Chapter 3. Vitamin K

Studies of vitamin K antagonists have more recently been studiedusing in vitro assays and are discussed in thesection Vitamin K-Dependent Carboxylase.SYNTHESIS OFVITAMINK The methods use

3 Vitamin K

John W Suttie

CONTENTS

History 112

Chemistry 112

Isolation 112

Structure and Nomenclature 113

Structures of Important Analogs, Commercial Forms, and Antagonists 114

Analogs and Their Biological Activity 114

Commercial Form of Vitamin K 114

Antagonists of Vitamin Action 115

Synthesis of Vitamin K 117

Physical and Chemical Properties 117

Analytical Procedures and Vitamin K Content of Food 118

Metabolism 120

Absorption and Transport of Vitamin K 120

Plasma and Tissue Concentrations of Vitamin K 121

Tissue Distribution and Storage of Vitamin K 122

Synthesis of Menaquinone-4 123

Metabolic Degradation and Excretion 123

Vitamin K-Dependent Proteins 125

Plasma-Clotting Factors 125

Calcified Tissue Proteins 125

Other Vitamin K-Dependent Proteins 127

Biochemical Role of Vitamin K 127

Discovery of g-Carboxyglutamic Acid 127

Vitamin K-Dependent Carboxylase 129

Vitamin K-Epoxide Reductase 131

Health Impacts of Altered Vitamin K Status 132

Methodology 132

Adult Human Deficiencies 133

Anticoagulant Therapy 134

Hemorrhagic Disease of the Newborn 134

Possible Role in Skeletal and Vascular Health 135

Other Factors Influencing Vitamin K Status 136

Vitamin K Requirements 137

Animals 137

Humans 138

Efficacy and Hazards of Pharmacological Doses of Vitamin K 139

References 140

The discovery of vitamin K was one of the outcomes of a series of experiments conducted byHenrik Dam who investigated the possible essential role of cholesterol in the diet of the chick.Dam [1] noted that chicks ingesting diets that had been extracted with nonpolar solvents toremove the sterols developed subdural or muscular hemorrhages and blood taken from theseanimals clotted slowly Subsequently, McFarlane et al [2] described a clotting defect seenwhen chicks were fed ether-extracted fish or meat meal, and Holst and Halbrook [3] observedscurvy-like symptoms including internal and external hemorrhages in chicks fed fish meal andyeast as a protein source Studies in a number of laboratories soon demonstrated that thisdisease could not be cured by the administration of any of the known vitamins Damcontinued to study the distribution and lipid solubility of the active component in vegetableand animal sources and in 1935 proposed [4,5] that the antihemorrhagic vitamin of the chickwas a new fat-soluble vitamin, which he called vitamin K Not only was K the first letter ofthe alphabet that was not used to describe an existing or postulated vitamin activity at thattime, but it was also the first letter of the German word koagulation Dam’s reporteddiscovery of a new vitamin was closely followed by an independent report of Almquist andStokstad [6,7] describing their success in curing the hemorrhagic disease with ether extracts ofalfalfa and clearly pointing out that microbial action in fish meal and bran preparations couldalso lead to the development of antihemorrhagic activity

A number of groups were involved in the attempts to isolate and characterize this newvitamin, and Dam’s collaboration with Karrer of the University of Zurich resulted in theisolation of the vitamin from alfalfa as a yellow oil Subsequent studies soon established thatthe active principle was a quinone and vitamin K1was characterized as 2-methyl-3-phytyl-1,4-naphthoquinone and synthesized by MacCorquodale et al in St Louis [8] Their identifica-tion was confirmed by independent synthesis of this compound by Karrer et al [9], Almquistand Klose [10], and Fieser [11] The Doisy group also isolated a form of the vitamin fromputrified fish meal, which in contrast to the oil isolated from alfalfa was a crystalline product.Subsequent studies demonstrated that this compound called vitamin K2, contained an unsat-urated side chain at the 3-position of the naphthoquinone ring Early investigators recognizedthat sources of this form of the vitamin, such as putrified fish meal, contained a number ofdifferent vitamins of the K2 series with differing chain length polyprenyl groups at the3-position The 1943 Nobel Prize in Physiology and Medicine was awarded to Dam andDoisy, and much of the early history of the discovery of vitamin K has been reviewed by them[12,13] and others [14,15]

CHEMISTRY

ISOLATION

Vitamin K can be isolated from biological material by standard methods used to obtainphysiologically active lipids The isolation is always complicated by the small amount ofdesired product in the initial extracts Initial extractions are usually made with the use of sometype of dehydrating conditions, such as chloroform–methanol, or by first grinding the wettissue with anhydrous sodium sulfate and then extracting it with acetone followed by hexane

or ether Large samples (kilogram quantities) of tissues can be extracted with acetone alone,and this extract can be partitioned between water and hexane to obtain the crude vitamin.Small samples, such as in vitro incubation mixtures or buffered subcellular fractions, can beeffectively extracted by shaking the aqueous suspension with a mixture of isopropanol andhexane The phases can be separated by centrifugation and the upper layer analyzed directly

Method s for the efficie nt extra ction of vitamin K from v arious foo d matrices have be endeveloped [16], and rather extens ive databas es of the vitamin K co ntent of foods are nowavailab le.

