloquinone levels are not dependable as measures of the status because of biases inherent to dietary intake questionnaires and to uctuations in intake. Instead, the undercarboxylated fractions of vitamin K-dependent proteins are considered as indicators of the vitamin K status of certain tissues. While uncarboxylated prothrombin a
VitaK, Maastricht University, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands. E-mail: [email protected]
b
Department of Pediatrics, University Medical Centre Utrecht, The Netherlands
This journal is © The Royal Society of Chemistry 2014
reects the hepatic vitamin K status, circulating uncarboxylated fractions of OC (uncarboxylated OC, ucOC) and MGP (desphospho-uncarboxylated MGP, dp-ucMGP) are recognized markers for the extra-hepatic vitamin K status and have been associated with osteoporotic fractures and arterial calcication, respectively.8–10 In line, long-term vitamin K deciency has been indicated as an independent, but modiable risk factor for the development of age-related diseases, including osteoporosis and cardiovascular disease (CVD).11 High circulating levels of ucOC are found in children and menopausal women, which are characterized by increased metabolic bone activity.5,12–18 Although elderly were previously described to have higher plasma dp-ucMGP levels,7 limited information exists on the extra-hepatic vitamin K status across age groups in terms of MGP carboxylation. In fact, circulating dp-ucMGP values in children are lacking. Several randomized controlled trials (RCTs) have shown that the extra-hepatic carboxylation of OC and MGP can be improved by increased vitamin K intake.4,6,19–21 Maximizing carboxylation of these markers by optimal vitamin K intake may reduce the risk of osteoporosis and CVD. Vitamin K occurs naturally in food as phylloquinone (vitamin K1) and menaquinones (MK-n, vitamin K2). Phylloquinone is the primary dietary form mainly found in green leafy vegetables.1 The short-chain menaquinone-4 (MK-4) is endogenously produced from menadione (synthetic form present in the animal feed) or converted from phylloquinone, and occurs in animal products, such as meat and eggs. The long-chain menaquinones (MK-7 to MK-10) generally are of microbial
Food Funct., 2014, 5, 229–234 | 229
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origin and are found in cheese and the Japanese fermented soybeans known as natto. Supplemental vitamin K comes in only three forms, i.e. phylloquinone, MK-4, and MK-7. Synthetic phylloquinone and MK-4 are given therapeutically to treat haemorrhagic disease of the new-born and osteoporosis, respectively. Phylloquinone is also used as an antidote to vitamin K antagonists (VKA) and in nutritional supplements, including multivitamins. MK-7 is, on the other hand, a natural fermentation product found in dietary supplements used almost invariably for maintaining bone health. The described forms of vitamin K have different chemical structures and pharmacokinetics probably accounting for differences in bioavailability. Two RCTs have been published showing higher bioavailability of MK-7 compared to phylloquinone and MK4.22,23 Sato et al. reported poor bioavailability of MK-4 at a nutritional level dose, whereas MK-7 was well absorbed in healthy women.22 Likewise, MK-7 was shown to be better absorbed and to be more potent in increasing carboxylation of both hepatic and extra-hepatic Gla-proteins than phylloquinone.23 The favourable effects of MK-7 supplementation on extra-hepatic carboxylation were conrmed in two independent short-term RCTs in healthy adults.4,6 Signicant associations were seen between plasma MK-7 concentrations and MK-7induced changes in circulating Gla-proteins; the higher the end MK-7 values, the larger the decrease in circulating ucOC and dpucMGP levels.4 Next to improved carboxylation, the long-term use of MK-7 supplements was recently shown to benecially affect bone strength24 and arterial stiffness (Braam et al., 2013, unpublished results). The rst aim of this paper was to measure circulating ucOC and dp-ucMGP levels across age groups in order to establish the tissue-specic vitamin K status during human life. Accordingly, target groups for vitamin K supplementation were appointed based on increased levels of these markers indicative of tissuespecic vitamin K deciency. Therefore, we measured circulating ucOC and dp-ucMGP in 896 samples of healthy volunteers. The second aim was to study the response to vitamin K supplements at different states of vitamin K deciency. Therefore, we examined the association between baseline dp-ucMGP levels and the vitamin K-induced effects on dp-ucMGP levels in healthy children and adults who participated in two independent randomized trials with MK-7 supplements.
Paper
Fig. 1
Circulating ucOC levels in healthy volunteers.
seen at 30 years but levels increased again at 40 years. Similar to men, levels were stable between 40 years and 70 years. Statistics showed that only the children had signicantly higher values as compared to the other study groups (including young adults). Accordingly, children were appointed as a target group for supplementation to improve their vitamin K status of bone. In contrast to circulating ucOC, low dp-ucMGP levels were found in children (mean 200 pmol l1, Fig. 2). A trend towards higher levels with increasing age was seen, with a statistical signicant increase from the age of 40 years (as compared to the young adults). A second signicant increase (as compared to 40 years) was observed aer the age of 70 years. Hence, adults >40 years were appointed as a target group for supplementation to improve their vitamin K status of the vasculature.
