Vitamin K Metabolism and Nutriture

M. J. Shearer S UMMA R Y. Vitamin K functions as a co-factor for the post-translational carhoxylation of specific glutamate residues to gamma-carboxyglutamate (Gla) residues in several blood coagulation factors (II, VII, IX and X) and coagulation inhibitors (proteins C and S) in the liver; as well as a variety of extrahepatic proteins such as the bone protein osteocalcin. This review outlines some recent advances in our understanding of the metabolism of vitamin K and its role in human nutriture. The introduction of new methodologies to measure the low endogenous tissue concentrations of K vitamins and circulating plasma levels of des-gamma-carboxyprothrombin (PIVKA-II) have provided correspondingly more refined indices for the assessment of human vitamin K status. The assays for vitamin K have also been used to study the sources, intestinal absorption, plasma transport, storage and transplacental transfer of K vitamins and the importance of phylloquinone (vitamin K,) versus menaquinones (vitamins K,) to human needs. The ability to biochemically monitor subclinical vitamin K deficiency has reaffirmedthe precarious vitamin K status of the newborn and led to an increased appreciation of the risk factors leading to haemorrhagic disease of the newborn and how this may be prevented. Biochemical studies are leading to an increased knowledge of the mode of action of traditional comnarin anticoagulants and how some unrelated compounds (e.g. antibiotics) may also antagonize vitamin K and cause bleeding. There is also an awareness of the possible deleterious effects of vitamin K antagonism or deficiency on non-hepatic Gla-proteins which may play some subtle role in calcium homeostasis.

It is more than 50 years since the antihaemorrhagic or Kougulation factor now known as vitamin K was discovered by Henrik Dam and the two major forms phylloquinone (vitamin K,) and menaquinones (vitamins K,) isolated and characterized from alfalfa (lucerne) and putrified fish meal respectively.‘.’ Since then, progress in determining the biochemical function and in understanding the metabolism and nutritional role of the vitamin has been erratic. Several phases may be briefly noted. The early phase of intensive study in the 1930’s and 1940’s saw the introduction of vitamin K into M. J. Shearer, Haematology Department, Clinical Science Laboratories, 18th Floor Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK. Bhd Reviews (1992) 6, 92-104 0 1992 Longman Group UK Ltd

clinical practice, initially for the correction of the clotting defect in obstructive jaundice and other biliary diseases and then for the prophylaxis and treatment of the bleeding syndrome in the newborn known as the haemorrhagic disease of the newborn.‘*2 Again, although the relationship between vitamin K deficiency and a decrease in the plasma concentration of prothrombin was established’ as early as 1936, it was not until the early to mid 1950’s that the three other procoagulant factors VII, IX and X were discovered and subsequently shown to require vitamin K for their biological activity and a further 20-25 years before the vitamin K-dependent coagulation inhibitors proteins C and S were discovered.3*4 The role of all these vitamin K-dependent factors in

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haemostasis has been discussed in a recent review in this journal. 5 The elucidation of the biochemical function of vitamin K came relatively late with the isolation in 1974 of a previously unknown acidic amino acid, gamma-carboxyglutamic acid (Gla) from bovine prothrombin and the parallel discovery that this was missing from the abnormal, functionally inactive molecules of prothrombin which circulate in the plasma of coumarin anticoagulated animals.6s7 It was equally realized that the Gla residues of prothrombin could provide the already well known calcium binding sites of the protein, that the abnormal prothrombin molecules might be similar to an already postulated liver precursor and that the role of vitamin K could be to act as an essential co-factor for a post-translational modification of prothrombin in which certain glutamic acid residues were transformed to gammacarboxyglutamic acid (Gla) residues. All these postulates were subsequently shown to be true (for reviews see refs8s9). The discovery of an amino acid with a strict requirement for vitamin K for its synthesis has provided a ready means of probing for vitamin K-dependent proteins in other tissues, either by analysing the tissue or isolated protein for its Gla content or by demonstrating the associated enzyme activity (vitamin K-dependent carboxylase) responsible for this amino acid modification.” These avenues have revealed the existence of many vitamin K-dependent proteins (or Gla-proteins) in a variety of calcified and non-calcified tissues but very few have been characterized as far as their amino acid sequence. The major exceptions are the two Gla-proteins osteocalcin (or bone Gla-protein) and matrix Gla-protein. It has been postulated that this class of non-hepatic vitamin K-dependent proteins may play a role, hereto undiscovered, in calcium homeostasis.“-I2 A detailed body of knowledge and understanding of the metabolism and nutritional role of vitamin K, such as exists for the other fat-soluble vitamins, has been held back firstly by the lack of sufficiently sensitive assays for vitamin K and its metabolites, and secondly, by the lack of sensitive criteria for monitoring subclinical states of vitamin K deficiency. These shortcomings are now being overcome by technological advances which have resulted in the development of chromatographic techniquesi3v14 to measure K vitamins at tissue concentrations and more sensitive coagulation or immunochemical methodsI for the detection of circulating species of abnormal prothrombin (des-gamma-carboxyprothrombin or PIVKA-II). These new methods offer new approaches to the study of several aspects of vitamin K metabolism and nutriture. Examples of outstanding problems are the nutritional origin and bioavailability of the various K vitamins (including the question of the utilization of enterically produced menaquinones), the mechanism of their intestinal absorption, plasma transport and tissue distribution

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at cellular and subcellular levels, and the utilization of different K vitamers by the vitamin K-dependent carboxylase. Finally, the application of these methods should provide a better understanding of vitamin K status in health and disease and the opportunity to re-examine the difficult question of dietary requirements and how these may be met by dietary intakes in individuals and population groups. Some of these questions will be addressed in this review. Nutritional

Sources and Intestinal Absorption

Chemical Structures

Naturally occurring compounds with vitamin K activity possess a common 2-methyl 1,Cnaphthoquinone ring structure but differ in the structure of their side chain at the 3-position (Fig. 1). The only form synthesized by plants and algae is phylloquinone (originally named vitamin K,) and this has the same phytyl side chain as chlorophyll. A second series, the menaquinones (originally named vitamins K2), are all synthesized by bacteria; these comprise a spectrum of molecular forms with side chains based on a number of repeating unsaturated 5-carbon (prenyl) units. The major forms are designated menaquinonen (MK-n) according to the number of prenyl units; the most commonly found forms in animal tissues are menaquinones 6- 13. Some bacteria synthesize menaquinones in which one or more of the prenyl units is saturated. The division between the plant vitamer phylloquinone and the bacterial menaquinones is somewhat artificial (since phylloquinone may be regarded as a partially saturated form of menaquinone-4) but serves to emphasize their different origins. Dietary Sources

The major dietary form of vitamin K is phylloquinone. Methods employing high-performance liquid 0

Fig. 1 Chemical structures of (top) phylloquinone or vitamin K, and (bottom) menaquinones or vitamins K,.

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VITAMIN K METABOLISM AND NUTRITURE

chromatography are now available for the accurate measurement of phylloquinone in plant and animal tissues,‘3*‘4*‘6 but comprehensive food values have not yet been published. There is a wide variation in the phylloquinone content of different foods. Approximate ranges are: green leafy vegetables (100-500 ug/lOOg); other vegetables and fruit (l-50 ug/lOO g); oils, fats and margarines (l-100 ug/lOO g); dairy produce (0.5-5 ug/lOO g); bread and cereal products (0.1-10 ug/lOO g). The bioavailability of phylloquinone from the above foods is unknown but is likely to vary widely being least efficient from green vegetables (where phylloquinone is tightly associated with chloroplasts) and being most efficient from processed foods. Intestinal Absorption The intestinal absorption of dietary phylloquinone (and probably menaquinones) is thought to be governed by the same principles established for other fat-soluble vitamins and highly lipid-soluble nutrients.‘7p’8 In the intraluminal phase of absorption, this involves the solubilization of vitamin K into mixed micelles composed of bile salts and the products of pancreatic lipolysis. Consistent with this model is the striking impairment of absorption seen in patients with extrahepatic cholestasis (obstructive jaundice) and severe pancreatic insufficiency. l7 Patients with biliary obstruction lack the detergent components of micelles, and the degree of absorption depends on the severity of the bile salt deficiency. In patients with chronic pancreatitis, the primary disturbance is a reduced generation of the solutes of mixed micelles, namely, 2-monoglycerides and fatty acids. This also impairs the absorption of phylloquinone, though this is usually less severe than in bile salt deficiency.r7 The essentiality of bile salts to the absorption of vitamin K is reflected in the higher incidence of vitamin K deficiency in cholestatic disease compared to most other gastrointestinal diseases including chronic pancreatitis. lg MicroJoral Synthesis of Menaquinones It has been commonly perceivedz0v2’ that the enormous microfloral population of the large intestine provides an important nutritional source of vitamin K for human needs. This belief seems to stem, very largely, from early studies in rats and chicks which first demonstrated the presence of menaquinones in faeces and intestinal contents and then their synthesis by specific cultures of microorganisms including many gut inhabitants. This view is reinforced by the infrequency of primary dietary vitamin K deficiency in most mammalian species including man and the long established association of human vitamin K deficiency with antibiotic therapy.‘gs22 As argued cogently by Doisy and Matschiner23*24 this post hoc, ergo prompter hoc reasoning does not stand up to

