key: cord-0766921-iuq8zrj0
authors: Mladěnka, Přemysl; Macáková, Kateřina; Kujovská Krčmová, Lenka; Javorská, Lenka; Mrštná, Kristýna; Carazo, Alejandro; Protti, Michele; Remião, Fernando; Nováková, Lucie
title: Vitamin K – sources, physiological role, kinetics, deficiency, detection, therapeutic use, and toxicity
date: 2021-09-02
journal: Nutr Rev
DOI: 10.1093/nutrit/nuab061
sha: c00ab4bd2bac804bd196755310ec37ff28205bfe
doc_id: 766921
cord_uid: iuq8zrj0

Vitamin K is traditionally connected with blood coagulation, since it is needed for the posttranslational modification of 7 proteins involved in this cascade. However, it is also involved in the maturation of another 11 or 12 proteins that play different roles, encompassing in particular the modulation of the calcification of connective tissues. Since this process is physiologically needed in bones, but is pathological in arteries, a great deal of research has been devoted to finding a possible link between vitamin K and the prevention of osteoporosis and cardiovascular diseases. Unfortunately, the current knowledge does not allow us to make a decisive conclusion about such a link. One possible explanation for this is the diversity of the biological activity of vitamin K, which is not a single compound but a general term covering natural plant and animal forms of vitamin K (K(1) and K(2)) as well as their synthetic congeners (K(3) and K(4)). Vitamin K(1) (phylloquinone) is found in several vegetables. Menaquinones (MK(4)–MK(13), a series of compounds known as vitamin K(2)) are mostly of a bacterial origin and are introduced into the human diet mainly through fermented cheeses. Current knowledge about the kinetics of different forms of vitamin K, their detection, and their toxicity are discussed in this review.

Vitamin K is well known as an essential factor in blood coagulation. Hence its name, vitamin K, which is derived from the German term for coagulation (Koagulation). Its importance was recognized by the Nobel Committee for Physiology or Medicine, since the Nobel Prize in Physiology or Medicine for 1943 was awarded to Henrik Carl Peter Dam and Edward Adelbert Doisy for their discoveries of vitamin K and its chemical nature, respectively. 1 Vitamin K is not a single compound but is a term for many similar compounds that have the physiological function of this vitamin (Fig. S1 in the Supporting Information online). They share a common structure, the 2-methyl-1,4-naphthoquinone core, also known as menadione. The simplest form, which contains only this core, is known as vitamin K 3 . In contrast to the natural forms, K 3 is hydrophilic and is not obtained through the diet. However, it acts as an intermediate in human metabolism. 2 Vitamin K obtained from the diet originates either from plant sources (in the form of vitamin K 1 , known as phylloquinone [phytomenadione, phytonadione]) or more commonly from animal sources in the form of vitamin K 2 (menaquinone, generally abbreviated as MK, see Fig. S1 in the Supporting Information online). Vitamin K 4 also exists, and this term is associated with other synthetic forms of vitamin K. It may be a reduced form of vitamin K 3 (menadiol) or its ester forms (eg, diacetate vitamin K 3 ).

Vitamin K 1 is a single compound found in photosynthetic organisms like cyanobacteria, algae, and green plants. [3] [4] [5] Due to the high vitamin K 1 content in the green parts of plants, phylloquinone was originally thought to be present only in chloroplasts, but further research has confirmed that it is also present in peroxisomes and plasma membranes. It is even present in some non-photosynthetic parasitic plants. Its function in plants is best seen in the chloroplasts, where it is tightly bound to the thylakoid membrane. Here, it serves as an electron acceptor during photosynthesis, forming part of the electron transport chain of photosystem I. The function of vitamin K 1 in other cellular compartments is also associated with its redox properties. [5] [6] [7] In absolute amounts, it constitutes approximately 75%-90% of all vitamin K in our diet, but dietary K 1 has low bioavailability, [8] [9] [10] [11] as will be discussed in more detail below. Green cruciferous vegetables (broccoli, brussels sprouts, cabbage, kale, kai-lan, etc.) are rich sources of vitamin K 1 , but spinach, chard, parsley, and various types of lettuce also have considerable phylloquinone content . 8, [12] [13] [14] In general, all edible green parts of plants can be considered an important source of vitamin K 1 . Of course, this also applies to wild edible plants. Examples include stinging nettles (Urtica dioica L.), wild garlic (Allium ursinum L.), dandelion leaves (Taraxacum campylodes G.E.Haglund), and ground elder (Aegopodium podagraria L.), a relative of parsley whose vitamin content is comparable with that of cultivated vegetables. 15 Edible greens also include culinary herbs, of which marjoram (Origanum majorana L.), mint (Mentha  piperita L.), and savoury (Satureja hortensis L.) have the highest content of vitamin K 1, reaching values of up to 1250lg/100 g when fresh and over 3000 lg/100 g in dried herb samples. The seeds of some species of Apiaceae plants that are used as spices also contain significant amounts of the vitamin K 1 . 16 Data on their vitamin K 1 content differ significantly from study to study because it can be affected by a number of factors, such as cultivar, cultivation method, cultivation site, climatic conditions, plant maturation, and the method of determination. [17] [18] [19] The richest vegetable sources of vitamin K 1 , kale and spinach, are good examples of this. Its content in these vegetables appears to vary greatly: 250-1139 lg/ 100 g and 240-1220 lg/100 g, respectively. 12, 15, 20 In most cases, neither boiling nor microwaving affects the vitamin K 1 content of vegetables. 12 Certain vegetable oils are also important dietary sources of phylloquinone for humans. Of these, soybean oil is the most significant source, followed by rapeseed oil and olive oil, containing on average about 180, 130, and 55 lg/100 g of phylloquinone, respectively. 8, 21, 22 Since these oils are frequently used when cooking vegetables, they can enrich the food with additional vitamin K 1 . Its content in vegetable oils is relatively stable when heated, reaching a maximum decrease of 15% after being heated for 40 minutes at [185] [186] [187] [188] [189] [190] C. On the other hand, vitamin K 1 is extremely sensitive to daylight and fluorescent light, and these light sources decrease the vitamin content by 46% and 87% after only 2 days of exposure, respectively. Therefore, it is recommended to store oils in dark containers. 22 Vitamin K 1 is the predominant form of vitamin K used in food supplements and drugs indicated in vitamin K deficiency, and therefore production of a large amount is required. Synthetic vitamin K 1 is manufactured by condensing naphthoquinone with isoprenoid precursors. 4, 23 Demand for vitamins of a natural origin and their sustainable production is increasing, so research is underway seeking efficient production strategies. One promising option is the use of microalgae and cyanobacteria, which can be grown in bioreactors under highly controlled conditions and which produce a significant amount of vitamin K 1. This is true even when the algae are cultivated using conventional aquaculture techniques. 3, 4, 24, 25 Vitamin K 2 is produced by certain obligate and facultative anaerobic bacteria. The form of vitamin K 2 generated depends on the strain of bacteria. The various forms principally differ in the number of 5-carbon prenyl units, which ranges from 4 to 13 (Fig. S1B in the Supporting Information online). The number of these units is given as a suffix(-n), ie, they are named MK-4 to MK-13. It should be also pointed out that there can be differences between natural and synthetic vitamin K 2 , as was demonstrated in the case of MK-7, for which the synthetic mono-cis form (2Z) was inactive or had little activity compared with the natural all-E form. Some bacteria also produce one or more saturated prenyl units, and this is indicated by an additional suffix, eg, MK-8(2H) in Fig. S1C in the Supporting Information online or MK-9(4H). 2,26-28 MK-4 (menatetrenone) is not produced by bacteria, but instead is formed in human and animal organisms from K 1. It can hence be both consumed in the diet or formed in the human body. The enzyme responsible for this conversion is UbiA prenyltransferase domain-containing protein-1 (also known as transitional epithelial response protein 1, TERE1 ). This enzyme can cleave the side chain to release vitamin K 3 and then prenylate it with geranylgeranyl pyrophosphate to produce MK-4. There is still some controversy as to whether or not vitamin K 3 is produced in the gastrointestinal tract (GIT), and whether the enzyme TERE1 catalyzes only prenylation. It is possible that both the production of K 3 in the GIT from human vitamin K by, eg, microflora, with subsequent metabolism to MK-4 by TERE1, and direct conversion of K 1 to MK-4 by TERE1 could be occurring. 2, 8, [29] [30] [31] [32] Major sources of vitamin K 2 in the human diet include dairy products. Cheeses are a particularly rich source, due to the bacterial fermentation that occurs when they are produced. In fact, about onehalf of all vitamin K 2 ingested by humans comes from cheese. 33, 34 Other important sources of MKs are fermented vegetables such as sauerkraut, and in Japan, natt o. 27 The concentrations of MK-4 to MK-12 can reach high values (eg, 5-50 mg/100 g), with MK-8, MK-9, MK-10, and MK-11 being the major forms in many different cheeses (Fig. S2 in the Supporting Information online 8, 27, [35] [36] [37] . It should be stressed, however, that apparently discrepant data have been reported in relation to MK-10 content. Its levels can be even higher than that of MK-8 in some cheeses. 36, 37 The type of vitamin K 2 is largely dependent on which bacterial species (eg, Lactococcus, Propionibacterium) is used in production. Ripening results in an increase in vitamin K 2 content. 37 Reduced-fat or fat-free cheeses contain less than onequarter of the vitamin K 2 compared with their full-fat equivalents. 36 Sauerkraut is a less rich source of vitamin K 2 (total MKs content is approximately 5 mg/100 g), but it contains a substantial quantity of vitamin K 1 , 8, 27 while natt o is by far the richest source of vitamin K 2 (the content of MK-7 is approximately 900 mg/100 g). Natt o is produced from soybeans through fermentation with Bacillus subtilis var. natto, which is responsible for the production of MK-7. Other important dietary sources of vitamin K 2 are bovine and pork liver, whereas vitamin K 2 is barely present in most fish, with the important exception of eel. 27, 37 Fermented beverages like beer and wine do not contain detectable amounts of MKs, because yeasts, in contrast to bacteria, do not produce MKs. 2, 8, 27 These theoretical nutritional data fit nicely into the assessment of human vitamin K 2 consumption in Europe. In a large epidemiological study, the intake of vitamin K 2 was more than 95% comprised of MK-4, MK-8, and MK-9; dietary MK-5 to MK-7 were detected in only low amounts. 10 It should, however, be pointed out that vitamin K consumption assessment based on a dietary questionnaire is not very precise. 38 Even though a large amount of research on vitamin K has been conducted, no precise daily requirement level for vitamin K (likely due to the wide range of vitamin K forms) has been established as yet. It has been suggested that the mean intake of vitamin K ranges from 70 mg/day to 300 mg/day, 10, 11, 31, [39] [40] [41] and the current recommendation by the European Food Safety Authority indicates 1 mg/kg per day is an adequate intake of total vitamin K in both children and adults, including pregnant women. 31 Interestingly, the current average human intake of vitamin K in developed countries is in general above this recommendation, notwithstanding the low frequency of use of vitamin K supplements. 28 An early study did not find substantial differences in the levels of vitamin K 1 , MK-7, and MK-8 between young and older persons. 42 However, regarding what constitutes adequate vitamin K level and how usable vitamin K is in the elderly, current knowledge is unsatisfactory. Other research rather suggests a level of insufficient vitamin K status in the elderly. 29, 43, 44 

