Vitamin K, an example of triage theory

Vitamin K, an example of triage theory: is micronutrient inadequacy linked to diseases of aging? ABSTRACT The triage theory posits that some functions of micronutrients (the ’40 essential vitamins, minerals, fatty acids, and amino acids) are restricted during shortage and that functions required for short-term survival take precedence over those that are less essential. Insidious changes accumulate as a consequence of restriction, which increases the risk of diseases of aging. For 16 known vitamin K–dependent (VKD) proteins, we evaluated the relative lethality of 11 known mouse knockout mutants to categorize essentiality. Results indicate that 5 VKD proteins that are required for coagulation had critical functions (knockouts were embryonic lethal), whereas the knockouts of 5 less critical VKD proteins [osteocalcin, matrix Gla protein (Mgp), growth arrest specific protein 6, transforming growth factor b–inducible protein (Tgfbi or big-h3), and periostin] survived at least through weaning. The VKD c-carboxylation of the 5 essential VKD proteins in the liver and the 5 nonessential proteins in nonhepatic tissues sets up a dichotomy that takes advantage of the preferential distribution of dietary vitamin K1 to the liver to preserve coagulation function when vitamin K1 is limiting. Genetic loss of less critical VKD proteins, dietary vitamin K inadequacy, human polymorphisms or mutations, and vitamin K deficiency induced by chronic anticoagulant (warfarin/coumadin) therapy are all linked to age-associated conditions: bone fragility after estrogen loss (osteocalcin) and arterial calcification linked to cardiovascular disease (Mgp). There is increased spontaneous cancer in Tgfbi mouse knockouts, and knockdown of Tgfbi causes mitotic spindle abnormalities. A triage perspective reinforces recommendations of some experts that much of the population and warfarin/coumadin patients may not receive sufficient vitamin K for optimal function of VKD proteins that are important to maintain long-term health. Am J Clin Nutr 2009;90:889–907. selection, metabolic resources are preferentially allocated to functions necessary for reproductive survival at the expense of those required for survival beyond reproductive age. The triage theory is unique in that it proposes a mechanistic trigger for the reallocation of micronutrient-dependent metabolic resources and suggests that age-related disease is easily preventable by ensuring an adequate supply of micronutrients. If the theory is correct, the implications for public health are enormous. Virtually every metabolic pathway includes one or more enzymes that require essential micronutrients for activity. Micronutrient intake below recommended concentrations, but not severe enough to cause overt clinical symptoms, is widespread not only in poor countries but also in the United States (especially in the poor, children, adolescents, the obese, and the elderly), in part because of the high consumption of calorie-rich, micronutrient-poor, unbalanced diets (1). Societal concern is low because no overt pathology has been associated with these levels of deficiency. But the triage theory suggests that insidious changes could be occurring, leading to an increased risk of diseases associated with aging. A variety of both active and passive homeostatic mechanisms could be involved in triage. Homeostatic mechanisms to combat micronutrient deficiency include tissue redistribution in response to deficiency (4), normal micronutrient tissue distribution patterns that favor some tissues or cell types over others (5), activation of stress responses leading to protective metabolic changes in essential processes (6), up-regulation of transporters (7), and different cofactor binding constants for isoenzymes as observed for polymorphic variants of many enzymes that bind micronutrient cofactors with different affinities (8). We have begun an analysis of available scientific evidence that is aimed at profiling the functional spectrum of individual micronutrients under different degrees of micronutrient adequacy INTRODUCTION The triage theory (1, 2) posits that, when the availability of a micronutrient is inadequate, nature ensures that micronutrient-dependent functions required for short-term survival are protected at the expense of functions whose lack has only longer-term consequences, such as the diseases associated with aging. The triage theory is similar to the ‘‘disposable soma’’ theory of aging (3), which suggests that, as a result of natural 1 From the Children’s Hospital Oakland Research Institute, Oakland, CA. Supported by the Bruce and Giovanna Ames Foundation and by a generous donation from Elizabeth B and J Burgess Jamieson. 3 Address correspondence to JC McCann or BN Ames, Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609. E-mail: jmccann@chori.org or bames@chori.org. Received April 14, 2009. Accepted for publication July 9, 2009. First published online August 19, 2009; doi: 10.3945/ajcn.2009.27930. 2 to test the central predictions of the triage theory. Because some micronutrients are required for numerous functions (eg, zinc, iron, riboflavin, and niacin), our initial focus is on micronutrients required for relatively few functions (eg, biotin, folate, iodine, molybdenum, selenium, thiamin, vitamin B-12, vitamin C, and vitamin K). In this article, results are presented for vitamin K. BACKGROUND An analysis of vitamin K appears to be relatively straightforward compared with virtually all other micronutrients because it has a single known major function. Vitamin K in its reduced form is a cofactor for one enzyme, c-glutamylcarboxylase, which is located in the endoplasmic reticulum of various tissues. The enzyme post-translationally c-carboxylates certain glutamic acid residues in a number of vitamin K–dependent (VKD) proteins (9). c-Carboxylation allows VKD proteins to bind calcium, is needed for proper folding of the Gla domain, and facilitates binding of Gla proteins to cell membranes (10, 11). It is required for the activity of coagulation and anticoagulation factors (12) and for osteocalcin binding to hydroxyapatite in bone (13) and is generally considered to be required for the function of growth arrest specific protein 6 (Gas6) and matrix Gla protein (Mgp) (14–16). During c-carboxylation, the reduced form of vitamin K is oxidized to the epoxide, which is then reduced by vitamin K epoxide reductase in a 2-step reaction to regenerate the active form (17), completing what is termed the vitamin K cycle. The common anticoagulant drug warfarin (Coumadin; Bristol-Myers Squibb Company, New York, NY) acts by inhibiting vitamin K epoxide reductase (9). For further general discussion of vitamin K and the vitamin K cycle, see several recent reviews (9, 18–22). A potentially complicating issue for this analysis is that vitamin K is not a single entity but a family of structurally related molecules derived from different sources. Major molecular forms, their primary dietary sources, and their relative contributions to vitamin K activity are shown in Table 1 (22–49). All molecules listed in Table 1 share the same methylated naph- thoquinone nucleus (menadione) but have side chains of differing composition and length (eg, reference 22), which results in different potencies and absorption efficiencies (20). Menadione, despite also being called vitamin K3, does not have vitamin K activity (30, 35) and has different chemical properties from the family of active vitamin Ks. Unlike phylloquinone and the menaquinones, menadione can undergo redox cycling leading to the production of reactive oxidants. It is used at high doses in cancer treatments to potentiate cell killing, usually in combination with radiation or chemotherapy (50–52). As shown in Table 1, phylloquinone (vitamin K1) is considered to be the primary dietary source of vitamin K activity in humans (23–25, 36). It is the major focus both of the experimental literature and of this analysis. MK-4 (also called menatetrenone) is of particular interest to this analysis because it is an endogenously produced form of vitamin K synthesized from vitamin K1 (22, 37, 40, 49, 53, 54) and may be more active than vitamin K1 in extrahepatic tissues (22, 42–44). For example, after intake by rats of a vitamin K1–enriched diet containing no MK-4, high concentrations relative to vitamin K1 are found in extrahepatic tissues (37, 55). MK-4 is also distinguished from vitamin K1 in that, in addition to its vitamin K activity, it has been shown at high doses to affect the expression of a number of genes (22). We discuss MK-4 in conjunction with its use at high doses (usually 45 mg/d) in treatment trials examining its effects on bone fragility and some cancers. MK-7, which has substantial vitamin K activity (56, 57), is found in small quantities in liver mitochondria and in some other tissues (33, 58). Natto is a soybean product fermented with Bacillus subtilis, which is rich in MK-7 (59). MK-7 is a possibly important source of vitamin K in individuals in Asian cultures who regularly consume natto. Thus, following expert opinion, we assume that vitamin K1 is the principal source of vitamin K activity in humans in the modern world, with the possible exception of individuals who regularly consume natto. During evolution, when mechanisms for dealing with micronutrient shortages were developed, vitamin K1 TABLE 1 Common vitamin K molecular forms, sources, and relative contributions to vitamin K activity1 Molecular form of vitamin K Phylloquinone (vitamin K1) Dihydrophylloquinone Long-chain menaquinones (eg, MK10-13) MK-4 MK-7 Primary sources (reference number) Green leafy vegetables (23–25) Partially hydrogenated oils (26) consumed in some processed foods (27) Colon bacteria (eg, 22) Synthesized in animals from menadione, a vitamin K–inactive (30) animal feed additive (35), and consumed in some meats and cheeses (24, 36) Synthesized from vitamin K1 (22, 32, 37–41); menadione most likely an intermediate (49) Colon and oral cavity bacteria (45, 46) Natto (24, 35, 48) Relative dietary contribution to vitamin K activity (reference number) Major (24) Minor (28) Minor (22, 29) [little vitamin K activity (30) and poorly absorbed (24)] Substantial amounts stored in liver mitochondria (31–34) Minor as a direct dietary source of vitamin K unless diets are very unbalanced (36) Probably major as a metabolic product of vitamin K1 in extra-hepatic tissues (22, 42–44) Minor [substantial vitamin K activity (30, 47), but small amounts in liver mitochondria and some extrahepatic tissues (33)]. Possibly major if large quantities of natto are regularly consumed (35) was most likely an even more dominant source of vitamin K activity, because leafy green vegetables were a major, although variable, component of the Paleolithic diet (60, 61). METHODS Standard search methods were used to retrieve published information, including PubMed, Google, and the ISI Web of Knowledge cited reference search tool (62). Together with published resources, a number of web-based data repositories were also used, particularly the Mouse Genomic Informatics (MGI) database (63), which was used to search for mouse knockout mutants; BRENDA: The Comprehensive Enzyme Information System (64) and Uniprot (65), 2 comprehensive protein information resources; SymAtlas (66), which contains tissuespecific gene expression information; and 2 resources of the National Library of Medicine (67): the Online Mendelian Inheritance in Man (OMIM) and Single Nucleotide Polymorphism (SNP) databases. RESULTS Known VKD-dependent proteins, tissue locations of ccarboxylation, and functions are listed in the first 3 columns of Table 2 (9, 12, 14–16, 18, 22, 42, 68–196). As shown, these VKD proteins form 3 general classes: 1) Four coagulation factors—prothrombin (FII), FX, FVII, and FIX—of which all but prothrombin are believed to be c-carboxylated exclusively in the liver (9, 12, 22, 79). Limited extrahepatic expression of prothrombin mRNA has been reported in several tissues, including the brain (176, 177), kidney (178), and cornea (179). This expression appears to be in conjunction with regulatory functions of thrombin in addition to its role in the coagulation pathway (180). 2) Three anticoagulation regulatory proteins with some additional functions (proteins C, S, and Z) that are c-carboxylated primarily in the liver but to some extent also in extrahepatic tissues (12, 18, 167, 168). 3) The remaining known VKD proteins—osteocalcin, Mgp, Gas6, the recently identified fasciclin I–like proteins periostin and transforming growth factor b–inducible protein (Tgfbi or big-h3, keratoepithelin), and 4 proline-rich Gla transmembrane proteins (Prrg1-4)—which are not involved in coagulation and are c-carboxylated exclusively or primarily in extrahepatic tissues (see references in Table 2). Categorizing VKD proteins according to survivability of mouse knockouts The degree of lethality of VKD protein mouse knockouts provides some indication of their relative necessity for survival. Mouse knockouts have been isolated for all known VKD proteins listed in Table 2, except protein S and Prrg1-4, and are listed in the fourth column of Table 2. As shown, knockout phenotypes for all 4 coagulation factors (FII, FX, FVII, and FIX) and for one of the anticoagulation factors (protein C) are either embryonic lethal or lethal soon after birth due to excessive bleeding (FII, FX, FVII, and FIX) or thrombosis (protein C), which suggests that these 5 proteins are critical for short-term survival. Not surprisingly, the c-glutamylcarboxylase knockout is also lethal because of hemorrhaging (197). Knockout phenotypes for all other VKD proteins are nonlethal at least through weaning (protein Z, Mgp, osteocalcin, Gas6, Tgfbi, and periostin), which suggests that they are less critical for survival than the coagulation factors. Mgp is marginally included in the nonlethal category, because offspring survive weaning but usually die by 2 mo of age. Thus, a major requirement of the triage theory—that the functional spectrum of individual micronutrients can roughly be categorized into functions required for survival in the short term (ie, survival for reproduction) and functions whose loss can be tolerated over the longer term—appears to be consistent with the above categorization based on the relative lethality of mouse knockout phenotypes. Not only do VKD-protein knockouts segregate into ‘‘more lethal’’ and ‘‘less lethal categories’’ but there is some logical consistency within these categories. That is, the most lethal knockouts are all coagulation or anticoagulation factors that are c-carboxylated exclusively or primarily (protein C) in the liver. The nonlethal knockouts are all proteins not involved in coagulation that are exclusively or primarily c-carboxylated in extrahepatic tissues, with the exception of one anticoagulation factor (protein Z), which is primarily c-carboxylated in the liver. Are VKD functions required for short-term survival more resistant to vitamin K scarcity than less critical functions? The first major prediction of the triage theory is that micronutrient-dependent functions required for short-term survival will be more resistant to micronutrient inadequacy than less essential functions. Several experiments comparing the sensitivity of c-carboxylation of the coagulation factor prothombin and the extrahepatic VKD protein osteocalcin to variations in vitamin K1 availability suggest that c-carboxylation of the more essential VKD protein (prothrombin) is more resistant to vitamin K1 inadequacy than that of the less essential VKD protein (osteocalcin) (198–201). For