Chemical structure and function of menaquinones

Vitamin K is a generic term for a number of structurally related compounds that are characterised by their common functional methylated naphthoquinone ring system, and an aliphatic side chain composed of a number of isoprenoid residues. All differences between the various forms of vitamin K originate from the differences in the length and the saturation degree of the side chain⁽Reference Shearer and Newman⁹⁾. Phylloquinone is a single compound with a side chain of four isoprenoid residues, three of which are saturated (Fig. 1). Menaquinones, commonly found in nature, have side chains of varying length between four and thirteen isoprene residues, most of which are unsaturated⁽Reference Collins and Jones¹⁰⁾. However, some bacteria produce isoprenologues in which one or more of the prenyl units are saturated⁽Reference Shearer and Newman⁹⁾. Menaquinones are generally denoted as MK-n, where n stands for the number of isoprene residues.

Fig. 1 Chemical structures of K vitamins. MK, menaquinone.

MK-4 is unique among the menaquinones in that it is not synthesised by bacteria. Instead, MK-4 is alkylated from menadione (vitamin K₃), a synthetic form of vitamin K that is present in animal feeds, or is the product of tissue-specific conversion directly from dietary phylloquinone, with menadione as the postulated intermediate⁽Reference Booth and Suttie^11, Reference Thijssen, Vervoort and Schurgers¹²⁾. There is also speculation that longer-chain menaquinones, such as MK-7, can be converted to MK-4 as well⁽Reference Schurgers, Teunissen and Hamulyak¹³⁾. The most abundant menaquinones in the human diet are the short-chain MK-4, which originates from animal products, and the long-chain MK-7, MK-8, MK-9 and MK-10.

All forms of vitamin K have one well-known function. They all serve as a cofactor for the post-translational enzyme γ-glutamate carboxylase, which is established by the common naphthoquinone ring structure⁽Reference Rishavy and Berkner¹⁴⁾. This enzyme converts certain protein-bound glutamate residues into γ-carboxyglutamate, generally known as Gla. Currently, seventeen members of the Gla protein family are known, including seven proteins involved in blood coagulation (all synthesised in the liver), osteocalcin (OC; bone), matrix Gla protein (MGP; mainly cartilage and vessel wall), growth arrest-specific protein 6, Gla-rich proteins, two proline-rich Gla proteins, two transmembrane Gla proteins, periostin and periostin-like factor⁽Reference McCann and Ames¹⁵⁾. With the exception of the clotting factors OC (bone formation) and MGP (inhibitor of soft tissue calcification), the physiological importance of these proteins is not yet fully understood⁽Reference McCann and Ames¹⁵⁾. At this time, conformation-specific assays are available for two extra-hepatic Gla proteins (OC and MGP).

Dietary intake of menaquinones

Menaquinones generally are of microbial origin. Important dietary sources are cheese, curd and natto (a traditional Japanese food composed of fermented soya beans)⁽Reference Schurgers and Vermeer¹⁶⁾, while dietary phylloquinone is mainly found in green vegetables, notably spinach, broccoli, kale and Brussels sprouts⁽Reference Bolton-Smith, Price and Fenton^17, Reference Shearer and Bolton-Smith¹⁸⁾. Estimated intake of phylloquinone and menaquinones in The Netherlands and Germany has suggested that between 10 and 25 % of total vitamin K intake are provided by menaquinones⁽Reference Schurgers and Vermeer^16, Reference Nimptsch, Rohrmann and Linseisen¹⁹⁾. Information on the dietary intake of menaquinones is, however, limited. This is mainly due to the lack of complete food composition tables that list menaquinone concentrations in common foods. Currently, most food composition data for menaquinones are restricted to single foods such as cheese or yogurt⁽Reference Schurgers and Vermeer^16, Reference Kamao, Suhara and Tsugawa²⁰⁾. However, a regularly updated and expanding food composition table of foods on the Dutch market is produced at VitaK's laboratories in The Netherlands.

