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Keywords:

  • Metabolism;
  • Vitamin K

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

Vitamin K is an essential fat-soluble micronutrient that is required for the post-translational γ-carboxylation of specific glutamic acid residues in hepatic and extra-hepatic proteins involved in blood coagulation and preventing cartilage and vasculature calcification. In humans, sources of vitamin K are derived from plants as phylloquinone and bacteria as the menaquinones. Menadione is a synthetic product used as a pharmaceutical but also represents an intermediate in the tissue-specific conversion of vitamin K to menaquinone-4, which preferentially resides in tissues such as brain. Research into vitamin K metabolism is essential for the understanding of vitamin K biology in health and disease. Progress in this area, driven by knowledge of vitamin K and the availability of markers of vitamin K status, has already proved beneficial in many areas of medicine and further opportunities present themselves. Areas of interest discussed in this review include prophylactic administration of vitamin K1 in term and preterm neonates, interactions between vitamins K and E, the industrial conversion of vitamin K to dihydro-vitamin K in foods, tissue-specific conversion of vitamin K to menaquinone-4, the biological activity of the five and seven carbon metabolites of vitamin K and circadian variations.

Abbreviations
ABC

ATP-biding cassette

DHK1

dihydrophylloquinone

IM

intramuscular

IV

intravenous

K1

vitamin K1 (phylloquinone)

K3

vitamin K3 (menadione)

K1O

vitamin K1 2,3-epoxide

MK-4

menaquinone-4

MK-n

vitamin K2 (menaquinone-n)

PIVKA-II

protein induced by vitamin K absence or antagonism-II (undercarboxylated prothrombin)

5C-metabolite

5 carbon aglycone vitamin K metabolite

7C-metabolite

7 carbon aglycone vitamin K metabolite

10C-metabolite

10 carbon aglycone vitamin K metabolite

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

Following the discovery of vitamin K by Henrik Dam in 1935 [1], the process of elucidating the mechanisms of vitamin K absorption, transport, action, storage and metabolism have yet to be completed. To date, milestones include the identification of dietary and bacterial sources of vitamin K, the discovery and clinical utilisation of vitamin K antagonists (and application as rodenticides), the identification of vitamin K epoxide reductase as the enzyme responsible for maintaining vitamin K in the biologically active form, the elucidation of the processes by which vitamin K is involved in protein modification, the discovery of extra-hepatic vitamin K dependent proteins and the inter-conversion and transport of vitamin K to brain tissue as menaquinone-4 (MK-4). The improved understanding of these processes will guide future research on the action of vitamin K in areas such as arterial calcification, bone disease and cancer. In this paper, on vitamin K metabolism we will summarise current knowledge outside of the more intensively studied areas, i.e. Gla-protein synthesis, vitamin K transport and the vitamin K cycle, and identify opportunities for research that will contribute to the further understanding of vitamin K biology.

The mechanisms involved in vitamin K catabolism and excretion remain poorly understood. The structures of vitamin K and the putative mechanisms of catabolism based on current knowledge are shown in Figs. 1 and 2, respectively. Originally it was speculated that vitamin K is incorporated into prothrombin to confer blood-clotting activity [2]. This suggestion was quickly dispelled when the administration of radioactive menadione (2-methyl-C14-1,4-naphthoquinone) (vitamin K3 (K3)) to rats [3, 4], and non-radioactive K3 to chickens, rabbits and humans [5-7] showed that K3 is rapidly metabolised with approximately 13–33% of the dose excreted as urinary conjugates. Three of these conjugate species, namely: diglucuronide (2-methyl-1,4-dihydroxynaphthalene-1,4-diglucuronide), monosulphate (2-methyl-1,4-hydroxy-1-naphthyl sulphate) and a conjugate referred to as ‘product 3’ were identified using paper chromatography by Hoskin et al. [3]. The glucuronide was later recognised as the most prevalent urinary conjugate of K3, and the phosphate (‘product 3’ from Hoskin's investigations) the least abundant, although a proportion of the conjugates remained uncharacterised [8].

image

Figure 1. Chemical structures of K vitamers and metabolites: a) Menadione (vitamin K3). b) Five carbon vitamin K metabolite. c) Seven carbon vitamin K metabolite. d) Vitamin K1 alcohol*. e) Vitamin K1 (phylloquinone). f) Vitamin K1 2,3-epoxide. g) Menaquinone-4 (vitamin K2). h) Menaquinone-7 (vitamin K2). *Vitamin K1 alcohol has only been detected in vitro.

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image

Figure 2. Putative mechanism for the metabolism of vitamin K, giving vitamin K1 (phylloquinone) as an example. Omega oxidation takes place in the endoplasmic reticulum before the ‘primed’ carboxylic acid product is transported to the mitochondrial matrix via a carnitine-dependent mechanism. Successive rounds of beta oxidation cleave two carbon units sequentially resulting in the two major excretory products, namely the five and seven carbon vitamin K metabolites (see Fig. 1).