There are apparent differences in the absorption of the various forms of vitamin K. In general, bile salts enable the formation of mixed micelles, which allow the uptake of natural lipophilic forms of vitamin K into the enterocytes. The vitamin is further packaged inside the enterocytes into the chylomicrons, and it then enters systemic circulation directly via the lymphatic system 31 (Fig. 1A) . The absorption of vitamin K 2 , in particular long-chain MKs, is excellent and may even be complete due to the co-presence of fat, eg, in dairy products. 8, 9 Vitamin K 1 is much less easily absorbed from the diet than dietary MK-7 or pure MK-4 are. 8, 45 However, pure vitamin K 1 has better bioavailability than dietary vitamin K 1 , pure MK-4, or pure MK-9, but lower than pure MK-7. 8, 23, 45, 46 Dietary vitamin K 1 is tightly bound to plant tissue, as mentioned, so pure vitamin K 1 is better absorbed than the dietary form. We note that there is no difference in the bioavailability of vitamin K 1 from various vegetables, and that cooking does not improve its absorption. 47 Fat, however, increases vitamin K 1 bioavailability from plant sources by about 3 times, but its bioavailability is still much lower than that of commercially solubilized vitamin K 1 . 45 One likely reason is that fat stimulates bile secretion. There are also differences in vitamin K 1 bioavailability between people with different types of diet. Diet involving fast-food eating resulted surprisingly in better absorption of vitamin K 1 than other diets with the same amount of fat ingested. It is possible that vegetable oil offers more easily absorbable vitamin K 1 than whole vegetables. 48 Surprisingly, at least according to in vitro everted intestinal sac experiments, unsaturated fatty acids can decrease the extent of vitamin K 1 absorption. 49 There is also substantial interindividual variability in vitamin K 1 absorption. 45, 48 Linear pharmacokinetics, ie, a linear relationship between dose and serum levels, were observed for both MK-7 and K 1 . 23 Long-term administration of MK-7 results in a marked increase in its serum levels, in contrast to vitamin K 1 , for which serum levels are only slightly above placebo. 23 This is because MK-7 has an elimination half-life of approximately 3 days, while that of vitamin K 1 is about 1-2 hours. 8, 23 Further, MK-9 has been shown to have a long elimination half-life (about 60 hours). 46 This is, however, less than that of MK-7, which suggests that prolongation of the side chain is A chylomicron loaded with vitamin K binds to low density lipoprotein receptor-related protein 1 (LRP1, 5a) or LDL particles loaded with vitamin K bind to LDL-receptors (LDLRs, 5 b), and this results in the uptake of vitamin K into the hepatocyte. A similar process can also be observed with VLDL in the peripheral tissues (not shown). 6: Vitamin K is x-hydroxylated by cytochrome 4F2 in the endoplasmic reticulum. 7: This metabolite is subsequently b-oxidized by mitochondrial trifunctional protein (MTP) to 5 C or 7 C or 10 C (not shown), which are subjected to glucuronidation by UDP-glucuronosyltransferase (GT, 8) in the endoplasmic reticulum. 9: Excretion of glucuronides in feces and urine not directly associated with increase in half-life. In contrast, MK-4 has a short half-life in systemic circulation, and, similarly to vitamin K 1 , its level dropped virtually to zero 24 hours after administration. 45 In rats, the oral administration of various forms of vitamin K resulted in increased plasma and liver levels of vitamin K 3 , and MK-4 to MK-11; MK-1 to MK-3 and MK-12 to MK-14, however, were apparently not absorbed. There was no correlation between plasma and liver levels since, surprisingly, the highest plasma levels were observed after the administration of vitamin K 3 and MK-8; MK-6 reached its highest levels in the liver. 50 Furthermore, vitamin K absorption can be decreased by certain drugs: cholestyramine can decrease vitamin K absorption likely due to the binding of bile acids; rifampicin can decrease vitamin K absorption due to the induction of metabolism; and orlistat forces the patient to decrease their intake of fat since it is associated with unpleasant GIT side effects. [51] [52] [53] [54] As mentioned, human microflora produces MKs. Many bacterial species are able to synthesize vitamin K 2 , and the form synthesized is species specific: Bacteroides produce mainly MK-10 and MK-11, Eubacterium lentum MK-6, Veillonella MK-7, and Enterobacter and Escherichia coli MK-8. 9, 27, 55, 56 There has been some discussion about the extent of absorption of MKs produced by human microflora. 9, 27, 31, 57 One study demonstrated that they are able to be absorbed and reach systemic circulation, at least on a small scale. 58 Furthermore, it is known that large-spectrum antibiotics can inhibit the growth of some vitamin Kproducing bacteria and increase the risk of vitamin K deficiency. 59, 60 It is unlikely that the vitamin K 2 forms with large side chains are absorbed in the distal colon, but they can be absorbed in the terminal ileum, where microflora is still present, and the absorption is further supported by the bile salts there. These large MKs are tightly bound to bacterial inner cytoplasmic membranes, and bile salts are hence required for their solubilization. 27, 61 After intestinal absorption, vitamin K 1 appears to be taken up primarily by the liver. The reason for the predominant clearance of vitamin K 1 in the liver might lie in the fact that vitamin K 1 is transported mainly by chylomicrons, which are taken up by the liver, whereas vitamin K 2 forms are also present in the very-low-density lipoprotein/low-density lipoprotein (VLDL/LDL) particles that are transported from the liver to the extrahepatic tissue. However, vitamin K 2 is also apparently taken up by the liver, where it can be used for carboxylation of proteins. 2,31,46,62 Cellular uptake of vitamin K is managed via lipoprotein receptors. 2 In humans, plasma and serum levels of vitamin K 1 , MK-4, MK-5, MK-6, and MK-8 are low, and are expressed in units of nM or even in lower concentrations. 42, 46, 48, [63] [64] [65] [66] [67] [68] The plasma concentrations of vitamin K 1 and MK-4 in mice and rats are apparently similar to those in humans. 32, 69 There is, as expected, a large difference in plasma levels of MK-7 between European and certain Japanese populations due to the Japanese consumption of natt o. In Europe, the plasma levels of MK-7 are below 1 nM or in the low nM range, 42, 65 while in one recent Japanese study, the mean plasma levels of MK-7 were 15.6 nM. 63 To enable a better comparison to be made, adjustments to triglyceride levels were recommended, but the results were essentially similar. 31, 65 The total body pool of vitamin K 1 is roughly 0.55 mg/kg, but no such data are as yet available for vitamin K 2 . 31

Experimental data has shown that vitamin K 1 and MK-4 are distributed differently. In rats, the same oral dose resulted in much higher levels in the liver of vitamin K 1 than of MK-4, while the opposite was found in the aorta. 70 This was also confirmed in human postmortem liver analyses, in which the amount of vitamin K 1 was always higher than of MK-4. 71 Surprisingly, the sum of the MK-7 to MK-11 levels in the human liver was mostly much higher than that of vitamin K 1 . 71, 72 One study found that there were no differences in the liver vitamin K 1 content and MK-7 to MK-9 content between non-cancerous liver samples and hepatitis or cirrhotic liver samples; however, a clear difference in the MK-10 to MK-13 content was observed. In that study, the subcellular localization of MK-10 and MK-11 was found to be mainly in the mitochondria. 72 The catabolism of vitamin K 1 and of vitamin K 2 shares common mechanisms, beginning with initial xhydroxylation mediated by CYP4F2, followed by shortening of the polyisoprenoic side chain via b-oxidation to carboxylic acids (in 5 C, 7 C or 10 C metabolites, Fig. 1B ), which are glucuronidated and excreted in urine and bile. 2, 31, 73 Hence, the previously mentioned localization of vitamin K 2 forms in the mitochondria could be linked to their metabolism via b-oxidation. The urinary excretion of these metabolites can be used as a marker of vitamin K body status. 74 