example, over a study period of 84 d, vitamin K1 intake was first reduced and then progressively increased in a group of older women in a controlled dietary setting (199). Undercarboxylated prothrombin and undercarboxylated osteocalcin (uc-osteocalcin) were monitored, and although both increased during the depletion phase, uc-osteocalcin increased over 2 wk before there was a statistically significant increase in undercarboxylated prothrombin. Other experiments reinforce this result (198, 200, 201). For additional discussion of the osteocalcin experiments, see 2 reviews (22, 202). Studies directly comparing the sensitivity of the c-carboxylation of other extrahepatic VKD proteins with that of prothrombin or other coagulation factors have not yet been published. Although results of the experiments discussed above are suggestive, they are not definitive. Because prothrombin is primarily c-carboxylated in the liver and osteocalcin in bone cells, it is not clear that forms circulating in the blood are the most direct measure of relative c-carboxylation efficiencies under vitamin K deficiency. That blood concentrations of c-carboxylation do not necessarily reflect tissue concentrations are shown by recent observations concerning Mgp, which is c-carboxylated in chondrocytes (cartilage) and vascular smooth-muscle cells. Although undercarboxylated Mgp is accumulated in sclerotic lesions compared with healthy arteries, blood concentrations appear to be lower in patients with coronary or other diseases characterized by arterial calcification compared with healthy controls (14, 203–207). TABLE 2 Vitamin K–dependent (VKD) proteins: tissue locations of c-carboxylation, functions, and phenotypes of mouse knockout (KO) strains and known human mutants1 VKD protein Tissue locations of c-carboxylation Functions Mouse KO phenotypes Human mutant phenotypes Coagulation factors (general reviews: references 9, 12, 22, 79) Factor II (prothrombin) Liver; limited EH (brain, kidney, cornea) (177–179) Primarily coagulation factor; some regulatory functions (180). Factor X Liver Coagulation factor Factor VII Liver Coagulation factor Factor IX Liver Coagulation factor Primarily liver (143) (15); some EH (eg, endothelial cells, keratinocytes)4 AC factor. Non-AC effects: antiinflammatory (90, 166, 167); autocrine growth factor (155); placental development (168, 169) AC factor. Also ligand for TAM RTKs (72, 96, 100) and C4b-binding protein (183, 184) Neonatal death (consumptive coagulopathy) (110) Homozygotes: severe neonatal purpura fulminans. Heterozygotes: increased risk of VTE (93, 113) No KO in MGI Homozygotes: severe neonatal purpura fulminans (84, 125, 128). Heterozygotes: ’10-fold increased risk of VTE (76, 93, 96), osteonecrosis (99, 136) SNPs: normal AC activity (132)6 AC factors (general reviews: references 12 and 18) Protein C Embryonic lethal: defects in yolk sac, internal bleeding, tissue necrosis; rarely, neonatal death (144, 154) Late embryonic or neonatal lethality—spontaneous bleeding (68, 86) 70% die within 24 h of birth from intraabdominal hemorrhaging (68, 140) Usually fatal; severe hemophilia (118, 120, 153) Protein S Primarily liver (15, 101, 143); some EH (eg, osteoblasts (126), endothelial cells (91) Protein Z Primarily liver (143); some EH (eg, vascular) (149) AC factor (114)5 No phenotypic abnormalities, but increased prothrombotic phenotype in factor V (Leiden) mice (158) EH: primarily bone, cartilage, vascular tissue (14)7 Negative regulator of vascular calcification (42, 83, 138)8 Nonlethal to weaning. Death by 2 mo of age. Arterial and other calcification.9 Other VKD proteins Mgp (42, 83, 138) Complete deficiency lethal (98). Prothrombin thrombophilia: increased risk of VTE (74, 137).2 Absence of factor X lethal (147). Congenital factor X deficiency (rare)—abnormal bleeding (73, 112). Abnormal bleeding: severe form is lethal (127) Hemophilia B: rare X-linked hereditary disease. Gene deletion causes extreme disability by early adulthood (81, 129, 148).3 Abnormal calcification (eg, Keutel syndrome, Singleton-Merten syndrome, SNPs)10 (Continued) TABLE 2 (Continued) VKD protein Tissue locations of c-carboxylation Osteocalcin (80, 102, 187) EH: primarily osteoblasts (80, 185).11 Gas6 (15, 77, 100, 152) EH (eg, smooth muscle, endothelial, NK cells) (15, 122, 134) Tgfbi (big-h3, keratoepithelin) (182)15 EH: widely expressed. Down-regulated in tumor cells (174, 182). Periostin (165, 170, 172, 181)18 EH [eg, heart (171, 172), osteoblasts, tissues undergoing remodeling, tumor cells (170)] Proline-rich Gla proteins 1–4 Variety of fetal and adult tissues [eg, spinal cord, thyroid (115, 116)] Functions ECM protein in bone (80, 135). Additional hormonal role for uc-osteocalcin in glucose homeotasis (92, 105, 119, 188). TAM RTK activating ligand14 ECM protein. Promotes microtubule stability (173). Complex role in cancer.16 ECM protein. Role in development (eg, bone, heart), wound healing. Complex role in cancer.19 Largely unknown21 Mouse KO phenotypes Human mutant phenotypes Nonlethal. BMD loss, bone fragility after ovariectomy (78, 88); disturbed glucose homeostasis (119).12 Nonlethal. Apparently normal phenotype (70). Differential effects on progression of several experimentally induced pathologies (see text). Nonlethal. Spontaneous cancer, chromosome abnormalities (174).17 SNPs: osteopenia (87), BMD in women (PM) (69, 82, 85, 107, 121, 139)13 Nonlethal. Developmental abnormalities [eg, latent heart valve disease (172, 186); poor recovery after induced myocardial infarction (162)].20 No KO in MGI None specified in OMIM SNPs: association with stroke (189), acute coronary syndrome (190) Corneal dystrophy (175) No information in OMIM 1 Several putative VKD proteins were not included in the table. Two fasciclin-like proteins in humans, stabilin-1 and stabilin-2, were recently shown to contain carboxylase recognition sites (CRS) but have not been further characterized (165). An additional Gla-rich protein (Grp) was recently isolated from sturgeon, with human homologs identified in an in silico analysis, but not further characterized (164). AC, anticoagulation; ECM, extracellular matrix; EH, extrahepatic; Gas6, growth arrest specific protein 6; MGI, Mouse Genomic Informatics; Mgp, matrix Gla protein; PM, postmenopausal; SNP, single nucleotide polymorphism; TAM RTKs, Tam, Axl, and Mer receptor tyrosine kinases; uc-osteocalcin, uncarboxylated osteocalcin; BMD, bone mineral density; VTE, venous thromoembolism; OMIM, Online Mendelian Inheritance in Man; Tgfbi, transforming growth factor b inducible protein. Information sources consisted of the published literature in MGI (63) and the OMIM (67) databases. 2 Common allele resulting in elevated plasma concentrations of prothrombin. 3 Classical hemophilia is hemophilia A, which is caused by factor VIII deficiency (133, 153). Factor VIII is not a VKD protein. 4 Endothelial cells, keratinocytes, and some hematopoietic cells (166). 5 Possibly other endothelial functions (149). 6 Mixed results suggesting an inverse association between protein Z blood concentrations and ischemic stroke (149). 7 Some synthesis also in heart, lung, and kidney (94). 8 Mechanism may involve inhibition of bone morphogenetic protein (151, 157). 9 Additional KO phenotypic characteristics include growth plate calcification resulting in small stature, osteopenia, and fractures (89, 123, 142). 10 Keutel syndrome is a rare inherited disease resulting from mutations that inactivate Mgp. Clinical characteristics include abnormal arterial and cartilage calcification, peripheral pulmonary stenosis, and midfacial hypoplasia (108, 130, 131). Singleton-Merten syndrome clinical features include progressive aortic calcification, abnormal dentition, muscular weakness, and widened medullary cavities of bone; possible involvement of Mgp has been suggested (97, 150). Mgp polymorphic variants are associated with coronary artery calcification in healthy men (191), increased plaque calcification in myocardial infarction patients (104), tooth loss (106), kidney stones (95), pseudoxanthoma elaticum (103), and loss of BMD (146). 11 Some expression in endothelial progenitor cells has recently been reported (185). 12 Increased early rate of bone formation in KOs, but increased fragility compared with wild type after ovariectomy (78, 88). Disturbed glucose homeostasis includes decreased b cell proliferation, greater glucose intolerance, and insulin resistance (119). 13 Evidence is mixed. Associations were not observed in premenopausal women (111, 141). 14 Promotes innate immune response, cell survival, differentiation, required for natural killer cell maturation, and various brain functions (15, 100, 122). 15 Tgfbi was recently discovered to contain CRS and c-glutamic acid residues (165). The extent to which known functions of Tgfbi depend on c-carboxylation is not yet known. 16 ECM functions include cell adhesion and spreading. Although properties of Tgfbi appear to be tumor suppressive, in some cases, Tgfbi also appears to be recruited in some cases during tumor metastasis (192). 17 Knockdown of Tgfbi results in mitotic spindle abnormalities (173) (see text). 18 It has recently been discovered that periostin contains CRS and c-glutamic acid residues (165). The extent to which known functions of periostin depend on c-carboxylation is not yet known. An isoform of periostin has also been identified (193). 19 Required for normal bone, heart, and dental ligament development; plays a role in remodeling processes after injury (161, 165, 171); and stimulates tumor progression (170) but appears to also have some tumor-suppressive properties (194–196). 20 KO offspring are growth retarded with several additional developmental abnormalities, including periodontal disease (161, 163) and abnormal incisor eruption (160). 21 One proline-rich Gla protein binds to YAP (117), a transcriptional coactivator identified as an oncogene (159). What is the mechanism? Because c-carboxylation of the most essential VKD proteins (coagulation factors) is localized in the liver, and that of the less essential VKD proteins in the extrahepatic tissues, preferential tissue distribution of vitamin K1 to the liver is one mechanism that could facilitate better tolerance of coagulation factors to vitamin K1 scarcity than extrahepatic c-carboxylation. Indeed, as is well documented, vitamin K1 preferentially accumulates in the liver of both humans (22, 83, 208) and rodents (55, 209). Orally administered vitamin K1 first dissolves in chylomicrons (lipoprotein particles), which are formed in the wall of the small intestine in response to dietary lipid intake, and on absorption rapidly enter the lymphatic circulation (210). Chylomicrons then undergo modification to become chylomicron remnants (CMRs) (211), which are smaller in size than chylomicrons, but retain dissolved lipid-soluble vitamins (212), such as vitamin K1. CMRs are primarily taken up by the liver (212–214), although some other tissues (215), including bone (216–219), have some capability of clearing CMRs. For example, mouse liver takes up ’5 times more CMRs than bone (218). After vitamin K1 has been removed from CMRs in hepatic tissue, a portion can then reenter the systemic circulation in very-low-density lipoprotein particles secreted from the liver (22, 210, 220). Vitamin K1 in this form then reaches extrahepatic tissues (22, 212). An additional mechanism by which c-carboxylation of some VKD proteins could be favored over others could involve different binding affinities for the c-glutamylcarboxylase. In fact, isolated peptides from the carboxylase recognition sites of various VKD proteins do bind with very different affinities to c-glutamylcarboxylase (221). However, because of the modulatory effects of secondary binding sites, the actual differences in affinities of the intact VKD proteins for the c-glutamylcarboxylase are not known, as recently discussed (9). CONSEQUENCES FOR LONGTERM HEALTH OF DECREASED FUNCTIONALITY OF VKD PROTEINS The second major prediction of the triage theory is that decreased functionality of VKD proteins not required for short-term survival can increase the risk of diseases associated with aging. In this section, 3 factors that modify VKD function are examined: genetic loss, dietary availability of vitamin K, and chronic anticoagulant therapy. Genetic loss Functional loss from genetic change is not expected to precisely mimic changes that might also be expected from vitamin K deficiency because mutations affect function throughout development and the life span. However, it is expected that functional loss resulting from mutations will to some extent prefigure categories of loss that might result from vitamin K deficiency. Below, functional changes resulting from genetic modification of VKD protein genes are briefly summarized, and interpretation of these changes from the perspective of the triage theory is discussed. Mouse and human mutant phenotypes These phenotypes are shown in the 4th and 5th columns of Table 2. Several points are evident from an examination of this table: 1) Coagulation and anticoagulation factors II (prothrombin), X, VII, IX, protein C, and protein S. The lethality of mouse knockouts is mirrored in human homozygous or deletion mutants that are also lethal or life threatening without treatment. Although a mouse knockout for protein S has not yet been isolated, it will probably prove to be lethal on the basis that homozygous human protein S mutants are characterized by severe neonatal purpura fulminans (125), which is similar to that observed in protein C homozygotes (109, 113). Less severe mutations that result in only partial loss of proteins C or S (93), or that result in overproduction of prothrombin (factor II) (74, 137), lead to increased risk of venous thrombosis, an age-associated condition. Less severe mutations in the other members of this class of VKD proteins result in lifelong, but not necessarily life-threatening, abnormal bleeding or thromboses. 2) Mgp. Keutel syndrome (108, 131) is the only known complete loss-of-function human mutation among VKD proteins with nonlethal knockouts. As in mice with Mgp knockouts, individuals with Keutel syndrome are born apparently normal but develop abnormal calcification early in life in multiple tissues, including arterial walls (130). Some evidence also suggests greater calcification in older healthy men (191) and myocardial infarction patients with Mgp polymorphisms (104), which is consistent with the known association of calcification with arterial disease (222). 3) Osteocalcin. As shown in Table 2, both mouse knockouts and human polymorphisms (the only human osteocalcin mutants so far identified) are characterized by increased bone fragility after estrogen loss. Note, however, the complexity of the mouse knockout phenotype compared with wild-type (ie, increased bone density before ovariectomy and greater bone fragility after ovariectomy) (88). To our knowledge, possible associations of osteocalcin polymorphisms with diabetes or other consequences of glucose dysregulation, as observed in mouse knockouts, have not yet been investigated in humans. 4) Gas6. As shown in Table 2, mouse knockouts appear to be normal but respond differently than wild type to several experimentally induced pathologic conditions. Some of these responses [eg, poor erythropoietic recovery from induced anemia (71), poor tissue repair after experimentally induced hepatitis (223), or greater loss of oligodendrocytes in a demyelination model (75)] suggest that wild-type Gas6 may protect against some disease processes. In contrast, other responses suggest the opposite [eg, decreased thrombus formation in a prothrombotic model (70, 145), less nephrotoxic nephritis in a disease model (156), and decreased inflammation in an induced atherosclerosis model (124)]. The multifaceted phenotype of Gas6 knockouts in these induced-disease models is not surprising given the wide range of Gas6-activated receptor tyrosine kinase (RTK) functions (Table 2). 