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The perfusion of isolated rat livers with radioactive K3 (2-[14C] methyl-1,4-naphthoquinone) confirmed that the metabolism of this compound is rapid, with approximately 93% metabolised within 5 h. The major conjugated product generated by the liver was the glucuronide of menadiol, predominantly excreted in bile (19% of dose), although 70% of the conjugated species produced during these experiments were not identified. The main conjugated metabolite in perfusing blood was the sulphate of menadiol (9.3% of the dose), a species that was absent in the bile [9].

Both intravenous (IV) and oral administration of K3 to normal rats and rats with biliary fistulas resulted in the urinary excretion of glucuronide and sulphate conjugates of menadiol. The glucuronide conjugate was most prevalent after oral administration suggesting both hepatic and extra-hepatic conjugation of menadiol [9]. No evidence of the conversion of K3 to menaquinones (vitamin K2 (MK-n)) was observed during these experiments [9].

Seminal studies on phylloquinone (vitamin K1 (K1)) metabolism in rats revealed that the major route of excretion of an IV-administered radioactive dose is the faeces, a proportion of which was unmodified K1 [10]. Losito et al. [11] compared the metabolism of K3-14C and K1-14C in normal and hepatectomised rats and established that K3 is quickly metabolised in both with 70% of the 14C from labelled K3 excreted in the urine within 24 h. Vitamin K1-14C was retained for longer by the liver and excreted at a far slower rate with just 10% of the radioactivity found in the urine of normal animals during the first 24 h. Radioactivity was virtually absent from the urine of the hepatectomised rats demonstrating that the metabolite in the normal urine is dependent upon the presence of a liver.

Human studies were later performed by Shearer and colleagues, employing radio-labelled tracer and unlabelled pharmacological doses of K1. It was shown that K1 is extensively catabolised to water-soluble metabolites, which quickly appear in the plasma, urine and bile [12-14]. In healthy subjects who ingested a 1 mg dose of [1′2′-3H2] phylloquinone with a meal, the proportion of radioactivity recovered from the faeces over a period of 3 days was 54–60%, of which 15–23% was identified as unmodified K1. The remaining lipid-soluble radioactive fraction consisted of polar metabolites [12]. The urinary excretion of these polar metabolites was studied after the injection of a 1 mg dose of [1′2′-3H2] phylloquinone in six normal subjects and was found to be virtually complete after 3 days, accounting for 8–26% of the administered dose. In two of these subjects faecal excretion was also determined and represented 34 and 38% of the dose, respectively [15], indicating that the biliary route is the major route of vitamin K metabolite excretion. In contrast, no detectable faecal levels of radioactivity were present when this protocol was repeated in a patient whose total bile was collected for a period of 3 days, suggesting that bile is probably the only route by which vitamin K metabolites can pass into the intestinal lumen [15].

The first attempts to identify the aglycone structures of K1 metabolites followed the identification of 2-(3-methyl-5-carboxypentyl)-3,5,6-trimethylbenzoquinone and its γ-lactone as products of α-tocopherol catabolism [16, 17]. On the supposition that quinones with different lengths and degrees of saturation of the side chain are catabolised by common metabolic pathways, Wiss and Gloor [18] fed physiological doses of radio-labelled K1 and related quinones (dl-α-tocopherylquinone, phytylplastoquinone and ubiquinone-9) to rats. In each case, a metabolite was isolated in which the side chain had been shortened to seven carbons with the formation of a γ-lactone metabolite and identified conjugated 2-methyl-3-(5′-carboxy-3′-hydroxy-3′-methylpentyl)-1,4-naphthoquinone lactone (vitamin K γ-lactone) as the major urinary catabolite of K1 (Fig. 1) [18]. The authenticity of vitamin K γ-lactone as a metabolite of K1 was questioned when Imada et al. [19] and Fujita et al. [20] identified two aglycone acids in urine and faeces with an intact quinone nucleus but with shortened side chain lengths of five and seven carbon atoms as major metabolites of ubiquinone-7 in rats and rabbits. The seven carbon aglycone was a γ,δ-unsaturated acid, which on exposure to the mineral acid conditions used during its isolation by Wiss and Gloor [18] had undergone lactonisation to form the experimental artefact, γ-lactone [19]. The ready lactonisation of γ,δ-unsaturated acids under acid conditions had previously been described [21].