Based on the content of menaquinones of certain foods, regional differences exist both for the form and the amount of menaquinones consumed. For example, in Japan, MK-7 intake has mostly been documented in the diet due to the consumption of natto, while long-chain menaquinones, MK-7 to MK-10, are predominantly consumed by high dairy intake populations, such as the Dutch population. In addition, cheese is the most important source of menaquinones in the European food supply, and therefore menaquinone concentrations in the cheeses of Dutch, German, Swiss, British and French origins were tested⁽Reference Schurgers and Vermeer¹⁶⁾. Since different lactic acid bacteria are used in European cheeses, a large variability in menaquinone content among the cheeses was found⁽Reference Fox and McSweeney²¹⁾. However, it remains to be seen whether these data are applicable to non-European countries.

The few studies that have provided estimates for menaquinone intakes have been mainly performed among Japanese or European populations for which menaquinone-rich foods are present in the diet. A study by Kamao et al. ⁽Reference Kamao, Suhara and Tsugawa²⁰⁾ measured the menaquinone content of foods and the estimated intake of MK-4 and MK-7 of 125 young Japanese women using a 3 d weighed food record. The MK-7 intake of this population was originally estimated at 57·4 (sd 83·7) μg/d and accounted for approximately 25 % of the total intake of vitamin K. However, this estimate is mainly driven by natto consumption, as it accounted for 99 % of the MK-7 intake and almost half of the population studied consumed natto. After stratifying by natto consumption, intake of MK-7 was estimated at 154·1 (sd 87·8) μg/d among natto consumers, but data on non-natto consumers were not provided.

Several Dutch studies investigating the associations of menaquinone intake with disease incidence obtained estimates for menaquinone intake⁽Reference Beulens, Bots and Atsma^2, Reference Geleijnse, Vermeer and Grobbee^3, Reference Beulens, Van der and Grobbee^22, Reference Gast, de Roos and Sluijs²³⁾ (Table 1) using FFQ. These intake estimates were based on direct measurements of menaquinones in foods in combination with published data⁽Reference Schurgers and Vermeer^16, Reference Elder, Haytowitz and Howe²⁴⁾. The self-reported mean intake of menaquinones was approximately 31 μg/d⁽Reference Geleijnse, Vermeer and Grobbee^3, Reference Beulens, Van der and Grobbee²²⁾. In the EPIC-Netherlands cohort, cheese contributed 53 % of menaquinone intake, while milk products and meat contributed 19 and 17 %, respectively. The most prominent long-chain menaquinone reported in the diet was MK-9. In the Rotterdam Study, MK-5 to MK-10 contributed 23·1 (sd 16·3) μg/d for men and 20·7 ± 13·8 μg/d for women. These data on food contents of menaquinones have also been applied to a German cohort of approximately 12 000 men⁽Reference Nimptsch, Rohrmann and Linseisen¹⁹⁾. Similar estimates of 34·7 μg/d (interquartile range 25·7–45·7) were reported for all menaquinones and 18·0 μg/d (11·7–27·0) for MK-5 to MK-9, with cheese being the most important food source of menaquinones⁽Reference Nimptsch, Rohrmann and Linseisen¹⁹⁾.

Table 1 Menaquinone intake, arterial calcification and risk of CHD

BAC, breast arterial calcification; CAC, coronary artery calcification.

Finally, the European Food Safety Authority reported data on UK intake of menaquinones based on the UK National Dietary and Nutrition Survey⁽Reference Bresson, Flynn and Heinonen²⁵⁾. This study used weighed dietary records, albeit based on the menaquinone content from a limited number of food items⁽Reference Bresson, Flynn and Heinonen²⁵⁾. The overall estimated intake of menaquinones ranged from 36 μg/d (female adults) to 54 μg/d (male teenagers). The mean estimated intake of menaquinones among male adults was 43 μg/d, which is similar to the intakes reported for other European countries.

It should be noted that estimates from the Dutch and German populations were obtained from FFQ that are designed to estimate the relative dietary intake of large populations, but not to estimate the absolute dietary intake. These limitations should be kept in mind when interpreting these data. In order to obtain more precise estimates of menaquinone intakes, studies using (weighed) food records and concentrations of individual menaquinones obtained from representative foods from different food supplies are required. Nonetheless, current studies have shown estimated intakes of menaquinones ranging between 30 and 50 μg/d, which account for up to 25 % of intake of total vitamin K.