certain experimental findings especially those of Barnes and Fiala25 who showed that a ‘dietary’ deficiency of vitamin K in the rat (originally thought to be resistant to dietary deprivation) can be readily induced by preventing coprophagy. Such experiments illustrate that the rat can only usefully utilize enterically synthesized menaquinones by a second passage through the bowel and presumably absorption from the upper region of the small bowel. The lack of significant absorption of enterically synthesized menaquinones is supported by a recent study2’j in which the menaquinone concentrations in the intestinal contents of rats fed different diets were measured directly by high-performance liquid chromatography; after 3 days the group fed a vitamin K-deficient diet developed signs of vitamin K deficiency but the amounts of menaquinones in the large intestine had actually increased compared to a control group of rats fed a normal diet. This study clearly showed that rats with a normal, or even increased, production of intestinal menaquinones could not maintain prothrombin synthesis from this source of vitamin K alone i.e. the diet must have been the major source of the vitamin. In the human too direct evidence of menaquinone absorption from the large intestine is presently lacking though it cannot be ruled out that small amounts are absorbed from this region. There is evidence that menaquinone absorption from the colon is possible by a passive diffusion process, .18,26 however given the evidence already cited, the amounts absorbed would seem to be insufficient to prevent the host from developing hypoprothrombinaemia under conditions of dietary deprivation. A potential confounding factor to the above controversy is the diet which in rats has been shown to have a major influence on the intestinal synthesis of menaquinones. 27-2g For example a low fibre diet based on boiled white rice consistently induced vitamin K deficiency in rats within 3 weeks, produced significantly lower faecal concentrations of several menaquinone-producing bacteria and lower hepatic levels of menaquinones. 28,2gThe above changes could be prevented by the dietary inclusion of black-eye beans. Since hepatic concentrations of phylloquinone were also reduced it is difficult to pinpoint to what degree the development of deficiency was due to a primary dietary deficiency of phylloquinone, a secondary dietary deficiency of menaquinones (by coprophagy) or the reduced availability of menaquinones for direct enteric absorption. Superficially the most persuasive evidence for the relevance of intestinal synthesis to human vitamin K nutriture comes from the finding of substantial concentrations of menaquinones in the liver. A more detailed consideration of the liver stores of menaquinones and their potential biological significance is given below. As to the argument of their origin, however, the clue may lie in the evidence that these highly lipophilic molecules are strongly retained by

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the liver. If the turnover is prolonged then the menaquinone content of human liver may arise from a relatively slow assimilation whether this be an inefficient (passive) process from the large intestine of the potentially high concentrations produced by bacterial synthesis or a more efficient (bile saltmediated) process from the small intestine of low concentrations present in certain foods. Tissue Distribution

and Storage

Assa_v MethodLy for Vitamin K So far vitamin K, with its extreme lipophilic character,

has proved resistant to immunochemical assay and the ability to measure tissue concentrations has only been possible since the advent of high-performance liquid chromatography leading particularly from the development of silica-based microparticulate packing materials, reliable solvent delivery systems and sensitive in-line detectors. Present methods are still time consuming and require a multi-stage purification procedure to removing interfering lipids.i3*14 The most recent advances have centered on the introduction of more selective detection systems which by exploiting the reversible redox reaction of quinone and quinol forms (Fig. 2) allow sensitive electrochemical30,31 or fluorometric32-34 measurements. Adult Plasma Concentrations

Plasma concentrations of vitamin K are considerably lower than other fat-soluble vitamins and comprise chiefly of phylloquinone.35 Detectable concentrations of menaquinone-7 and possibly menaquinone-8 have also been reported 36-38 but longer chain menaqui-

uv

QUINONE

FLUORESCENCE

\

QUINOL

REDUCTIVE

-

REDOX

\-

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nones are beyond the limits of detection of current methodologies. Despite the technological advances of the last decade reported values for the mean circulating concentration of phylloquinone in healthy adult populations show a IO-fold variation.14.3s This variation almost certainly reflects methodological differences rather than any great geographic or ethnic influences. Thus a number of laboratories on changing their original procedures 3g-41 have reported decreased values.33,42,43 As a result there has been a discernible downwards trend towards a normal fasting value of about 0.5 ng/ml (1.1 nmol/l). Because of the wide interlaboratory differences little is yet known of how plasma concentrations vary in health and disease. Some comparative measurements from the author’s laboratory are shown in the Table. They reveal how both diet and metabolic disorders, particularly those affecting lipid metabolism, may influence circulating levels of the vitamin. The most powerful metabolic determinant are plasma lipids leading to significantly raised concentrations in primary hyperlipidaemia or in conditions associated with secondary hyperlipidaemia (e.g. diabetes and haemodialysis patients). Among these patient groups highly significant statistical correlations of circulating phylloquinone in the fasting state are seen with total plasma triglyceride but not with cholestero135~~*4s even though patients with discrete hypercholesterolaemia (ie without hypertriglyceridaemia) do tend to have raised phylloquinone levels (Table). A similar positive association of plasma phylloquinone with plasma triglycerides in the fasting state has been reported in a population survey of over 300 healthy (but not necessarily normolipaemic) adults.46 Plasma

Transport

Unlike vitamins A and D, no specific plasma carrier protein for vitamin K has been identified. Instead vitamin K is probably transported entirely by lipoproteins, a property it shares with vitamin E. Current evidence suggests that dietary K vitamins are incorporated chemically unchanged into chylomicrons in the intestinal mucusa, secreted into the lymph, and

\ Fig. 2 The reversible reaction between vitamin K quinone and quinol forms showing the options available for their detection using high-performance liquid chromatography. The stable quinone form (as occurs in the diet) may be detected by ultraviolet (UV) detection though this lacks sensitivity and selectivity. The reduction of the quinone to the quinol allows the sensitive detection of the quinol by fluorescence measurements; this reduction may be achieved in line by chemical, photochemical or electrochemical methods. In electrochemical detection (ECD) the vitamin may be detected by measuring the current produced either by straight reduction to the quinol (reductive mode) or with greater sensitivity and selectivity by first reducing the quinone at an upstream electrode and then re-oxidising the quinol back to the quinone at a second downstream electrode (redox mode). Further theoretical and practical details are provided in refs.‘3.‘4.3’

Table Mean plasma disease

phylloquinone

concentrations

Group”

n

Plasma Mean

Healthy newborn (cords) Healthy adults Healthy adults (non-fasting) Hypercholesterolaemia Hypertriglyceridaemia Mixed hyperlipidaemia Diabete? (normal lipids) Diabete? (raised lipids) Haemodialysis patients

20 45 22 10 7 13 42 38 20

0.02 0.41 0.66 0.76 2.55 4.60 0.62 0.88 0.74

a All subjects were fasting except where stated b Untreated non-insulin dependent

in health and

K, (ng/ml) Median 0.02 0.37 0.53 0.71 1.97 1.90 0.35 0.72 0.41

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eventually enter the liver cells via chylomicron remnant particles. Detailed knowledge of the interrelationship of vitamin K with various lipoproteins remains to be established but phylloquinone has been located in each of the three major classes, namely very low density, low density and high density lipoproteins.35*44 Fractionation studies have also revealed that the excess phylloquinone in hyperlipidaemic patients with raised plasma concentrations of the vitamin is carried by the triglyceride-rich very low density lipoproteins. 3s,44 This would explain the strong statistical association with triglycerides.