Using RP partition thin-layer chromatography, Shearer et al. [12] isolated sufficient quantities of methylated derivatives of the two major urinary aglycone metabolites of vitamin K to verify their chemical structures by NMR and MS [12]. They established that the major metabolic endpoints of K1 catabolism are two side chain shortened carboxylic acids with the structures 2-methyl-3-(3′-3′-carboxymethylpropyl)-1,4-naphthoquinone (side chain length five carbon atoms (5C-metabolite)) and 2-methyl-3-(5′-carboxy-3′-methyl-2′-pentenyl)-1,4-naphthoquinone (side chain length seven carbon atoms (7C-metabolite)), respectively (Fig. 1), both were conjugated mainly (approximately 90%) with glucuronic acid. The side chain structures of the authentic urinary K1 metabolites were identical to those previously identified for ubiquinone-7, and in common with ubiquinone-7 metabolism [19], the γ-lactone metabolite described by Wiss and Gloor [18] was shown to be an experimental artefact arising from the exposure of the 7C-metabolite to mineral acid.

The ingestion of a large pharmacological dose of K1 (400 mg) whilst attempting to isolate ‘abnormal’ urinary K1 metabolites from subjects treated with warfarin, resulted in the isolation of a third aglycone metabolite that was tentatively identified as 2-methyl-3-(7′-carboxy-3′,7′-dimethyl-2′-heptenyl)-1,4-naphthoquinone (side chain length ten carbon atoms (10C-metabolite)) [14]. A fourth compound with similar properties to K3 was also identified although whether this originated from impurities present in the pharmaceutical K1 preparation used for the supplementation of this individual during this experiment, or an undescribed metabolic K1 intermediate [22] or was in fact K3 that is now known to be a metabolite of K1 [23] is unclear. The identification of the 10C-metabolite in urine added weight to the degradation pathway discussed by Imada et al. [19]. Here the initial step of ubiquinone-7 metabolism was proposed as ω-oxidation resulting in the formation of a carboxylic acid metabolite followed by a series of side chain shortening reactions by β-oxidation cleaving two carbon units per oxidation round. A similar pathway is envisaged for the metabolism of vitamin K and is consistent with the existence of a general degradation pathway for isopreniod quinones as originally proposed by Wiss and Gloor [18].

The effects of warfarin on the urinary excretion of vitamin K aglycone metabolites has also been studied after IV administration of a 1 mg dose of [1′2′-3H2] phylloquinone [22]. In subjects maintained on a therapeutic dose of warfarin, there was a twofold increase in the excretion of urinary radioactivity. Also an increase in the warfarin dose resulted in a corresponding increase in the urinary radioactivity. In one subject, the radioactivity in faeces was also determined and was found to fall from 51 to 27% of the administered dose (45 μg [1′2′-3H2] K1) in response to a therapeutic dose of warfarin.

Treatment with warfarin changed both the chemical nature and conjugation pattern of the metabolites. In response to a therapeutic dose of warfarin, sustained for several days, there was a reduction in the excretion of the aglycone metabolites identified in normal subjects (to approximately 10% of vitamin K aglycone metabolite excretion) and three additional major urinary aglycone metabolites were identified. These were more polar than those identified in normal subjects possibly due to the presence of hydroxyl groups. A number of other minor urinary aglycones were also identified but were not further characterised. The proportion of vitamin K aglycone metabolites excreted as glucouronides fell to 50%, and there was an increase in an unidentified conjugated species that was resistant to hydrolysis with both β-glucuronidase and phenolsulphatase [22].

2 Putative phase-I transformations

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

The precise biochemical pathways of vitamin K metabolism have not been elucidated or the metabolic intermediates described. A working model analogous to that of fatty acids is presented here (Fig. 2), as a definitive characterisation of the associated mechanisms is beyond the scope of this review.

ω-Oxidation is initiated within the endoplasmic reticulum by oxygen attack at the ω-carbon or at the penultimate carbon of straight chain fatty acids by a cytochrome P450 mixed function oxidase. Subsequent oxidation of the alcohol by alcohol and aldehyde dehydrogenases results in the formation of a metabolic intermediate, which is referred to here as ‘vitamin K carboxylic acid’.

The aglycone structures of the K1 urinary metabolites previously identified [12, 14] suggest that vitamin K carboxylic acid undergoes a series of β-oxidation reactions. β-Oxidation in mammalian cells occurs both in the mitochondria and peroxisomes with overlapping specificity. Fatty acids with side chain lengths of greater than 12 carbons (e.g. PUFAs, dicarboxylic fatty acids, prostaglandins and the side chain of cholesterol) are in general preferentially or exclusively oxidised in peroxisomes [24]. It is possible that both β-oxidation systems contribute to vitamin K catabolism, however the relative physiological contribution of these two systems is unknown and may depend on species and metabolic conditions.

The accumulation of K1 in the mitochondria in response to administration of labelled K1 has been shown previously by sub-cellular studies in rats [25, 26]. This increase led Thierry and Suttie [26] to suggest a storage or metabolic role for mitochondria, therefore an outline of mitochondrial β-oxidation is given in this review.