5) Protein Z. There is limited information on protein Z, but the possible linkage of protein Z deficiency and its polymorphisms in humans to stroke and other arterial diseases (149) suggests that there could be a connection between protein Z deficiency and age-associated disease. 6) Tgfbi. The striking characteristics of Tgfbi knockouts are increased spontaneous cancer and mitotic abnormalities (174). In an important series of experiments, Ahmed et al (173) show that Tgfbi is required to maintain microtubule stability. Knockdown of Tgfbi causes mitotic spindle abnormalities and centrosome duplication (173). Genomic instability is associated with increased cancer risk (224) and also correlates with the risk of metastasis (174, 225). TGF-b, which induces Tgfbi, is known to suppress genomic instability (226) [inhibition of TGF-b increases centrosome aberration frequency, tetraploidy, and aneuploidy (227)], but the mechanism is not known. Because expression of Tgfbi requires induction by TGF-b, it seems possible that Tgfbi maintenance of microtubule stability may be the mechanism by which TGF-b suppresses genomic instability. It is of interest that TGF-b null cells are also more sensitive to alkylating agents due to lack of expression of O6-methylguanine DNA methyltransferase (228), suggesting that TGF-b induces the expression of this DNA repair enzyme. Although Tgfbi may act as a tumor suppressor, it also appears to promote extravasation (leakage of cancer cells into tissues from capillaries), an essential step in metastasis, at least in colon cancer (192). Hence, similar to TGF-b, which appears to transition from a tumor suppressor in the early stages of carcinogenesis to an oncogene in tumor progression (229–231), the role of Tgfbi in cancer may vary depending on the stage of carcinogenesis and tumor type. As shown in Table 2, the only known consequence in humans of mutations at the Tgfbi locus is corneal dystrophy. On the basis of the mouse knockout phenotype, however, it may be productive to examine polymorphism phenotypes and families with corneal dystrophy for evidence of genomic instability or increased cancer risk. 7) Periostin. The knockout phenotype suggests a role for periostin in cardiovascular health and recovery from myocardial infarction (162, 172). Periostin also appears to play a role in cancer progression. Periostin is, like Tgfbi, induced by TGF-b (172), and, like Tgfbi, periostin appears to have both tumor suppressive and oncogenic properties. For example, the rate of growth in periostin knockouts of tumors initiated by subcutaneous injection of several cancer cell lines was greater than in wild-type (194), which suggests tumor-suppressive properties (195, 196). On the other hand, periostin is well known to be up-regulated in a wide variety of metastatic tumors (232). Human periostin mutants have not yet been identified. However, it is of interest that it was recently reported (233) that periostin and Tgfbi are binding partners and that a Tgfbi mutation resulting in corneal dystrophy prevents their interaction. This raises the question of whether binding to periostin is required for Tgfbi effects on the mitotic spindle. cruitment of Tgfbi and periostin in cancer progression provide a third example. These effects, within the context of ongoing pathology, are somewhat similar to ‘‘antagonistic pleiotropy,’’ an evolutionary theory of aging suggested many years ago (234). Antagonistic pleiotropy suggests that some genes that have a positive function early in life may have a deleterious function late in life. In the Gas6 example, the health-promoting functions of Gas6, such as stabilizing platelet aggregates in hemostasis, can become disease promoting when recruited by an ongoing pathological process, such as thrombosis. Although the triage theory predicts that partial loss of Gas6 function due to vitamin K deficiency could lead to the initiation of age-associated disease in otherwise healthy individuals, it does not preclude the possibility that the partial loss of function could also lead to greater resistance to the progression of some diseases (see Discussion). Because Gas6 knockouts do not appear to have been followed into old age (see Table 2), it is not known whether they might have been more susceptible to the development of any age-associated diseases. Dietary availability of vitamin K As shown in Table 3 (16, 22, 42, 52, 80, 102, 135, 204, 235– 278), vitamin K deficiency has been linked to a variety of ageassociated conditions, including loss of bone mineral density (BMD) or increased fracture risk (80, 102, 135, 235–252), arterial calcification or cardiovascular disease (16, 42, 253–262), cancer (22, 52, 263–268), insulin resistance (269, 270), osteoarthritis (80, 271), chronic kidney disease (42, 272–274) [frequently accompanied by vascular calcification (279)], and inflammation (22, 275). Bone-related conditions, arterial calcification or other cardiovascular conditions, and cancer have been most widely studied, and evidence linking them to vitamin K availability is briefly discussed below. Bone-related conditions Triage theory perspective The phenotypes discussed above raise several points that help to clarify the triage theory and bring it to bear in the context of disease progression. First, venous thrombosis is considered to be an age-related disease. Although the partial loss-of-function protein C and protein S mutants are linked to increased risk of this condition, the triage theory would predict that venous thrombosis would be unlikely to result from vitamin K deficiency because these 2 VKD proteins are required for short-term survival and are predominantly c-carboxylated in the liver. Thus, the theory predicts that vitamin K deficiency would have to be severe enough to adversely affect coagulation function to interfere with their c-carboxylation. Second, in contrast, triage theory would predict that vitamin K deficiency that is not severe might be linked to bone fragility after menopause (osteocalcin), arterial calcification (Mgp), and genomic instability and cancer because these conditions are linked to genetic loss of VKD proteins not required for short-term survival. These proteins are c-carboxylated exclusively in extrahepatic tissues and thus are likely to require higher intakes of vitamin K for maximum function compared with hepatic VKD proteins. The complex phenotype of apparently normal Gas6 knockouts that is revealed in response to pathologic stress and the re- For extensive discussion of this topic, see several reviews (80, 135, 235, 236, 280, 281). During pregnancy, a rare condition resulting in severe reduction of maternal vitamin K results in serious bone and cartilage abnormalities in the fetus (42, 237). In young girls, an association between low vitamin K1 status and several markers indicating increased bone turnover has also been reported (238). In adults, vitamin K deficiency has been linked to increased risk of bone-related conditions in many studies. Vitamin K1 intake (239–241), status (242, 243), or treatment (244); MK-4 treatment at pharmacologic doses (245); and MK7-rich natto intake (246, 247) have all been linked positively to variables indicative of bone health (particularly decreased fracture risk, increased BMD, and bone mineral content). Despite the considerable number of positive studies, most (80, 235, 236), although not all (135, 282), expert reviewers consider that a causal relation between vitamin K and bone health has not been shown, primarily because results of treatment trials have not been consistently reproducible. Several new treatment trials involving high doses of MK-4 (45 mg/d) (248–250) and vitamin K1 (500–1000 lg/d) (250–252) have appeared since those discussed by reviewers, and results of these trials are mixed as well. Thus, despite a large body of work linking vitamin K availability to bone health, a definitive causal relation has not been established in reproducible randomized controlled treatment trials. TABLE 3 Age-related disease or conditions linked to dietary vitamin K inadequacy Representative references Age-associated condition Studies Reviews Positive: references 237–249 Negative: references 250–252 Positive: references 255–260, 276 Negative: references 261, 262 References 269, 270 References 271, 277 References 80, 102, 135, 235, 236 Specific endpoints (eg, bone mineral density) not consistently replicated Substantial design (eg, duration of treatment or follow-up) differences among studies References 16, 42, 253, 254 Most positive results are for menaquinone intake or vitamin K1 supplementation Confounding food components in vitamin K1–containing foods complicate interpretation of vitamin K1 intake study results Reference 269 Chronic kidney disease Reference 272 Inflammation Reference 275 References 42, 273, 274 Reference 22 Cancer References 263–268 Increase in insulin sensitivity in both a randomized vitamin K1 intervention trial and a cross-sectional, food-frequency questionnaire–based intake study Low vitamin K1 status associated with osteoarthritis Results in a randomized intervention trial most evident in individuals deficient in vitamin K at baseline Suboptimal vitamin K status and elevated undercarboxylated Mgp (204) in hemodialysis patients in one study; no association reported in another (278) Vitamin K status inversely associated with several circulating markers of inflammation Some case reports and several small pilot trials suggest that treatment with high doses of MK-4 may be therapeutic in patients with preexisting cancer. A prospective epidemiologic and nested case-control study of the same population presents marginally statistically significant results suggesting an inverse relation between menaquinone, but not vitamin K1 intake, and subsequent prostate cancer. Bone abnormalities (primarily increased fracture risk or loss of bone mineral density) Arterial calcification or cardiovascular disease (atherosclerosis, other coronary heart disease) Insulin resistance Osteoarthritis Reference 80 References 22, 52 Although we do not disagree with this conclusion, note that there are substantial design (eg, duration of treatment, whether subjects were also treated with vitamin D and calcium, whether subjects had existing evidence of bone disease, outcome measures) and other protocol differences among trials that prevent precise comparisons. In addition, treatment trials usually extend over a relatively short time period (1–3 y) compared with dietary inadequacy, which may last for many years. Arterial calcification and cardiovascular disease Fewer studies have examined relations between vitamin K and cardiovascular health [see several recent reviews (16, 42, 254, 280)]. Cross-sectional (255, 256) and population follow-up (257, 258) studies from the Netherlands reported statistically significant inverse associations between total vitamin K intake (vitamin K1 1 menaquinone) and aortic atherosclerosis (255) and between low menaquinone intake and coronary (256) and aortic (258) calcification and diagnosis of (257) or mortality from (258) coronary heart disease, and all-cause mortality (258). We are aware of 2 vitamin K1 intervention trials that examined cardiovascular outcomes (259, 276). One trial (259) reported a positive effect of vitamin K1 supplementation (1000 lg/d) for 3 y in postmenopausal women on elasticity of the carotid artery, although significant changes were not detected in other indicators of arterial health, as pointed out by reviewers (254). A recent 3-y vitamin K1 (500 lg/d) supplementation study in older men and postmenopausal women did not measure carotid elas- Comments ticity but did report less progression of coronary artery calcification that was statistically significant in subjects with 85% adherence to the supplement and in those with preexisting coronary artery calcification (276). It has proven more difficult to detect a relation between vitamin K1 and cardiovascular disease by using epidemiologic techniques for several reasons. Experts have pointed out that foods rich in vitamin K1 are also rich in other substances associated with heart health, so that it is difficult, if not impossible, to tease out any effect due specifically to vitamin K1 (254, 261, 262). In addition, the food-frequency questionnaires used in several studies from the Netherlands that indicated statistically significant inverse relations between menaquinone, but not vitamin K1, intake and calcification or coronary heart disease (256–258) only poorly reflected vitamin K1 intake. Furthermore, in these studies (256–258), vitamin K1 intakes were quite high, and the range of intakes was narrow. If an inverse relation was observed for menaquinone intake, one might expect that an inverse association would also be observed for vitamin K1 intake, provided that a well-validated foodfrequency questionnaire was used and that the cohort examined had a wide range of vitamin K1 intake. Important additional evidence linking vitamin K to cardiovascular health comes from 2 studies that observed a relation between vitamin K (vitamin K1 1 vitamin K2) intake and serum concentrations of carboxylated osteocalcin with cardiovascular and bone health in the same individuals (255, 260). In the first case, both low vitamin K intake and high circulating uc-osteocalcin (possibly a surrogate indicating poor c-carboxylation of Mgp) were associated with atherosclerosis (255). In the second case, women with both atherosclerosis and relatively high serum concentrations of uc-osteocalcin also had low BMD (260). The association of aortic calcification with indicators of poor bone health has been widely reported (eg, 283–286), and possible mechanisms have recently been reviewed (287). However, a recent large prospective cohort analysis did not find an association (288). Cancer Pharmacologic doses of MK-4 inhibit the growth of a number of cancer cell lines in vitro, and some in vivo evidence also suggests a therapeutic effect on cancer progression (22, 52). The likely mechanism is modulation of gene transcription, which may in part be mediated by the binding of MK-4 to the SXR/ PXR receptor (the human and rodent, respectively, versions of receptors that bind steroids and a wide variety of molecules perceived by the organism as xenobiotics) (22, 289). It is not known whether these effects occur under normal physiologic conditions. Some case reports suggest that high-dose treatment with MK-4 in patients with different types of cancer may be therapeutic (52), and the possible protective effects of daily high-dose MK-4 treatment on the recurrence of hepatocellular carcinoma in successfully treated patients have been reported in several small trials (263–266). For additional discussion of some of these studies, see several reviews (22, 290, 291). The only attempt of which we are aware to examine the possible relations between cancer incidence and vitamin K intake in normal physiologic ranges is a recent prospective epidemiologic analysis of a large cohort of .11,000 men (267). Marginally statistically significant evidence was provided that suggested an inverse relation between new cases of advanced prostate cancer diagnosed during a mean follow-up period of 8.6 y and dietary intake (determined by food-frequency