Response to vitamin K supplements at different states of vitamin K deciency While circulating dp-ucMGP levels increased (non-signicantly, P ¼ 0.053) in the control group of children (placebo capsules), a signicant decrease was found in the children that received the MK-7 supplements (P ¼ 0.013, Table 1, Fig. 3). The change in the placebo group differed signicantly from the change in the MK-
Results Vitamin K status during life time In children, extremely high serum ucOC levels were found (35 ng ml1, Fig. 1). These values were >6 times higher as compared to the other age groups (range 1.5–5.5 ng ml1). In the total group, a trend towards lower ucOC levels with increasing age was seen from 20 years to 80 years. In the male adults, the highest levels were seen in the young adults with a rst decline in serum ucOC at 30 years (Fig. 1, upper right box, black bars). Between 40 years and 70 years, stable levels were seen with a second decrease in circulating ucOC levels at 70 years. In the female adults, the highest levels were also seen in the young adults (Fig. 1, upper right box, grey bars). Further, a decline was
230 | Food Funct., 2014, 5, 229–234
Fig. 2
Circulating dp-ucMGP levels in healthy volunteers.
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7 group (P ¼ 0.002). A large variability was seen in the baseline values and MK-7 responses in our population of healthy children. Both in children and adults, MK-7-induced changes in dpucMGP levels were dependent on the baseline dp-ucMGP values. Higher baseline values gave larger decreases in circulating dp-ucMGP levels (Fig. 4).
Discussion In this paper, we investigated the vitamin K status during life time by measuring circulating levels of two extra-hepatic Glaproteins, namely circulating ucOC and dp-ucMGP, across several age groups. High ucOC levels were seen in the group of children, indicating an unusually poor vitamin K status of growing bone, which may increase osteoporosis risk in later life. Previous studies consistently showed elevated ucOC values in healthy children,12,14–16,25 and the higher metabolic activity of growing bone probably accounts for the observed vitamin K deciency in children. Thus, maximizing the accretion of bone during growth by optimal nutrition (including vitamin K intake) may reduce the risk of developing osteoporosis.26
Table 1 Circulating dp-ucMGP values in healthy children (before and after treatment)a
Placebo (n 21)
dp-ucMGP (pmol l1, week 0) dp-ucMGP (pmol l1, week 8) D dp-ucMGP (pmol l1) D dp-ucMGP (%)
MK-7 (n 21)
Median
Range (min; max)
156a
(55; 370)
172a
(32; 546)
178
(97; 340)
138
(45; 401)
24a
(111; 97)
21b
(145; 44)
18a
(30; 76)
18b
(46; 45)
Median
Range (min; max)
a
Between-group differences were assessed using the Mann–Whitney U test. Median values within a row with unlike superscript letters were signicantly different (P < 0.05).
Changes in circulating dp-ucMGP levels after treatment in 42 healthy children.
Fig. 3
This journal is © The Royal Society of Chemistry 2014
MK-7-induced changes in circulating dp-ucMGP levels in relation to baseline values in children and young adults.
Fig. 4
Unfortunately, the importance of vitamin K during growth has only been studied to a limited extent. Kalkwarf et al. found that a better vitamin K status (high plasma phylloquinone and low %ucOC) was associated with lower bone turnover (BAP, NTX) in healthy girls (3–16 years).27 The vitamin K status was however not consistently associated with the bone mineral content (BMC). O'Connor et al. showed in healthy Danish girls (11–12 years) that a better vitamin K status (low %ucOC) did relate to increased BMC, but not to bone turnover [OC, pyridinoline (PYD) and deoxypyridinoline DPD].28 van Summeren et al. conrmed the association between vitamin K status (the ratio of ucOC to carboxylated OC) and (total-body) BMC in healthy Dutch children (8–14 years).14 Consistently, reduced bone density was seen in vitamin K-decient (high ucOC) children as caused by (long-term) VKA therapy.29,30 Children using VKA presented higher levels of bone resorption markers [DPD/ creatinine, cross-linked telopeptide of type I collagen (ICTP)] and lower levels of bone formation markers [carboxylated OC, type I procollagen N-terminal propeptide (PINP)].30 We earlier reported that MK-7 (45 mg per day, i.e. RDA for children) supplementation improved the vitamin K status (28% reduction in ucOC levels) in healthy children, but data on bone mass are lacking.13 Altogether, these ndings suggest potentially adverse effects of low vitamin K status on growing bone, but the question remains whether higher vitamin K intake improves bone health in healthy children. In adults, on the other hand, the association between vitamin K status and bone health has been extensively studied.31,32 Observational studies have indicated that vitamin K deciency (increased circulating ucOC) leads to age-related bone loss.33 Data from RCTs in adults, however, gave contradictory results for effects on bone mass, although the vitamin K status improved (decreased circulating ucOC) consistently in all studies.31,32 It could be that improving vitamin K status may have more pronounced effects on bone quality, i.e. microarchitecture of bone, than on bone mass. Moreover, carboxylation-independent mechanisms have been suggested for vitamin K's role in bone health.33 Largely based on the observational ndings, the European Food Safety Authorities (EFSA) approved the claim: “a cause and effect relationship has been established between vitamin K and maintenance of normal bone”.34 Our recent ndings that long-term MK-7 supplementation decreases Food Funct., 2014, 5, 229–234 | 231