Products of ω-oxidation are activated to enable transportation to the site of β-oxidation. By analogy to known transformations of fatty acids, a thioester linkage is formed between the carboxyl group of the ω-oxidation product and the sulfhydryl group of CoA. Acyl-CoA is subsequently transported into the mitochondrial matrix by transferring the acyl group of CoA to carnitine, a zwitterionic derivative of lysine. Carnitine acyltransferase I, located on the inner mitochondrial membrane, catalyses the transport of activated products to the inner mitochondrial membrane, with the transfer of the acyl group from the sulphur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine.

On entering the mitochondrial matrix it may be envisaged that the ‘activated’ vitamin K carboxylic acid undergoes recurring rounds of β-oxidation, hydration, oxidation and thiolysis with the resultant loss of two carbon units from the isoprene side chain for each cycle. This model is consistent with the known structures of vitamin K metabolites excreted in the urine.

Metabolism of vitamin K carboxylic acid by β-oxidation is favourable until a side chain length of five carbon units is reached. At this point further cleavage is inhibited, possibly via mechanism of steric hindrance by the naphthoquinone nucleus that is analogous to other quinones metabolised by this route [19].

3 Putative phase-II transformations

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

Glucuronidation is the most common transformation in mammals and previous studies suggest it to be the predominant conjugate of vitamin K [12, 13]. Although there is no direct evidence, it is probable that the glucuronic moiety of glucuronic acid, formed from glucose by uridine diphosphoglucose dehydrogenase, is transferred to one or more of the naphthoquinone hydroxyl groups formed by the reduction of the quinone to the quinol in microsomes. The diglucuronide and monoglucuronide conjugates have previously been identified in the urine of rats following the administration of K3 [3] and bile [27], respectively. Hydroxyl groups are also target functional groups for sulphate conjugation, and a urinary monosulphate K3 metabolite was identified by Hoskin et al. in rats [3], and as the predominant conjugated metabolite in perfusing blood following K3 administration to isolated rat livers [9]. It is not unusual for a number of metabolic pathways to operate simultaneously producing molecules conjugated to multiple conjugating agents.

Other phase-II conjugating agents utilise target functional groups present in previously isolated aglycones of vitamin K. Carboxylic acids can be conjugated to amino acids (most commonly glycine) via their CoA thioesters by the appropriate amino acid N-transferase and under the influence of the enzyme glutathione S-transferase, glutathione can react with a wide range of substrates including epoxides. This may be of significance in the catabolism of vitamin K 2,3-epoxide.

4 Measurement of vitamin K metabolites as markers of vitamin K status

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

As the number of vitamin K dependent proteins identified and the knowledge of their widespread tissue distribution increases, there is a need to re-evaluate the role of vitamin K in terms of general physiological function and maintenance of health. To support this, biochemical measures to assess vitamin K status have been developed [28]. Current measures include the direct measurement of circulating vitamin K (as an indicator of tissue stores) and functional assessments of the γ-carboxylation status of specific Gla proteins such as prothrombin and osteocalcin representing hepatic and bone γ-carboxylation capacity, respectively. In addition, measurements of urinary free Gla offer an overall assessment of the γ-carboxylation status of Gla proteins. Each of these status assessments has a number of methodological and interpretational caveats [29]. The measurement of circulating vitamin K to assess tissue stores has the major disadvantage that only K1 is commonly measured to the detriment of the MK series. MK-n from the diet [30] and possibly from intestinal synthesis [31-33], make a significant contribution to total daily vitamin K intake, and liver stores of vitamin K are predominately long chain MKs [31, 32].

The measurement of the urinary 5C- and 7C-metabolites is attractive because K1 and the MK-n share common metabolites; thus potentially reflecting a summation of the availability of total vitamin K. An assay for the routine measurement of the 5C- and 7C-metabolites under physiological conditions has been developed [34]. In brief, urinary salts are removed by RP (C18) SPE and the predominantly conjugated vitamin K metabolites subsequently hydrolysed with methanolic hydrochloric acid. The resultant carboxylic acid aglycones are then quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis is by RP (C18) HPLC with a methanol-aqueous mobile phase. Metabolites are detected by amperometric, oxidative electrochemical detection of their quinol forms, generated by post-column coulometric reduction at an upstream electrode. This method was used to construct a reference range (median (inner 95% reference range)) for the urinary excretion of the 5C- and 7C-metabolites by 159, free-living, young, healthy adults eating self-selected diets. This was 5.2 (1.7–11.0) μg/day and 1.1 (0.2–2.7) μg/day, respectively. The 5C-metabolite was the predominant excretory product in 155 (97%) of the 159 adults (unpublished data). Using this methodology, a series of supplementation experiments using K1, MK-4, MK-7 and K3 were performed to confirm that the 5C- and 7C-metabolites are common to all K vitamers [34]. This methodology has subsequently been extended for the analysis of bile, plasma and faecal matter.