The only well-known function of vitamin K in humans is its involvement in the c-carboxylation of a number of proteins (Fig. 2) . Vitamin K is a crucial coenzyme for the posttranslational c-carboxylation of glutamic acid residues on the luminal side of the rough endoplasmic reticulum ( Fig. 3 [75] [76] [77] [78] ). This process requires 2 enzymes [c-glutamyl carboxylase (vitamin K-dependent carboxylase) and vitamin K epoxide reductase (vitamin K epoxide reductase complex subunit 1, VKORC1), which are likely located in close proximity to one another in the membrane of the endoplasmic reticulum 62,79-81 ] as well as carbon dioxide and oxygen. Proteins that are undergoing c-carboxylation contain a homologous sequence of about 18 amino acids long located immediately upstream of the carboxylated domain. This domain binds to the c-glutamyl carboxylase and markedly facilitates the enzymatic reactions catalyzed by this enzyme. Interestingly, there are different affinities between the various vitamin K-dependent proproteins and the enzyme, which might explain some differences in their processing. 75,78,82 VKORC1 must first reduce vitamin K, which is found in food in its quinone form. The reduced form of vitamin K (hydroquinone) is modified by the carboxylase, by deprotonation, and by the subsequent incorporation of oxygen into the alkoxide. This form is a strong base that reacts with the targeted protein and forms a carbanion in the c-position of glutamic acid. This enables carboxylation by c-glutamyl carboxylase. The vitamin K epoxide must then be reduced again into quinone and subsequently hydroquinone by VKORC1, and the reaction continues. 75, 76 Through this process, vitamin K is recycled, so the physiological requirements of vitamin K are relatively low. 83 Formed c-carboxylated proteins are then likely transported via the classical secretory pathway through Figure 2 The general fate of vitamin K-dependent proteins at the site of production. A: Under normal conditions. B: Under vitamin K depletion. 1: Preproprotein is synthesized from mRNA by ribosomes. 2: Preproprotein is targeted to the endoplasmic reticulum (ER), where the ER signaling sequence is removed and the protein is processed by (vitamin K-dependent) c-glutamyl carboxylase. 3: Proprotein is carboxylated when vitamin K is present. 4: Carboxylated or 4a: uncarboxylated proprotein is transported to the Golgi apparatus, where the PRO-sequence is mostly cleaved (some proteins are also glycosylated there, not shown). 4b: In some cases under vitamin K deficiency, the uncarboxylated protein is cleft by proteasome. 5: Mature protein is extruded via a secretory vesicle into the extracellular space the Golgi apparatus, where the prosequence is mostly cleaved, as has been demonstrated for prothrombin by a Ca 2þ -dependent serine proteinase. 84 There are, however, some differences: (1) matrix Gla-protein is exceptional, since the proprotein sequence is also maintained in the mature protein; 85 and (2) the impact of carboxylation on secretion is different (Factor II and Factor X can be secreted both in the uncarboxylated and carboxylated forms, while proteins C and Z are secreted only in the carboxylated forms). 86, 87 In the case of protein C, the uncarboxylated form is decomposed by proteasome, which explains its inability to be excreted. 87 Although mutated c-glutamic acid carboxylated proteins can be cleft intracellularly, 87 secreted c-carboxyglutamated proteins cannot be reused and are excreted in urine. 61 As a result, they have attracted great interest as being potentially useful for diagnostic purposes, as will be discussed later in this article.

There are some differences in the carboxylation process related to the form of vitamin K. Vitamin K 3 Figure 3 Probable steps in the carboxylation process mediated by vitamin K in the endoplasmic reticulum. A protein that contains a PRO-sequence is targeted and subsequently bound to the carboxylase in the first step (A). This binding markedly increases the enzymatic function of the carboxylase. The quinone form of vitamin K is reduced to hydroquinone by VKORC1. Hydroquinone is deprotonated by carboxylase in the next step (B). Oxygen reacts with deprotonated vitamin K hydroquinone to produce alkoxide (C). This strong base deprotonates the c-carbon of glutamyl residue to form a carbanion, which reacts with carbon dioxide (D-E). At the same time, vitamin K epoxide is formed (E). c-glutamyl carboxylation is accomplished and the formed protein is released from the enzyme and further transported to the Golgi apparatus (not shown), while vitamin K epoxide is converted first to vitamin K quinone and then to vitamin K hydroquinone (F) by VKORC1. Data for this figure were taken from Rishavy et al (2004), 75 Down et al (1995), 76 Ayombil et al (2020) 77 and Berkner (2000) 78 Abbreviations: carboxylase, vitamin K-dependent c-glutamyl carboxylase; VKORC1, vitamin K epoxide reductase (MK-0) is inactive and vitamin K 1 is less active than MK-4; with increasing numbers of isoprene units, from MK-4 to MK-10, the carboxylation activity decreases. 88 Also, MK-7 has been shown to be more active than vitamin K 1 in the recovery of vitamin K-dependent synthesis of coagulation factors. 23 In accordance with this, the best catalytic effects on carboxylation in rats were observed for MK-4 to MK-7. Another experiment showed MK-9 also to be active, but the lag time between administration and the effect was longer. Interestingly, vitamin K 3 is not significantly active, even after intravenous administration. 50 This information fits the reported trend in enzymatic vitamin K reduction, since vitamin K 3 was again here inactive. At a concentration of 10 mM, there was no difference between vitamin K 1 and MK-4 reduction, but at 100 mM, vitamin K 1 was a better substrate. MK-7 was much less reduced than either vitamin K 1 or MK-4. 89 Interestingly, there was little difference between the epoxide, hydroquinone, and quinone forms, suggesting that the reduction process is more rapid than carboxylation itself. 88 In the absence of vitamin K, or with a blockade of VKORC-1 by coumarin anticoagulants, vitamin K quinone can be reduced to vitamin K hydroquinone by cytosolic NAD(P)H-dependent oxidoreductase (flavoprotein DT-diaphorase), but vitamin K epoxide is not a substrate for this enzyme, so vitamin K recycling is not enabled. 61 Moreover, at least in rats, DTdiaphorase has very low activity in the arterial wall compared with the liver. 90 This seems to explain why the effects of vitamin K anticoagulants on blood coagulation are easily reversed by the administration of vitamin K 1 , which is rapidly taken up by the liver, whereas the extrahepatic inhibition of protein c-carboxylation is not so easily reversed. Contrarily, MK-4 is able to normalize blood coagulation and reverse aortic calcification. 70 It should be mentioned that, in extrahepatic tissues, vitamin K epoxide reductase like 1 (VKORL1) can also be involved in vitamin K recovery; however, its importance under physiological conditions seems to be low. 2, 81 c-carboxyglutamate proteins Currently, there are at least 18 or 19 human physiological proteins and 1 pathological protein for which posttranslational maturation is enabled by vitamin Kdependent c-glutamyl carboxylation 29, 91 (Table 1) . These proteins are known as Gla proteins (from c-carboxyglutamic acid). They can be tentatively subclassified into a few categories according to their major (or more precisely their most well-known) effects. Many of these proteins have, however, more complex effects. There are 7 proteins involved in blood coagulation (coagulation factors II, VII, IX, and X and proteins C, S, and Z), 4 or 5 proteins primarily have effects on connective tissue mineralization or have a closely related role (4 well-documented Gla proteins: matrix Gla protein (MGP), osteocalcin, Gla-rich protein, and nephrocalcin; if the fifth, periostin, is a Gla protein is disputable), 4 are transmembrane receptors (prolinerich Gla proteins 1-4), 1 is a growth-factor-like signaling molecule, 1 binds to hyaluronic acid in the extracellular matrix, and the last is c-glutamyl carboxylase itself. 92 Given the necessity of this enzyme for other Gla protein formation, it is not surprising that in contrast to other Gla proteins, it does not necessitate the proprotein activation of the enzyme. The individual vitamin K-dependent enzymes will be briefly characterized in Table 1 and the following text.