Relationship of Plasma Levels to Vitamin K Status With the present inadequate knowledge of the mechanism of transport of vitamin K the question of whether plasma concentrations are an accurate indicator of tissue reserves or vitamin K ‘status’ cannot be convincingly answered at this time. One obvious limitation, already discussed, is the influence of plasma lipids. Two important consequences follow. The first is that plasma measurements should be made, wherever possible, in the fasting state; the second is that an improved correlation with vitamin status might be obtained by expressing plasma concentrations as a ratio of phylloquinone to triglycerides.35,46 The possible unmasking effect of the latter procedure is illustrated by the fact that although plasma concentrations of phylloquinone show a moderate correlation with age which is more pronounced in men than in women46*47 when plasma phylloquinone is corrected for triglycerides both elderly men and women (65-92 years) show significantly reduced values compared to a younger age range (20-49 years).46 This may indicate that the elderly have lower reserves of phylloquinone. Even without correcting for triglycerides there is some evidence that plasma concentrations of phylloquinone may be a useful indicator of tissue reserves. Thus, low plasma concentrations have been found in hospitalized patients who had other evidence of a poor nutritional status4’ or were being fed parenterally;49 in both studies the presentation of a low plasma phylloquinone was a risk factor for the subsequent development of antibiotic-induced hypoprothrombinaemia. More direct evidence of the responsiveness of plasma levels to dietary deprivation of vitamin K has come from studies showing a rapid decline in plasma phylloquinone by dietary restriction either in normals50q51or in patients undergoing surgery.37 In the surgical group a low vitamin K intake for only three days resulted in a 4-fold lowering of liver concentrations compared to a group on a normal diet.37 This rapid decline in both tissues suggests that in situations of reduced dietary intake, plasma concentrations mirror hepatic stores quite closely.

Adult Liver Reserves With the exception of protein S, vitamin K-dependent coagulation factors are entirely synthesized and carboxylated in the liver. An assessment of liver stores of vitamin K should therefore give more precise information of the vitamin K status of individuals, at least as far as their coagulation function is concerned. The distribution of molecular forms of vitamin K in the liver is quite different from plasma in that long chain menaquinones, undetectable or barely detectable in plasma, are now known to be the predominant hepatic forms. Until quite recently the little available evidence had suggested that phylloquinone comprised about half of the total human liver content of vitamin K and that the other half was made up of menaquinones with side chains of 7-13 isoprene units.52 The measurement of K vitamins in small tissue samples obtained at post-mortem26~35~53or biopsy37 has suggested that the proportion of hepatic stores as phylloquinone in most people is much lower and on average accounts for only about 10% of total reserves. The remaining 90% stores of menaquinones show wide inter-subject variations. Some generalisations may be made. Menaquinones with side chains below six isoprene units are less common in nature and were less evident in human livers. Of the higher menaquinone homologues the small UK survey of 10 postmortem samples 35s3 showed that the concentrations of menaquinone-6 averaged 11 pmol/g, menaquinones 7-9 averaged about 30 pmol/g and menaquinone-10 averaged 50 pmol/g; measurements beyond menaquinone-10 were not made. However in two similar small Japanese surveys the predominant form was menaquinone-1 1 at average concentrations of 73 pmol/g26 and 120 pmol/g37 respectively. One study also detected respectable concentrations of menaquinones-12 and 13.37 The concentrations of menaquinones 7-9 in the Japanese studies were comparable to those found in the UK except that menaquinone9 seems to be a less abundant hepatic form in Japan than in the UK. The detection of quite large concentrations of menaquinone-9 in dairy produce, especially cheese,s4 and the more common consumption of the latter in the UK may explain this difference.

Biological SigniJicance of Hepatic Menaquinones The biological relevance of the relatively large hepatic stores of menaquinones is as mysterious as their origin. Early bioassay data in which various forms of vitamin K were given intracardially to deficient rats suggested that long chain menaquinones (7, 9 and 10) were more efficient in reversing hypoprothrombinaemia and up to 25 times as active as phylloquinone itself. 5s It is also known that both phylloquinone and menaquinones may serve as a co-factor for the hepatic carboxylase in vitro.” The ability of humans to efficiently utilize the large

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endogenous hepatic menaquinone pool has been put into some doubt by the study of Suttie et alsl who found that biochemical and coagulation abnormalities consistent with signs of a mild deficiency could be rapidly induced in human volunteers by a fairly modest dietary restriction of phylloquinone-rich foods. On the other hand overt vitamin K deficiency with bleeding is rarely seen, even in severe malnutrition, lg which does suggest that the ability to utilize menaquinones may prevent the appearance of severe deficiency symptoms. A key question is whether there are mechanisms by which the menaquinones which undoubtedly are present in many membranous organelles may be transported to and utilized by the microsomal carboxylase in situations of dietary lack. This question is made more relevant by the first direct evidence that whereas phylloquinone hepatic stores are rapidly depleted by dietary restriction, menaquinone stores are not.37 Another key to the above inconsistencies may lie in the differences in disposition and rates of metabolism of various molecular forms of vitamin K. For example the relative bioassay data in rats55 were made by comparing the response in prothrombin levels at 18 h. As Matschine? later pointed out the much higher activity of long chain menaquinones in these experiments could be partly explained by their sustained response. Thus the response to phylloquinone peaked at 5 h and by 18 h had declined substantially. These results and the more direct evidence from human liver measurements37 discussed above suggest that phylloquinone is utilized and metabolized at a faster rate than long chain menaquinones. This slower turnover of higher molecular weight members of the menaquinone series is presumably due to their high degree of lipophilicity and greater affinity for membranes.

Vitamin K Metabolism

in the Newborn

Interest in the metabolism and nutriture of vitamin K in the perinatal period stems from the increased risk that babies have of developing vitamin K deficiency which at its most extreme manifests as the bleeding syndrome known as haemorrhagic disease of the newborn. Some recent research using the new sensitive assays for K vitamins and des-gammacarboxyprothrombin has sought to pinpoint the metabolic/nutritional reasons for this susceptibility. The progress made in this area is reviewed below. Gradient. The first direct evidence for the existence of a placental barrier to the transport of vitamin K to the human fetus came from early high-performance liquid chromatographic measurements which showed that although phylloquinone was readily detectable in maternal plasma, the concentrations in cord plasma were undetectable.57 These findings of very low endogenous cord plasma concentrations have been disputed by some40*41,58but conMaternal-cord

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firmed by others. 42,5g-61 This variation in literature values, which at their most extreme differ by several lOOO-fold, suggests serious methodological discrepancies. As with adult values, however. there has been a trend towards lower values, several groups now agreeing that cord concentrations are below 50 pg/ ml (110 pmol/l) and that the average maternal/fetal concentration gradient of phylloquinone is within the range of 20: 1 to 40: 1.35.42,43.61This is by far the highest placental blood gradient of any of the fatsoluble vitamins. Further evidence that phylloquinone does not readily cross the placenta is shown by the similar high maternal/fetal concentration gradient when the vitamin is administered to mothers shortly before delivery. 42*57.61 The extent to which cord blood concentrations were raised depended on the dose, route and time interval between maternal administration and cord sampling, ranging from 2 to 5-fold when single doses of l-5 mg were given intravenously or intramuscularly57~61 to about 40-fold when mothers received 20 mg/day orally for at least 3 days.42 Despite the fact that supplementation procedures do raise cord blood levels significantly, in one study42 to the endogenous maternal range, there was no corresponding effect on the vitamin K-dependent clotting factor activities. 42.61 These results offer strong evidence that the well known low concentrations of vitamin K-dependent clotting factors in the newborn are not the consequence of vitamin K deficiency but rather the result of the reduced synthesis of their precursor proteins possibly due to reduced mRNA transcription or translation.‘2q62 This does not of course discount the possibility of vitamin K deficiency occuring in individual babies at birth (see under haemorrhagic disease of the newborn). One group of babies who may benefit from the maternal route of vitamin K supplementation are those born to mothers on anticonvulsant therapy. Such babies are known to run a higher risk of developing early vitamin K deficiency63 and there is some unconfirmed evidence that antenatal vitamin K can substantially reduce this risk.64 Plasma Concentrations. Plasma vitamin K concentrations which are very low at birth start to be detectable at around 12-24 h after delivery43 and by 3-4 days breast-fed infants have plasma concentrations in the same range as adults.43*60 In contrast babies fed on vitamin K supplemented milk formulas have significantly higher concentrations of phylloquinone at 3-4 days. 43V60These high plasma levels result from the differential concentrations of phylloquinone in human milk (about 2 ug/16’) compared to formulas which in the studies cited above43,60 contained about 50 us/l. Despite the marked differences in serum phylloquinone concentrations there is no detectable difference in coagulation between breast-fed and formula-fed infants in the first month of life.43 On the Infant