questionnaire) of menaquinones but not of vitamin K1 (267). A subsequent nested case-control study using the same cohort (268) reported that higher ratios of uc-osteocalcin to total osteocalcin were also marginally associated with advanced or high-grade prostate cancer cases. This latter result was based on a single model-dependent P value that achieved only borderline significance and was most likely driven by outliers in the 4th quartile. Results of both of these studies are very weakly statistically significant and, in our opinion, are uncertain unless replicated in an independent cohort. As investigators discuss, interpretation is also complicated by the fact that MK-4 is endogenously synthesized from vitamin K1, and c-carboxylation of uc-osteocalcin is well known to be sensitive to vitamin K1 intake (198–201). Chronic anticoagulant therapy Because warfarin blocks vitamin K epoxide reductase, it is expected that warfarin treatment will decrease c-carboxylation of all VKD proteins, not just coagulation factors. Indeed, a broad spectrum of effects has been reported in humans and rodents, including effects of treatment on bone health, arterial calcification, and cancer, which are briefly described below. Bone-related conditions Use of warfarin during the early stages of pregnancy can lead to fetal warfarin syndrome, which is characterized by abnormal bone development and cartilage calcification (80, 253, 292), similar to the effects of severe maternal vitamin K deficiency on offspring discussed above (237). Ostopenia was reported in 52% of children and adolescents in long-term anticoagulant therapy (293). In adults, results of the significant number of studies examining possible linkages between warfarin-induced vitamin K deficiency and osteoporosis, loss of BMD, or increased fracture risk are mixed (80). However, as recently discussed in a thorough review of the field (135), association of anticoagulant use with poor bone health is most consistently reported in individuals in long-term therapy (eg, reference 294). Studies that failed to segregate subjects based on duration of therapy were generally negative [see a recent example (295)]. Adverse effects of chronic warfarin use in humans are reinforced by experiments in rats in which warfarin treatment of 4 mo decreased BMD and increased bone fragility after ovariectomy (296). Arterial or aortic valve calcification It is not clear whether the possibility of arterial calcification in fetal warfarin syndrome (discussed above) has been investigated. A rare adverse reaction to long-term warfarin therapy is tracheobronchial calcification (297), which may account for depressed arterial hemodynamics in patients at higher cardiovascular risk in long-term warfarin therapy (298). Several observational studies have also reported associations between aortic valve calcification and long-term anticoagulant therapy (205, 274, 299–301) in humans; warfarin treatment also induces arterial calcification in rats (43, 302). This effect may be exacerbated by the fact that warfarin also blocks the biosynthesis of MK-4 from vitamin K1 (43). Cancer As discussed above, there is a high rate of spontaneous cancer in mouse Tgfbi knockouts. Because warfarin treatment is expected to interfere with c-carboxylation of Tgfbi, it is of interest to ask whether long-term warfarin treatment in humans increases cancer risk. Few studies have looked explicitly at this question because research on warfarin and cancer has been driven primarily by the fact that cancer triggers the coagulation cascade and is a major risk factor for venous thromboembolism (VTE) (303, 304), which can occur even before the clinical detection of cancer (305, 306). Cancer incidence after a VTE event has been tracked over the short term in many studies, and in fewer cases for periods longer than a year (305, 307–310). Results consistently suggest a much greater risk of occult cancers detected relatively soon after VTE, but a small increased risk persisting for 10 y (307, 310). Although very few studies have examined effects of warfarin use per se on cancer risk (311, 312), there is no indication to date that long-term warfarin use contributes to this residual risk, and in fact a recent report suggested that chronic warfarin treatment had a protective effect on urogenital cancer in men (313). Because of the complex relation between cancer and coagulation, it may be very difficult to design experiments to explicitly target any effects on cancer risk of anticoagulation therapy due to interference with c-carboxylation of VKD proteins. A body of evidence, although mixed, suggests that warfarin treatment of patients with metastatic cancer can increase survival, as recently reviewed (304, 311). Inhibitory effects of warfarin on c-carboxylation of VKD proteins known to be recruited during metastasis—gas6, tgfbi, and periostin (discussed above)—should be included in discussions of possible mechanisms. The fact that all 3 causes of loss (genetic, dietary intake, and anticoagulant therapy) are associated with increased bone fragility after estrogen loss (suggesting osteocalcin involvement) is compelling, despite poor reproducibility among treatment trials and other uncertainties discussed above. Similarly, linkage of all 3 types of loss to increased arterial calcification (suggesting Mgp involvement), which is known to be associated with cardiovascular disease (222), is also compelling, although fewer studies have been conducted. The variously health- or disease-promoting phenotypes of Gas6 knockouts when challenged by different induced pathologies raises the important point that loss of function may, in some circumstances, have health-promoting consequences. This point is reinforced by the recruitment of other VKD-dependent proteins, including coagulation factors, Tgfbi, and periostin in cancer progression. And finally, the propensity of Tgfbi mouse knockouts to develop spontaneous cancer suggests that further studies that examine possible linkages between vitamin K intake and cancer risk should be undertaken. IS THERE A PUBLIC HEALTH PROBLEM? In the United States, average intake of vitamin K1 is 70–80 lg/d (314), which is below the currently recommended Adequate Intake of 90–120 lg/d (25). Generally low intakes are also reported in Ireland (315, 316) and the United Kingdom (317– 319), where the general guideline for vitamin K1 intake is ’70 lg/d (1 lg
kg21
d21) (320). Recommended intakes of vitamin K1 are based solely on amounts required to maintain coagulation function (25, 320). It is doubtful that average intakes, although below the Adequate Intake, hinder coagulation because the Adequate Intake includes a safety factor (25). However, concern has been expressed among some experts that current intake recommendations for vitamin K1 may not be high enough to ensure adequate function of VKD proteins not involved in coagulation (eg, references 253, 321, 322). This concern is primarily based on the fact that, although prothrombin is essentially 100% c-carboxylated under normal conditions (47), ’10–40% of serum osteocalcin remains undercarboxylated (eg, references 251, 323); percentages are considerably higher in children (324). Interpretation of the significance of these results must take into account recent evidence that osteocalcin, in addition to its role as an extracellular matrix protein in bone, may, in an uncarboxylated form, also play a hormonal role in glucose homeostasis (92, 105, 119). Thus, the percentage of fully c-carboxylated osteocalcin in blood that is required for optimal health cannot be assumed to be 100% and remains to be determined. Nevertheless, it is clear that c-carboxylation of osteocalcin in blood is substantially more sensitive to vitamin K1 deficiency than prothrombin, as discussed above. On the basis of triage theory, we would predict that c-carboxylation of all extrahepatic VKD proteins (eg, Mgp, Gas6, Tgfbi, and periostin) is more sensitive to vitamin K1 deficiency than is prothrombin. Because, to the extent they have been studied, there are no known functions of the uncarboxylated forms of these proteins, concentrations of vitamin K1 intake required for their optimal c-carboxylation may be a more helpful guideline for setting new intake recommendations than concentrations required for full c-carboxylation of osteocalcin in blood. Clearly, more research is needed to quantify the percentage of c-carboxylation required for optimal activity of these proteins and to better understand relations between circulating and tissue forms of proteins whose main activities are not in blood. Some experts have suggested that revised recommendations should include consideration of MK-4 or MK-7 supplements as alternative sources of vitamin K in addition to vitamin K1 for optimal health (47). After dietary intake, both MK-4 and MK-7 are distributed more rapidly and more broadly to extrahepatic tissues than vitamin K1 and thus could circumvent the higher intake requirement of vitamin K1 due to its triage allocation to the liver. Observations supporting the use of these alternatives are briefly discussed below, along with some cautionary notes that suggest that safety studies should precede any change in official dietary intake recommendations. MK-4 is more active than vitamin K1 in extrahepatic tissues (22, 42–44) and possibly has health-promoting effects (in addition to its role in c-carboxylation) on the expression of certain genes (22, 325). In addition, some evidence (discussed above) suggests that normal dietary intakes of menaquinones (of which MK-4 is a major component) may be inversely related to the risk of cardiovascular disease (256–258). On the other hand, MK-4 is naturally synthesized from vitamin K1 in the body, and supplying additional MK-4 could alter this natural balance. Of particular interest relevant to the possible use of MK-7 as an additional source of vitamin K are the decreased risks of fractures and bone loss among Japanese women consuming natto, which contains high concentrations of MK-7 (246, 247, 326) (see reference 47 for further discussion). Natto consumption is conceivably partially responsible for the dramatically lower prevalence of atheroscerlosis (327–329) and bone fragility (330–332) in Japan compared with Western countries. A causal role for MK-7, however, is not definitive and interpretation of these studies should take into account the observation that, even in a region of Japan with relatively high natto consumption [Saitama Prefecture located in Kanto I region discussed by Yaegashi et al (326)], vitamin K1 is still the predominant dietary form of vitamin K (333). Although MK-7 has a longer half-life and accumulates to higher concentrations in serum than vitamin K1 (47), experts have also cautioned that intakes of MK-7 that are relatively low compared with vitamin K1 may interfere with anticoagulant therapy due to its potency (47). A second area of possibly serious public health concern is functional vitamin K deficiency induced by long-term anticoagulant therapy with warfarin drugs, as previously discussed (22, 42, 135, 253). These drugs are among the most prescribed in the United States: .30 million prescriptions are dispensed each year (334). As discussed above, long-term warfarin use is linked to increased arterial calcification (299–301) and bone loss or fragility (80, 135). It seems desirable, as previously suggested (42), to seriously explore the use of alternatives to warfarin that do not interfere with c-carboxylation in extrahepatic tissues. And finally, many individuals do not meet recommended intakes for more than one micronutrient. Suboptimal concentrations of other micronutrients could exacerbate effects of vitamin K deficiency. For example, both calcitriol, the active form of vitamin D, and vitamin A are known to modulate Mgp expression (42). Low vitamin D status, which is quite common (335), has been linked in several studies to vascular calcification, which could be due to effects on Mgp expression (336). Calcitriol also modulates expression of the osteocalcin gene (337). DISCUSSION In this article, vitamin K serves as an example to test the predictions of a new theory (the triage theory) that explains why modest micronutrient deficiencies may cause age-related diseases such as osteoporosis, cardiovascular disease, and cancer. The evidence presented here is consistent with a system that prioritizes the protection of VKD functions when vitamin K1 is scarce according to their essentiality for short-term survival at the expense of functions required to maintain long-term health. Our analysis highlights what appears to be the primary mechanism that accomplishes this prioritization: the separation of coagulation factors from less essential VKD proteins by localizing their c-carboxylation in the liver, where ingested vitamin K1 is preferentially distributed. An additional mechanism also suggests preferential protection of c-carboxylation in the liver. As indicated above, long-chain menaquinones (and small amounts of shorter-chain menaquinones with vitamin K activity) are stored in mitochondria in the liver but not in the extrahepatic tissues. Although these menaquinones are not considered by most experts to contribute substantially to vitamin K activity, as discussed in the text, it is difficult to rule out some role for them, possibly as a back-up system when other sources of vitamin K are scarce, as originally suggested 16 y ago (338) and as recently reviewed (22). Evidence supporting the 3 major predictions of the triage theory The evidence discussed here is consistent with the major predictions of the triage theory, although there are some information gaps and uncertainties. Below, the strengths and weaknesses of the evidence relative to each of these predictions are briefly discussed. Categories of essentiality for short-term survival according to mouse knockout lethality To conduct an analysis from the triage theory perspective, the spectrum of functions of a particular micronutrient must be categorizable according to their degree of essentiality for shortterm survival. Because information on mouse knockout phenotypes was available for almost all VKD proteins with known functions, we chose to use mouse knockout lethality as a categorizing guide. Although the use of mouse KO lethality to achieve this categorization appears reasonable, the method is not perfect. It cannot be assumed that essentiality in mice will always correlate with essentiality in humans. In this case, however, the similarity of mouse knockout phenotypes and human mutant phenotypes (Table 2) suggests that the method worked reasonably well. Furthermore, in a knockout mutant, essentiality for embryogenesis trumps later metabolic needs. Thus, knockouts of genes required for embryogenesis will be lethal, even if those genes are not required for short-term survival after development. This limitation does not appear to be a factor in the case of vitamin K because all lethal knockouts were coagulation or anticoagulation factors, which are known to be critical for hemostasis throughout life. Comparative sensitivity of VKD protein c-carboxylation to vitamin K1 availability A key prediction of the triage theory is that VKD proteins required for short-term survival (coagulation factors) will be more resistant to loss of vitamin K1 than those that are less essential (Mgp, osteocalcin, Gas6, Tgfbi, and periostin). As discussed, c-carboxylation of osteocalcin is more sensitive to decreased vitamin K1 availability than prothrombin, although these are the only pair of essential and nonessential VKD proteins for which results of studies comparing