Absorption and metabolism of menaquinones

Phylloquinone is primarily obtained from green, leafy vegetables in which it is tightly bound to the membranes of plant chloroplasts, and thus less bioavailable compared with phylloquinone obtained from plant oils and/or dietary supplements⁽Reference Booth and Suttie¹¹⁾. Menaquinones, which are primarily derived from animal-based sources, are consumed in food matrices containing more fat that may improve absorption and lead to higher bioavailability than phylloquinone⁽Reference Gijsbers, Jie and Vermeer²⁶⁾. However, this has yet to be systematically tested for all menaquinones.

Following intestinal absorption, all vitamin K forms are incorporated into TAG-rich lipoproteins and transported primarily to the liver, but also to other target tissues. Circulating TAG-bound forms of vitamin K peak at around 4–10 h after intake and the majority of phylloquinone and MK-4 are removed from the circulation by 24 h postprandially⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Schurgers and Vermeer^16, Reference Sato, Schurgers and Uenishi²⁷⁾. Currently, human data on the absorption of menaquinones from food sources are limited to MK-7. These MK-7 data show similar peaks at 4 h after intake, but MK-7 does not appear to be completely removed from the circulation after 72–96 h⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Schurgers and Vermeer¹⁶⁾. The different pharmacokinetics among various vitamin K forms also result in very different plasma half-life times. Whereas phylloquinone has a relatively short half-life time⁽Reference Novotny, Kurilich and Britz²⁸⁾, MK-7 has a reported half-life time of several days⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Sato, Schurgers and Uenishi²⁷⁾. Available data indicate higher absorption and bioavailability of MK-7 than phylloquinone, which may facilitate its uptake by various target tissues.

Another difference between the short-chain forms of vitamin K (phylloquinone and MK-4) and the long-chain forms relates to tissue distribution. For example, one study showed that MK-9 is preferentially incorporated in LDL, which facilitates its transport to non-hepatic target tissues⁽Reference Schurgers and Vermeer²⁹⁾. It is not known whether other long-chain menaquinones have similar transport differences. MK-4 is unique among the menaquinones in its tissue distribution, which relates to its non-bacterial origin. As recently shown in a rodent model using stable isotopes⁽Reference AL Rajaba, Booth and Peterson³⁰⁾, phylloquinone consumed in the form of leafy greens is converted to MK-4 in some, but not all, tissues. The labelled MK-4 was most abundant in the brain, kidney, fat and reproductive organs. In contrast to phylloquinone as the sole dietary source of vitamin K, there was no conversion of phylloquinone to MK-4 in the liver nor were there detectable amounts of labelled MK-4 in serum. These data confirm earlier rodent studies that have reported differences in tissue distribution between phylloquinone and MK-4⁽Reference Ronden, Thijssen and Vermeer^31, Reference Thijssen and Drittij-Reijnders³²⁾.

A caveat to these conclusions is that the data for phylloquinone are much more comprehensive than those for menaquinones. Many investigators have studied phylloquinone pharmacokinetics using different study designs, including stable isotopes⁽Reference Fu, Peterson and Hdeib^33–Reference Jones, Bluck and Wang³⁵⁾. In contrast, menaquinone pharmacokinetic data are limited: (1) in the forms studied (mainly limited to MK-7); (2) from a lack of replication by independent laboratories; (3) by an absence of using stable isotope technology. More research is clearly required to quantify the differences in absorption and bioavailability among the various forms of vitamin K in order to set nutrient requirements.