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other hand sensitive immunoassays for des-gammacarboxyprothrombin revealed a higher frequency of detection of this marker for vitamin K deficiency in breast-fed infants than in formula-fed infants, none of whom had received vitamin K at birth.60766 Fetal and Neonatal Liver Reserves.

Phylloquinone has been detected in the liver of the human fetus as early as 10 weeks gestation at concentrations of l-2 rig/g (2.2-4.4 pmol/g).35 Similar concentrations were detected throughout fetal development, the median hepatic concentrations at pre-term (1.4 rig/g)) and term (1 .O rig/g)) being about one fifth the value in adults.35*53 Although significantly reduced these fetal reserves are not as low as perhaps expected from previous findings of exceedingly low concentrations in fetal and cord plasma. The major difference between fetal/neonatal reserves and those of adults is that the normal menaquinone complement, which makes up the majority of the total adult stores, are very low in the perinatal period. 35,67,68In fact the concentrations of all menaquinones throughout gestation, at birth and in the first week of life are so low that they are assay undetectable by current normally methods.35*67*68 From limited analyses of postmortem samples it would seem that there is a gradual build-up of hepatic stores of menaquinones in the neonate but that adult concentrations have still not been reached by 1 month of age.35*68 One implication of these findings is that the needs of the human fetus and neonate are met largely by phylloquinone. It is not known whether this relative deficit of menaquinones is a major factor in the susceptibility of the newborn to vitamin K deficiency or whether these bacterial forms of vitamin K derive from the diet, gut flora or a combination of both sources. A gradual increase in the hepatic stores of menaquinones would of course be consistent with the known colonization of the neonatal gut by enteric microflora but in view of the evidence suggesting their low hepatic turnover, the slow accumulation of menaquinones could also be explained by quite low dietary intakes taken over a prolonged period. Low fetal reserves of menaquinones are not unexpected. Not only are the concentrations in maternal blood available to the placenta lower than phylloquinone but the efficiency of placental transport itself is likely to be very low for these high molecular weight, lipophilic forms.

Haemorrhagic Disease of the Newborn Haemorrhagic disease of the newborn (HDN) is a bleeding syndrome caused by a deficiency of vitamin K in early life.‘j’*” Until about 20 years ago HDN was always thought to present in the first week of life; this syndrome is now known as classical HDN to distinguish it from a late onset form, which was first reported in 1967, and which has a peak incidence

at 3-6 weeks.‘l A worldwide resurgence of HDN in developed countries, mainly of the late onset form, and its frequent association with intracranial haemorrhagic still makes the disease a serious cause of infant morbidity and mortality. 6g Two major risk factors for HDN are breast feeding and not giving vitamin K at birth. As already discussed the protection afforded by formula feeds derives from their higher vitamin K content than breast milk. Detection and Incicence of Subclinical Dejiciency

Several attempts have been made to document the incidence of subclinical vitamin K deficiency amongst normal babies using the detection of the precursor molecule, des-gamma-carboxyprothrombin as a biochemical marker. These abnormal molecules (also known as PIVKA or proteins induced by vitamin K absence or antagonism) are released into plasma in situations of vitamin K deficiency or antagonism. Although functional assays have been used to detect des-gamma-carboxyprothrombin the most useful assays are immunoassays either using crossed immunoelectrophoresis or even more sensitive enzyme-linked immunoabsorbent methods. The use of several assay methods with different sensitivities15 often makes the interpretation of these studies difficult.” Some trends however can be seen. Whether measured by immunoelectrophoresis73 or enzyme-linked immunoabsorbent assay74 the incidence of detection of des-gammacarboxyprothrombin is lower in cord plasma than 3-5 days after birth. In many respects more relevant results may be obtained using a less sensitive method such as crossed immunoelectrophoresis since the detection of very small amounts of des-gammacarboxyprothrombin by some immunoassays may reflect some other facet of liver metabolism rather than vitamin K deficiency. For instance abnormal prothrombin molecules are rarely detected in cord plasma by immunoelectrophoresis but at 4-5 days the incidence of detection rises to about 50% in all babies and about 70% in solely breast fed babies.@j These results suggest that while the fetus derives sufficient transplacental transfer of vitamin K to ensure that prothrombin is fully or almost fully carboxylated at birth the existing reserves are not normally sufficient to meet the demands on this posttranslational function through the first few days of life. As would be expected an important determinant of vitamin K status immediately after birth is the volume of milk ingested. It has been shown that the average daily intake in babies with des-gammacarboxyprothrombin is significantly lower than in babies without this marker73 and that babies with des-gamma-carboxyprothrombin who also had low factor II and VII activities had failed to increase their milk intake beyond 100 ml/day by the third and fourth day of life. 66 The delay which is often seen before lactation becomes established would thus account for the temporal dip in vitamin K status. A

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cultural change in developed countries which may have contributed to the resurgence of HDN has been the widespread abandonment of early cow’s milk supplements because of the risk of cow’s milk protein intolerance.

Aetiology

of Late HDN

By l-2 months of age the incidence of detection of des-gamma-carboxyprothrombin in healthy breastfed infants (not given vitamin K at birth) falls to about 10% using the monoclonal immunoabsorbent assay60x75 and to less than 1% by immunoelectrophoresis. Once again the protective effect of formula feeding is seen by the inability of the very sensitive monoclonal assay to detect abnormal prothrombin molecules in some 50 formula-fed infants at l-month and beyond.60 The aetiology of late HDN seems to be different from that of the classical syndrome. For example although breast-feeding is a strong risk factor the volume of milk intake is unlikely to be so important since the peak incidence occurs well beyond the time taken for breast feeding to become established. Also mothers of affected infants do not necessarily have particularly low breast milk concentrations of vitamin K.70 Some studies have reported mild abnormalities of liver function which may be transient, mild and self correcting but sufficient to create a temporary cholestasis and impairment of vitamin K absorption. 77--79 Affected infants have no accompanying sign of deficiencies of other fat-soluble vitamins except for evidence of lower than normal plasma levels.78 Both infectious77.7g and environmental” agents have been listed as possible triggering factors of liver dysfunction leading to late HDN but the evidence is still largely speculative.

Prophylaxis Because of the high risk and potentially disastrous consequences of intracranial haemorrhage, and the inability to detect individual infants at risk, many countries are now favouring a policy of vitamin K prophylaxis for all babies.69,79,81,82 Predictionss3 made from the first survey in the British Isless suggest that without prophylaxis some 40 cases of intracranial haemorrhage due to vitamin K deficiency would occur annually. Both epidemiologica170~8’ and biochemical evidence of the incidence of des-gammathat parenteral carboxyprothrombin6’ suggest prophylaxis gives almost total protection against late HDN. On the other hand there is equally clear epidemiologica170*81 and biochemical75 evidence that a single oral dose at birth, though protecting against classical HDN, gives less protection against the late onset disease than a single parenteral dose. Comparative blood measurements in infants have shown that the bioavailability of phylloquinone after oral administration is much lower than after intramuscular

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administration.s5 Of more concern is the great variability in the efficiency of intestinal absorption of phylloquinone from traditional oil-based preparations. Despite its efficacy the practice of intramuscular prophylaxis is not ideal; there are proper concerns about the medical risks and social acceptability of injecting newborn babies and even that the very high regimens tissue levels53s85 achieved by intramuscular may be excessive. 84 Future policies should aim at finding more efficient regimens of oral prophylaxis probably involving newer, better absorbed vitamin K preparations and possibly repeated dosages.