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the age-related decline in bone strength in healthy postmenopausal women are consistent with the EFSA's opinion.24 Women in menopause are, like children, characterized by high bone metabolism with increased levels of ucOC.5,17,18,35 Women at their 20s and 50s were determined by Kim et al. as having elevated serum ucOC levels suggesting that vitamin K supplements may improve their bone metabolism.18 Aer a decline in circulating ucOC levels at their thirties, we found higher ucOC levels from 40 years in our population of healthy women. Unfortunately, levels were not signicantly different from those of other adult age groups. It should be noted that we excluded osteoporotic women, which may explain the relatively small augmentation in circulating ucOC levels from 40 years and the lack of an extra elevation with menopause. In healthy men, a trend towards lower levels with increasing age was seen, including a plateau phase between 40 years and 70 years. Previous studies also showed a decreasing trend in circulating ucOC levels with increasing age in men.5,17 This is the rst study in which circulating dp-ucMGP levels were measured in children. In contrast to high ucOC levels, children had relatively low levels of dp-ucMGP, but in all children levels were well above the detection limit. MGP is mainly found in cartilage and the vascular wall, where it is produced by chondrocytes and vascular smooth muscle cells, respectively. MGP is an important inhibitor of vascular calcication and its uncarboxylated form has been associated with arterial calcication in healthy adults and in patients at increased cardiovascular risk.7 The consequences of dysfunctional or lack of MGP are shown by two human syndromes. The rare autosomal recessive Keutel syndrome is caused by mutations in the MGP gene leading to excessive cartilage calcication.36 Administration of VKA to women during pregnancy can cause embryopathy and skeletal defects, known as foetal warfarin syndrome.37 MGP is therefore thought to play a role in promoting growth and skeletal modelling in children. As indicated, no studies on dpucMGP values in children have been published thus far. We found a broad range of plasma dp-ucMGP values (32–546 pmol l1) in our population of healthy children. In order to explain the discrepancy in ucOC and dp-ucMGP levels, we could hypothesize that in children the vasculature is less vitamin Kdecient than bone. We have shown in this study that shortterm MK-7 supplementation signicantly decreased circulating levels of dp-ucMGP in the group of children. Despite the different degrees of vitamin K deciency in bone (high levels of ucOC) and the vasculature (low levels of dp-ucMGP) in children, the effective level of MK-7 administration was similar for ucOC13 and dp-ucMGP, i.e. net changes (as compared to the placebo group) of 38% and 36%, respectively. We further found larger responses to supplementation in children with higher baseline dp-ucMGP values. This was conrmed in a cohort of healthy adults, indicating that more pronounced vitamin K deciency (higher baseline dp-ucMGP values) gives larger responses to MK-7 supplements. We found that in healthy adults circulating dp-ucMGP levels gradually increased with age; a signicant rise was seen above 40 years of age. In line, Cranenburg et al. showed a trend of increasing values with age with signicant higher values for
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elderly (66–80 years) as compared to young adults (25–40 years).7 Four different randomized trials are published, all showing improvements in circulating dp-ucMGP levels aer phylloquinone20 or menaquinone4,6,38 (Braam et al., 2013, unpublished results) supplementation. Effective daily doses ranged from 90 mg to 500 mg with effective levels of 30% to 80%, respectively. The question remains whether improved carboxylation of MGP benecially affects cardiovascular health. Only three published RCTs investigated the health effects of vitamin K supplementation on vascular parameters (CAC and arterial stiffness)20,39 (Braam et al., 2013, unpublished results). All showed positive results, but individual changes in circulating dp-ucMGP levels could not be linked to changes in vascular measures. These ndings are in contrast with the results of case-control studies that have reported higher plasma dpucMGP levels among patients at increased cardiovascular risk, including patients with aortic valve disease, aortic stenosis, and chronic kidney disease.10 So, the association between MGP and vascular measures may be stronger in less healthy populations. Furthermore, it is plausible that vitamin K inuences cardiovascular health through carboxylation-independent mechanisms. In vitro studies suggested that vitamin K may decrease gene expression of proinammatory cytokines.40
Experimental Vitamin K status during life time Study population. Blood samples from 896 apparently healthy children (age range, 6–19 years) and adults (age range, 20–80 years) were analysed retrospectively. Only healthy volunteers were selected as determined by interviews and questionnaires. Exclusion criteria were: abnormal weight or height (children: weight or height outside p3–p97, adults: BMI > 30 kg m2), use of vitamin K supplements, use of oral anticoagulants (VKA), use of drugs that inuence bone metabolism, corticoid treatment, pregnancy (when applicable), gastrointestinal diseases, osteoporosis, chronic diseases, anaemia, and/or recent blood donation (