5C- and 7C-metabolite excretion is ideally determined from complete 24-h urine collections. If 24-h sample collections are not feasible then the analysis of the 5C- and 7C-metabolites in spot urine collections can be performed. However, when taking this approach it is important to recognise and control factors that contribute to analytical intra- and inter-day variation in the urinary excretion of the 5C- and 7C-metabolites. Circadian variation in the circulatory concentration of K1 has previously shown maximal and minimal levels at 10:00 and 22:00 h, respectively [35, 36]. The principal determinate of this rhythm is likely to be dietary vitamin K1 intake, given that peak levels of the vitamin are found 4–6 h postprandially [37]. To correct for variations in renal fluid excretion, the interpretation of 5C- and 7C-metabolite laboratory data from the analysis of spot urine samples should be expressed as a function of urinary creatinine concentration. The daily excretion of creatinine also typically follows a circadian rhythm with a 14% higher value in the late afternoon relative to the 24-h mean [38].

In one study, spot urines were collected from eight healthy subjects (4 male, 4 female, aged 19–27, mean = 22.3) who consumed identical, synchronised meals. Urine samples were collected at 09:00 h (0 h) on day 1 post 14-h overnight fast and represented the 2-h second morning void and at 5, 8, 11, 14, 18 and 22 h (non-fasting). Further morning urines were collected post 14-h fast at 09:00 h on days 2, 8, 15, 22, 29 and 57 (non-fasting). All urine samples were divided into 0.5 mL aliquots and stored at −30°C until analysis (unpublished data).

When expressed as a molar ratio with creatinine, the urinary vitamin K metabolites followed a circadian variation. Maximal urinary levels of the 5C- and 7C-metabolites occurred at 09:00 h with levels of 61 (±12.9) and 24 (±7.58) nmoL/mmoL creatinine, respectively. Levels then declined to a trough of 45.09 (±8.07) nmoL/mmoL creatinine (5C) and 9.88 (±2.19) nmoL/mmoL creatinine (7C) at 23:00 h before returning to near baseline levels during the early hours of the morning. There was a significant difference between the peak and trough levels of 5C-metabolite excretion (p = 0.011), but not between peak and trough 7C-metabolite excretion (p = 0.151). 7C-metabolite excretion as a proportion of total vitamin K metabolite excretion peaked at 38.3% at 09:00 h. The lowest proportion of 7C-metabolite excretion (24.9%) coincided with the trough (23:00 h) in total (5C + 7C) metabolite excretion. The mean 24-h variation in urinary vitamin K metabolites was 26.7% (±8%) and 58.1% (±2%) for the 5C- and 7C-metabolites, respectively.

The mean, 2-month temporal variation in the urinary excretion of the 5C- and 7C-metabolites was 13.8 and 8.0%, respectively. The inter-subject variability in urinary creatinine excretion over the 2-month duration of this study was 25.7%, which compares well with the 26% inter-subject variation for first morning void urines previously reported for healthy adults [38].

During this study, the mean total daily urinary 5C- and 7C-metabolite excretion was 17.7 nmoL/day (range = 7.4–24.8 nmoL/day), within the normal range for healthy young adults (6.6–47.1 nmoL/day). The 7C-metabolite accounted for 26.5% (range = 15.9–48.9%) of total urinary excretion.

5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

The differences in metabolism of vitamin K in its naturally occurring dietary form (K1) and the form produced during hydrogenation of vegetable oils in food industry processes (dihydrophylloquinone (DHK1)) has been investigated [39]. During this study, the urinary excretion of the 5C- and 7C-metabolites in response to the sequential ingestion of diets designed to provide an adequate, followed by a restricted supply of K1 with a subsequent period of K1 or DHK1 administration was determined [39]. Manipulation of dietary K1 and DHK1 intake was reflected by a rapid and corresponding response in the urinary excretion of the 5C-metabolite. A steady corresponding change in excretion of the 7C-metabolite in response to manipulation of dietary K1 intake was also observed. In line with previously established values [39], the 5C-metabolite accounted for 75% of the total daily metabolite excretion with diets containing 11–206 μg/d K1, consistent with documented K1 intakes in UK and US populations [40-42].

Excretion of the 7C-metabolite did not respond to dietary DHK1 repletion and continued to fall as in the period of depletion. Two possible explanations present themselves; a yet to be identified intermediate 7C-DHK1 metabolite is excreted, or the 7C-DHK1 has altered kinetic properties and is further catabolised to the common 5C-metabolite prior to excretion. 7C-metabolite excretion during DHK1 repletion therefore reflects the contribution of combined menaquinone and tissue-store K1 metabolism and is equivalent to a state of continued K1 restriction. In DHK1 replete subjects, the 5C-metabolite excretion was significantly greater than in K1 replete subjects, which may support the theory that an equivalent 7C-metabolite is not excreted but further metabolised to the 5C metabolite prior to excretion. However, plasma DHK1 concentrations have previously been found to be lower than those of K1 for an equivalent dietary intake [43], suggesting a lower rate of intestinal absorption and/or a shorter clearance half-life.