efficiencies of c-carboxylation have been published. Experiments to compare the sensitivity of c-carboxylation of the other extrahepatic VKD proteins to vitamin K1 availability relative to prothrombin or other coagulation factors are needed to further confirm this prediction. Linkage of decreased functionality of VKD proteins to age-related conditions The most important outcome of this part of the analysis is that genetic impairment in osteocalcin or Mgp in both mice and humans, limited vitamin K availability, and chronic warfarin therapy are all variously linked to the same set of age-associated conditions (bone deterioration and fragility late in life and arterial calcification or other cardiovascular conditions). Although both genetic impairment in mice and vitamin K1 deficiency are linked to glucose dysregulation or insulin resistance, to our knowledge, experiments have not been conducted to examine possible linkages of chronic warfarin therapy or human osteocalcin polymorphisms to this condition. Tgfbi (big-h3) is an extracellular matrix protein only recently recognized to be c-carboxylated (165). The observations that its mouse knockout is characterized by spontaneous cancer (174) and its knockdown by mitotic spindle abnormalities and centrosome amplification (173) are potentially of great importance. As discussed above, few studies have been designed to specifically target possible linkages between vitamin K intake and cancer risk. Tgfbi mutations in humans are linked to corneal dystrophy (175), and examination of the many families identified with these mutations for possible evidence of mitotic abnormalities or increased cancer risk appears warranted. Micronutrition in disease prevention compared with disease progression The promotion of, or resistance to, the progression of different induced diseases in otherwise apparently normal Gas6 knockout mutants and the recruitment of Tgfbi and periostin, both of which have important health-promoting functions, to further the metastatic progression of cancer (192, 232) raises an important issue concerning the potentially contradictory roles of micronutrients in disease prevention compared with disease progression. These knockout phenotypes serve as reminders that ‘‘good’’ functions can be recruited by pathologic processes for ‘‘bad’’ ends (339). In addition to the examples cited in the text, some members of the TAM (Tam, Axl, Mer) subfamily of RTKs, of which Gas6 is the primary activating ligand, are up-regulated in cancer progression (122). In fact, there is ongoing research to develop chemotherapeutic strategies to inhibit these receptors (122). Modest vitamin K deficiency could offer a therapeutically effective nutritional alternative. The triage theory, supported by evidence discussed in this review, predicts that a relatively modest degree of vitamin K deficiency that is not severe enough to interfere with coagulation could be effective in retarding these recruitments because c-carboxylation of these VKD proteins occurs extrahepatically. Possibly improved survival of cancer patients treated with warfarin is assumed to be due to prevention of cancer-induced VTE (311), but the possibility that inhibition of c-carboxylation of extrahepatic VKD proteins recruited for cancer progression should not be discounted as a possible mechanism. A similar mechanism involving inhibition of gas6 function has been suggested to explain the protective effects of warfarin treatment against diabetic nephropathy in streptozotocin-induced diabetes in rodents (340) and in cyclophosphamideinduced diabetes in NOD (nonobese diabetic) mice (341). In contrast, in cases where disease progression may be hindered by a functional Gas6 [demyelination (75) and recovery from chronic anemia (71), as discussed in the text], vitamin K supplementation could be beneficial. In the absence of ongoing disease, one would expect that modest vitamin K1 deficiency would adversely affect the broad spectrum of TAM RTK functions that are enabled by Gas6 binding, as well as the healthpromoting functions of Tgfbi and periostin (see text and Table 2). Chronic warfarin therapy would also be expected to have an adverse effect on these extrahepatic functions and could have an even greater effect on Gas6 functions because it would be expected to impair c-carboxylation of protein S (an alternate ligand for the TAM RTK receptors) as well as Gas6. CONCLUSIONS The functional spectrum of vitamin K viewed through the lens of the triage theory may provide a helpful way to think about the potential effects of vitamin K1 deficiency on age-associated disease. Vitamin K1 is an excellent example of a micronutrient for which the severe and immediate clinical consequences of deficiency (bleeding) have dominated its history. This is also the case for almost all other micronutrients—eg, vitamin C and scurvy, thiamine and beriberi, niacin and pellagra, or vitamin D and rickets. In recent years, more probing scientific investigation has begun to unearth subtle long-term health effects of modest deficiencies of many micronutrients, some of which we have previously discussed (eg, references 1, 342–346). The triage theory supplies a unifying framework explaining why a crop of diseases associated with aging is emerging for so many micronutrients. It is our hope that this analysis will stimulate further efforts to redefine micronutrient adequacy on the basis of longterm effects. Methods to determine optimal micronutrient intakes on the basis of long-term needs should allow recommended intakes to be set more accurately and with less reliance on uncertain safety factors. The result may be decreased intake recommen- dations for some micronutrients and increased recommendations for others. This greater certainty should stimulate more aggressive public health efforts to remedy deficiencies. REFERENCES 1. Ames BN. Low micronutrient intake may accelerate the degenerative diseases of aging through allocation of scarce micronutrients by triage. Proc Natl Acad Sci USA 2006;103:17589–94. 2. Ames BN, McCann JC. Forword: Prevention of cancer, and the other degenerative diseases of aging, through nutrition. In: S. Knasmuller DMD, I. Johnson, C. Gerhauser, eds. Chemoprevention of cancer and DNA damage by dietary factors. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co KGaA, 2009:xxxi–vii. 3. Kirkwood TB. Understanding ageing from an evolutionary perspective. J Intern Med 2008;263:117–27. 4. Georgieff MK, Landon MB, Mills MM, et al. Abnormal iron distribution in infants of diabetic mothers: spectrum and maternal antecedents. J Pediatr 1990;117:455–61. 5. Wastney ME, House WA. Development of a compartmental model of zinc kinetics in mice. J Nutr 2008;138:2148–55. 6. DePasquale-Jardieu P, Fraker PJ. Further characterization of the role of corticosterone in the loss of humoral immunity in zinc-deficient A/J mice as determined by adrenalectomy. J Immunol 1980;124:2650–5. 7. Sekler I, Sensi SL, Hershfinkel M, Silverman WF. Mechanism and regulation of cellular zinc transport. Mol Med 2007;13:337–43. 8. Ames BN, Elson-Schwab I, Silver EA. High-dose vitamin therapy stimulates variant enzymes with decreased coenzyme binding affinity (increased K(m)): relevance to genetic disease and polymorphisms. Am J Clin Nutr 2002;75:616–58. 9. Berkner KL. Vitamin K-dependent carboxylation. Vitam Horm 2008; 78:131–56. 10. Freedman SJ, Blostein MD, Baleja JD, Jacobs M, Furie BC, Furie B. Identification of the phospholipid binding site in the vitamin K-dependent blood coagulation protein factor IX. J Biol Chem 1996; 271:16227–36. 11. Freedman SJ, Furie BC, Furie B, Baleja JD. Structure of the metal-free gamma-carboxyglutamic acid-rich membrane binding region of factor IX by two-dimensional NMR spectroscopy. J Biol Chem 1995;270: 7980–7. 12. Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988;53:505–18. 13. Shearer MJ, Vitamin K. Lancet 1995;345:229–34. 14. Cranenburg EC, Vermeer C, Koos R, et al. The circulating inactive form of matrix Gla protein (ucMGP) as a biomarker for cardiovascular calcification. J Vasc Res 2008;45:427–36. 15. Bellido-Martin L, de Frutos PG. Vitamin K-dependent actions of Gas6. Vitam Horm 2008;78:185–209. 16. Berkner KL, Runge KW. The physiology of vitamin K nutriture and vitamin K-dependent protein function in atherosclerosis. J Thromb Haemost 2004;2:2118–32. 17. Garcia AA, Reitsma PH. VKORC1 and the vitamin K cycle. Vitam Horm 2008;78:23–33. 18. Sadler JE. Medicine: K is for koagulation. Nature 2004;427:493–4. 19. Kaneki M, Hosoi T, Ouchi Y, Orimo H. Pleiotropic actions of vitamin K: protector of bone health and beyond? Nutrition 2006;22:845–52. 20. Booth SL, Al Rajabi A. Determinants of vitamin K status in humans. Vitam Horm 2008;78:1–22. 21. Stafford DW. The vitamin K cycle. J Thromb Haemost 2005;3:1873–8. 22. Shearer MJ, Newman P. Metabolism and cell biology of vitamin K. Thromb Haemost 2008;100:530–47. 23. Booth S, Sadowski J, Pennington J. Phylloquinone (vitamin K1) content of foods in the U.S. Food and Drug Administration’s total diet study. J Agric Food Chem 1995;43:1574–9. 24. Schurgers L, Geleijnse J, Grobbee D, et al. Nutritional intake of vitamins K1 (phylloquinone) and K2 (menaquinone) in the Netherlands. J Nutr Environ Med 1999;9:115–22. 25. NAS/DRI. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press, 2000. 26. Davidson KW, Booth S, Dolnikowski G, Sadowski JA. Conversion of vitamin K1 to 2#,3#-dihydrovitamin K1 during the hydrogenation of vegetable oils. J Agric Food Chem 1996;44:980–3. 27. Booth SL, Webb DR, Peters JC. Assessment of phylloquinone and dihydrophylloquinone dietary intakes among a nationally representative sample of US consumers using 14-day food diaries. J Am Diet Assoc 1999;99:1072–6. 28. Booth SL, Lichtenstein AH, O’Brien-Morse M, et al. Effects of a hydrogenated form of vitamin K on bone formation and resorption. Am J Clin Nutr 2001;74:783–90. 29. Suttie JW. The importance of menaquinones in human nutrition. Annu Rev Nutr 1995;15:399–417. 30. Buitenhuis HC, Soute BA, Vermeer C. Comparison of the vitamins K1, K2 and K3 as cofactors for the hepatic vitamin K-dependent carboxylase. Biochim Biophys Acta 1990;1034:170–5. 31. Shearer MJ, Bach A, Kohlmeier M. Chemistry, nutritional sources, tissue distribution and metabolism of vitamin K with special reference to bone health. J Nutr 1996;126:1181S–6S. 32. Thijssen HH, Drittij-Reijnders MJ. Vitamin K distribution in rat tissues: dietary phylloquinone is a source of tissue menaquinone-4. Br J Nutr 1994;72:415–25. 33. Thijssen HH, Drittij-Reijnders MJ. Vitamin K status in human tissues: tissue-specific accumulation of phylloquinone and menaquinone-4. Br J Nutr 1996;75:121–7. 34. Reedstrom CK, Suttie JW. Comparative distribution, metabolism, and utilization of phylloquinone and menaquinone-9 in rat liver. Proc Soc Exp Biol Med 1995;209:403–9. 35. Schurgers LJ, Vermeer C. Determination of phylloquinone and menaquinones in food. Effect of food matrix on circulating vitamin K concentrations. Haemostasis 2000;30:298–307. 36. Elder SJ, Haytowitz DB, Howe J, Peterson JW, Booth SL. Vitamin K contents of meat, dairy, and fast food in the U.S. Diet. J Agric Food Chem 2006;54:463–7. 37. Schurgers LJ, Dissel PE, Spronk HM, et al. Role of vitamin K and vitamin K-dependent proteins in vascular calcification. Z Kardiol 2001; 90(suppl 3):57–63. 38. Davidson RT, Foley AL, Engelke JA, Suttie JW. Conversion of dietary phylloquinone to tissue menaquinone-4 in rats is not dependent on gut bacteria. J Nutr 1998;128:220–3. 39. Thijssen HH, Vervoort LM, Schurgers LJ, Shearer MJ. Menadione is a metabolite of oral vitamin K. Br J Nutr 2006;95:260–6. 40. Thijssen HH, Drittij-Reijnders MJ, Fischer MA. Phylloquinone and menaquinone-4 distribution in rats: synthesis rather than uptake determines menaquinone-4 organ concentrations. J Nutr 1996;126:537–43. 41. Groenen-van Dooren MM, Soute BA, Jie KS, Thijssen HH, Vermeer C. The relative effects of phylloquinone and menaquinone-4 on the blood coagulation factor synthesis in vitamin K-deficient rats. Biochem Pharmacol 1993;46:433–7. 42. Schurgers LJ, Cranenburg EC, Vermeer C. Matrix Gla-protein: the calcification inhibitor in need of vitamin K. Thromb Haemost 2008; 100:593–603. 43. Schurgers LJ, Spronk HM, Soute BA, Schiffers PM, DeMey JG, Vermeer C. Regression of warfarin-induced medial elastocalcinosis by high intake of vitamin K in rats. Blood 2007;109:2823–31. 44. Spronk HM, Soute BA, Schurgers LJ, Thijssen HH, De Mey JG, Vermeer C. Tissue-specific utilization of menaquinone-4 results in the prevention of arterial calcification in warfarin-treated rats. J Vasc Res 2003;40:531–7. 45. Liljemark WF, Gibbons RJ. Ability of Veillonella and Neisseria species to attach to oral surfaces and their proportions present indigenously. Infect Immun 1971;4:264–8. 46. FAO/WHO. Vitamin K. Human vitamin and mineral requirements. Report of a joint FAO/WHO expert consultation. Bangkok, Thailand: Food and Nutrition Division, FAO Rome, 2002:133–150. 47. Schurgers LJ, Teunissen KJ, Hamulyak K, Knapen MH, Vik H, Vermeer C. Vitamin K-containing dietary supplements: comparison of synthetic vitamin K1 and natto-derived menaquinone-7. Blood 2007; 109:3279–83. 48. Sakano T, Notsumoto N, Nagaoka N. Measurement of K vitamins in food by high-performance liquid chromatography with fluorometric detection. Vitamins (Japan) 1988;62:393–8. 49. Okano T, Shimomura Y, Yamane M, et al. Conversion of phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) in mice: two possible routes for menaquinone-4 accumulation in cerebra of mice. J Biol Chem 2008;283:11270–9. 50. Osada S, Tomita H, Tanaka Y, et al. The utility of vitamin K3 (menadione) against pancreatic cancer. Anticancer Res 2008;28:45–50. 51. Takahashi K, Shibata T, Oba T, et al. Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells. Life Sci 2009;84:211–7. 52. Lamson DW, Plaza SM. The anticancer effects of vitamin K. Altern Med Rev 2003;8:303–18. 53. Sakamoto N, Kimura M, Hiraike H, Itokawa Y. Changes of phylloquinone and menaquinone-4 concentrations in rat liver after oral, intravenous and intraperitoneal administration. Int J Vitam Nutr Res 1996;66:322–8. 54. Yamamoto R, Komai M, Kojima K, Furukawa Y, Kimura S. Menaquinone-4 accumulation in various tissues after an oral administration of phylloquinone in Wistar rats. J Nutr Sci Vitaminol (Tokyo) 1997;43:133–43. 55. Ronden JE, Thijssen HH, Vermeer C. Tissue distribution of K-vitamers under different nutritional regimens in the rat. Biochim Biophys Acta 1998;1379:16–22. 56. van Summeren MJ, Braam LA, Lilien MR, Schurgers LJ, Kuis W, Vermeer C. The effect of menaquinone-7 (vitamin K2) supplementation on osteocalcin carboxylation in healthy prepubertal children. Br J Nutr 2009;19:1–8. 57. Sogabe N, Tsugawa N, Maruyama R, et al. Nutritional effects of gamma-glutamyl carboxylase gene polymorphism on the correlation between the vitamin K status and gamma-carboxylation of osteocalcin in young males. J Nutr Sci Vitaminol (Tokyo) 2007;53:419–25. 58. Suttie JW. Vitamin D. In: Zempleni J, Rucker R, McCormick D, Suttie J, eds. Handbook of vitamins. 4th ed. (Clinical Nutrition in Health and Disease). Boca Raton, FL: CRC Press (Taylor & Francis), 2007:111–52. 59. Ikeda H, Doi Y. A vitamin-K2-binding factor secreted from Bacillus subtilis. Eur J Biochem 1990;192:219–24. 60. Eaton SB, Eaton SB 3rd, Konner MJ, Shostak M. An evolutionary perspective enhances understanding of human nutritional requirements. J Nutr 1996;126:1732–40 61. Eaton SB, Konner M. Paleolithic nutrition. A consideration of its nature and current implications. N Engl J Med 1985;312:283–9. 62. ISI Web of Knowledge Cited Reference Search. Available from: http: //apps.isiknowledge.com (cited 9 June 2009). 