The first well-described proteins dependent on vitamin K were 7 players in the coagulation cascade, including (pro)coagulatory factors II (thrombin), VII, IX, and X, as well as anticoagulant proteins C, S, and Z. [93] [94] [95] [96] [97] Coagulation factors VII, IX, and X, and protein C have high structural homology both in gene and protein structure and organization. 94, 97 In addition to c-glutamyl carboxylation, they also undergo another specific posttranslational modification -the b-hydroxylation of aspartic acid or asparagine. 94 In general, we can classify them as proenzymes (the majority being zymogens) or co-factors (protein S and Z). Proenzymes act as serine proteases upon activation. In fact, they share several features with the digestive enzymes chymotrypsinogen/ chymotrypsin and trypsinogen/trypsin. In contrast to these nonselective enzymes, the coagulation factors with enzymatic activity mentioned here have an additional polypeptide chain, which procures narrower substrate specificity. 94, 97 All of these anticoagulation/ coagulation factors, with the exception of protein S, 98 are synthesized mainly in the liver. The Gla content ranges between 9 and 13. Binding to calcium leads to modifications in these Gla proteins, and the resultant structural changes cause the exposure of the phospholipid-binding domain, which anchors them in membranes, exposing in particular phosphatidylserine. 61, 94, 97, [99] [100] [101] This is apparently the crucial step, since in absence or inhibition of vitamin K regeneration, the coagulation process is inhibited. The roles of the vitamin K-dependent coagulation factors are shown in Fig. 4 . Since protein Z is the newest member, it will be discussed here briefly. This anticoagulant protein shares similarities with other vitamin K-dependent proteins involved in the coagulation cascade, but lacks a functional serine protease activity. 100, 102 Its complex with protein Z-dependent protease inhibitor (PZI) binds to factor Xa on the phospholipid surface and blocks it. It seems to be important during pregnancy, since low levels of protein Z are associated with several pregnancy complications. 103 Another important c-carboxyglutamic acid protein, osteocalcin (also known as BGP and bone Gla protein, Fig. 5 ), has been largely associated with its effect on bones. Its physiological role is apparently more complex and clearly not fully understood. Most research has been carried out in mice, which however, have 2 genes for osteocalcin, together with 1 osteocalcin-related gene. This is an important difference from rats and humans, which have only 1 osteocalcin gene. For this reason, caution should be taken when interpreting animal studies. Some, but not all, results have been confirmed in limited number of human studies. [104] [105] [106] [107] [108] [109] Osteocalcin is synthesized mainly in osteoblasts, with a lesser contribution from odontoblasts and hypertrophic chondrocytes. Three glutamic acid residues are c-carboxylated, [104] [105] [106] and the signal sequence is removed by peptidases. 77 This carboxylated protein is secreted from the intracellular vesicles into the bone matrix. This form has, as is the case for coagulation factors, a high affinity for calcium ions, and hence it binds to these ions in hydroxyapatite. It should be mentioned that it represents about 15%-20% of the noncollagen proteins in the matrix, which renders it the most frequent noncollagen protein in bones. 108 It was once thought that carboxylated osteocalcin initiated hydroxyapatite formation, whereas the current data rather point out that it regulates the hydroxyapatite size and is crucial for the alignment of apatite crystals to collagen fibers. This ability is considered to be the reason for osteocalcin's effect on bone strength and its protective properties against bone fractures. 104, 107 The data on other systemic effects of osteocalcin are less conclusive. Regardless, during bone resorption, the pH in bones decreases, which leads to osteocalcin decarboxylation and its release into the circulation. 105, 106 Un(der)carboxylated osteocalcin is considered by most authors to be a hormone. 104, 105, 107, 108 It has been shown, at least in animal studies, to affect glucose and lipid metabolism and to modulate fertility; it even has central nervous system effects, and could be involved in some types of cancer. [104] [105] [106] 108 The largest data are on the cross-link between osteocalcin and insulin, and un(der)carboxylated osteocalcin has been shown to improve insulin sensitivity and glucose tolerance. It causes the release of insulin, mainly via incretin glucagon-like peptide-1. Insulin, on the other hand, stimulates bone resorption and promotes the decarboxylation of osteocalcin, enabling its systemic effects. However, these data have been only partly confirmed in human studies; apparently, various factors such as gender, concomitant disease, and age influence the final effect. [105] [106] [107] [108] [109] In addition, the information available on un(der)carboxylated osteocalcin receptors is not fully conclusive. Un(der)carboxylated osteocalcin has been shown to bind to GPRC6A, a Gprotein-coupled receptor family C group 6-member A, in most tissues, and many of the above-mentioned effects are likely mediated by its binding to this receptor. 108 At least in mice, another receptor mediates its effects in the brain. 104, 105 One candidate, Gpr158, has recently been suggested. 110 In addition, un(der)carboxylated osteocalcin is considered to be a marker of bone quality, since its levels correlate with bone mineral density and are increased in bone fractures. 111, 112 Supplementation with vitamins K 1 or K 2 decreases the serum un(der)carboxylated osteocalcin level or the ratio of un(der)carbocylated to total osteocalcin 68, 109, [113] [114] [115] [116] ; hence, un(der)carboxylated osteocalcin is considered one of the markers of vitamin K deficiency. 74 As carboxylated osteocalcin modulates extracellular matrix mineralization in bones, another protein that is dependent on vitamin K, MGP, blocks this process in other tissues where it would be pathological. In fact, it is synthesized in vascular smooth muscles and chondrocytes. It protects arteries and cartilage against mineralization. 85, 117 Apparently, both carboxylated osteocalcin and activated MGP act at the local level, ie, at the site where they are produced. 85, 118 In contrast to uncarboxylated osteocalcin, it is not known whether uncarboxylated MGP has any systemic effects. Moreover, MGP is not only carboxylated in 5 of 9 glutamate A: Resting state: coagulation factors are found in inactive forms in circulation, phosphatidylserine is not at the surface of the platelets (see the upper magnification), and tissue factor (TF) is not in direct contact with the blood (see the lower magnification). B: Activation of blood coagulation: in case of vascular damage, TF on subendothelial cells is now available for factor VII (FVII), which is activated. Platelet activation leads to modifications in the structure of the plasmatic membrane, with the exposure of phosphatidylserine on its surface. C: Activation of vitamin K-dependent coagulation factor: activated factor VII (FVIIa) cleaves factor IX (FIX) into an active enzyme (factor IXa, FIXa), which needs activated factor VIII (FVIIIa) for its activity. This complex activates factor X (FX) into an active enzyme (FXa), which needs activated factor V (FVa) for its activity. The whole FXa, FVa, calcium and phospholipid complex is also known as prothrombinase, and it activates thrombin (factor II, FII). Factor V (FV) or factor VIII (FVIII) are activated either by FXa or thrombin (not shown). D: Regulatory anticoagulant vitamin Kdependent factors: thrombin with thrombomodulin (TM) cleaves inactive plasma protein C (PC) into the active enzyme APC. For its enzymatic activity, APC also needs protein S (PS). PS does not require proteolysis in order to be active in PC-catalyzed lysis. This complex cleaves both FVa and FVIIIa. Protein Z (PZ) is a cofactor of PZ-dependent protease inhibitor (PZI), which blocks the enzymatic activity of FXa residues, it also undergoes serine phosphorylation in 3 of 5 serine residues (Fig. S3 in the Supporting Information online 85 ). Carboxylation is again carried out by c-glutamyl carboxylase, while phosphorylation occurs by the Golgi casein kinase. 85, 119 The function of phosphorylation is currently unknown, but it might be responsible for regulating trafficking. The inhibition of soft tissue calcification might be accomplished by its binding both to calcium and to bone morphogenic protein-2 (BMP-2). 85, 117 Solid evidence of the importance of MGP as an inhibitor of soft tissue calcification can be derived from MGP-deficient animals, which generally die within 2 months due to the calcification and subsequent rupture of the thoracic or abdominal aorta. 120 Extensive arterial calcification is also observed in patients with Keutel syndrome, in which mutation of the MGP gene is present. 85, 121 Since the MGP can be presented as uncarboxylated-unphosphorylated, or uncarboxylated-phosphorylated and vice versa, or carboxylated-phosphorylated, it has been tested for diagnostic purposes. The results are ambiguous, but high levels of uncarboxylated-unphosphorylated MGP is considered to be a marker of vitamin K deficiency. It decreases after administration of vitamin K, but is dependent on age . 68, 83, 85, 91, 117, 122 Periostin: Periostin is another suggested c-carboxyglutamyl protein, or more precisely glycoprotein, with an effect on bones. It was initially discovered in a mouse osteoblastic cell line and was formerly named osteoblastic-specific factor 2 (OSF-2). 123 The current name derives from its presence in the periosteum of long bones. It is, however, widely expressed both in the fetus (6). This is a crucial step in bone formationthe correct alignment of collagen fibers with hydroxyapatite (7). When osteoresorption takes place, osteoclasts decrease the pH level in the bones (8) ; this can lead to the decarboxylation of osteocalcin to form un(der)carboxylated osteocalcin, which is released into the systemic circulation (9), where it could exert its hormonal effect, in particular by binding to the GRP6CA receptor (10) and in adults, and its effects are clearly not limited to the bones. It plays an important role in the growth and repair of damaged connective tissues (eg, bones, teeth, skin, tendons, and valves). [124] [125] [126] [127] It stimulates inflammatory reactions (eg, by the activation of NF-jB), 125, 126, 128 which is likely linked to the healing process. On the other hand, it is also associated with fibrosis. 125, 127, 128 On the molecular level, it is secreted into the extracellular matrix, where it can bind to several integrins and initiate signaling cascades associated with protein kinases. 124, 126 Interestingly, the c-carboxylation of the possible 28 glutamyl residues has not been widely investigated, and there is a report indicating that gamma-glutamyl residues were not detected in periostin, either in lungs from patients suffering from idiopathic fibrosis or in a cell culture. 129 It is, hence, not clear whether it is a Gla protein. Periostin has been suggested to be a marker of type II immune reactions in chronic inflammatory diseases such as asthma and atopic dermatitis, as well as a predictor of the effect of biologic drugs acting on this level. It is also thought to be a useful marker when making risk assessments of developing fractures and when investigating left ventricular function after myocardial infarction and chronic kidney disease. [124] [125] [126] [127] 130 Given the diversity of these processes, it cannot be considered a selective marker. Moreover, it is highly expressed in youth, most likely due to its impact on bone growth. 125 Contrarily, smoking decreases its expression. 126 There have been some attempts to develop drugs that influence periostin or its receptors. 125, 127, 131 Periostin has 4 isoforms designated 1-4, which differ in the presence/absence of particular exons, leading to varied (sometimes opposite) effects. 91 This can complicate the deciphering of its physiological function and its possible use as a therapeutic target.

Nephrocalcin: Nephrocalcin is another acidic glycoprotein containing a Gla sequence. It is produced in the human kidneys. Nephrocalcin has 4 similar forms that differ in Gla content. Normal form A contains 3 Gla residues, while B and C only contain 2, and D none. In particular, normal form A binds 4 atoms of calcium and has a high affinity to calcium oxalate. It inhibits calcium oxalate crystallization. Interestingly, uncarboxylated nephrocalcin has been isolated from the urine and even from the kidney stones of patients suffering from them. Increased nephrocalcin has been detected in the urine of patients with renal cell carcinoma. [132] [133] [134] [135] [136] Gas6: Gas6, a product of growth arrest-specific gene 6, shares 43% homology with protein S and has the same protein organization. 137, 138 In contrast to protein S, plasma Gas6 levels are very low, in subnanomolar concentrations, and it is not produced by the liver. 98, 138 Its target is the so-called TMA, three tyrosine kinase receptors that gave name to this family-Tyro3 (Sky), MerTK (c-mer), and Axl. Gla residues bind to calcium ions, and this causes a structural modification, which likely enables the binding of Gas6/protein S to the TMA receptors. Protein S binds only to the former 2 receptors and contains a thrombin-sensitive cleavage site, in contrast to Gas6. 98, 138 In fact, Gas6 is likely not involved in coagulation cascade-like protein S, but on the other hand it is involved in platelet aggregation. 139, 140 The pathophysiological function of Gas6 is extensive. 138, 139, 141, 142 It inhibits inflammatory reactions and plays a role as a growth factor, which may reflect its impact on cancer growth. Gas6 overexpression is observed in several cancers, and in general its expression predicts a poor prognosis. 142 In addition to the aforementioned receptor binding, the Gla residues (via calcium and phosphatidylserine) are attached to damaged tissues in line with its role as a growth factor. 138, 139 It is also involved in tissue fibrosis. 141 Gas6 has also been suggested to affect vascular calcification. However, this has recently been refuted. 143 Since protein S binds to the same 2 receptors as Gas6, there may be an overlap between some of their functions, but decisive data on this are missing. The crucial involvement of protein S in blood coagulation is undisputable. 98 One good example is the clear risk of venous thromboembolism in humans with rare cases of protein S deficiency. 144 Interestingly, no other apparent important symptoms in these patients are described, which belies the other physiological role(s) of protein S. In any case, protein S is involved in immune and vascular system regulation. It should be emphasized that approximately 60% of protein S circulates with complement component C4b. It has been speculated that the C4b-protein S complex can bind via its Gla domain to the surface of certain membranes and in this way regulate the complement activity. 98 Heavy chain 2 of inter-a-trypsin inhibitors: Two cglutamic acid carboxylated residues have also been discovered in heavy chain 2 of inter-a-trypsin inhibitors (gene ITIH2). The function of heavy chains of the intera-trypsin inhibitor family is interaction with hyaluronic acid, with a possible impact on maintaining the integrity of the extracellular matrix. Originally, the inter-atrypsin inhibitor was designated a protein inhibitor. Its enzyme inhibition properties are rather weak, especially when compared with other physiological inhibitors. The structure is specific. It is composed of a common light chain (known as bikunin) to which 1 or 2 heavy chains are attached by a unique ester bond between the carboxyl group of the aspartic acid terminal residue and a C6-hydroxy group of the N-acetyl galactosamine residue of the chondroitin sulphate of the light chain. The 6 heavy chains that are currently known are structurally related. The function of the Gla residues of heavy chain 2 is unknown, since 3 other characterized heavy chains (1, 3, and 4) do not contain them . [145] [146] [147] [148] [149] Proline-rich Gla proteins and transmembrane Gla proteins: Targeted amino acid sequence research resulted in the discovery of other Gla proteins. First, 2 proteins with specific proline-rich cytoplasmic regions were found. They were, hence, named proline-rich Gla proteins 1 and 2 (PRGP1 and PRGP2). They are widely distributed, with the highest expression of PRGP1 being in the spinal cord and of PRGP2 being in the thyroid gland. 150 PRGP1 expression is limited to some tissues, whereas PRGP2 expression is more extensive. 151 Further research showed that carboxylated PRGP2 is localized on the cell surface, and it was suggested that it has an intracellular binding partner YAP (Yes-associated protein). 152 Later, other structurally similar members of the vitamin K protein family were discovered. They were initially named transmembrane Gla proteins 3 and 4 (TMG3 and TMG4). They are also known as PRGP3 and PRGP4. In fact, TMG3 is similar to PRGP1 and TMG4 to PRGP2. Both TMG3 and TMG4 are widely expressed in both fetal and adult human tissue. 151 Gla-rich protein: The last-discovered member of the vitamin K-dependent proteins is Gla-rich protein (GRP). In comparison with other Gla proteins, it contains the highest number of c-glutamic acid moieties, which span over the entire protein. It was initially discovered in the calcified cartilage of Adriatic sturgeon. Its ortholog is found in many species, including humans. 153 In humans, GRP is widely expressed. Carboxylated GRP apparently acts as an inhibitor of tissue calcification, in particular of vascular and chondrocyte calcification, possibly by direct interaction with calcium. [154] [155] [156] [157] [158] Its anti-inflammatory properties have also been documented. 157 Given its similar function to osteocalcin and MGP, it is not surprising that it has a similar expression pattern, at least in some pathologies. 154, 157 Higher GRP accumulation has been observed in calcified aortic valves and atheromatous regions. Interestingly, foam cells contain lower levels of carboxylated and higher levels of uncarboxylated GRP. 154 Similarly, higher GRP gene expression but only uncarboxylated GRP were detected in fibroblast-like synoviocytes from osteoarthritic patients. At the same time, lower levels of c-glutamyl carboxylase and VKOR1 were found in these samples. 157 Transthyretin: There is one other Gla protein that is contrarily considered to be pathological. Physiologically, transthyretin, a protein involved in the transport of the thyroid hormone thyroxine and retinol, is not a Gla protein. But in rare cerebral steno-occlusive Moyamoya disease, its c-glutamic acid carboxylated counterpart was observed in cerebrospinal fluid. It has been observed in 71% of patients suffering from this disease. [159] [160] [161] Some researchers have suggested that vitamin K might play an additional role(s) in the human body, such as directly binding to intracellular receptors, interfering with enzymes, or having antioxidant and/or antiinflammatory activities. 2, 44, 162 