Microbiotic production of menaquinones

Most aerobic Gram-positive bacteria and the majority of anaerobic bacteria produced by the gut use menaquinones in their electron transport pathways. The length of the side chain, as indicated by different menaquinones, is controlled by specific bacteria⁽Reference Collins and Jones¹⁰⁾. The reasons for this are not entirely clear, but the length and degree of saturation of the menaquinone side chain are often dependent on the growth temperature of a given species⁽Reference Nowicka and Kruk³⁶⁾. Based on qualitative bacteriological analyses, several bacteria have been identified to produce specific menaquinones. Menaquinones produced by the gut flora have been tabulated by previous studies⁽Reference Fernandez and Collins^37–Reference Mathers, Fernandez and Hill³⁹⁾. For example, Bacteroides fragilis produces MK-10 to MK-12, while Eubacterium lentum produces MK-6. Likewise, bacteria used as starter cultures for the production of foods such as cheese may also produce specific menaquinones. For example, the lactic acid bacteria Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris produce mainly MK-8 and MK-9⁽Reference Morishita, Tamura and Makino⁴⁰⁾, while propionibacteria produce mainly MK-9⁽Reference Hojo, Watanabe and Mori⁴¹⁾. The implications for the relative bioavailability of dietary menaquinones produced by bacteria in the food supply need to be considered relative to the bioavailability of menaquinones produced by bacteria in the human intestine.

It was once stated that up to 50 % of the human requirement for vitamin K was fulfilled by the intestinal production of menaquinones⁽Reference Conly and Stein^42, Reference Suttie⁴³⁾. The 50 % estimate was based on semi-quantitative measurements of the vitamin K content of the human liver, in which one-half of the vitamin K content was phylloquinone and the other half was a mixture of long-chain menaquinones⁽Reference Duello and Matschiner⁴⁴⁾. However, subsequent studies indicated that phylloquinone accounted for less than 10 % of the vitamin K content in the human liver, with a greater preponderance of MK-10, MK-11 and MK-12 than previously assumed⁽Reference Suttie^43, Reference Usui, Tanimura and Nishimura⁴⁵⁾. Based on these hepatic menaquinone concentrations, one would predict that circulating long-chain menaquinones would be in much higher concentrations than phylloquinone should these menaquinones have a major contribution to the human requirement for vitamin K. This, however, does not appear to be the case, and the route of absorption of bacterially produced menaquinones is still unclear. The absorption of all vitamin K forms takes place in the small intestine via a process requiring bile salts⁽Reference Olson⁴⁶⁾. However, bile salts are absent in the colon where the majority of menaquinones are produced, suggesting a low absorption of these vitamin K forms⁽Reference Conly and Stein⁴⁷⁾. This was confirmed by Ichihashi et al. ⁽Reference Ichihashi, Takagishi and Uchida⁴⁸⁾, who showed that the absorption of intestinally produced menaquinones in rats is low and that the absorption rates decrease markedly with the length of the side chain. A study in infants also indicated that intestinally produced menaquinones are not well absorbed⁽Reference Fujita, Kakuya and Ito⁴⁹⁾. This study compared faecal and serum concentrations of phylloquinone and menaquinones of formula-fed infants with breast-fed infants. Formula-fed infants had higher serum and faecal phylloquinone concentrations as well as a higher MK-5 to MK-9 faecal concentration compared with breast-fed infants. Serum menaquinones were undetected in most formula-fed infants, suggestive of poor absorption⁽Reference Fujita, Kakuya and Ito⁴⁹⁾.

Another consideration is that most bacterially produced menaquinones are within the bacterial membranes, hence not readily bioavailable. It has been postulated that these bacterially synthesised menaquinones may be important in maintaining normal coagulation among severely ill patients with prolonged vitamin K deficiency⁽Reference Ramotar, Conly and Chubb⁵⁰⁾; however, current data are inconclusive regarding the relative contribution of menaquinones to fulfilling the dietary requirements for vitamin K.

The amount of menaquinones required to prevent deficiency and maintain optimal body stores

To understand the impact of menaquinones on health, it is necessary to demonstrate the link between the intakes of menaquinones and the nutritional status of vitamin K. Several biochemical markers of vitamin K status are available and all have their strengths and weaknesses, as detailed elsewhere⁽Reference Booth and AL Rajaba⁵¹⁾. However, measures of plasma or tissue menaquinone concentrations are needed to isolate the effects of menaquinones from those of phylloquinone on human health. Other markers of vitamin K status, which include urinary metabolites of vitamin K⁽Reference AL Rajaba, Peterson and Choi^52, Reference Harrington, Booth and Card⁵³⁾, coagulation times and uncarboxylated Gla proteins, cannot differentiate the effects of menaquinones from phylloquinone. Therefore, differences between menaquinones and phylloquinone can only be determined through the use of study designs that directly compare the response of individual biomarkers with the intakes of individual forms of vitamin K.