63. Mouse Genomics Informatics (MGI/4,21). Available from: http: //www.informatics.jax.org (cited 15 May 2009). 64. Chang A, Scheer M, Grote A, Schomburg I, Schomburg D. BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acids Res 2009;37:D588–92. Available from: http://www.brenda-enzymes (cited 15 May 2009). 65. The Uniprot Consortium. The Universal Protein Resource (UniProt). Release 15.6. Nucleic Acids Res 2009;37:D169–74. Available from: http://www.uniprot.org (cited 15 May 2009). 66. SymAtlas. Available through the Novartis Research Foundation (GNF) BioGPS database website: http://biogps.gnf.org (cited 15 May 2009). 67. National Library of Medicine. Available from: http://www.ncbi.nlm. nih.gov/ (cited 9 June 2009). 68. Aasrum M, Prydz H. Gene targeting of tissue factor, factor X, and factor VII in mice: their involvement in embryonic development. Biochemistry (Mosc) 2002;67:25–32. 69. Andrew T, Mak YT, Reed P, MacGregor AJ, Spector TD. Linkage and association for bone mineral density and heel ultrasound measurements with a simple tandem repeat polymorphism near the osteocalcin gene in female dizygotic twins. Osteoporos Int 2002;13:745–54. 70. Angelillo-Scherrer A, de Frutos P, Aparicio C, et al. Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis. Nat Med 2001;7:215–21. 71. Angelillo-Scherrer A, Burnier L, Lambrechts D, et al. Role of Gas6 in erythropoiesis and anemia in mice. J Clin Invest 2008;118:583–96. 72. Benzakour O, Gely A, Lara R, Coronas V. [Gas-6 and protein S: vitamin K-dependent factors and ligands for the TAM tyrosine kinase receptors family (in French)]. Med Sci (Paris) 2007;23:826–33. 73. Bereczky Z, Bardos H, Komaromi I, et al. Factor XDebrecen: Gly204Arg mutation in factor X causes the synthesis of a nonsecretable protein and severe factor X deficiency. Haematologica 2008; 93:299–302. 74. Bezemer ID, Rosendaal FR. Predictive genetic variants for venous thrombosis: what’s new? Semin Hematol 2007;44:85–92. 75. Binder MD, Cate HS, Prieto AL, et al. Gas6 deficiency increases oligodendrocyte loss and microglial activation in response to cuprizoneinduced demyelination. J Neurosci 2008;28:5195–206. 76. Borgel D, Gandrille S, Aiach M. Protein S deficiency. Thromb Haemost 1997;78:351–6. 77. Borgel D. Gas6 inflames cell interactions. Blood 2008;111:3915. 78. Boskey AL, Gadaleta S, Gundberg C, Doty SB, Ducy P, Karsenty G. Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone 1998;23:187–96. 79. Bristol JA, Ratcliffe JV, Roth DA, Jacobs MA, Furie BC, Furie B. Biosynthesis of prothrombin: intracellular localization of the vitamin K-dependent carboxylase and the sites of gamma-carboxylation. Blood 1996;88:2585–93. 80. Bugel S. Vitamin K and bone health in adult humans. Vitam Horm 2008;78:393–416. 81. Carcao MD, Aledort L. Prophylactic factor replacement in hemophilia. Blood Rev 2004;18:101–13. 82. Chen HY, Tsai HD, Chen WC, Wu JY, Tsai FJ, Tsai CH. Relation of polymorphism in the promotor region for the human osteocalcin gene to bone mineral density and occurrence of osteoporosis in postmenopausal Chinese women in Taiwan. J Clin Lab Anal 2001;15:251–5. 83. Cranenburg EC, Schurgers LJ, Vermeer C. Vitamin K: the coagulation vitamin that became omnipotent. Thromb Haemost 2007;98:120–5. 84. Crowther MA, Kelton JG. Congenital thrombophilic states associated with venous thrombosis: a qualitative overview and proposed classification system. Ann Intern Med 2003;138:128–34. 85. Deng HW, Shen H, Xu FH, et al. Tests of linkage and/or association of genes for vitamin D receptor, osteocalcin, and parathyroid hormone with bone mineral density. J Bone Miner Res 2002;17:678–86. 86. Dewerchin M, Liang Z, Moons L, et al. Blood coagulation factor X deficiency causes partial embryonic lethality and fatal neonatal bleeding in mice. Thromb Haemost 2000;83:185–90. 87. Dohi Y, Iki M, Ohgushi H, et al. A novel polymorphism in the promoter region for the human osteocalcin gene: the possibility of a correlation with bone mineral density in postmenopausal Japanese women. J Bone Miner Res 1998;13:1633–9. 88. Ducy P, Desbois C, Boyce B, et al. Increased bone formation in osteocalcin-deficient mice. Nature 1996;382:448–52. 89. El-Maadawy S, Kaartinen MT, Schinke T, Murshed M, Karsenty G, McKee MD. Cartilage formation and calcification in arteries of mice lacking matrix Gla protein. Connect Tissue Res 2003;44(Suppl 1):272–8. 90. Esmon CT. Role of coagulation inhibitors in inflammation. Thromb Haemost 2001;86:51–6. 91. Fair DS, Marlar RA, Levin EG. Human endothelial cells synthesize protein S. Blood 1986;67:1168–71. 92. Ferron M, Hinoi E, Karsenty G, Ducy P. Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice. Proc Natl Acad Sci USA 2008;105:5266–70. 93. Franco RF, Reitsma PH. Genetic risk factors of venous thrombosis. Hum Genet 2001;109:369–84. 94. Fraser JD, Price PA. Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase. J Biol Chem 1988;263: 11033–6. 95. Gao B, Yasui T, Itoh Y, Tozawa K, Hayashi Y, Kohri K. A polymorphism of matrix Gla protein gene is associated with kidney stones. J Urol 2007;177:2361–5. 96. GarciadeFrutos P, Fuentes-Prior P, Hurtado B, Sala N. Molecular basis of protein S deficiency. Thromb Haemost 2007;98:543–56. 97. Gay BB Jr, Kuhn JP. A syndrome of widened medullary cavities of bone, aortic calcification, abnormal dentition, and muscular weakness (the Singleton-Merten syndrome). Radiology 1976;118:389–95. 98. Girolami A, Santarossa L, Scarparo P, Candeo N, Girolami B. True congenital prothrombin deficiency due to a ’new’ mutation in the pre-propeptide (ARG-39 GLN). Acta Haematol 2008;120:82–6. 99. Glueck CJ, Freiberg RA, Wang P. Heritable thrombophiliahypofibrinolysis and osteonecrosis of the femoral head. Clin Orthop Relat Res 2008;466:1034–40. 100. Hafizi S, Dahlback B. Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily. FEBS J 2006; 273:5231–44. 101. He X, Shen L, Bjartell A, Dahlback B. The gene encoding vitamin K-dependent anticoagulant protein S is expressed in multiple rabbit organs as demonstrated by Northern blotting, in situ hybridization, and immunohistochemistry. J Histochem Cytochem 1995;43:85–96. 102. Heiss C, Hoesel LM, Wehr U, et al. Diagnosis of osteoporosis with vitamin K as a new biochemical marker. Vitam Horm 2008;78:417–34. 103. Hendig D, Zarbock R, Szliska C, Kleesiek K, Gotting C. The local calcification inhibitor matrix Gla protein in pseudoxanthoma elasticum. Clin Biochem 2008;41:407–12. 104. Herrmann SM, Whatling C, Brand E, et al. Polymorphisms of the human matrix gla protein (MGP) gene, vascular calcification, and myocardial infarction. Arterioscler Thromb Vasc Biol 2000;20:2386–93. 105. Hinoi E, Gao N, Jung DY, et al. The sympathetic tone mediates leptin’s inhibition of insulin secretion by modulating osteocalcin bioactivity. J Cell Biol 2008;183:1235–42. 106. Hirano H, Ezura Y, Ishiyama N, et al. Association of natural tooth loss with genetic variation at the human matrix Gla protein locus in elderly women. J Hum Genet 2003;48:288–92. 107. Huang QY, Recker RR, Deng HW. Searching for osteoporosis genes in the post-genome era: progress and challenges. Osteoporos Int 2003;14: 701–15. 108. Hur DJ, Raymond GV, Kahler SG, Riegert-Johnson DL, Cohen BA, Boyadjiev SA. A novel MGP mutation in a consanguineous family: review of the clinical and molecular characteristics of Keutel syndrome. Am J Med Genet A 2005;135:36–40. 109. Hurtado B, Munoz X, Mulero MC, et al. Functional characterization of twelve natural PROS1 mutations associated with anticoagulant protein S deficiency. Haematologica 2008;93:574–80. 110. Jalbert LR, Rosen ED, Moons L, et al. Inactivation of the gene for anticoagulant protein C causes lethal perinatal consumptive coagulopathy in mice. J Clin Invest 1998;102:1481–8. 111. Jiang D-K, Xu F-H, Liu M-Y, et al. No evidence of association of the osteocalcin gene HindIII polyorphism with bone mineral density in Chinese women. J Musculoskelet Neuronal Interact 2007;7:149–54. 112. Karimi M, Menegatti M, Afrasiabi A, Sarikhani S, Peyvandi F. Phenotype and genotype report on homozygous and heterozygous patients with congenital factor X deficiency. Haematologica 2008;93: 934–8. 113. Khan S, Dickerman JD. Hereditary thrombophilia. Thromb J 2006;4:15. 114. Koren-Michowitz M, Rahimi-Levene N, Volcheck Y, Garach-Jehoshua O, Kornberg A. Protein Z and its role in venous and arterial thrombosis. Isr Med Assoc J 2006;8:53–5. 115. Kulman JD, Harris JE, Haldeman BA, Davie EW. Primary structure and tissue distribution of two novel proline-rich gammacarboxyglutamic acid proteins. Proc Natl Acad Sci USA 1997;94: 9058–62. 116. Kulman JD, Harris JE, Xie L, Davie EW. Identification of two novel transmembrane gamma-carboxyglutamic acid proteins expressed broadly in fetal and adult tissues. Proc Natl Acad Sci USA 2001;98: 1370–5. 117. Kulman JD, Harris JE, Xie L, Davie EW. Proline-rich Gla protein 2 is a cell-surface vitamin K-dependent protein that binds to the transcriptional coactivator Yes-associated protein. Proc Natl Acad Sci USA 2007;104:8767–72. 118. Kundu RK, Sangiorgi F, Wu LY, et al. Targeted inactivation of the coagulation factor IX gene causes hemophilia B in mice. Blood 1998; 92:168–74. 119. Lee NK, Sowa H, Hinoi E, et al. Endocrine regulation of energy metabolism by the skeleton. Cell 2007;130:456–69. 120. Lin HF, Maeda N, Smithies O, Straight DL, Stafford DW. A coagulation factor IX-deficient mouse model for human hemophilia B. Blood 1997;90:3962–6. 121. Lin GT, Tseng HF, Chang CK, et al. SNP combinations in chromosomewide genes are associated with bone mineral density in Taiwanese women. Chin J Physiol 2008;51:32–41. 122. Linger RM, Keating AK, Earp HS, Graham DK. TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer. Adv Cancer Res 2008;100:35–83. 123. Luo G, Ducy P, McKee MD, et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 1997;386: 78–81. 124. Lutgens E, Tjwa M, de Frutos PG, et al. Genetic loss of Gas6 induces plaque stability in experimental atherosclerosis. J Pathol 2008;216: 55–63. 125. Mahasandana C, Suvatte V, Marlar RA, Manco-Johnson MJ, Jacobson LJ, Hathaway WE. Neonatal purpura fulminans associated with homozygous protein S deficiency. Lancet 1990;335:61–2. 126. Maillard C, Berruyer M, Serre CM, Dechavanne M, Delmas PD. Protein-S, a vitamin K-dependent protein, is a bone matrix component synthesized and secreted by osteoblasts. Endocrinology 1992;130: 1599–604. 127. Mariani G, Herrmann FH, Dolce A, et al. Clinical phenotypes and factor VII genotype in congenital factor VII deficiency. Thromb Haemost 2005;93:481–7. 128. Marlar RA, Neumann A. Neonatal purpura fulminans due to homozygous protein C or protein S deficiencies. Semin Thromb Hemost 1990;16:299–309. 129. Matthews RJ, Peake IR, Bloom AL, Anson DS. Carrier detection through the use of abnormal deletion junction fragments in a case of haemophilia B involving complete deletion of the factor IX gene. J Med Genet 1988;25:779–80. 130. Meier M, Weng LP, Alexandrakis E, Ruschoff J, Goeckenjan G. Tracheobronchial stenosis in Keutel syndrome. Eur Respir J 2001;17: 566–9. 131. Munroe PB, Olgunturk RO, Fryns JP, et al. Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome. Nat Genet 1999;21:142–4. 132. Ndonwi M, Lu L, Tu Y, Phillips M, Broze G Jr. Functional analysis of protein Z (Arg255His) and protein Z-dependent protease inhibitor (Lys25Arg and Ser40Gly) polymorphisms. Br J Haematol 2008;143: 298–300. 133. Oldenburg J. Mutation profiling in haemophilia A. Thromb Haemost 2001;85:577–9. 134. Park IK, Giovenzana C, Hughes TL, Yu J, Trotta R, Caligiuri MA. The Axl/Gas6 pathway is required for optimal cytokine signaling during human natural killer cell development. Blood 2009;113:2470–7. 135. Pearson DA. Bone health and osteoporosis: the role of vitamin K and potential antagonism by anticoagulants. Nutr Clin Pract 2007;22:517–44. 136. Pierre-Jacques H, Glueck CJ, Mont MA, Hungerford DS. Familial heterozygous protein-S deficiency in a patient who had multifocal osteonecrosis. A case report. J Bone Joint Surg Am 1997;79:1079–84. 137. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3#-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698–703. 138. Proudfoot D, Shanahan CM. Molecular mechanisms mediating vascular calcification: role of matrix Gla protein. Nephrology (Carlton) 2006;11:455–61. 139. Raymond MH, Schutte BC, Torner JC, Burns TL, Willing MC. Osteocalcin: genetic and physical mapping of the human gene BGLAP and its potential role in postmenopausal osteoporosis. Genomics 1999; 60:210–7. 140. Rosen ED, Chan JC, Idusogie E, et al. Mice lacking factor VII develop normally but suffer fatal perinatal bleeding. Nature 1997;390:290–4. 141. Sowers M, Willing M, Burns T, et al. Genetic markers, bone mineral density, and serum osteocalcin levels. J Bone Miner Res 1999;14: 1411–9. 142. Speer MY, McKee MD, Guldberg RE, et al. Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo. J Exp Med 2002;196:1047–55. 143. Su AI, Cooke MP, Ching KA, et al. Large-scale analysis of the human and mouse transcriptomes. Proc Natl Acad Sci USA 2002;99:4465–70. 144. Sun WY, Witte DP, Degen JL, et al. Prothrombin deficiency results in embryonic and neonatal lethality in mice. Proc Natl Acad Sci USA 1998;95:7597–602. 145. Tjwa M, Bellido-Martin L, Lin Y, et al. Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes. Blood 2008;111:4096–105. 146. Tsukamoto K, Orimo H, Hosoi T, et al. Association of bone mineral density with polymorphism of the human matrix Gla protein locus in elderly women. J Bone Miner Metab 2000;18:27–30. 147. Uprichard J, Perry DJ. Factor X deficiency. Blood Rev 2002;16:97–110. 148. van den Berg H, DeGroot P, Fischer K. Phenotypic heterogeneity in severe hemophilia. J Thromb Haemost 2007;5(suppl 1):151–6. 149. Vasse M. Protein Z, a protein seeking a pathology. Thromb Haemost 2008;100:548–56. 150. Villines TC, Hatzigeorgiou C, Feuerstein IM, O’Malley PG, Taylor AJ. Vitamin K1 intake and coronary calcification. Coron Artery Dis 2005; 16:199–203. 151. Wallin R, Schurgers L, Wajih N. Effects of the blood coagulation vitamin K as an inhibitor of arterial calcification. Thromb Res 2008;122: 411–7. 152. Walzer T, Vivier E. NK cell development: gas matters. Nat Immunol 2006;7:702–4. 153. Wang L, Zoppe M, Hackeng TM, Griffin JH, Lee KF, Verma IM. A factor IX-deficient mouse model for hemophilia B gene therapy. Proc Natl Acad Sci USA 1997;94:11563–6. 154. Xue J, Wu Q, Westfield LA, et al. Incomplete embryonic lethality and fatal neonatal hemorrhage caused by prothrombin deficiency in mice. Proc Natl Acad Sci USA 1998;95:7603–7. 155. Xue M, Campbell D, Jackson CJ. Protein C is an autocrine growth factor for human skin keratinocytes. J Biol Chem 2007;282: 13610–6. 156. Yanagita M. Gas6, warfarin, and kidney diseases. Clin Exp Nephrol 2004;8:304–9. 157. Yao Y, Shahbazian A, Bostrom KI. Proline and gamma-carboxylated glutamate residues in matrix Gla protein are critical for binding of bone morphogenetic protein-4. Circ Res 2008;102:1065–74. 158. Yin ZF, Huang ZF, Cui J, et al. Prothrombotic phenotype of protein Z deficiency. Proc Natl Acad Sci USA 2000;97:6734–8. 159. Zhao B, Ye X, Yu J, et al. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev 2008;22:1962–71. 160. Kii I, Amizuka N, Minqi L, Kitajima S, Saga Y, Kudo A. Periostin is an extracellular matrix protein required for eruption of incisors in mice. Biochem Biophys Res Commun 2006;342:766–72. 161. Rios H, Koushik SV, Wang H, et al. Periostin null mice exhibit dwarfism, incisor enamel defects, and an early-onset periodontal disease-like phenotype. Mol Cell Biol 2005;25:11131–44. 