The symptoms of vitamin K deficiency are spontaneous cutaneous purpura, epistaxis, gastrointestinal, genitourinary, gingival, or other bleeding. Although vitamin K deficiency is not common in adults, it can occur for several reasons: (a) poor vitamin K dietary content, (b) many pathological states (eg, liver disease, cholestasis, cystic fibrosis, alcoholism, malabsorption states (including inflammatory bowel disease), and bariatric surgical intervention), and (c) pharmacotherapy with several drugs (mainly coumarin-based anticoagulants, but also the drugs previously mentioned that interfere with the absorption, metabolism, and synthesis of vitamin K, such as rifampicin and antibiotics). 2, 29, 54, 111, 163, 164 In new-born babies, there is also a risk of hypovitaminosis, since (a) they have poor vitamin K stores, (b) vitamin K levels are low in breast milk, and (c) immature GIT microflora is likely not a source of the vitamin. In line with these findings, exclusively breast-fed infants are more susceptible to the development of vitamin K hypovitaminosis than formula-fed children. 31, 165, 166 

Making an accurate bioanalysis of vitamin K deficiency is not an easy task. Because it is among the most lipophilic and least abundant fat-soluble vitamins, it is extremely difficult to determine in human biological samples, even when using the most modern instrumentation. It can be measured directly in plasma, but there are many forms of vitamin K, and an abnormal lipid profile may also affect the result. 74 Chromatographic methods have become dominant for directly measuring vitamin K. An example of such analysis is shown in Fig. S4 in the Supporting Information online. 167 Unfortunately, the complex preparation of the samples also relates to the detection methods, such as mass spectrometry (MS), which has become the detection method of choice in vitamin K determination. There are many methods used for measuring systemic vitamin K levels. Some of them are briefly characterized in Table 2 . Vitamin K deficiency will lead to a state in which Gla proteins cannot be carboxylated and are hence mostly released in their uncarboxylated form. 61 Uncarboxylated vitamin K proteins can thus be alternative targets for quantitation, but these assays are not yet standardized, and some suffer from important drawbacks 74, 163 or there are other aspects to consider. These proteins were formerly known as PIVKA (proteins induced by vitamin K absence). However, not all uncarboxylated proteins are secreted. 86, 87 Furthermore, elevated expression of proteins can overwhelm the intrinsic carboxylation system and lead to the production of uncarboxylated proteins, as was documented with PRGP2. 152 Additionally, in contrast to coagulation Gla proteins, other Gla proteins Abbreviations:K 1 , Vitamin K 1 (phylloquinone); K 2 , Vitamin K 2 (either the methodology does not allow differentiation between individual K 2 forms or not specified); LC-PDA/UV, liquid chromatography with photodiode/ultraviolet detection; LC-FLD, liquid chromatography with fluorescence detection; LC-EC, liquid chromatography with electrochemical detection; LC-CL, liquid chromatography with chemiluminescence detection; CL, chemiluminescence; LC-MS, liquid chromatography with mass spectrometry detection; SFC-MS, supercritical fluid chromatography with mass spectrometry detection are apparently not fully carboxylated, even in healthy organisms. 28, 109, 156 The time aspect should also be considered, since some coagulation factors have shorter or longer half-lives. 188 PIVKA II (des-carboxyprothrombin) is an earlier-known marker of vitamin K status with low sensitivity. 68 It is (1) increased in many infants, and (2) its normalization takes months. In adults, it is clearly diagnostically better than prothrombin time or partial thromboplastin time, which are also affected by liver diseases, hematological diseases, and some other diseases, and their sensitivity in mild vitamin K deficiency is also low. 31, 74, 163 Factor VII has also been tested, due to its short half-life, but it is not a sensitive marker. 31 Another tested marker of poor vitamin K content in the body, and in particular in the bones, is uncarboxylated osteocalcin. The levels of total osteocalcin vary, so the percentage of uncarboxylated osteocalcin seems to be a more suitable marker. However, the available data do not fully support this, and estrogen levels might influence it. 31, 163 Uncarboxylatedunphosphorylated MGP as a marker of vitamin K deficiency was mentioned above. A sensitive marker of vitamin K 1 dietary depletion is the urinary concentration of Gla residues. However, data on vitamin K 2 deficiency in relation to this marker are not available, no parameter cut-off for vitamin K deficiency has yet been established, and the measurement usually requires 24 h urinary collection. 31, 68, 74 Moreover, supplementation of vitamin K did not modify urinary Gla residues, in contrast to the clear increment in the carboxylated:total osteocalcin ratio. 64 Another possibility is the measurement of urinary menadione (vitamin K 3 ) excretion, which also changes in response to vitamin K depletion or repletion. 68 