6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

In blood, the predominant circulating form of vitamin K is K1, however in certain extra-hepatic tissues, e.g. pancreas, salivary gland and brain, MK-4 has been found to predominate [44, 45]. K3 has been demonstrated to be a metabolite of K1, MK-4 and MK-7 [23] generated via a process that is independent of gut bacteria action [46, 47]. Recently UBIAD1, a prenyltranferase, was identified as the enzyme responsible for the conversion of vitamin K to MK-4 [48]. This enzyme might catalyse the removal of the side chain of K1 and MK-n, though this is still to be confirmed. However, it is thought that UBIAD1 is responsible for the prenylation of K3 with geranylgeranyl pyrophosphate adding the side chain to convert to MK-4. The mechanism by which vitamin K is transported to specific tissues for conversion to MK-4 and in which form remains unclear.

UBIAD1 function is not effected by warfarin and is encoded by UBIAD1 (MIM 611632), a 15.2 kb gene located at 1p36.22, encoding the 338 amino acid protein that localises to mitochondria. Bacterial MK-4 is known to be a membrane-bound electron carrier. Whether MK-4 has a similar carrier function in eukaryotic cells was studied by Vos [49], who identified Drosophila UBIAD1(Heix) as a modifier of pink1 (MIM 608309), a gene associated with mitochondrial function that is mutated in Parkinson's disease. They proposed that mitochondrial dysfunction in PINK1 deficiency is rescued by MK-4, which functions as a mitochondrial electron carrier and restores ATP production. Mutations in UBIAD1 have been associated with Schnyder corneal dystrophy, which is linked to the accumulation of cholesterol and phosopholipid deposits in the cornea [50-52].

Recently in vitro studies by McDonald et al. indicated that ω-hydroxyvitamin K1 (Fig. 1), a metabolite of K1 can be produced independently of β-oxidation generated by CYP4F2 acting as a K1 oxidase [53]. Further analysis indicated that a V433M polymorphism (rs2108622) in the CYP4F2 gene (MIM 604426), which is associated with higher warfarin dose requirements in humans, causes approximately 40% reduction in capacity to metabolise K1, thus explaining the increased warfarin dose requirement in carriers of this polymorphism. This polymorphism has a minor allele frequency of 5.8–26.7% in different ethnic groups [54], suggesting an evolutionary advantage in its preservation. The significance of this reaction in terms of metabolic function is unclear. The authors suggest that it prevents excess accumulation of K1 in vivo although as CYP4F2 oxidises many other lipids, it may be metabolised as an after effect of these more dominant processes. ω-Hydroxyvitamin K1 is likely be metabolised and excreted via the same mechanism as other K vitamers as this compound is also an intermediate in the putative phase-I ω-oxidation conversion of K1.

7 Vitamin K metabolism in newborn infants

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

The opportunity to apply vitamin K metabolite excretion as a non-invasive marker of total vitamin K status is particularly attractive when studying newborn infants. As in healthy adults, the 5C-metabolite is the predominant metabolite excreted by term infants and the majority of preterm infants pre- and post-vitamin K prophylaxis. However, exposure to a large pharmacological dose of vitamin K to protect against vitamin K deficiency bleeding can disturb the normal pattern of metabolite excretion [55].

The rate of 5C- and 7C-metabolite excretion by term infants pre-K1 prophylaxis is far lower than in healthy young adults (25-fold) or in adults fed with a K1-restricted diet of 11 μg/day for 15 days (14-fold) [55]. This finding is in keeping with the notion that infants are born with vitamin K insufficiency as evidenced by low K1 and K2 concentrations in plasma [56] and liver tissue of foetuses and neonates [57]. However, it does not exclude the possibility that low urinary output may also reflect the immaturity of the vitamin K catabolic pathway. In healthy adults, the typical 5C:7C-metabolite ratio is 4:1 [34, 39]. Term infants in this study predominately excreted the 5C-metabolite in urine, albeit with a reduced ratio of 2:1. Determination of the urinary 5C- and 7C-metabolites in neonates demonstrated a dose-dependent relationship consistently responding to prophylaxis within 24 h. Comparison of equivalent K1 dosages via intramuscular (IM) and IV routes confirmed the slower excretion of K1 metabolites post-IM administration. This supports the concept of a muscular depot effect for this route, which would be consistent with the extended protection against vitamin K deficiency bleeding conferred by IM relative to the IV route [58]. In comparison, Shearer et al. previously determined that in adults a 1 mg tritiated dose of K1 given IV is rapidly eliminated via the urine with 2% of the dose excreted within 2 h [15]. Harrington et al. determined that in term infants only 0.03% of a parenterally administered K1 dose was excreted as urinary metabolites within the first 24 h post-prophylaxis [55]. Compared to Shearer's data from adults where 14–20% of an IV dose was excreted within the first 24 h [15], it appears the rate of K1 clearance in neonates is significantly slower. This is supported by the prolonged presence of K1 in term neonate blood after oral administration of K1 [59, 60] and is in contrast to the more rapid rate of clearance seen in the plasma of adults [13, 15]. Investigations in adults indicate that the relative excretory proportions of absorbed K1 are approximately 20–30% urinary and 30–40% faecal [13, 40].