Hepatic Metabolism

and Vitamin K Antagonism

Vitamin K antagonists based on 4-hydroxycoumarin or indanedione structures were introduced into clinical practice long before their biochemical basis was understood. Leading from the finding in 1970 that oral anticoagulants caused the hepatic accumulation of the metabolite, vitamin K 2,3-epoxide, it is now known that all oral anticoagulants interfere with a metabolic cycle in the liver known as the vitamin K-epoxide cycle (Fig. 3).87 Although the dietary form of vitamin K has the stable quinone structure the vitamin K-dependent carboxylase (denoted E, in Fig. 3) which catalyses the conversion of glutamate residues to gammacarboxyglutamate residues can only utilize the quinol form. During gamma-glutamyl carboxylation vitamin K quinol is simultaneously converted to vitamin K 2,3-epoxide by vitamin K epoxidase (E,). The similarities in location and requirements of the carboxylase and epoxidase suggest that the same enzyme (E,) may be responsible for both activitiesa The epoxide produced is recycled by two enzyme activities; in the first step the epoxide is reduced to vitamin K quinone by a dithiol-dependent vitamin K epoxide reductase (E,) while in the second step vitamin K quinone is reduced back to the active quinol form. Although this vitamin K reductase activity is denoted in Figure 3 by a separate enzyme (E3) there is evidence88q89 that this conversion may be effected by the same enzyme that reduces the epoxide to the quinone (E2). Warfarin and other oral anticoagulants are strong inhibitors of the dithiol-dependent vitamin K epoxide reductase (EJ and vitamin K reductase (E3) activities. This accounts for both their pharmacological action and the accumulation of vitamin K epoxide. The dithiol-dependent activity, however, is not the only pathway by which vitamin K can be reduced to the quinol. 9o A second NAD(P)H-dependent pathway (E4) is relatively insensitive to warfarin. During warfarin anticoagulation this enzyme is able to bypass the inhibited dithiol-dependent enzyme.91p93 When the epoxide cycle is completely blocked, as in anticoagulant overdose, the NAD(P)H-dependent enzyme

100

VITAMIN K METABOLISM AND NUTRITURE

NADH+H+

NAD+

Gla

Glu

QUINONE Fig. 3 Scheme showing the cyclic metabolism of vitamin K in relation to the conversion of glutamate residues (Glu) to gammacarboxy glutamate (Gla) residues in vitamin K-dependent proteins. Known enzyme activities are denoted by E,, E,, E, and E,. The active form of vitamin K needed for carboxylation is the reduced form, vitamin K quinol. The carboxylation reaction is driven by a vitamin K-dependent carboxylase activity (E,) coupled to a vitamin K-epoxidase activity (E,) which simultaneously converts vitamin K quinol to vitamin K 2,3-epoxide. Vitamin K 2,3-epoxide is reduced back to the quinone by vitamin K epoxide reductase (EJ. The cycle is completed by the reduction of recycled vitamin K quinone by a vitamin K reductase activity (Es). Both vitamin K epoxide and vitamin K reductase activities E, and E, are dithiol-dependent (X-(SH,) and X-S, denote reduced and oxidised dithiols), and are inhibited by coumarin anticoagulants such as warfarin. Exogenous vitamin K may enter the cycle via an NAD(P)H-dependent vitamin K reductase activity (E,) which is not inhibited by war-farm.

provides the only route by which the antidotal effect of vitamin K may be mediated.93 In humans the inhibition of the vitamin K epoxide reductase by coumarins results in the dose-dependent plasma accumulation of the epoxide metabolite when a pharmacological dose of the vitamin is administered.94*95 This effect provides a ready screening test for possible vitamin K antagonists even when other coagulation indices are normal. In the early 1980’s a group of compounds which were suspected of having antivitamin K activity and which caused the release of vitamin K epoxide into plasma49g96were antibiotics containing an N-methyl-thiotetrazole (NMTT) side chain (e.g. moxalactam, cefamandole etc). Experimental studies in animals have since confirmed that the target of this inhibition is vitamin K epoxide reductase though the active inhibitor is not known.97 These drugs were initially investigated because they had been shown to be associated with an increased risk of bleeding amongst hospitalized patients. The fact that patients at greatest risk were those with low serum phylloquinone concentrations and/or other indications of malnourishment48*49 and that antibiotic-induced hypoprothrombinaemia could not be induced in healthy volunteers9’ shows the importance of vitamin K status to the pharmacological expression of these unusual vitamin K antagonists. As shown in Figure 3, in the presence of an inhibitor of vitamin K epoxide reductase, the supply of the quinol to the carboxylase can only be augmented by the input of dietary vitamin K quinone into the cycle and its conversion to the quinol by an alternative NAD(P)Hdependent reductase; therefore in states of dietary depletion even weak inhibitors can sufficiently interrupt the supply of quinol co-factor to cause hypoprothrombinaemia and bleeding. The enzyme vitamin K epoxide reductase seems to be particularly susceptible to various antagonists with widely different structures. Other compounds with weak antagonistic activity include salicylate,99-‘01

lapachollo2 and sulphaquinoxaline.lo3 A group of more powerful and potentially dangerous inhibitors of vitamin K epoxide reductase are the so called superwarfarins developed to combat warfarin resistance. Such compounds (e.g. difenacoum, brodifacoum) are characterized by the introduction of tetralin and aromatic moieties at the 3-position providing a more bulky and lipophilic character with apparently much greater binding characteristics than conventional anticoagulant drugs (Fig. 4). This results in very long biological half-lives. Several cases of accidental poisoning with superwarfarins have been reported’04-‘08 including multiple fatalities when poisoned rice bait was eaten by peasant farmers in Indonesia.“’ Poisoning with these rodenticides requires very careful clinical management because the requirement for vitamin K to maintain carboxylation is enormously increased and can only be met by frequent transfusion and/or administration of massive doses of vitamin K over a prolonged period of time.86 Non-hepatic Metabolism

Gla-containing proteins and/or vitamin K-dependent carboxylase activity have been located in a number

Fig. 4 Chemical structures of two ‘superwarfarin’ (left) difenacoum and (right) brodifacoum.

rodenticides

BLOOD REVIEWS

of extrahepatic tissues including bone, cartilage, dentin, kidney, placenta, pancreas, spleen, lung and testis.“*” Likewise vitamin K reductase, vitamin K epoxide reductase and/or vitamin K epoxidase activities have also been detected in various extrahepatic tissues or cell lines.“0-“3 This evidence suggests that the vitamin K-epoxide cycle is operational in all tissues capable of synthesizing vitamin K-dependent (Gla-containing) proteins. Little is known however of the function of noncoagulation Gla-proteins or indeed other aspects of vitamin K metabolism in extrahepatic tissues. Vermeer and colleagues reasoned that anticoagulant drugs may also inhibit vitamin K-dependent processes in extrahepatic tissues with possible deleterious consequences in patients on long term anticoagulant therapy. l2 Although their first results indicated very little inhibition of vitamin K epoxide reductases of extrahepatic soft tissues’ lo more recent data” suggest that coumarin drugs do inhibit hepatic and extrahepatic enzymes to a similar extent. Oral anticoagulants also affect circulating levels of the bone protein osteocalcin leading to both a decrease in total antigenic protein released into the bloodstream1’4~“5 and a decreased Gla-content. 114-1l6 Despite these demonstrable biochemical effects, there has been little to suggest that even long term treatment with oral anticoagulants has any adverse effect on the structure and function of mature bone. Two recent reports suggest that anticoagulant therapy may increase urinary calcium loss” or reduce bone mass’17 in some patients but this requires confirmation. On the other hand there is an increasing realization and clear clinical evidence that warfarin interferes with the normal process of mineralization in rapidly growing bone. Fetuses whose mothers have been exposed to warfarin in early pregnancy may present with congenital defects known collectively as the fetal warfarin syndrome characterized by excessive calcification of the epiphyses and irregular growth of the facial and long bones. Strong circumstantial evidence that the bony defects of warfarin embryopathy are mediated via its antagonism of the vitamin K-epoxide cycle has come from detailed studies in an infant, who in addition to a selective deficiency of the vitamin K-dependent coagulation factors had identical clinical features to the fetal warfarin syndrome and a specific biochemical defect consistent with a congenital deficiency of vitamin K epoxide reductase.“’ A deficiency of this enzyme would be expected to mimic the antagonistic effect of warfarin and prevent the normal recycling of vitamin K. It should perhaps be emphasized that the inability to ascribe a function to the vitamin K-dependent proteins osteocalcin and matrix Gla-protein is not unusual; these are just two of several non-collagenous proteins present in bone tissue (others are sialoprotein, proteoglycans, phosphoproteins etc) for which no proven metabolic or structural function are yet known but which many investigators believe are