162. Shimazaki M, Nakamura K, Kii I, et al. Periostin is essential for cardiac healing after acute myocardial infarction. J Exp Med 2008;205: 295–303. 163. Oka T, Xu J, Kaiser RA, et al. Genetic manipulation of periostin expression reveals a role in cardiac hypertrophy and ventricular remodeling. Circ Res 2007;101:313–21. 164. Viegas CS, Simes DC, Laize V, Williamson MK, Price PA, Cancela ML. Gla-rich protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates. J Biol Chem 2008;283:36655–64. 165. Coutu DL, Wu JH, Monette A, Rivard GE, Blostein MD, Galipeau J. Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells. J Biol Chem 2008;283: 17991–8001. 166. Jackson CJ, Xue M. Activated protein C—an anticoagulant that does more than stop clots. Int J Biochem Cell Biol 2008;40:2692–7. 167. Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007;109:3161–72. 168. Isermann B, Sood R, Pawlinski R, et al. The thrombomodulin-protein C system is essential for the maintenance of pregnancy. Nat Med 2003; 9:331–7. 169. Lay AJ, Liang Z, Rosen ED, Castellino FJ. Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities. J Clin Invest 2005;115:1552–61. 170. Kudo Y, Siriwardena BS, Hatano H, Ogawa I, Takata T. Periostin: novel diagnostic and therapeutic target for cancer. Histol Histopathol 2007;22:1167–74. 171. Lorts A, Schwanekamp JA, Elrod JW, Sargent MA, Molkentin JD. Genetic manipulation of periostin expression in the heart does not affect myocyte content, cell cycle activity, or cardiac repair. Circ Res 2009;104:e1–7. 172. Conway SJ, Molkentin JD. Periostin as a heterofunctional regulator of cardiac development and disease. Curr Genomics 2008;9:548–55. 173. Ahmed AA, Mills AD, Ibrahim AE, et al. The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel. Cancer Cell 2007;12:514–27. 174. Zhang Y, Wen G, Shao G, et al. TGFBI deficiency predisposes mice to spontaneous tumor development. Cancer Res 2009;69:37–44. 175. Kannabiran C, Klintworth GK. TGFBI gene mutations in corneal dystrophies. Hum Mutat 2006;27:615–25. 176. Dihanich M, Kaser M, Reinhard E, Cunningham D, Monard D. Prothrombin mRNA is expressed by cells of the nervous system. Neuron 1991;6:575–81. 177. Kim S. Characterization of the prothrombin gene expression during nerve differentiation. Biochim Biophys Acta 2004;1679:1–9. 178. Stapleton AM, Timme TL, Ryall RL. Gene expression of prothrombin in the human kidney and its potential relevance to kidney stone disease. Br J Urol 1998;81:666–71 (discussion 671–2). 179. Ayala A, Warejcka DJ, Olague-Marchan M, Twining SS. Corneal activation of prothrombin to form thrombin, independent of vascular injury. Invest Ophthalmol Vis Sci 2007;48:134–43. 180. Di Cera E. Thrombin. Mol Aspects Med 2008;29:203–54. 181. Litvin J, Zhu S, Norris R, Markwald R. Periostin family of proteins: therapeutic targets for heart disease. Anat Rec A Discov Mol Cell Evol Biol 2005;287:1205–12. 182. Thapa N, Lee BH, Kim IS. TGFBIp/betaig-h3 protein: a versatile matrix molecule induced by TGF-beta. Int J Biochem Cell Biol 2007; 39:2183–94. 183. Dahlback B. The tale of protein S and C4b-binding protein: a story of affection. Thromb Haemost 2007;98:90–6. 184. Maurissen LF, Thomassen MC, Nicolaes GA, et al. Re-evaluation of the role of the protein S-C4b binding protein complex in activated protein C-catalyzed factor Va-inactivation. Blood 2008;111: 3034–41. 185. Gossl M, Modder UI, Atkinson EJ, Lerman A, Khosla S. Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis. J Am Coll Cardiol 2008;52:1314–25. 186. Snider P, Hinton RB, Moreno-Rodriguez RA, et al. Periostin is required for maturation and extracellular matrix stabilization of noncardiomyocyte lineages of the heart. Circ Res 2008;102:752–60. 187. Hauschka PV, Lian JB, Cole DE, Gundberg CM. Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiol Rev 1989;69:990–1047. 188. Fukumoto S, Martin TJ. Bone as an endocrine organ. Trends Endocrinol Metab 2009;20:230–6. 189. Munoz X, Obach V, Hurtado B, de Frutos PG, Chamorro A, Sala N. Association of specific haplotypes of GAS6 gene with stroke. Thromb Haemost 2007;98:406–12. 190. Jiang L, Liu CY, Yang QF, Wang P, Zhang W. Plasma level of growth arrest-specific 6 (GAS6) protein and genetic variations in the GAS6 gene in patients with acute coronary syndrome. Am J Clin Pathol 2009; 131:738–43. 191. Crosier MD, Booth SL, Peter I, et al. Matrix Gla protein polymorphisms are associated with coronary artery calcification in men. J Nutr Sci Vitaminol (Tokyo) 2009;55:59–65. 192. Ma C, Rong Y, Radiloff DR, et al. Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation. Genes Dev 2008;22:308–21. 193. Litvin J, Selim AH, Montgomery MO, et al. Expression and function of periostin-isoforms in bone. J Cell Biochem 2004;92:1044–61. 194. Shimazaki M, Kudo A. Impaired capsule formation of tumors in periostin-null mice. Biochem Biophys Res Commun 2008;367:736–42. 195. Erin N, Wang N, Xin P, et al. Altered gene expression in breast cancer liver metastases. Int J Cancer 2009;124:1503–16. 196. Kim CJ, Yoshioka N, Tambe Y, Kushima R, Okada Y, Inoue H. Periostin is down-regulated in high grade human bladder cancers and suppresses in vitro cell invasiveness and in vivo metastasis of cancer cells. Int J Cancer 2005;117:51–8. 197. Zhu A, Sun H, Raymond RM Jr, et al. Fatal hemorrhage in mice lacking gamma-glutamyl carboxylase. Blood 2007;109:5270–5. 198. Booth SL, O’Brien-Morse ME, Dallal GE, Davidson KW, Gundberg CM. Response of vitamin K status to different intakes and sources of phylloquinone-rich foods: comparison of younger and older adults. Am J Clin Nutr 1999;70:368–77. 199. Booth SL, Martini L, Peterson JW, Saltzman E, Dallal GE, Wood RJ. Dietary phylloquinone depletion and repletion in older women. J Nutr 2003;133:2565–9. 200. Sokoll LJ, Booth SL, O’Brien ME, Davidson KW, Tsaioun KI, Sadowski JA. Changes in serum osteocalcin, plasma phylloquinone, and urinary gamma-carboxyglutamic acid in response to altered intakes of dietary phylloquinone in human subjects. Am J Clin Nutr 1997;65: 779–84. 201. Bach AU, Anderson SA, Foley AL, Williams EC, Suttie JW. Assessment of vitamin K status in human subjects administered ‘‘minidose’’ warfarin. Am J Clin Nutr 1996;64:894–902. 202. Booth SL, Suttie JW. Dietary intake and adequacy of vitamin K. J Nutr 1998;128:785–8. 203. Schurgers LJ, Teunissen KJ, Knapen MH, et al. Novel conformationspecific antibodies against matrix gamma-carboxyglutamic acid (Gla) protein: undercarboxylated matrix Gla protein as marker for vascular calcification. Arterioscler Thromb Vasc Biol 2005;25:1629–33. 204. Cranenburg EC, Brandenburg VM, Vermeer C, et al. Uncarboxylated matrix Gla protein (ucMGP) is associated with coronary artery calcification in haemodialysis patients. Thromb Haemost 2009;101:359–66. 205. Koos R, Krueger T, Westenfeld R, et al. Relation of circulating matrix Gla-protein and anticoagulation status in patients with aortic valve calcification. Thromb Haemost 2009;101:706–13. 206. Hermans MM, Vermeer C, Kooman JP, et al. Undercarboxylated matrix GLA protein levels are decreased in dialysis patients and related to parameters of calcium-phosphate metabolism and aortic augmentation index. Blood Purif 2007;25:395–401. 207. Parker BD, Ix JH, Cranenburg EC, Vermeer C, Whooley MA, Schurgers LJ. Association of kidney function and uncarboxylated matrix Gla protein: data from the Heart and Soul Study. Nephrol Dial Transplant 2009;24:2095–101. 208. Schurgers LJ, Vermeer C. Differential lipoprotein transport pathways of K-vitamins in healthy subjects. Biochim Biophys Acta 2002;1570: 27–32. 209. Sato T, Ohtani Y, Yamada Y, Saitoh S, Harada H. Difference in the metabolism of vitamin K between liver and bone in vitamin K-deficient rats. Br J Nutr 2002;87:307–14. 210. Lamon-Fava S, Sadowski JA, Davidson KW, O’Brien ME, McNamara JR, Schaefer EJ. Plasma lipoproteins as carriers of phylloquinone (vitamin K1) in humans. Am J Clin Nutr 1998;67:1226–31. 211. Rohlmann A, Gotthardt M, Hammer RE, Herz J. Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants. J Clin Invest 1998;101:689–95. 212. Cooper AD. Hepatic uptake of chylomicron remnants. J Lipid Res 1997;38:2173–92. 213. Havel RJ. Postprandial lipid metabolism: an overview. Proc Nutr Soc 1997;56:659–66. 214. Hussain MM, Goldberg IJ, Weisgraber KH, Mahley RW, Innerarity TL. Uptake of chylomicrons by the liver, but not by the bone marrow, is modulated by lipoprotein lipase activity. Arterioscler Thromb Vasc Biol 1997;17:1407–13. 215. Karpe F, Humphreys SM, Samra JS, Summers LK, Frayn KN. Clearance of lipoprotein remnant particles in adipose tissue and muscle in humans. J Lipid Res 1997;38:2335–43. 216. Newman P, Bonello F, Wierzbicki AS, Lumb P, Savidge GF, Shearer MJ. The uptake of lipoprotein-borne phylloquinone (vitamin K1) by osteoblasts and osteoblast-like cells: role of heparan sulfate proteoglycans and apolipoprotein E. J Bone Miner Res 2002;17:426–33. 217. Niemeier A, Kassem M, Toedter K, et al. Expression of LRP1 by human osteoblasts: a mechanism for the delivery of lipoproteins and vitamin K1 to bone. J Bone Miner Res 2005;20:283–93. 218. Niemeier A, Niedzielska D, Secer R, et al. Uptake of postprandial lipoproteins into bone in vivo: impact on osteoblast function. Bone 2008;43:230–7. 219. Saupe J, Shearer MJ, Kohlmeier M. Phylloquinone transport and its influence on gamma-carboxyglutamate residues of osteocalcin in patients on maintenance hemodialysis. Am J Clin Nutr 1993;58:204–8. 220. Erkkila AT, Lichtenstein AH, Dolnikowski GG, et al. Plasma transport of vitamin K in men using deuterium-labeled collard greens. Metabolism 2004;53:215–21. 221. Berkner KL. The vitamin K-dependent carboxylase. Annu Rev Nutr 2005;25:127–49. 222. Mackey RH, Venkitachalam L, Sutton-Tyrrell K. Calcifications, arterial stiffness and atherosclerosis. Adv Cardiol 2007;44:234–44. 223. Lafdil F, Chobert MN, Deveaux V, et al. Growth arrest-specific protein 6 deficiency impairs liver tissue repair after acute toxic hepatitis in mice. J Hepatol 2009;51:55–66. 224. Jeggo PA. Genomic instability in cancer development. Adv Exp Med Biol 2005;570:175–97. 225. Bakhoum SF, Thompson SL, Manning AL, Compton DA. Genome stability is ensured by temporal control of kinetochore-microtubule dynamics. Nat Cell Biol 2009;11:27–35. 226. Glick AB, Weinberg WC, Wu IH, Quan W, Yuspa SH. Transforming growth factor beta 1 suppresses genomic instability independent of a G1 arrest, p53, and Rb. Cancer Res 1996;56:3645–50. 227. Maxwell CA, Fleisch MC, Costes SV, et al. Targeted and nontargeted effects of ionizing radiation that impact genomic instability. Cancer Res 2008;68:8304–11. 228. Yamada H, Vijayachandra K, Penner C, Glick A. Increased sensitivity of transforming growth factor (TGF) beta 1 null cells to alkylating agents reveals a novel link between TGFbeta signaling and O(6)-methylguanine methyltransferase promoter hypermethylation. J Biol Chem 2001;276:19052–8. 229. Wakefield LM, Roberts AB. TGF-beta signaling: positive and negative effects on tumorigenesis. Curr Opin Genet Dev 2002;12:22–9. 230. Fleisch MC, Maxwell CA, Barcellos-Hoff MH. The pleiotropic roles of transforming growth factor beta in homeostasis and carcinogenesis of endocrine organs. Endocr Relat Cancer 2006;13:379–400. 231. Barcellos-Hoff MH, Akhurst RJ. Transforming growth factor-beta in breast cancer: too much, too late. Breast Cancer Res 2009;11:202. 232. Ruan K, Bao S, Ouyang G. The multifaceted role of periostin in tumorigenesis. Cell Mol Life Sci 2009;66:2219–30 233. Kim BY, Olzmann JA, Choi SI, et al. Corneal dystrophy-associated H124H mutation disrupts TGFBI interaction with periostin and causes mislocalization to the lysosome. J Biol Chem 2009;284:19580–91. 234. Williams GC. Pleiotropy, natural selection, and the evolution of senescence. Evolution Int J Org Evolution 1957;11:398–411. 235. Shea MK, Booth SL. Update on the role of vitamin K in skeletal health. Nutr Rev 2008;66:549–57. 236. Cashman KD, O’Connor E. Does high vitamin K1 intake protect against bone loss in later life? Nutr Rev 2008;66:532–8. 237. Brunetti-Pierri N, Hunter JV, Boerkoel CF. Gray matter heterotopias and brachytelephalangic chondrodysplasia punctata: a complication of hyperemesis gravidarum induced vitamin K deficiency? Am J Med Genet A 2007;143:200–4. 238. Kalkwarf HJ, Khoury JC, Bean J, Elliot JG. Vitamin K, bone turnover, and bone mass in girls. Am J Clin Nutr 2004;80:1075–80. 239. Booth SL, Tucker KL, Chen H, et al. Dietary vitamin K intakes are associated with hip fracture but not with bone mineral density in elderly men and women. Am J Clin Nutr 2000;71:1201–8. 240. Booth SL, Broe KE, Gagnon DR, et al. Vitamin K intake and bone mineral density in women and men. Am J Clin Nutr 2003;77: 512–6. 241. Feskanich D, Weber P, Willett WC, Rockett H, Booth SL, Colditz GA. Vitamin K intake and hip fractures in women: a prospective study. Am J Clin Nutr 1999;69:74–9. 242. Tsugawa N, Shiraki M, Suhara Y, et al. Low plasma phylloquinone concentration is associated with high incidence of vertebral fracture in Japanese women. J Bone Miner Metab 2008;26:79–85. 243. Booth SL, Broe KE, Peterson JW, et al. Associations between vitamin K biochemical measures and bone mineral density in men and women. J Clin Endocrinol Metab 2004;89:4904–9. 244. Braam LA, Knapen MH, Geusens P, et al. Vitamin K1 supplementation retards bone loss in postmenopausal women between 50 and 60 years of age. Calcif Tissue Int 2003;73:21–6. 245. Cockayne S, Adamson J, Lanham-New S, Shearer MJ, Gilbody S, Torgerson DJ. Vitamin K and the prevention of fractures: systematic review and meta-analysis of randomized controlled trials. Arch Intern Med 2006;166:1256–61. 246. Ikeda Y, Iki M, Morita A, et al. Intake of fermented soybeans, natto, is associated with reduced bone loss in postmenopausal women: Japanese Population-Based Osteoporosis (JPOS) Study. J Nutr 2006;136: 1323–8. 247. Kaneki M, Hodges SJ, Hosoi T, et al. Japanese fermented soybean food as the major determinant of the large geographic difference in circulating levels of vitamin K2: possible implications for hip-fracture risk. Nutrition 2001;17:315–21. 248. Hirao M, Hashimoto J, Ando W, Ono T, Yoshikawa H. Response of serum carboxylated and undercarboxylated osteocalcin to alendronate monotherapy and combined therapy with vitamin K2 in postmenopausal women. J Bone Miner Metab 2008;26:260–4. 249. Knapen MH, Schurgers LJ, Vermeer C. Vitamin K2 supplementation improves hip bone geometry and bone strength indices in postmenopausal women. Osteoporos Int 2007;18:963–72. 250. Binkley N, Harke J, Krueger D. Vitamin K treatment reduces undercarboxylated osteocalcin but does not alter bone turnover, density or geometry in healthy postmenopausal North American women. J Bone Miner Res 2009;24:983–91. 251. Booth SL, Dallal G, Shea MK, Gundberg C, Peterson JW, DawsonHughes B. Effect of vitamin K supplementation on bone loss in elderly men and women. J Clin Endocrinol Metab 2008;93:1217–23. 252. Cheung AM, Tile L, Lee Y, et al. Vitamin K supplementation in postmenopausal women with osteopenia (ECKO trial): a randomized controlled trial. PLoS Med 2008;5:e196. 253. Vermeer C, Hamulyak K. Vitamin K: lessons from the past. J Thromb Haemost 2004;2:2115–7. 254. Erkkila AT, Booth SL. Vitamin K intake and atherosclerosis. Curr Opin Lipidol 2008;19:39–42. 255. Jie KS, Bots ML, Vermeer C, Witteman JC, Grobbee DE. Vitamin K intake and osteocalcin levels in women with and without aortic atherosclerosis: a population-based study. Atherosclerosis 1995;116:117–23. 256. Beulens JW, Bots ML, Atsma F, et al. High dietary menaquinone intake is associated with reduced coronary calcification. Atherosclerosis 2009;203:489–93. 