In the study by Harrington et al. [55], 3 of 13 preterm infants studied (23%) predominantly excreted the 7C-metabolite rather than the 5C-metabolite, vitamin K1 2,3-epoxide (K1O) was also present at detectable levels in the plasma of all preterm infants who predominately excreted the 7C-metabolite was but not detectable in the plasma of any of the preterm infants predominately excreting the 5C-metabolite. K1O is typically undetectable in plasma from unsupplemented adults (∼<0.12 μg/L) and seen at elevated levels post-pharmacological doses of K1 or post-exposure to vitamin K antagonists, e.g. warfarin. The combined evidence of proportionally increased 7C-metabolite excretion and unusually elevated K1O could indicate that hepatic vitamin K metabolism is immature and/or overloaded in preterm neonates. It was noted that predominant excretion of the 7C-metabolite was seen in two of four infants receiving the higher 500 μg K1 dose and in one of four infants who received 200 μg IV whereas the 5C-metabolite predominated in all preterm infants receiving the lower 200 μg IM dose. Therefore, this study may indicate that metabolic overload in the immature hepatocytes of preterm neonates is exacerbated by K1 administration at birth. Infants that predominantly excreted the 7C-metabolite also had the highest concomitant plasma K1 concentrations, which also supports a decreased rate of clearance or would be consistent with these infants having the greatest exposure to vitamin K. In a previous study, the 7C-metabolite was also shown to be the predominant urinary metabolite in adults given very high doses (45 mg) of MK-4 [34]. This pattern of results could indicate saturation of various hepatic processes including those linked to and directly involved in vitamin K metabolism [39]. Other evidence of possible hepatic overload includes the finding that preterm neonatal plasma K1 concentrations remained grossly elevated on day 5 postpartum, reaching concentrations several hundred-fold higher than endogenous concentrations in healthy adults [61], where an equivalent dose would be cleared by this time [12]. This finding indicates a significantly increased plasma half-life of K1 in preterm neonates consistent with data reported elsewhere [62, 63].

Faecal vitamin K metabolite analysis has been successfully applied to neonatal meconium and faecal samples in order to compare patterns of vitamin K metabolism in term and preterm infants and assess the efficacy of the postpartum prophylactic administration of K1 [64].

8 Vitamin K and E interactions

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

Bleeding has for a long time been known as a complication of vitamin E overdose. High-dose vitamin E supplementation has been shown to result in a prolonged prothrombin time and activated partial thromboplastin time and lowered vitamin K dependent coagulation factors that could be prevented by infusing K3 prior to α-T administration [65]. It has also been shown to induce the appearance of undercarboxylated prothrombin in the blood of healthy adults [66]. A large-scale study demonstrated that long-term vitamin E supplementation has a mild anti-thrombotic effect [67] comparable with the therapeutic use of vitamin K antagonists such as warfarin. Vitamins E and K are thought to share common metabolic pathways including blood transport via lipoproteins, catabolism via ω- and β-oxidation and biliary excretion. Therefore, it follows that upregulation of one or more of these pathways in response to increased vitamin E intake could result in an increased rate of vitamin K catabolism and/or excretion, thereby inducing vitamin K deficiency. The historical background to this field is described in more detail by Traber [68].

To investigate the mechanism through which vitamin K depletion is conferred in response to high-dose vitamin E administration, animal studies have been conducted. Rats administered high-dose α-tocopherol were found to be MK-4 deplete in brain, lung, kidney and heart and K1 deplete in lung tissue when compared to controls. Tissue K1 concentrations however, were not significantly altered in liver and heart. These changes were not accompanied by a detectable increase in urinary vitamin K metabolite excretion indicating an alternative pathway was upregulated. ATP-binding cassette (ABC) transporters were modified in response to vitamin E administration possibly showing that vitamin K is lost via biliary excretion [69].

Although the metabolic pathways of vitamins E and K show many similarities, a number of key areas of difference are apparent. It is known that increased dietary α-tocopherol increases the expression of genes, which encode proteins involved in phase-I and phase-II transformations as well as phase-III excretion pathways. However, studies by Mustacich showed that elevated α-tocopherol does not modulate CYPF2 expression in rodents [70]. α-Tocopherol is β-oxidised to produce α-CEHC, and although this has been detected in extra-hepatic tissues, including brain, at concentrations 20-fold higher than circulating vitamin K1 concentrations, there is no evidence α-CEHC inhibits vitamin K recycling in vivo.