10 1

essential to the mineralization process.’ l9 By analogy function that the essential gammato carboxyglutamate residues play in calcium binding of the blood coagulation factors it is not unreasonable to assume that the Gla-containing moieties of osteocalcin and matrix Gla-protein may play an important subtle role in bone mineralization. This raises the question of whether the vitamin K status of an individual can influence normal bone metabolism or affect the progression of disease. Some tantalizing observations are worthy of further exploration and explanation. Firstly it has been shown that circulating phylloquinone was low in osteoporotic patients who had sustained either spinal crush-fractures or fractures of the neck of femur. lzo Secondly some findings in post-menopausal women indicate that supplementation with pharmacological doses of phylloquinone increases both the total and the Gla-content of circulating osteocalcin and may decrease urinary calcium loss particularly when this is already high.12’ In the last decade then, our conception of the role of vitamin K has changed from that of a rather specific regulatory function in the synthesis of six blood coagulation proteins to the possibility that the vitamin has a more diverse role in calcium homeostasis. A major problem, however, is that whereas the essential functions of the coagulation Gla-proteins have been delineated those of the non-coagulation Gla-proteins remain to be discovered. References 1. Dam H 1966 Historical survey and introduction. Vitamins and Hormones 24: 295-306 2. Almquist H J 1941 Vitamin K. Physiological Reviews 21: 194-216 3. Stenflo J 1976 A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization. Journal of Biological Chemistry 251: 355-363 4. Di Scipio R G, Hermodson M A, Yates S G, Davie E W 1977 A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S. Biochemistry 16: 698-706 5. Mackie I J. Bull H A 1989 Normal haemostasis and its regulation. ‘Blood Reviews 3: 237-250 6. Stenflo J, Femlund P, Egan W, Roepstorff P 1974 Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proceedings of the National Academy of Sciences of the United States of America 71: 2730-2733 7. Nelsestuen G L, Zytkovicz T H, Howard J B 1974 The mode of action of vitamin K. Identification of ycarboxyglutamic acid as a component of prothrombin. Journal of Biological Chemistry 249: 634776350 8. Suttie J W 1985 Vitamin K. In: Diplock A T (ed) FatSoluble Vitamins. Heinemann, London, pp. 225-31 I 9. Furie B, Furie B C 1990 Review article: molecular basis of vitamin K-dependent y-carboxylation. Blood 75: 1753-1762 10. Vermeer C 1990 Review article: y-Carboxyglutamatecontaining proteins and the vitamin K-dependent carboxylase. Biochemical Journal 266: 625-636 11. Lian J B 1988 Osteocalcin: functional studies and postulated role in bone resorption. In: Suttie J W (ed) Current advances in vitamin K research. Elsevier, New York, pp. 245-257 12. Venneer C, Hamulyak K 1991 Pathophysiology of vitamin K-deficiency and oral anticoagulants. Thrombosis and Haemostasis 66: 153- 159 13. Shearer M J 1983 High-performance liquid

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chromatography of K vitamins and their antagonists. Advances in Chromatography 21: 243-301 De Leenheer A P, Nelis H J, Lambert W E, Bauwens R M 1988 Chromatography of fat-soluble vitamins in clinical chemistry. Journal-of-Chromatography 429: 3-58 Widdershoven J. van Munster P. De Abreu R et al. 1987 Four methods compared for measuring des-carboxyprothrombin (PIVKA-II). Clinical Chemistry 33: 2074-2078 Ball G F M 1988 Fat-Soluble Vitamin Assays in Food Analysis: A Comprehensive Review. Elsevier, London, p. 326 Shearer M J, McBurney A, Barkhan P 1974 Studies on the absorption and metabolism of phylloquinone (vitamin K,) in man. Vitamins and Hormones 32: 513-542 Hollander D 1981 Intestinal absorption of vitamins A,E,D, and K. Journal of Laboratory and Clinical Medicine 97: 449-462 Savage D, Lindenbaum J 1983 Clinical and experimental human vitamin K deficiency. In: Lindenbaum J (ed) Nutrition in Hematology. Churchill Livingstone, New York, pp. 271-320 Lakshmanan M R, Cama H R 1970 Malnutrition in respect of fat-soluble vitamins: vitamin K. In: Morton R A (ed) Fat-soluble Vitamins. International Encyclopaedia of Food and Nutrition. (vol 9) Pergamon Press, Oxford, pp. 435-447 Passmore R, Eastwood M A 1986 Davidson and Passmore Human Nutrition and Dietetics, 8th ed. Churchill Livingstone, Edinburgh Lipsky J J 1988 Review: antibiotic-associated hypoprothrombinaemia. Journal of Antimicrobial Chemotherapy 21: 281-300 Doisy E A Jr, Matschiner J T 1965 Nutritional aspects with special reference to hypoprothrombinemia and vitamin K. In: Morton R A (ed) Biochemistry of Quinones. Academic Press, London, pp. 317-353 Doisy E A Jr, Matschiner J T 1970 Biochemistry of vitamin K. In: Morton R A (ed) Fat-soluble Vitamins. International Encyclopaedia of Food and Nutrition. (vol 9) Pergamon Press, Oxford, pp. 293-331 Barnes R H, Fiala G 1959 Effects of the prevention of coprophagy in the rat. VI. Vitamin K. Journal of Nutrition 68: 603-614 Uchida K, Komeno T 1988 Relationships between dietary and intestinal vitamin K, clotting factor levels, plasma vitamin K, and urinary Gla. In: Suttie J W (ed) Current Advances in Vitamin K Research. Elsevier, New York, pp. 477-492 Ramotar K, Krulicki W, Gray G, Louie T J 1988 Studies on intestinal and hepatic concentrations of menaquinone and hypoprothrombinemia in vitamin K-deficient-rats. In: Suttie J W (ed) Current Advances in Vitamin K Research. Elsevier, New York, pp. 493-498 Mathers J C, Fernandez F, Hill M J, McCarthy P T, Shearer M J. Oxlev A 1990 Dietarv modification of potential vitamin K supply from enteric bacterial menaquinones in rats. British Journal of Nutrition 63: 639-652 Shearer M J, Kazim N, Mathers J C 1991 Measurement and significance of hepatic menaquinones in rats fed on low-fibre diets. Proceedings of the Nutrition Society 50: 71 A Haroon Y, Schubert C A W, Hauschka P V 1984 Liquidchromatographic dual electrode detection system for vitamin K compounds. Journal of Chromatographic - _ Science 22: 89-93 Shearer M J 1986 Vitamins. In: Lim C K (ed) Hplc of Small Molecules: A Practical Approach. IRL Press, Oxford pp. 157-219 Langenberg J P, Tjaden U R 1984 Determination of (endogenous) vitamin K, in human plasma by reversedphase high-performance liquid chromatography using fluorometric detection after post-column electrochemical reduction: comparison with ultraviolet, single and dual electrochemical detection. Journal of Chromatography 305: 61-72 Lambert W E, De Leenheer A P, Lefevere M F 1986 Determination of vitamin K in serum using HPLC with post-column reaction and fluorescence detection. Journal of Chromatographic Science 24: 76-79