Under controlled conditions of dietary intake, circulating menaquinone concentrations increase in response to the high intake of menaquinones and decline over time when the dietary source of menaquinones is removed⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Gijsbers, Jie and Vermeer²⁶⁾. However, data are limited, since the HPLC and MS techniques are limited to a few qualified laboratories and the long-chain menaquinones are often below the detection limit in the circulation when measured in the general population. Only a few studies have measured plasma menaquinones in response to the intake of individual menaquinones, and these have been limited to MK-7 or MK-4 supplementation⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Sato, Schurgers and Uenishi^27, Reference Bruge, Bacchetti and Principi⁵⁴⁾. For plasma MK-7, two studies showed a clear dose–response effect on circulating MK-7 concentrations after supplementation with doses ranging between 45 and 420 μg⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Bruge, Bacchetti and Principi⁵⁴⁾. In contrast, MK-4 was not detected in the circulation following a single dose of 420 μg⁽Reference Sato, Schurgers and Uenishi²⁷⁾.

Only two studies have investigated the response of vitamin K urinary metabolites to single oral doses of menaquinones. Both studies showed a good response of urinary menadione⁽Reference Thijssen, Vervoort and Schurgers¹²⁾ or side-chain catabolite⁽Reference Harrington, Soper and Edwards⁵⁵⁾ excretion to relatively high doses of 15 or 45 mg of MK-4 or 1 mg of MK-7. Both studies included a direct comparison of menaquinones with phylloquinone, and showed similar results for both vitamin K forms. Harrington et al. ⁽Reference Harrington, Booth and Card⁵³⁾ showed that the excretion of the 5- and 7-carbon side-chain metabolites responds to the depletion and repletion of phylloquinone. A similar response to menaquinones would be expected, but no studies of similar design have been conducted.

Prothrombin time (PT), also expressed as an international normalised ratio, is a test of coagulation that can reflect clinical deficiency of vitamin K due to frank deficiency or the antagonism of vitamin K. However, PT is non-specific because abnormal values are also indicative of diseases unrelated to vitamin K deficiency. PT changes only when prothrombin concentrations drop below 50 % of normal, demonstrating its low sensitivity for detecting the deficiency of vitamin K⁽Reference Suttie⁵⁶⁾. To date, only two studies⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Schurgers, Shearer and Hamulyak⁵⁷⁾ have reported the effects of MK-7 and MK-9 on coagulation parameters. In these studies, the antidotal effect of single doses of MK-7 and MK-9 was studied in volunteers stabilised on oral vitamin K antagonists. Both studies showed that MK-7 and MK-9 decreased the international normalised ratio and the concentrations of coagulation factors, and this effect was stronger for MK-7 than phylloquinone⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Schurgers, Shearer and Hamulyak⁵⁷⁾. However, in one study⁽Reference Schurgers, Shearer and Hamulyak⁵⁷⁾, menaquinones were provided as different food sources with differing doses and bioavailability, which may have influenced the results. Although these studies are informative in the clinical context of the reversal of oral anticoagulation, they are not suitable for determining the amount of menaquinones required to prevent deficiency or maintain optimal body stores. Although the effects of coagulation factors on depletion or repletion with menaquinones have not been investigated to date, sustained intakes as low as 10 μg/d of phylloquinone for several weeks do not prolong PT in otherwise healthy adults^(7, Reference Frick, Riedler and Brogli^58–Reference Udall⁶⁰⁾.