Approved indications of vitamin K administration are not extensive. Vitamin K is indicated for the prevention of hemorrhagic disease in newborns or as an antidote to correct vitamin K antagonist overshooting or poisoning, since vitamin K antagonists are also used as rodenticides. Oral administration is preferred in most cases. Prophylaxis with vitamin K markedly decreased the vitamin K deficiency in new-born babies due to their above-mentioned poor vitamin K stores. 31, 165, 166 It has been suggested that preterm infants have an even greater risk of vitamin K deficiency, but there are currently insufficient data in relation to the prevention of this deficiency. 189 Importantly, the effect of vitamin K administration on coagulation normalization is very rapid. 166 Analyzing the 18 or 19 Gla proteins formed by presence of vitamin K, it is apparent that the effects of vitamin K are not limited to blood coagulation. First, their effects on bone and vascular calcification are becoming more apparent. It is, hence, not surprising that a large amount of research has been dedicated to investigating whether increased intake and/or supplementation with vitamin K can positively affect osteoporosis, fractures, and cardiovascular diseases. 29, 31, 163, 165 An effect on cancer has also been suggested, and there are reports on a possible relationship between vitamin K and cognitive function, highlighting its role in brain physiology. 190 There are solid background data justifying testing of the possible use of vitamin K in the treatment of osteoporosis and the prevention of bone fractures: (1) there is the above-described positive effect of osteocalcin on bones, (2) a number of studies have reported that low serum vitamin K 1 levels or low vitamin K 1 intake and high un(der)carboxylated osteocalcin levels are associated with an increased risk of fractures, (3) the pharmacological inhibition of vitamin K reduction due to binding to VKORC1 by coumarin anticoagulants such as warfarin may affect bone quality, (4) similarly, the absence of VKORC1 is associated with calcification abnormalities, and (5) vitamin K was a successful treatment in many animal studies (eg, in the tail-suspension rat model of bone volume loss, or in a sciatic neurectomized rat model of osteoporosis). 2, 111, 163, [191] [192] [193] [194] [195] Most epidemiological as well as clinical studies have demonstrated that the intake of vitamin K can have a positive effect on the bones. On the other hand, this was not found in all studies. Additionally, 2 of the positive effect trials were retracted , so the real effect of vitamin K on osteoporosis remains rather elusive. 196 A meta-analysis of 4 prospective cohort studies and 1 nested control study, including a total of almost 81 000 subjects, suggested that the population with the highest intake of dietary vitamin K 1 had a 22% lower risk of fractures. A subgroup analysis documented that the effect was observed after 10 years. The authors also observed that an increase of daily vitamin K 1 intake by 50 mg was associated with a 3% lower risk of fracture. 197 There are also meta-analyses reporting the effect of vitamin K supplementation. The first meta-analysis of 17 studies showed that vitamin supplementation had a positive effect on bone mineral density in the lumbar spine, but not at the femoral neck. Moreover, the net effect on the lumbar spine was questioned by the authors, since it was observed only in Asian and not in Western studies. It was also limited to women and to vitamin K 2 . 198 The latter is not so surprising, given the observed differences in the pharmacokinetics and pharmacodynamics between these forms. The authors speculated that a greater effect might be observed in secondary bone loss caused, eg, by pharmacotherapy with glucocorticoids. 198 Additionally, in another meta-analysis, it was shown that the effect of vitamin K 2 is observed mainly in postmenopausal women with osteoporosis. 113 This investigation of 19 randomized controlled trials with 6759 participants reported the improvement of lumbar and forearm bone mineral density, in particular, after long-term use by osteoporotic women. There was no significant improvement in non-osteoporotic women. The doses in the analyzed studies were mostly 45-90 mg/day of MK-4 or 100-180 mg/day of MK-7 . The overall effect on fractures was numerically high (a decrease in risk by 36%), but not significant. However, a significant decrease of 53% was reported after rejecting 1 study that induced heterogeneity. Interestingly, an older meta-analysis, which included mainly trials with MK-4, showed a massive and even more potent reduction of risk of hip, vertebral, and nonvertebral fractures. 199 This analysis was, however, apparently influenced by 2 retracted trials. 196 Also, the newest meta-analyses did not clarify the issue, since when clinical trials with different vitamin K forms were assessed, vitamin K supplementation decreased the risk of clinical fractures but not vertebral fractures, although, when assessing the effect of randomized studies with solely MK-4, there was no significant effect on any fracture incidence. Both meta-analyses, however, confirmed a significant effect of vitamin K supplementation on lumbar bone mineral density. 116, 200 The net effect could also have been influenced by different concomitantly used drugs and comparators (eg, vitamin D, bisphosphonates). Summing up the available information, some positive effect of vitamin K on osteoporosis and decreased frequency of bone fractures seems to be possible, but apparently a long period of treatment is needed to achieve this effect. In addition, vitamin K may not be able to stop a reduction in bone mineral density or content in some parts of the skeleton, but may slow down its progression. Again, this effect may require long-term treatment. The effect of MK-7 supplementation on femoral bone is one example. It was not present after 1 or 2 years of treatment; 3 years were needed to document its positive influence. 115 Based on this information, longterm supplementation with vitamin K in postmenopausal women with osteoporosis might have some potential utility, in particular, given its negligible risk of serious side effects. This is also supported by the fact that MK-4 in relatively high doses is registered in Japan for postmenopausal osteoporosis. 61, 111 The main reason for speculating that vitamin K has positive cardiovascular effects stems from its involvement in the synthesis of MGP. As has been described here in detail, MGP blocks the calcification of arteries. It need not be emphasized that the calcification of arteries is an independent risk factor for the development of coronary heart disease and coronary events. 201, 202 In addition, several clinical trials have reported that vitamin K antagonists promote atherosclerotic calcification. 29 There has been a lot of discussion about whether there is subclinical hypovitaminosis in certain population groups. A substantial under-c-carboxylation of vitamin K-dependent proteins has been found in postmenopausal women, 64, 114 and about one-quarter of a cohort ranging from 42 years to 89 years had very low plasma vitamin K 1 levels. 66 If this theory is correct, then vitamin K supplementation should reduce the risk of arterial calcification and coronary artery disease. Epidemiological studies from the Netherlands have found that vitamin K 2 , but not vitamin K 1 , limits coronary calcification and decreases the incidence of and mortality due to coronary artery disease. 10, 11, 203 Also, a very recent Norwegian study has found that higher intake of vitamin K 2 decreased the incidence of coronary artery disease, but vitamin K 1 had no effect, notwithstanding the fact that higher quartiles of vitamin K 1 intake, compared with vitamin K 2 intake, were associated with higher physical activity. 204 The lack of effectiveness of vitamin K 1 was confirmed by the extension of the Dutch EPIC study with a median follow-up of 16.8 years. The same study, however, also reported only a tendency of vitamin K 2 , particularly its long chain forms, to reduce cardiovascular mortality. 205 Considering the differences between the pharmacokinetic and pharmacodynamic data on vitamins K 1 and K 2 encompassing: the forms of vitamin K 2 having longer half-lives, a more potent effect, and higher concentrations in extrahepatic tissues, a stronger effect of vitamin K 2 over vitamin K 1 would be expected. On the other hand, a similarly designed Spanish epidemiological study found a different outcome. Both forms of vitamin K decreased cardiovascular mortality, but the decreases were insignificant. However, an increased intake of vitamin K 1 , but not vitamin K 2, during the study significantly attenuated cardiovascular mortality. 206 Explaining this difference is not easy. One should emphasize that the intake of vitamin K 1 was apparently much higher in the Spanish study, which is quite logical, given the difference in vegetable consumption between these two countries. Although the populations were different, this was likely not the reason: on the one hand, a higher consumption of vitamin K 1 was linked with a healthier lifestyle in the Spanish study, but in the Dutch EPIC study, it was linked with hypertension, hypercholesterolemia, and diabetes. On the other hand, higher vitamin K 2 consumers in the Rotterdam Study had a rather healthier lifestyle. There is also an American epidemiological study in which the average consumption of vitamin K 1 was even lower. This study reported that vitamin K 1 decreased the incidence of coronary heart disease, but had no effect on mortality due to this disease or on the risk of stroke. 39 Interestingly, the authors themselves reported that a positive impact was seen in connection with dietary patterns associated with a high phylloquinone diet. Indeed, higher intake of vitamin K 1 generally reflects healthier diets and lifestyles. 68 Another study found that patients suffering from chronic kidney disease with inadequate total vitamin K intake (K 1 þ K 2 ) had higher cardiovascular and allcause mortality than those with adequate intake. 40 A recent meta-analysis of 21 observational studies found that higher intake of either vitamin K 1 or vitamin K 2 was associated with a decrease in incidence of coronary artery diseases, with the impact of vitamin K 2 being numerically higher. Regardless, neither form of the vitamin impacted the cardiovascular fatalities. 207 Another meta-analysis confirmed that neither form was found to have an impact on cardiovascular mortality. 208 It should also be emphasized that all of these studies were epidemiological studies using questionnaires to assess dietary habits with its known limits 38, 68 ; for a definite conclusion on the effect of vitamin K on cardiovascular diseases, prospective clinical studies are needed. The current clinical studies have not brought decisive data: supplementation with vitamin K 1 slightly reduced coronary artery calcification in older persons, while supplementation with MK-7 did not influence femoral arterial calcification in diabetic patients. 67, 209 A meta-analysis of 3 controlled trials also found that vitamin K supplementation decreased vascular calcification. It should, however, be mentioned that this effect was largely driven by one single study. 122 The most recent systematic review was not conclusive about the possible impact of vitamin K on calcification of major vessels. 210 On the other hand, higher levels of uncarboxylated dephosphorylated MGP, as the above-mentioned marker of vitamin K deficiency, were found to be associated with higher incidence of coronary artery disease and even cardiovascular mortality. 207 This was, however, not fully confirmed in another meta-analysis. 122 It should also be mentioned that higher levels of uncarboxylated dephosphorylated MGP can be a consequence of administration of vitamin K antagonists and can, therefore, simply reflect that these patients are more ill. 207, 211, 212 Hence, positive cardiovascular effects are not clearly documented for either form of vitamin K, and clinical longterm studies are definitely needed. Based on their negligible toxicity, vitamin K 1 and/or vitamin K 2 might be potentially administered to patients suffering from coronary artery disease in cases where there is an assumption of low vitamin K levels. They should not, however, be treated with vitamin K anticoagulants. 28 There are additional data concerning the effects of vitamin K on all-cause mortality, cancer, and diabetes. Higher levels of uncarboxylated dephosphorylated MGP are associated with all-cause mortality, and this supports the possible impact of vitamin K. 207, 208, 212 The Spanish PREDIMED epidemiological study found that a higher intake of vitamin K 1 or vitamin K 2 decreased both all-cause mortality and cancer mortality. 206 The effect of vitamin K 1 on all-cause mortality was contrarily not observed in the most recent study data obtained from the extension of the Dutch EPIC study, which also reported a positive influence of long-chain vitamin K 2 forms only, and then only in 1 of 3 models. 205 Also, a recent meta-analysis did not find an impact from either form of vitamin K on all-cause mortality. 208 The EPIC-Heidelberg study did not find any relationship between vitamin K 1 and cancer incidence or mortality, but it did document that vitamin K 2 decreased mortality due to cancer. This finding was likely driven by the effect of vitamin K 2 on lung cancer. Vitamin K 2 also decreased the risk of developing lung and prostate cancer, but had no effect on colorectal or breast cancer. 33 The existing literature has some data on the anticancer effects of vitamin K. Most data relate to synthetic vitamin K 3 , which (on a molecular basis) is apparently a much more active anticancer drug than either vitamin K 1 or vitamin K 2 . Vitamin K 3 can redox-cycle, and this represents the major difference from the natural forms of vitamin K, which have a side chain blocking this pro-oxidation mechanism. Interestingly, although the natural forms of vitamin K are less active, a few clinical phase I/II studies have reported some promising data for both. 213 Higher vitamin K 2 has been suggested to slightly decrease the risk of developing diabetes mellitus type II, while vitamin K 1 had only a tendency in this respect. Vitamin K 2 also very slightly increased HDL cholesterol and decreased systemic inflammation. 34 Since the scientific evidence is limited in such cases, no recommendations can be given at the moment.