It is likely that the excretion of vitamins E and K in bile is facilitated by the same, or related, transporter proteins. The ABC superfamily of transporters is responsible for excretion of xenobiotic compounds and their metabolites from the liver. ABC transporters expressed on hepatic canalicular membranes include the multidrug resistance protein P-glycoprotein (MDR1) and the multidrug resistance-related protein MRP6 (ABCC6), which is a member of the OATP family of organic anion transporting polypeptides.

The MDR1 gene (MIM171050) codes for P-glycoprotein, a large transmembrane protein that is associated with an increased efficiency of vitamin K excretion in bile. The gene is located at chromosome 7q21. A 3435C>T polymorphism in MDR1 has been shown to effect P-glycoprotein activity most likely by inducing a conformation change in the polymorphic protein, thereby altering the structure of substrate and inhibitor interaction sites [71]. Reduced α-tocopherol and hepatic α-CEHC levels have been observed with a concurrent increase in MDR1 expression, which may be due to upregulation of the nuclear receptors, CAR and PXR, as analysis of rodents receiving vitamin E supplementation by Mustacich showed a 1.6- to 1.8-fold increase in their expression [72].

9 Biological activity of vitamin K metabolites

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

Vitamin K has been shown to reduce inflammatory response in both in vitro and animal studies [73] and in large-scale human studies [74]. This could be through production of vitamin K dependent proteins with anti-inflammatory properties [75] and/or through direct action as an antioxidant [76]. The possibility that metabolites of vitamin K possess inherent biological activity should also be considered. Previous unpublished studies have demonstrated that the 5C- and 7C-metabolites have no functionality in the γ-carboxylation of proteins. The biotransformation of vitamin K during its elimination increases its water solubility and thus the rate of excretion. These changes result in the shortened circulatory half-life of the 5C- and 7C-metabolites and potentially minimise any biological activity, since there is a general relationship between the concentration of a substance and the potency of its biological activity. However there are many exceptions, especially in phase-I biotransformation reactions. Preliminary data have demonstrated that the 7C-metabolite may be a natural modulator of cytokines in vitro [77] and is more potent than selected analgesics in a rat model of inflammation and pain [78, 79]. The investigation of the biological activity of vitamin K metabolites is in its infancy and presents many opportunities for future research.

10 Conclusions

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References

Analytical methods are available to support the further investigation of vitamin K metabolism and excretion. In both unsupplemented and supplemented adults, the excretion of the 5C-metabolite is greater than for the 7C. Whereas physiological quantities of K1 are largely metabolised to the terminal 5C-metabolite, vitamin K supplementation leads to a greater urinary excretion of the less extensively metabolised 7C-metabolite [12, 55], and supra-doses of vitamin K1 may result in excretion of a 10C-metabolite [14].

Supplementation studies with K1, MK-4 and MK-7 clearly indicate that the 5C- and 7C-metabolites are common products of the major naturally occurring forms of vitamin K. Excretion of the 5C- and 7C-metabolites also increases in response to supplementation with K3 and is likely to reflect the process of inter-conversion to MK-4 [34].

The measurement of the urinary 5C- and 7C-metabolites show promise as markers of vitamin K status and hepatic function and have several potential advantages over other markers. At the present time, the only available indicator of vitamin K status that may be said to directly reflect storage and transport (static indicator) are serum K1 concentrations. Other markers such as undercarboxylated prothrombin (PIVKA-II), undercarboxylated osteocalcin (GluOC) or, the less commonly measured, urinary free Gla may be said to be functional indicators. The measurement of serum K1 has a number of drawbacks as a status biomarker: one major problem being that it is transported in association with serum lipids, particularly triglyceride-rich lipoproteins. Associations have been demonstrated with both serum triglycerides and serum cholesterol. Thus, true vitamin K1 stores are overemphasised by a relative hyperlipidaemia and underemphasised by a relative hypolipidaemia. The large inter-subject normal variability in lipid lipoprotein and metabolism (and aging effects) represents major confounders to serum K1 measurements. Even imbibing alcohol the night before taking a fasting blood sample can cause sufficient hypertriglyceridaemia to artificially raise serum K1. Also, serum K1 assays will not reflect the known metabolic conversion of dietary K1 to MK-4 in humans. Most important of all, serum K1 measurements reflect only one of several vitamin K compounds known to contribute to vitamin K status. In contrast, the measurement of urinary vitamin K metabolites reflects excretion of all K vitamers.

The authors have declared no conflict of interest.

12 References

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Putative phase-I transformations
  5. 3 Putative phase-II transformations
  6. 4 Measurement of vitamin K metabolites as markers of vitamin K status
  7. 5 Comparison of vitamin K1 and dihydro-vitamin K1 metabolism
  8. 6 Conversion of vitamin K to MK-4 and ω-hydroxyvitamin K1
  9. 7 Vitamin K metabolism in newborn infants
  10. 8 Vitamin K and E interactions
  11. 9 Biological activity of vitamin K metabolites
  12. 10 Conclusions
  13. Acknowledgements
  14. 12 References
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