Thus far, conformation-specific tests have been developed for prothrombin, OC and MGP to evaluate the extent to which the various Gla proteins are carboxylated in healthy subjects. Advantages of measuring uncarboxylated vitamin K-dependent proteins are that insufficiencies measured in circulating forms theoretically reflect what occurs at the tissue level. Undercarboxylated prothrombin, also known as PIVKA-II, detects abnormalities in prothrombin before the prolongation of PT, but does not have the sensitivity to detect the variability of usual vitamin K intakes observed in normal healthy populations. PIVKA-II has been used as a marker of vitamin K status in healthy people and has been shown to respond to both dietary depletion and subsequent repletion with phylloquinone⁽Reference Booth, Lichtenstein and O'Brien-Morse^61, Reference Booth, Martini and Peterson⁶²⁾. However, only one study⁽Reference Furukawa, Nakanishi and Okuda⁶³⁾ investigated the effect of a single intravenous dose of 10 mg MK-4 in vitamin K-deficient cancer patients, and showed a decrease in PIVKA-II levels 1–3 d after ingestion.

The effect of menaquinones on the proportion of OC that is uncarboxylated (ucOC) has been more frequently studied. ucOC is highly responsive to supplementation with either MK-4 or MK-7 in doses ranging from 45 μg/d to 45 mg/d (MK-4 only)⁽Reference Schurgers, Teunissen and Hamulyak^13, Reference Bruge, Bacchetti and Principi^54, Reference Emaus, Gjesdal and Almas^64–Reference van Summeren, Braam and Lilien⁶⁶⁾. Only a low dose of 45 μg MK-7/d did not lead to a significant reduction in ucOC⁽Reference Bruge, Bacchetti and Principi⁵⁴⁾. A direct comparison of phylloquinone with MK-7 supplementation indicated that MK-7 is more effective in carboxylating OC than phylloquinone⁽Reference Schurgers, Teunissen and Hamulyak¹³⁾. Assays to measure desphospho-uncarboxylated MGP only recently became available. Since that time, several intervention studies have shown clear dose–response effects of desphospho-uncarboxylated MGP to MK-7 supplementation with doses ranging between 10 and 360 μg/d⁽Reference Cranenburg, Koos and Schurgers^67–Reference Westenfeld, Krueger and Schlieper⁷⁰⁾. However, a direct comparison between menaquinones and phylloquinone in the response of desphospho-uncarboxylated MGP to supplementation by the individual vitamin K forms has not been made.

The amount of menaquinones required to prevent chronic diseases

Menaquinones and bone

In bone, three vitamin K-dependent proteins have been isolated: protein S; MGP; OC. The anticoagulant protein S is synthesised by osteoblasts (bone-forming cells), but its role in bone metabolism is unclear. MGP has been found in bone, dentine, cartilage and soft tissue, including blood vessels, and is associated with the organic matrix and mobilisation of bone Ca. The results of animal studies suggest that MGP prevents the calcification of soft tissue and cartilage, while facilitating normal bone growth and development⁽Reference Booth⁷⁴⁾. OC is a protein synthesised by osteoblasts. The synthesis of both OC and MGP is regulated by calcitriol and retinoic acid. Higher ucOC concentrations, indicating a low vitamin K status, were associated with a higher hip fracture risk and lower bone mineral density (BMD) in adults⁽Reference Booth, Tucker and Chen^75–Reference Vergnaud, Garnero and Meunier⁸⁵⁾ and children⁽Reference Kalkwarf, Khoury and Bean^86–Reference van, Braam and Noirt⁸⁹⁾. Unfortunately, the proportion of ucOC does not differ between phylloquinone and menaquinones in terms of the form responsible as an enzyme cofactor. However, serum menaquinone levels were lower in patients with osteoporosis, osteopenia and osteoporotic fractures compared with controls⁽Reference Hodges, Pilkington and Stamp^90–Reference Tamatani, Morimoto and Nakajima⁹³⁾. In addition, an inverse association was found between circulating MK-7 levels and the incidence of vertebral fractures in Japanese women, although this association was stronger for phylloquinone and fracture risk⁽Reference Tsugawa, Shiraki and Suhara⁹⁴⁾.