34. Haroon Y, Bacon D S, Sadowski J A 1986 Liquid chromatographic determination of vitamin K, in plasma with fluorometric detection. Clinical Chemistry 32: 19255 1929 35. Shearer M J, McCarthy P T, Crampton 0 E, Mattock M B 1988 The assessment of human vitamin K status from tissue measurements. In: Suttie J W (ed) Current Advances in Vitamin K Research. Elsevier, New York, pp. 437-452 36. Hodges S J, Pilkington M J, Shearer M J, Bitensky L, Chayen J 1990 Age related changes in the circulating levels of congeners of vitamin K,, menaquinone-7 and menaquinone-8. Clinical Science 78: 63-66 37. Usui Y, Tanimura H, Nishimura N, Kobayashi N, Okanoue T, Ozawa K 1990 Vitamin K concentrations in the plasma and liver of surgical patients. American Journal of Clinical Nutrition 51: 846-852 38. Plantalech L, Guillaumont M, Vergnaud P, Leclercq M, Delmas P D 1991 Impairment of gamma carboxylation of circulating osteocalcin (bone gla protein) in elderly women. Journal of Bone and Mineral Research 6: 1211-1216 39. Lefevere M F, De Leenheer A P, Claeys A E, Claeys I V, Steyaert H 1982 Multidimensional liquid chromatography: a breakthrough in the assessment of physiological vitamin K levels. Journal of Lipid Research 23: 1068-1072 40. Sann L, Leclercq M, Guillaumont M, Trouyez R, Bethenod M, Bourgeay-Causse M 1985 Serum vitamin K, concentrations after oral administration of vitamin K, in low birth weight infants. Journal of Pediatrics 107: 608-611 41. Pietersma-de Bruyn A L J M, van Haard P M M 1985 Vitamin K, in the newborn. Clinica Chimica Acta 150: 95-101 42. Mandelbrot L, Guillaumont M, Leclercq M et al 1988 Placental transfer of vitamin K, and its implications in fetal hemostasis. Thrombosis and Haemostasis 60: 39-43 43. Pietersma-de Bruyn A L J M, van Haard P M M, Beunis M H. Hamulvak K. Kuiiners J C 1990 Vitamin K, levels and coagulatcon factors in healthy term newborns till 4 weeks after birth. Haemostasis 20: 8-14 44. Mattock M, Shearer M J, Rahim S, Redmond S, ElGohari R, Barkhan P 1983 The plasma transport of vitamin K, (phylloquinone) in hyperlipoproteinaemia. Clinical Science 64: 63P 45. Saupe J, Bennhold I, Bonello E et al Vitamin K, (phylloquinone) concentrations in hemodialysis patients. Journal of Electrolyte and Mineral Metabolism (in press) 46. Sadowski J A, Hood S J, Dallal G E, Garry P J 1989 Phylloquinone in plasma from elderly and young adults: factors influencing its concentration. American Journal of Clinical Nutrition 50: 100-108 47. Lambert W E, De Leenheer A P, Baert E J 1986 Wetchemical post-column reaction and fluorescence detection analysis of the reference interval of endogenous serum vitamin K,(,,,. Analytical Biochemistry 158: 257-261 48. Cohen H, Scott S D, Mackie I J et al 1988 The development of hypoprothrombinaemia following antibiotic therapy in malnourished patients with low serum vitamin K, levels. British Journalof Haematology 68: 63-66 49. Shearer M J. Bechtold H. Andrassv K et al 1988 Mechanism of cephalosporin-induced hypoprothrombinemia: relation to cephalosporin side chain, vitamin K metabolism, and vitamin K status. Journal of Clinical Pharmacology 28: 88-95 L L, Kindberg C G, 50. Allison P M, Mummah-Schendel Harms C S, Bang N U, Suttie J W 1987 Effects of a vitamin K-deficient diet and antibiotics in normal human volunteers. Journal of Laboratory and Clinical Medicine 110: 180-188 51. Suttie J W, Mummah-Schendel L L, Shah D V, Lyle B J. Greger J L 1988 Vitamin K deficiency from dietary vitamin K restriction in humans. American Journal of Clinical Nutrition 47: 475-480 52. Duello T J, Matschiner J T 1972 Characterization of vitamin K from human liver. Journal of Nutrition 102: 331-335 0 E, 53. McCarthy P T, Shearer M J, Gau G, Crampton Barkhan P 1986 Vitamin K content of human liver at different ages. Haemostasis 16 (Supplement 5): 84-85 aspects of 54. Shearer M J, Kries R V, Saupe J Comparative human vitamin K metabolism and nutriture. Journal of Nutritional Science and Vitaminology (in press)

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55. Matschiner J T, Taggart W V 1968 Bioassay of vitamin K by intracardial injection in deficient adult male rats. Journal of Nutrition 94: 57-59 56. Matschiner J T 1971 Isolation and identification of vitamin K from animal tissue. In: The Biochemistry, Assay and Nutritional Value of Vitamin K and Related Compounds, Association of Vitamin Chemists, Chicago, pp. 21-37 51. Shearer M J, Rahim S. Barkhan P, Stimmler L 1982 Plasma vitamin K, in mothers and their newborn babies. Lancet ii: 460-463 58. Greer F R, Mummah-Schendel L L. Marshall S, Suttie J W 1988 Vitamin K, (phylloquinone) and vitamin K, (menaquinone) status in newborns during the first week of life. Pediatrics 81: 137-140 59. Hiraike H, Kimura M, ltokawa Y 1988 Distribution of K vitamins (phylloquinone and menaquinones) in human placenta and maternal and umbilical cord plasma. American Journal of Obstetrics and Gynecology 158: 564-569 60. Widdershoven J, Lambert W, Motohara K et al 1988 Plasma concentrations of vitamin K, and PIVKA-II in bottle-fed and breast-fed infants with and without vitamin K prophylaxis at birth. European Journal of Pediatrics 148: 139-142 61 Yang Y-M, Simon N. Maertens P, Brigham S, Liu P 1989 Maternal-fetal transport of vitamin K, and its effect on coagulation in premature infants. Journal of Pediatrics 115: 1009-1013 62 Kisker C T, Perlman S. Bohlken D, Wicklund B 1988 Measurement of prothrombin mRNA during gestation and early neonatal development. Journal of Laboratory and Clinical Medicine 112: 407-412 63 Laosombat V 1988 Hemorrhagic disease of the newborn after maternal anticonvulsant therapy: a case report and literature review. Journal of the Medical Association of Thailand 71: 643-648 64. Deblay M F, Vert P. Andre M, Marchal F 1982 Transplacental vitamin K prevents haemorrhagic disease of infant of epileptic mother (Letter) Lancet i: 1247 65. Haroon Y, Shearer M J, Rahim S, Gunn W G, McEnery G. Barkhan P 1982 The content of phylloquinone (vitamin K,) in human milk, cows’ milk and infant formula foods determined by high-performance liquid chromatography. Journal of Nutrition 112: 1105-l 117 66. van Kries R, Becker A. Gobel U 1987 Vitamin K in the newborn: influence of nutritional factors on acarboxyprothrombin detectability and factor II and VII clotting activity. European Journal of Pediatrics 146: 123-127 67. Kayata S, Kindberg C. Greer F R, Suttie J W 1989 Vitamin K, and K, in infant human liver. Journal of Pediatric Gastroenterology and Nutrition 8: 304-307 68. Shirahata A. Nakamura T, Ariyoshi N 1991 Vitamin K, and K, contents in blood, stool. and liver tissues of neonates and young infants, In: Suzuki S. Hathaway W E, Bonnar J, Sutor A H (eds) Perinatal Thrombosis and Hemostasis. Springer-Verlag, Tokyo, pp. 213-223 69. Lane P A. Hathaway W E 1985 Vitamin K in infancy. Journal of Pediatrics 106: 351-359 70. von Kries R, Shearer M J, Giibel U 1988 Vitamin K in infancy. European Journal of Pediatrics 147: 106-l 12 71. Shearer M J 1990 Annotation: vitamin K and vitamin K-dependent proteins, British Journal of Haematology 75: 156-162 12 Shapiro A D, Jacobson L J, Armon M E et al 1986 Vitamin K deficiency in the newborn infant: prevalence and perinatal risk factors. Journal of Pediatrics 16: 675-680 73 von Kries R. Gobel U. Maase B 1985 Vitamin K deficiencv in the newborn (Letter). Lancet ii: 728-729 74 Motohara K, Endo F, Matsuda I 1985 Effect of vitamin K administration on acarboxv nrothrombin (PIVKA-II) levels in newborns. Lancet ii: 242-244 75 Motohara K, Endo F. Matsuda I 1986 Vitamin K deficiency in breast-fed infants at one month of age. Journal of Pediatric Gastroenterology and Nutrition 5: 931-933 76 von Kries R 1991 Vitamin K deficiency and breast-feeding. In: Suzuki S, Hathaway W E, Bonnar J, Sutor A H (eds) Perinatal Thrombosis and Hemostasis. Springer-Verlag, Tokyo, pp. 239-247 11 von Kries R, Reifenhauser A, Giibel U, McCarthy P,