257. Gast GC, de Roos NM, Sluijs I, et al. A high menaquinone intake reduces the incidence of coronary heart disease in women. Nutr Metab Cardiovasc Dis 2009;19:504–10. 258. Geleijnse JM, Vermeer C, Grobbee DE, et al. Dietary intake of menaquinone is associated with a reduced risk of coronary heart disease: the Rotterdam Study. J Nutr 2004;134:3100–5. 259. Braam LA, Hoeks AP, Brouns F, Hamulyak K, Gerichhausen MJ, Vermeer C. Beneficial effects of vitamins D and K on the elastic properties of the vessel wall in postmenopausal women: a follow-up study. Thromb Haemost 2004;91:373–80. 260. Jie KG, Bots ML, Vermeer C, Witteman JC, Grobbee DE. Vitamin K status and bone mass in women with and without aortic atherosclerosis: a population-based study. Calcif Tissue Int 1996;59:352–6. 261. Erkkila AT, Booth SL, Hu FB, Jacques PF, Lichtenstein AH. Phylloquinone intake and risk of cardiovascular diseases in men. Nutr Metab Cardiovasc Dis 2007;17:58–62. 262. Erkkila AT, Booth SL, Hu FB, et al. Phylloquinone intake as a marker for coronary heart disease risk but not stroke in women. Eur J Clin Nutr 2005;59:196–204. 263. Mizuta T, Ozaki I, Eguchi Y, et al. The effect of menatetrenone, a vitamin K2 analog, on disease recurrence and survival in patients with hepatocellular carcinoma after curative treatment: a pilot study. Cancer 2006;106:867–72. 264. Hosho K, Okano J, Koda M, Murawaki Y. Vitamin K2 has no preventive effect on recurrence of hepatocellular carcinoma after effective treatment. Yonago Acta Med 2008;51:95–9. 265. Hotta N, Ayada M, Sato K, et al. Effect of vitamin K2 on the recurrence in patients with hepatocellular carcinoma. Hepatogastroenterology 2007;54:2073–7. 266. Kakizaki S, Sohara N, Sato K, et al. Preventive effects of vitamin K on recurrent disease in patients with hepatocellular carcinoma arising from hepatitis C viral infection. J Gastroenterol Hepatol 2007;22: 518–22. 267. Nimptsch K, Rohrmann S, Linseisen J. Dietary intake of vitamin K and risk of prostate cancer in the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Heidelberg). Am J Clin Nutr 2008;87:985–92. 268. Nimptsch K, Rohrmann S, Nieters A, Linseisen J. Serum undercarboxylated osteocalcin as biomarker of vitamin K intake and risk of prostate cancer: a nested case-control study in the Heidelberg cohort of the European prospective investigation into cancer and nutrition. Cancer Epidemiol Biomarkers Prev 2009;18:49–56. 269. Yoshida M, Jacques PF, Meigs JB. Effect of vitamin K supplementation on insulin resistance in older men and women. Diabetes Care 2008;31: 2092–96. 270. Yoshida M, Booth SL, Meigs JB, Saltzman E, Jacques PF. Phylloquinone intake, insulin sensitivity, and glycemic status in men and women. Am J Clin Nutr 2008;88:210–5. 271. Neogi T, Booth SL, Zhang YQ, et al. Low vitamin K status is associated with osteoarthritis in the hand and knee. Arthritis Rheum 2006; 54:1255–61. 272. Pilkey RM, Morton AR, Boffa MB, et al. Subclinical vitamin K deficiency in hemodialysis patients. Am J Kidney Dis 2007;49: 432–9. 273. Holden RM, Booth SL. Vascular calcification in chronic kidney disease: the role of vitamin K. Nat Clin Pract Nephrol 2007;3:522–3. 274. Danziger J. Vitamin K-dependent proteins, warfarin, and vascular calcification. Clin J Am Soc Nephrol 2008;3:1504–10. 275. Shea MK, Booth SL, Massaro JM, et al. Vitamin K and vitamin D status: associations with inflammatory markers in the Framingham Offspring Study. Am J Epidemiol 2008;167:313–20. 276. Shea M, O’Donnell C, Hoffmann U, et al. Vitamin K supplementation and progression of coronary artery calcium in older men and women. Am J Clin Nutr 2009;89:1799–807. 277. Neogi T, Felson DT, Sarno R, Booth SL. Vitamin K in hand osteoarthritis: results from a randomised clinical trial. Ann Rheum Dis 2008; 67:1570–3. 278. Schlieper G, Brandenburg V, Djuric Z, et al. Risk factors for cardiovascular calcifications in non-diabetic Caucasian haemodialysis patients. Kidney Blood Press Res 2009;32:161–8. 279. Ishimura E, Okuno S, Taniwaki H, et al. Different risk factors for vascular calcification in end-stage renal disease between diabetics and nondiabetics: the respective importance of glycemic and phosphate control. Kidney Blood Press Res 2008;31:10–5. 280. Booth S. Roles for vitamin K beyond coagulation. Ann Rev Nutr 2009; 29:5.1–5.22. 281. Vermeer C, Gijsbers BL, Craciun AM, Groenen-van Dooren MM, Knapen MH. Effects of vitamin K on bone mass and bone metabolism. J Nutr 1996;126:1187S–91S. 282. Iwamoto J, Sato Y, Takeda T, Matsumoto H. High-dose vitamin K supplementation reduces fracture incidence in postmenopausal women: a review of the literature. Nutr Res 2009;29:221–8. 283. Kiel DP, Kauppila LI, Cupples LA, Hannan MT, O’Donnell CJ, Wilson PW. Bone loss and the progression of abdominal aortic calcification over a 25 year period: the Framingham Heart Study. Calcif Tissue Int 2001;68:271–6. 284. Hak AE, Pols HA, van Hemert AM, Hofman A, Witteman JC. Progression of aortic calcification is associated with metacarpal bone loss during menopause: a population-based longitudinal study. Arterioscler Thromb Vasc Biol 2000;20:1926–31. 285. Chow JT, Khosla S, Melton LJ III, Atkinson EJ, Camp JJ, Kearns AE. Abdominal aortic calcification, BMD, and bone microstructure: a population-based study. J Bone Miner Res 2008;23:1601–12. 286. Naves M, Rodriguez-Garcia M, Diaz-Lopez JB, Gomez-Alonso C, Cannata-Andia JB. Progression of vascular calcifications is associated with greater bone loss and increased bone fractures. Osteoporos Int 2008;19:1161–6. 287. Hofbauer LC, Brueck CC, Shanahan CM, Schoppet M, Dobnig H. Vascular calcification and osteoporosis: from clinical observation towards molecular understanding. Osteoporos Int 2007;18:251–9. 288. Samelson EJ, Cupples LA, Broe KE, Hannan MT, O’Donnell CJ, Kiel DP. Vascular calcification in middle age and long-term risk of hip fracture: the Framingham Study. J Bone Miner Res 2007;22: 1449–54. 289. Kakizaki S, Yamazaki Y, Takizawa D, Negishi M. New insights on the xenobiotic-sensing nuclear receptors in liver diseases—CAR and PXR. Curr Drug Metab 2008;9:614–21. 290. Mizuta T, Ozaki I. Hepatocellular carcinoma and vitamin K. Vitam Horm 2008;78:435–42. 291. Tamori A, Habu D, Shiomi S, Kubo S, Nishiguchi S. Potential role of vitamin K(2) as a chemopreventive agent against hepatocellular carcinoma. Hepatol Res 2007;37(Suppl 2):S303–7. 292. Sathienkijkanchai A, Wasant P. Fetal warfarin syndrome. J Med Assoc Thai 2005;88(suppl 8):S246–50. 293. Avgeri M, Papadopoulou A, Platokouki H, et al. Assessment of bone mineral density and markers of bone turnover in children under long-term oral anticoagulant therapy. J Pediatr Hematol Oncol 2008; 30:592–7. 294. Gage BF, Birman-Deych E, Radford MJ, Nilasena DS, Binder EF. Risk of osteoporotic fracture in elderly patients taking warfarin: results from the National Registry of Atrial Fibrillation 2. Arch Intern Med 2006; 166:241–6. 295. Woo C, Chang LL, Ewing SK, Bauer DC. Single-point assessment of warfarin use and risk of osteoporosis in elderly men. J Am Geriatr Soc 2008;56:1171–6. 296. Hara K, Kobayashi M, Akiyama Y. Influence of bone osteocalcin levels on bone loss induced by ovariectomy in rats. J Bone Miner Metab 2007;25:345–53. 297. Thoongsuwan N, Stern EJ. Warfarin-induced tracheobronchial calcification. J Thorac Imaging 2003;18:110–2. 298. Krishnan S, Chawla N, Ezekowitz MD, Peixoto AJ. Warfarin therapy and systolic hypertension in men with atrial fibrillation. Am J Hypertens 2005;18:1592–9. 299. Schurgers LJ, Aebert H, Vermeer C, Bultmann B, Janzen J. Oral anticoagulant treatment: friend or foe in cardiovascular disease? Blood 2004;104:3231–2. 300. Koos R, Mahnken AH, Muhlenbruch G, et al. Relation of oral anticoagulation to cardiac valvular and coronary calcium assessed by multislice spiral computed tomography. Am J Cardiol 2005;96:747–9. 301. Holden RM, Sanfilippo AS, Hopman WM, Zimmerman D, Garland JS, Morton AR. Warfarin and aortic valve calcification in hemodialysis patients. J Nephrol 2007;20:417–22. 302. Price PA, Faus SA, Williamson MK. Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler Thromb Vasc Biol 1998;18:1400–7. 303. Zwicker JI, Furie BC, Furie B. Cancer-associated thrombosis. Crit Rev Oncol Hematol 2007;62:126–36. 304. Adcock DM, Fink LM, Marlar RA, Cavallo F, Zangari M. The hemostatic system and malignancy. Clin Lymphoma Myeloma 2008;8: 230–6. 305. Douketis JD, Gu C, Piccioli A, Ghirarduzzi A, Pengo V, Prandoni P. The long-term risk of cancer in patients with a first episode of venous thromboembolism. J Thromb Haemost 2009;7:546–51. 306. Iodice S, Gandini S, Lohr M, Lowenfels AB, Maisonneuve P. Venous thromboembolic events and organ-specific occult cancers: a review and meta-analysis. J Thromb Haemost 2008;6:781–8. 307. Sorensen HT, Mellemkjaer L, Steffensen FH, Olsen JH, Nielsen GL. The risk of a diagnosis of cancer after primary deep venous thrombosis or pulmonary embolism. N Engl J Med 1998;338:1169–73. 308. Murchison JT, Wylie L, Stockton DL. Excess risk of cancer in patients with primary venous thromboembolism: a national, population-based cohort study. Br J Cancer 2004;91:92–5. 309. Piccioli A, Lensing AW, Prins MH, et al. Extensive screening for occult malignant disease in idiopathic venous thromboembolism: a prospective randomized clinical trial. J Thromb Haemost 2004;2:884–9. 310. Baron JA, Gridley G, Weiderpass E, Nyren O, Linet M. Venous thromboembolism and cancer. Lancet 1998;351:1077–80. 311. Tagalakis V, Blostein M, Robinson-Cohen C, Kahn SR. The effect of anticoagulants on cancer risk and survival: systematic review. Cancer Treat Rev 2007;33:358–68. 312. White RH. Cancer begets venous thromboembolism, but is venous thromboembolism a risk factor for cancer? J Thromb Haemost 2009;7: 543–5. 313. Tagalakis V, Tamim H, Blostein M, Collet JP, Hanley JA, Kahn SR. Use of warfarin and risk of urogenital cancer: a population-based, nested case-control study. Lancet Oncol 2007;8:395–402. 314. Booth SL, Pennington JA, Sadowski JA. Food sources and dietary intakes of vitamin K-1 (phylloquinone) in the American diet: data from the FDA Total Diet Study. J Am Diet Assoc 1996;96:149–54. 315. Collins A, Cashman KD, Kiely M. A preliminary assessment of vitamin K1 intakes and serum undercarboxylated osteocalcin levels in 11-13 year old Irish girls. Int J Vitam Nutr Res 2006;76:385–90. 316. Collins A, Cashman KD, Kiely M. Phylloquinone (vitamin K1) intakes and serum undercarboxylated osteocalcin levels in Irish postmenopausal women. Br J Nutr 2006;95:982–8. 317. Prynne CJ, Thane CW, Prentice A, Wadsworth ME. Intake and sources of phylloquinone (vitamin K(1)) in 4-year-old British children: comparison between 1950 and the 1990s. Public Health Nutr 2005;8: 171–80. 318. Thane CW, Bolton-Smith C, Coward WA. Comparative dietary intake and sources of phylloquinone (vitamin K1) among British adults in 1986–7 and 2000–1. Br J Nutr 2006;96:1105–15. 319. Thane CW, Paul AA, Bates CJ, Bolton-Smith C, Prentice A, Shearer MJ. Intake and sources of phylloquinone (vitamin K1): variation with socio-demographic and lifestyle factors in a national sample of British elderly people. Br J Nutr 2002;87:605–13. 320. UK Department of Health. Dietary reference values for food energy and nutrients for the United Kingdom. Report on health and social subjects no. 41. London, United Kingdom: HMSO, 1991. 321. Rucker RB. Improved functional endpoints for use in vitamin K assessment: important implications for bone disease. Am J Clin Nutr 1997;65:883–4. 322. Binkley NC, Krueger DC, Kawahara TN, Engelke JA, Chappell RJ, Suttie JW. A high phylloquinone intake is required to achieve maximal osteocalcin gamma-carboxylation. Am J Clin Nutr 2002;76:1055–60. 323. Binkley NC, Krueger DC, Engelke JA, Foley AL, Suttie JW. Vitamin K supplementation reduces serum concentrations of under-gammacarboxylated osteocalcin in healthy young and elderly adults. Am J Clin Nutr 2000;72:1523–8. 324. van Summeren M, Braam L, Noirt F, Kuis W, Vermeer C. Pronounced elevation of undercarboxylated osteocalcin in healthy children. Pediatr Res 2007;61:366–70. 325. Igarashi M, Yogiashi Y, Mihara M, Takada I, Kitagawa H, Kato S. Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene. Mol Cell Biol 2007;27:7947–54. 326. Yaegashi Y, Onoda T, Tanno K, Kuribayashi T, Sakata K, Orimo H. Association of hip fracture incidence and intake of calcium, magnesium, vitamin D, and vitamin K. Eur J Epidemiol 2008;23:219–25. 327. Kitamura A, Iso H, Imano H, et al. Prevalence and correlates of carotid atherosclerosis among elderly Japanese men. Atherosclerosis 2004; 172:353–9. 328. Solberg LA, Ishii T, Strong JP, et al. Comparison of coronary atherosclerosis in middle-aged Norwegian and Japanese men: an autopsy study. Lab Invest 1987;56:451–6. 329. Sims FH, Yoshida Y, Sakurai I, Tsuda Y, Wakasugi C. A comparison of coronary arteries from Japanese and NZ subjects. Pathology 1995;27: 215–20. 330. Iki M, Kagamimori S, Kagawa Y, Matsuzaki T, Yoneshima H, Marumo F. Bone mineral density of the spine, hip and distal forearm in representative samples of the Japanese female population: Japanese Population-Based Osteoporosis (JPOS) Study. Osteoporos Int 2001;12: 529–37. 331. Dennison E, Yoshimura N, Hashimoto T, Cooper C. Bone loss in Great Britain and Japan: a comparative longitudinal study. Bone 1998;23: 379–82. 332. Barrett-Connor E, Siris ES, Wehren LE, et al. Osteoporosis and fracture risk in women of different ethnic groups. J Bone Miner Res 2005; 20:185–94. 333. Kamao M, Suhara Y, Tsugawa N, et al. Vitamin K content of foods and dietary vitamin K intake in Japanese young women. J Nutr Sci Vitaminol (Tokyo) 2007;53:464–70. 334. Wysowski DK, Nourjah P, Swartz L. Bleeding complications with warfarin use: a prevalent adverse effect resulting in regulatory action. Arch Intern Med 2007;167:1414–9. 335. Looker AC, Pfeiffer CM, Lacher DA, Schleicher RL, Picciano MF, Yetley EA. Serum 25-hydroxyvitamin D status of the US population: 1988–1994 compared with 2000–2004. Am J Clin Nutr 2008;88:1519–27. 336. Zittermann A, Koerfer R. Vitamin D in the prevention and treatment of coronary heart disease. Curr Opin Clin Nutr Metab Care 2008;11:752–7. 337. St-Arnaud R. The direct role of vitamin D on bone homeostasis. Arch Biochem Biophys 2008;473:225–30. 338. Conly JM, Stein K. The production of menaquinones (vitamin K2) by intestinal bacteria and their role in maintaining coagulation homeostasis. Prog Food Nutr Sci 1992;16:307–43. 339. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100: 57–70. 340. Arai H, Nagai K, Doi T. Role of growth arrest-specific gene 6 in diabetic nephropathy. Vitam Horm 2008;78:375–92. 341. Kurohara M, Yasuda H, Moriyama H, et al. Low-dose warfarin functions as an immunomodulator to prevent cyclophosphamide-induced NOD diabetes. Kobe J Med Sci 2008;54:E1–13. 342. Ames BN. Micronutrients prevent cancer and delay aging. Toxicol Lett 1998;102–103:5–18. 343. McCann JC, Ames BN. An overview of evidence for a causal relation between iron deficiency during development and deficits in cognitive or behavioral function. Am J Clin Nutr 2007;85:931–45. 344. McCann JC, Hudes M, Ames BN. An overview of evidence for a causal relationship between dietary availability of choline during development and cognitive function in offspring. Neurosci Biobehav Rev 2006;30:696–712. 345. McCann JC, Ames BN. Is there convincing biological or behavioral evidence linking vitamin D deficiency to brain dysfunction? FASEB J 2008;22:982–1001. 346. McCann JC, Ames BN. Is docosahexaenoic acid, an n23 long chain polyunsaturated fatty acid, required for the development of normal brain function? An overview of evidence from cognitive and behavioral tests in humans and animals. Am J Clin Nutr 2005;82: 281–95.