Intervention studies using pharmacological doses of MK-4 showed beneficial effects on bone parameters⁽Reference Cockayne, Adamson and Lanham-New¹⁾. However, these intervention studies used very high doses of MK-4 (generally 45 mg) that cannot be obtained from the habitual diet. Since this paper is focused on dietary doses that are relevant for nutritional requirements, we will focus on intervention studies that used doses that can be nutritionally obtained. Although natto contains more than 100 times as much menaquinones as various cheeses, studies on natto or its equivalent amount of MK-7 are considered within the dietary intake range.

Intervention studies investigating the effect of menaquinone supplementation on bone markers are shown in Table 2. Whereas MK-7 at low doses did not affect bone formation⁽Reference Emaus, Gjesdal and Almas^64, Reference Tsukamoto, Ichise and Kakuda⁶⁵⁾, intake of natto three times per week increased bone-specific alkaline phosphatase when compared with once-per-week natto intake⁽Reference Katsuyama, Ideguchi and Fukunaga⁹⁵⁾. Only one study showed decreased bone resorption due to a combination of vitamin K forms, vitamin D₃, Ca and lifestyle recommendations⁽Reference Kanellakis, Moschonis and Tenta⁹⁶⁾. This finding was independent of the form of vitamin K taken, although on a molecular basis, the daily intake of MK-7 (0·154 μmol) in this study was about 30 % less than that of phylloquinone (0·221 μmol).

Table 2 Intervention studies on the dietary levels of vitamin K, bone markers and bone mineral density (BMD)

DBPC, double-blind placebo-controlled; MK-7, menaquinone-7; NA, not applicable.

* Urinary deoxypyridinoline.

† Bone-specific alkaline phosphatase.

‡ Degradation products of C- or N-terminal telopeptides of type I collagen.

A few cross-sectional studies investigated the association between menaquinone intake and bone maintenance. For example, two Japanese studies showed that the usual dietary intake of natto was effective in maintaining bone stiffness⁽Reference Katsuyama, Ideguchi and Fukunaga⁹⁷⁾ and was positively associated with a 3-year change in BMD at the femoral neck⁽Reference Ikeda, Iki and Morita⁹⁸⁾. Within Norwegian individuals, no linear association was found between dietary menaquinones and BMD of the total hip; however, fractional polynomial regression analyses for the detection of non-linear associations showed a small, positive association between dietary menaquinones and BMD among women⁽Reference Apalset, Gjesdal and Eide⁹⁹⁾. Of note, these studies described a proposed effect of dietary menaquinones via natto consumption. Although MK-7 is an important nutrient of natto, natto also contains other ingredients that have been postulated to promote bone health. Hence MK-7 may be a surrogate marker for other bone-promoting ingredients in these studies.

As shown in Table 2, three intervention studies on menaquinones and BMD used doses of menaquinones that are attainable with the diet. Only one study⁽Reference Kanellakis, Moschonis and Tenta⁹⁶⁾ observed a beneficial effect of vitamin K on BMD of the lumbar spine. The two other studies did not show an effect on bone stiffness or the rate of bone loss. The main differences between these three studies are the inclusion of vitamin D as part of the treatment and the regional differences in the prevalence of vitamin D deficiency⁽Reference Lips¹⁰⁰⁾. These vitamin D-related disparities in study designs may have influenced the effect of menaquinones.

Only one observational study specifically examined the association between dietary menaquinone intake and fracture risk, reporting that a low intake of phylloquinone, but not menaquinones, was associated with an increased risk of hip fractures in Norwegian individuals⁽Reference Apalset, Gjesdal and Eide¹⁰¹⁾. To date, no intervention study has evaluated the efficacy of menaquinones in doses attainable in the diet in reducing fracture risk.

Only a few studies have made a direct comparison between menaquinones and phylloquinone for bone health. These studies indicated no differences⁽Reference Kanellakis, Moschonis and Tenta⁹⁶⁾ or showed stronger effects for phylloquinone than menaquinones⁽Reference Tsugawa, Shiraki and Suhara^94, Reference Apalset, Gjesdal and Eide¹⁰¹⁾.