An emerging issue is the impact of vitamin K on age-related diseases. Low vitamin K levels are suggested to be associated with functional decline, osteoarthrosis, sarcopenia, mobility limitation and disability, and frailty in older persons. These conditions might be at least partly related to low-grade inflammation and pathological calcification. Both of these processes can be related. Vitamin K can, hence, bring some benefit when they occur. The mechanism has not been elucidated, but it might be associated with inhibition of soft tissue mineralization, anti-inflammatory effects, and impact on the mitochondria. 29, 44 Very recent research on vitamin K has brought a surprising discovery: vitamin K 2 (form not specified) was found in a docking study to be the best ligand for free fatty acid binding pockets in the SARS-CoV-2 spike protein, and hence could potentially impact its binding to human cells via the ACE2 receptor. 214 

There have been almost no reported cases of natural vitamin K systemic toxicity, either in animals or in humans. There has been some concern that a high intake of vitamin K could result in overcoagulation. However, this has not been observed, possibly due to the limited sites for c-carboxylation. Contrarily, very high doses of vitamin K can paradoxically cause hypoprothrombinemia, as has been documented in rare human case reports. In animals, massive doses have led to hemorrhages and anemia. 215 Available data in humans show that 10 mg/day of vitamin K 1 given for 1 month is not associated with any adverse effects. This is in line with the findings from animal experiments, in which even 2 g/kg for the same period of time was safe. 31 In general, the reported side effects of vitamin K have only been local, eg, minor gastrointestinal complaints and skin rashes after vitamin K 2 , which disappear after discontinuing administration. 9, 28, 113, 116 Clinically, intramuscular administration of vitamin K 1 is not very convenient due to pain at the site of injection and skin bruising. Therefore, more modern approaches such as microneedles are being examined. 29 It should be mentioned, however, that vitamin K 3 in high doses can lead, in a dose-dependent manner, to a toxic reaction, including hemolytic anemia, particularly in new-born infants. This reaction is likely associated with the redoxcycling of this vitamin, which does not occur in natural vitamins K 1 and K 2 due to the absence of unsubstituted position 3, which is highly reactive and binds thiol groups to form thioethers. 2, 216 Furthermore, preparations of vitamin K 1 available for intravenous administration are sometimes associated with anaphylactoid reactions. Because of the lipid solubility of vitamin K, the preparations are aqueous colloidal suspensions that can induce anaphylactoid reactions. However, liposomal preparations can avoid this adverse reaction. 217 CONCLUSIONS Vitamin K has been traditionally linked with coagulation. Although this connection is correct, vitamin K has many other roles in human physiology. Vitamin K is necessary for the posttranslational modification of 18 or 19 proteins, among which 11 or 12 are not related to coagulation. In particular, its involvement in connective tissue calcification has stimulated intense research, although the data are still inconclusive. Vitamin K is not a single compound. Indeed, the diversity and the varied biological properties of the various forms of vitamin K can potentially explain the failure of research to date to clearly establish the role vitamin K plays in human beings. Apparently, additional (and particularly prospective clinical) studies are needed in order to clarify its non-coagulation-related effects in humans. Regardless, supplementation with natural forms of vitamin K is largely safe and hence is acceptable under specific conditions, such as in postmenopausal osteoporosis.

English expression in this article was revised by the agency "theBESTtranslation." Declaration of interest. The authors have no relevant interests to declare.

The following Supporting Information is available through the online version of this article at the publisher's website.

Figure S1 Chemical structures of vitamin K Figure S2 Vitamin K content in selected food Figure S3 Structure of matrix Gla protein (MGP) Figure S4 An HPLC analysis of vitamin K standards and a spiked human fecal sample

The Nobel Chronicles

Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis

Pilot-scale production of phylloquinone (vitamin K1) using a bubble column photo-bioreactor

A potential biotechnological process for the sustainable production of vitamin K1

Vitamin K in plants

Biosynthesis of vitamin K1 (phylloquinone) by plant peroxisomes and its integration into signaling molecule synthesis pathways

A role for phylloquinone biosynthesis in the plasma membrane as revealed in a non-photosynthetic parasitic plant. bioRxiv

Determination of phylloquinone and menaquinones in food. Effect of food matrix on circulating vitamin K concentrations

The role of menaquinones (vitamin K2) in human health

A high menaquinone intake reduces the incidence of coronary heart disease

Dietary intake of menaquinone is associated with a reduced risk of coronary heart disease: the Rotterdam Study

Phylloquinone (vitamin K1) content of vegetables

Simultaneous determination of carotenoids, tocopherols and phylloquinone in 12 Brassicaceae vegetables

Determination of vitamin K1 content in olive oil, chard and human plasma by RP-HPLC method with UV-Vis detection

Phylloquinone content from wild green vegetables may contribute substantially to dietary intake

Phylloquinone content of herbs, spices and seasonings

Vitamin K1 (phylloquinone) content of green vegetables: effects of plant maturation and geographical growth location

Summer (Subarctic) versus winter (Subtropic) production affects spinach (Spinacia oleracea L.) leaf bionutrients: vitamins (C, E, Folate, K1, provitamin A), lutein, phenolics, and antioxidants

Glucosinolates, carotenoids, and vitamins E and K variation from selected kale and collard cultivars

Vitamin K1 (phylloquinone) content of foods: a provisional table

The UK food data-base for vitamin K and why we need it

Vitamin K1 (phylloquinone) content of edible oils: effects of heating and light exposure

Vitamin K-containing dietary supplements: comparison of synthetic vitamin K-1 and natto-derived menaquinone-7

Microalgae as a source of vitamin K1

Vitamin analysis of five planktonic microalgae and one macroalga

Characterization of MK 8 (H 2 ) from Rhodococcus sp. B7740 and its potential antiglycation capacity measurements

Vitamin K2 -Vital for Health and Wellbeing

US Pharmacopeial Convention safety evaluation of menaquinone-7, a form of vitamin K

Vitamin K as a diet supplement with impact in human health: current evidence in age-related diseases

Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme

Dietary reference values for vitamin K

Conversion of phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) in mice: two possible routes for menaquinone-4 accumulation in cerebra of mice

Dietary vitamin K intake in relation to cancer incidence and mortality: results from the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition

Dietary phylloquinone and menaquinones intakes and risk of type 2 diabetes

Determination of phylloquinone and menaquinones in animal products with fluorescence detection after postcolumn reduction with metallic zinc

Multiple vitamin K forms exist in dairy foods

Reproducibility and relative validity of a food frequency questionnaire to estimate intake of dietary phylloquinone and menaquinones

Phylloquinone intake as a marker for coronary heart disease risk but not stroke in women

Vitamin K intake and mortality in people with chronic kidney disease from NHANES III

Vitamin K adequacy in a representative sample of Spanish adults. Dietary determinants

Age-related changes in the circulating levels of congeners of vitamin-K2, menaquinone-7 and menaquinone-8

Vitamin K status in the elderly

Vitamin K as a powerful micronutrient in aging and age-related diseases: pros and cons from clinical studies

Effect of food composition on vitamin K absorption in human volunteers

Differential lipoprotein transport pathways of K-vitamins in healthy subjects

Comparison of phylloquinone bioavailability from food sources or a supplement in human subjects

The effect of different meals on the absorption of stable isotope-labelled phylloquinone

Factors affecting the absorption of vitamin K-1 in vitro

Comparison of intestinal absorption of vitamin K2 (menaquinone) homologues and their effects on blood coagulation in rats with hypoprothrombinaemia

Orlistat enhances warfarin effect

Vitamin K deficiency and bleeding after long-term use of cholestyramine

Severe rifampicin-induced vitamin K deficiency coagulopathy in a child

The Complete Drug Reference

Metabolic engineering of menaquinone-8 pathway of Escherichia coli as a microbial platform for vitamin K production

Production of menaquinones by intestinal anaerobes

Fecal concentrations of bacterially derived vitamin K forms are associated with gut microbiota composition but not plasma or fecal cytokine concentrations in healthy adults

The contribution of vitamin K2 (menaquinones) produced by the intestinal microflora to human nutritional requirements for vitamin K

Use of hypoprothrombinemia-inducing cephalosporins and the risk of hemorrhagic events: a nationwide nested case-control study

Dose response and minimal daily requirement for vitamin K in man

A comprehensive review of vitamin K and vitamin K antagonists

The vitamin K cycle

Plasma phylloquinone, menaquinone-4 and menaquinone-7 levels and coronary artery calcification

Effect of phylloquinone supplementation on biochemical markers of vitamin K status and bone turnover in postmenopausal women

Vitamin K, vertebral fractures, vascular calcifications, and mortality: Vitamin K Italian (VIKI) dialysis study

Low vitamin K status is associated with osteoarthritis in the hand and knee

Vitamin K supplementation and progression of coronary artery calcium in older men and women

Concepts and controversies in evaluating vitamin K status in population-based studies

Phylloquinone and menaquinone-4 distribution in rats: synthesis rather than uptake determines menaquinone-4 organ concentrations

Tissue-specific utilization of menaquinone-4 results in the prevention of arterial calcification in Warfarin-treated rats

Vitamin K status in human tissues: tissuespecific accumulation of phylloquinone and menaquinone-4

Measurement of vitamin-K in humanliver by gradient elution high-performance liquid-chromatography using platinum-black catalyst reduction and fluorimetric detection

Excretion of the urinary 5C-and 7C-aglycone metabolites of vitamin K by young adults responds to changes in dietary phylloquinone and dihydrophylloquinone intakes

Laboratory assessment of vitamin K status

A new model for vitamin K-dependent carboxylation: the catalytic base that deprotonates vitamin K hydroquinone is not Cys but an activated amine

The mechanism of action of vitamin K

Insights into vitamin K-dependent carboxylation: home field advantage

The vitamin K-dependent carboxylase

Assembly of the warfarin-sensitive vitamin K 2,3-epoxide reductase enzyme complex in the endoplasmic reticulum membrane

A conformational investigation of propeptide binding to the integral membrane protein c-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry

Structural and functional insights into enzymes of the vitamin K cycle

The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase

Role of vitamin K and vitamin K-dependent proteins in vascular calcification

Processing of prothrombin in the secretory pathway

Matrix Gla-protein: the calcification inhibitor in need of vitamin K

Unique secretion mode of human protein Z: its Gla domain is responsible for inefficient, vitamin K-dependent and warfarinsensitive secretion

Secretion, gamma-carboxylation, and endoplasmic reticulum-associated degradation of chimeras with mutually exchanged Gla domain between human protein C and prothrombin

Comparison of the vitamins K1, K2 and K3 as cofactors for the hepatic vitamin K-dependent carboxylase

Structural insights into phylloquinone (vitamin K1), menaquinone (MK4, MK7), and menadione (vitamin K3) binding to VKORC1

Matrix Gla protein synthesis and gammacarboxylation in the aortic vessel wall and proliferating vascular smooth muscle cells-a cell system which resembles the system in bone cells

Vitamin K-dependent proteins involved in bone and cardiovascular health

Vitamin K-dependent carboxylation of the carboxylase

The coagulation cascade: initiation, maintenance, and regulation

The molecular basis of blood coagulation

Coagulation testing in the core laboratory

The role of tissue factor and factor VIIa in hemostasis

Surface-dependent reactions of the vitamin K-dependent enzyme complexes

Vitamin K-dependent protein S: beyond the protein C pathway

The role of Ca 2þ ions in the complex assembling of protein Z and Z-dependent protease inhibitor: a structure and dynamics investigation

Phosphatidylserine and phosphatidylethanolamine bind to protein Z cooperatively and with equal affinity

Efficient thrombin generation requires molecular phosphatidylserine, not a membrane surface

Computational study of the putative active form of protein Z (PZa): sequence design and structural modeling

Protein Z, an anticoagulant protein with expanding role in reproductive biology

New insights into the biology of osteocalcin

Osteocalcin and its endocrine functions

Osteocalcin-a versatile bone-derived hormone

Osteocalcin promotes bone mineralization but is not a hormone

GPRC6A: an update of its clinical and biological multi-organic interactions

The role of osteocalcin in human glucose metabolism: marker or mediator?