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Shearer M J, Barkhan P 1985 Late onset haemorrhagic disease of newborn with temporary malabsorption of vitamin K, (Letter) Lancet i: 1035 Matsuda I, Nishiyama S, Motohara K, Endo F, Ogata T, Futagoishi Y 1989 Late neonatal vitamin K deficiency associated with subclinical liver dysfunction in human milkfed infants. Journal of Pediatrics 114: 602-605 Nagao T, Hanawa Y 1991 The third nationwide survey on vitamin K deficiency in infancy in Japan. In: Suzuki S, Hathaway W E. Bonnar J, Sutor A H (eds) Perinatal Thrombosis and Hemostasis. Springer-Verlag. Tokyo, pp. 249-255 Koppe J G, Pluim E, Olie K 1989 Breast milk. PCBs. dioxms and vitamin K deficiency: discussion paper. Journal of the Roval Societv of Medicine 82: 416-419 ._, Gobel U,-von Kries R, Petrich C 1991 Vitamin K prophylaxis and late onset hemorrhagic disease of the newborn in West Germany during 1988. In: Suzuki S, Hathaway W E, Bonnar J, Sutor A H (eds) Perinatal Thrombosis and Hemostasis. Springer-Verlag. Tokyo, DD. 231-238 Handel J, Tripp J H 1991 Vitamin K prophylaxis against haemorrhagic disease of the newborn in the United Kingdom. British Medical Journal 303: 1109 von Kries R 1991 Neonatal vitamin K: prophylaxis for all. British Medical Journal 303: 1083-1084 McNinch A W, Tripp J H 1991 Haemorrhagic disease of the newborn in the British Isles: two year prospective study. British Medical Journal 303: 1105-l 109 McNinch A W, Upton C, Samuels M et al 1985 Plasma concentrations after oral or intramuscular vitamin K, in neonates. Archives of Disease in Childhood _ 60. ___8_.14-X_._ 1X Park B K, Scott A K, Wilson A C. Haynes B P, Breckenridge A M 1984 Plasma disposition of vitamin K, in relation to anticoagulant poisoning. British Journal of Clinical Pharmacology 18: 655-662 Suttie J W 1987 Recent advances in hepatic vitamin K metabolism and function. Hepatology 7: 3677376 Suttie J W. Preusch P C 1986 Studies of the vitamin K-dependent carboxylase and vitamin K epoxide reductase in rat liver. Haemostasis 16: 193-215 Preusch P C, Brummet S R 1988 Steady state kinetics of microsomal vitamin K epoxide reduction. In: Suttie J W (ed) Current Advances in Vitamin K Research. Elsevier, New York, pp. 75-82 Wallin R, Hutson S 1982 Vitamin K-dependent carboxylation: evidence that at least two microsomal dehydrogenases reduce vitamin K, to support carboxylation. Journal of Biological Chemistry 257: 15831586 Wallin R, Martin L F 1985 Vitamin K-dependent carboxylation and vitamin K metabolism in liver: effects of warfarin. Journal of Clinical Investigation 76: 187991884 Wallin R 1986 Vitamin K antagonism of coumarin anticoagulation: a dehydrogenase pathway in rat liver is responsible for the antagonistic effect. Biochemical Journal 236: 685-693 Wallin R, Patrick S D, Ballard J 0 1986 Vitamin K antagonism of coumarin intoxication in the rat. Thrombosis and Haemostasis 55: 235-239 Shearer M J. McBurney A. Breckenridge A M, Barkhan P 1977 Effect of warfarin on the metabolism of phylloquinone (vitamin K,): dose-response relationships in man. Clinical Science and Molecular Medicine 52: 621-630 Choonara I A, Scott A K, Haynes B P, Cholerton S, Breckenridge A M, Park B K 1985 Vitamin K, metabolism in relation to pharmacodynamic response in anticoagulated patients. British Journal of Clinical Pharmacoloav 20: 643-648 Bechtold H, Andrassy K, JHhnbhen E et al 1984 Evidence for impaired hepatic vitamin K, metabolism in oatients treated with N-methyl-thiotetrazole cephalosporms. Thrombosis and Haemostasis 51: 358-361 Creedon K A, Suttie J W 1986 Effect of N-methylthiotetrazole on vitamin K epoxide reductase. Thrombosis Research 44: 147- 153 Bang N U, Tessler S S. Heidenreich R 0. Marks C A. Mattler L E 1982 Effects of moxalactam on blood coagulation and platelet function. Reviews of Infectious Diseases 4 (Suppl): S546-S554 “_I

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99. Park B K, Leek J B 1981 On the mechanism of salicylateinduced hypoprothrombinaemia. Journal of Pharmacy and Pharmacology 33: 25-28 100. Hildebrandt E, Suttie J W 1984 Indirect inhibition of vitamin K epoxide reduction by salicylate. Journal of Pharmacy and Pharmacology 36: 5866591 101. Takahashi 0 1988 Inhibition of phylloquinone epoxide reductase by BHT quinone methide, salicylic acid and CLtocopherolquinone. Biochemical Pharmacology 37: 28572859 102. Preusch P C, Suttie J W 1984 Lapachol inhibition of vitamin K epoxide reductase and vitamin K quinone reductase. Archives of Biochemistry and Biophysics 234: 405-412 103. Preusch P C, Hazelett S E, Lemasters K K 1989 Sulphaquinoxaline inhibition of vitamin K epoxide and quinone reductase. Archives of Biochemistry and Biophysics 269: 18-24 104. Park B K, Choonara I A, Haynes B P, Breckenridge A M, Malia R G, Preston F E 1986 Abnormal vitamin K metabolism in the presence of normal clotting factor activity in factory workers exposed to 4-hydroxycoumarins. British Journal of Clinical Pharmacoloav 21: 289-294 105. Barlow A M, Gay A L, Park B K 198Zbifenacoum (Neosorexa) poisoning. British Medical Journal 285: 541 106. Lipton R A, Klass E M 1984 Human ingestion of a ‘superwarfarin’ rodenticide resulting in a prolonged anticoagulant effect. Journal of the American Medical Association 252: 3004-3005 107. Jones E C, Growe G H, Naiman S C 1984 Prolonged anticoagulation in rat poisoning. Journal of the American Medical Association 252: 3005-3007 108. Watts R G, Castleberry R P, Sadowski J A 1990 Accidental poisoning with a superwarfarin compound (brodifacoum) in _ a child. Pediatrics 86: 883-887 109. Anonvmous 1983 Rat ooison kills 18. New Scientist 98: 764 110. De Boer-van den Berg: Thijssen H H W, Vermeer C 1988 The in vivo effects of oral anticoagulants in man: comparison between liver and non-hepatic tissues. Thrombosis and Haemostasis 59: 147-150 111. Canfield L M, Johnson T M, Martin G S, Gunn J M 1987 Absorption and metabolism of vitamin K in Swiss 3T3

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Vitamin K metabolism and nutriture.

Vitamin K functions as a co-factor for the post-translational carboxylation of specific glutamate residues to gamma-carboxyglutamate (Gla) residues in...
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