Requirements across the life cycle

Dietary intake has historically been considered the primary determinant of vitamin K status⁽Reference Booth and Suttie¹¹⁾. However, other non-dietary factors are emerging as determinants, such as age and ethnicity⁽Reference Booth and AL Rajaba⁵¹⁾. To develop recommendations for dietary intakes⁽⁸⁾, sufficient data are needed to evaluate requirements across the life cycle. The data for phylloquinone are sparse⁽⁷⁾, but there are even less data for menaquinones. Currently, data on the intake or supplementation of menaquinones are limited to the assessment in children and teenagers in the UK National Dietary and Nutrition Survey⁽Reference Bresson, Flynn and Heinonen²⁵⁾.

The only clinically indicated use of vitamin K is as a prophylactic against vitamin K deficiency bleeding in otherwise healthy-appearing neonates⁽Reference Shearer¹⁰²⁾. The low content of vitamin K in breast milk, low placental transfer of vitamin K, low levels of clotting factors at birth and a sterile gut are all contributing factors to the risk of vitamin K deficiency bleeding in the first few months of life. Prevention of vitamin K deficiency bleeding by oral or intramuscular administration of vitamin K at birth is standard practice in many countries. Whereas most countries use phylloquinone, certain Asian countries, including Japan, use MK-4 prophylactically⁽Reference Shearer¹⁰²⁾. At no other point in the life cycle is frank deficiency of vitamin K a concern among an otherwise healthy population.

Safety of high vitamin K intake

There is no documented case of toxicity for phylloquinone or menaquinones^(7, Reference Bresson, Flynn and Heinonen²⁵⁾. The European Food Safety Authority's safety assessment of menaquinones as a source of vitamin K added for nutritional purposes concluded that low doses of menaquinones presented no safety concerns⁽Reference Bresson, Flynn and Heinonen²⁵⁾. Similarly, an animal study reported no toxicity associated with synthetic MK-7 administered in a single oral dose up to 2000 mg/kg or for 90 d of oral administration of 10 mg/kg per d⁽Reference Pucaj, Rasmussen and Moller¹⁰³⁾.

It is often postulated that excessive vitamin K may result in overcoagulation, i.e. increased thrombosis risk. However, vitamin K-dependent proteins have a limited number of Glu residues capable of γ-carboxylation per molecule, beyond which there can be no further γ-carboxylation or excessive coagulation. Despite this, it is critical to demonstrate that a high intake of menaquinones does not increase thrombosis risk. It was shown in rats that thrombosis risk is not increased at doses up to 250 mg/kg of MK-4⁽Reference Ronden, Groenen-van Dooren and Hornstra¹⁰⁴⁾. In human subjects, the endogenous thrombin potential, which is the most sensitive marker to evaluate thrombosis risk in plasma⁽Reference Hemker, AL Dieri and De Smeat¹⁰⁵⁾, was not affected by MK-7 intakes as high as 360 μg/d for 6 weeks⁽Reference Westenfeld, Krueger and Schlieper⁷⁰⁾. The only exception to this is observed in individuals on coumarin-based oral anticoagulants, for whom dietary supplementation with vitamin K can influence the stability of the international normalised ratio⁽Reference Suttie, Mummah-Schendel and Shah^59, Reference Holmes, Hunt and Shearer¹⁰⁶⁾. MK-7 has the potential to interfere with oral anticoagulants at doses greater than 50 μg/d⁽Reference Schurgers, Teunissen and Hamulyak¹³⁾. However, there is little collective experience on the potential toxicity or adverse events associated with sustained menaquinone supplementation among individuals with normal coagulation.

In Asia, MK-4 is routinely used for osteoporosis treatment in doses of 45 mg/d without reported toxicity. Reported adverse effects associated with these high doses are limited to skin rashes that subside with cessation of the MK-4 dosing⁽Reference Bunyaratavej, Penkitti and Kittimanon¹⁰⁷⁾. As concluded by the European Food Safety Authority⁽Reference Bresson, Flynn and Heinonen²⁵⁾, phase I clinical trials have not yet been designed to test the safety of menaquinones nor has any form of vitamin K been adequately tested for mutagenicity. However, it is biologically implausible to attain such high levels of intake and sufficient bioavailability from menaquinones obtained from food sources to present a risk to health.