Gpr158 mediates osteocalcin's regulation of cognition

Vitamin K and bone health: a review on the effects of vitamin K deficiency and supplementation and the effect of non-vitamin K antagonist oral anticoagulants on different bone parameters

Correlation of serum osteocalcin fractions with bone mineral density in women during the first 10 years after menopause

Does vitamin K2 play a role in the prevention and treatment of osteoporosis for postmenopausal women: a meta-analysis of randomized controlled trials

A high phylloquinone intake is required to achieve maximal osteocalcin gamma-carboxylation

Three-year low-dose menaquinone-7 supplementation helps decrease bone loss in healthy postmenopausal women

The efficacy and safety of menatetrenone in the management of osteoporosis: a systematic review and meta-analysis of randomized controlled trials

Matrix Gla-Protein (MGP) not only inhibits calcification in large arteries but also may be renoprotective: connecting the dots

Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins

Matrix Gla protein, a new c-carboxyglutamic acidcontaining protein which is associated with the organic matrix of bone

Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein

Long term follow-up of four patients with Keutel syndrome

Vitamin K status, supplementation and vascular disease: a systematic review and meta-analysis

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I

Periostin action in bone

Periostin in inflammation and allergy

Periostin: an emerging biomarker for allergic diseases

Periostin in cardiovascular disease and development: a tale of two distinct roles

Periostin in kidney diseases

Absence of vitamin K-dependent c-carboxylation in human periostin extracted from fibrotic lung or secreted from a cell line engineered to optimize c-carboxylation

Serum periostin is associated with fracture risk in postmenopausal women: a 7-year prospective analysis of the OFELY study

the COMET Investigators. Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis

Role of urinary inhibitors and promoters in calcium oxalate crystallisation

Properties and function of nephrocalcin: mechanism of kidney stone inhibition or promotion

Purification and characterization of the principal inhibitor of calcium oxalate monohydrate crystal growth in human urine

Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. Evidence that nephrocalcin from patients with calcium oxalate nephrolithiasis is deficient in gamma-carboxyglutamic acid

Role of nephrocalcin in inhibition of calcium oxalate crystallization and nephrolithiasis

Crystal structure of a C-terminal fragment of growth arrest-specific protein Gas6. Receptor tyrosine kinase activation by laminin G-like domains

Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily

GAS6/TAM pathway signaling in hemostasis and thrombosis

In vivo thrombus formation in murine models

Gas6/TAM system: a key modulator of the interplay between inflammation and fibrosis

Molecular insights of Gas6/TAM in cancer development and therapy

Gas6 protein: its role in cardiovascular calcification

Protein S deficiency: a clinical perspective

Complementary DNA and derived amino acid sequence of the precursor of one of the three protein components of the inter-a-trypsin inhibitor complex

Inhibitors. In: Human Blood Plasma Proteins: Structure and Function

Structure and function of inter-alpha-trypsin inhibitor heavy chains

Inter-alpha-trypsin inhibitor proteoglycan family-a group of proteins binding and stabilizing the extracellular matrix

Inter-alpha-trypsin inhibitor, a covalent proteinglycosaminoglycan-protein complex

Primary structure and tissue distribution of two novel proline-rich gamma-carboxyglutamic acid proteins

Identification of two novel transmembrane gamma-carboxyglutamic acid proteins expressed broadly in fetal and adult tissues

Proline-rich Gla protein 2 is a cell-surface vitamin K-dependent protein that binds to the transcriptional coactivator Yesassociated protein

Gla-rich protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates

Gla-rich protein acts as a calcification inhibitor in the human cardiovascular system

A dual role for GRP in cardiovascular disease

Gla-rich protein is a potential new vitamin K target in cancer: evidences for a direct GRP-mineral interaction

Gla-rich protein is involved in the crosstalk between calcification and inflammation in osteoarthritis

Laiz e V. Gla-rich protein, a new player in tissue calcification?

Transthyretin: a multifaceted protein

Detection of a gamma-carboxy-glutamate as novel post-translational modification of human transthyretin

Moyamoya disease and moyamoya syndrome

Effects of vitamin K 2 on the expression of genes involved in bile acid synthesis and glucose homeostasis in mice with humanized PXR

Vitamin K and osteoporosis: myth or reality?

Cardiovascular effects of coumarins besides their antioxidant activity

The role of vitamin K2 in osteoporosis and cardiovascular disease prevention

Rise in late onset vitamin K deficiency bleeding in young infants because of omission or refusal of prophylaxis at birth

Quantification of phylloquinone and menaquinones in feces, serum, and food by high-performance liquid chromatography-mass spectrometry

Development of a validated liquid chromatography method for the simultaneous determination of eight fat-soluble vitamins in biological fluids after solid-phase extraction

A concise review of quantification methods for determination of vitamin K in various biological matrices

Analytical techniques for the determination of biologically active quinones in biological and environmental samples

Determination of vitamins K 1 , MK-4, and MK-7 in human serum of postmenopausal women by HPLC with fluorescence detection

A novel salting-out assisted extraction coupled with HPLC-fluorescence detection for trace determination of vitamin K homologues in human plasma

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

Determination of plasma vitamin K by high-performance liquid chromatography with fluorescence detection using vitamin K analogs as internal standards

Quantification of fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry

Rapid, high performance method for the determination of vitamin K 1 , menaquinone-4 and vitamin K 1 2,3-epoxide in human serum and plasma using liquid chromatography-hybrid quadrupole linear ion trap mass spectrometry

Selective chemiluminescence method for monitoring of vitamin K homologues in rheumatoid arthritis patients

Routine supercritical fluid chromatography tandem mass spectrometry method for determination of vitamin K1 extracted from serum with a 96-well solid-phase extraction method

LC-MS/MS quantitative analysis of phylloquinone, menaquinone-4 and menaquinone-7 in the human serum of a healthy population

A simple, sensitive, and high-throughput LC-APCI-MS/ MS method for simultaneous determination of vitamin K1, vitamin K1 2,3-epoxide in human plasma and its application to a clinical pharmacodynamic study of warfarin

Ba s B. A new electrochemical sensor with the Refreshable Silver Liquid Amalgam Film multi-Electrode for sensitive voltammetric determination of vitamin K2 (menaquinone)

Quantification of vitamins A, E, and K and carotenoids in submilliliter volumes of human milk

Determination of vitamins E, D3, and K1 in plasma by liquid chromatography-atmospheric pressure chemical ionizationmass spectrometry utilizing a monolithic column

Chapter 5 -Methods for assessment of vitamin K

Human vitamin K1 ELISA. User instruction

Human vitamin K1 (VK1) ELISA kit

Human vitamin K2 (VK2) ELISA kit

Decrease in protein C antigen and formation of an abnormal protein soon after starting oral anticoagulant therapy

Prophylactic vitamin K for the prevention of vitamin K deficiency bleeding in preterm neonates

The relationships between vitamin K and cognition: a review of current evidence

Fetal warfarin syndrome

Fetal warfarin syndrome

Bone quality and vitamin K2 in type 2 diabetes: review of preclinical and clinical studies

Combination use of vitamin K 2 further increases bone volume and ameliorates extremely low turnover bone induced by bisphosphonate therapy in tail-suspension rats

Effects of vitamin K2 on cortical and cancellous bone mass, cortical osteocyte and lacunar system, and porosity in sciatic neurectomized rats

Caution to readers about systematic review on vitamin K and prevention of fractures that included problematic trials

Vitamin K intake and the risk of fractures: a metaanalysis

Effect of vitamin K on bone mineral density: a metaanalysis of randomized controlled trials

Vitamin K and the prevention of fractures: systematic review and meta-analysis of randomized controlled trials

Effect of vitamin K on bone mineral density and fractures in adults: an updated systematic review and meta-analysis of randomised controlled trials

Cardiovascular event prediction and risk reclassification by coronary, aortic, and valvular calcification in the Framingham Heart Study

Value of progression of coronary artery calcification for risk prediction of coronary and cardiovascular events: result of the HNR Study

High dietary menaquinone intake is associated with reduced coronary calcification

Association of dietary vitamin K and risk of coronary heart disease in middle-age adults: the Hordaland Health Study Cohort

Vitamin K intake and all-cause and cause specific mortality

Dietary intake of vitamin K is inversely associated with mortality risk

Association of vitamin K with cardiovascular events and all-cause mortality: a systematic review and meta-analysis

Vitamin K status and cardiovascular events or mortality: a meta-analysis

The effect of menaquinone-7 supplementation on vascular calcification in patients with diabetes: a randomized, double-blind, placebo-controlled trial

Vitamin K supplementation for the prevention of cardiovascular disease: where is the evidence? A systematic review of controlled trials

Dephosphorylated-uncarboxylated Matrix Gla protein concentration is predictive of vitamin K status and is correlated with vascular calcification in a cohort of hemodialysis patients

Impact of stopping vitamin K antagonist therapy on concentrations of dephospho-uncarboxylated Matrix Gla protein

The anticancer effects of vitamin K

Molecular simulations suggest vitamins, retinoids and steroids as ligands of the free fatty acid pocket of the SARS-CoV-2 spike protein

Toxicity of vitamin K: induced hypoprothrombinemia and altered liver function

Effects of oxidative stress on phospholipid signaling in rat cultured astrocytes and brain slices

Anaphylactic shock and vitamin K1