Crude nonpolar solvent extra cts of tissues contai n large amounts of con taminatin g lipi d

in add ition to the desir ed vita min Further pur ification and identifi cation of vita min K in thisextra ct can be facilitated by a prelimina ry fract ionation of the crud e lipi d e xtract on hydrate dsilici c acid [17] A numb er of the forms of the vitamin can be separat ed from each other an dfrom other lipid s by revers ed-phase partiti on ch romatograp hy, as descri bed by Matsc hinerand Tag gart [18] Thes e general procedures app ear to extra ct the major ity of vita min K fromtissues Following separat ion of the total vitamin K fract ion from much of the contam inate dlipid, the various forms of the vitamin can be separat ed by the procedures describ ed in the

STRUCTURE AND NOMENCLATURE

The nomencla ture of compo unds possess ing vita min K activity has been modified a num ber

of times since the discove ry of the vitamin The nomencla ture in general use at the presenttime is that of the most recent ly ado pted IUPA C–IUB Subc ommitt ee Report on Nomencl a-ture of Quinones [19] The term vitamin K is used as a generic descrip tor of 2 -methyl-1,4-n aphthoqu inone and all derivativ es of this compo und that exhibi t an antihe morrha gicactivit y in anima ls fed a vitamin K-deficien t diet The co mpound 2-me thyl-3-phy tyl-1,4-n aphthoqu inone is pro duced in green plants and is general ly called vitamin K1, but isprefer ably called phylloqui none The USP nomencla ture for phy lloquin one is phy tonadione.The compo und first isolated from putrifie d fish meal and ca lled at that time, vita min K2 isone of a series of vita min K compoun ds with unsatur ated side chains call ed multipreny l-menaqu inones that are synthes ized by a number of facul tative and obliga te an aerobic ba cteria[20] The particular menaquino ne shown in Figure 3.1 (2-methy l-3-far nesylgeran ylgeranyl-1,4-n aphthoqu inone) has 7 isopre noid units , or 35 carbons in the side chain and was onc ecalled vitamin K2 but now is call ed menaq uinone-7 (MK- 7) Vitamins of the menaqu inoneseries with up to 13 prenyl groups have been identified, as well as several partially saturatedmembers of this series The parent compound of the vitamin K series, 2-methyl-1,4-naphtho-quinone, has often been called vitamin K3but is more commonly and correctly designated asmenadione MK-4 is a minor bacterial product but can be formed by animals by thealkylation of menadione or through the degradation of phylloquinone by a pathway notyet eluci dated (see secti on Synthesi s of Menaqu inone-4 )

4

1 2

3

3 6

FIGURE 3.1 Structures of some compounds with vitamin K activity

S TRUCTURES OF IMPORTANT A NALOGS, COMMERCIAL FORMS , AND A NTAGONISTS

Analo gs and The ir Biolog ical Activ ity

Follow ing the discove ry of vita min K, a num ber of related compound s were synthes ized invarious labo ratories and their biologic al activity compared with that of the isolated forms[21, 22] Structu ral featu res found to be essent ial for signi ficant biological activit y included : anap hthoquinon e ring, a 2-Me group on the ring, a n uns aturated isopre noid uni t adjacent tothe ring, and trans -configu ration of the polyisop renoid side chain The vitamin K analogsillustr ated in Figure 3.2 have all been sho wn to ha ve low or mini mal activit y relat ive to transphy lloquino ne in whol e-ani mal assays

The activit y of various struc tural an alogs of vita min K in whol e-animal assay systems is,

of course, a summ ation of the relative absorpt ion, trans port, meta bolism, and effecti veness ofthis comp ound at the active site as compared with that of the referen ce co mpound Much ofthe data on biological activit y of various compoun ds wer e obtaine d by the use of an 18 h oraldose c urative test using vita min K-defici ent ch ickens It was fou nd that when administe redorall y, isopre na logs wi th 3–5 isopre noid group s had maxi mum activit y [22] The lack ofeffe ctiveness of higher isopre nalogs in this type of assay may be due to the relative ly poorabsorp tion of these compoun ds Matsc hiner and Tag gart [23] ha ve shown that when intra-cardia l injection of vita min K to de ficient rats is used as a pro tocol, the very high mo lecularweigh t isopre nalogs of the menaquinon e seri es are the most active; maximum activit y wasobserved with MK-9 Struct ure–func tion relat ionship s of v itamin K analogs ha ve also beenstudi ed using in vitr o assays of the vitamin K-depe nde nt g-glutamy l carboxyl ase, and theseare discus sed in the section Vitam in K-Depen dent Car boxylas e

Comme rcia l Form of Vitam in K

Only a few form s of vitamin K are commercial ly impor tant The major use of vitamin K inthe anima l indust ry is in poul try an d swine diets Chicks are ve ry sensitiv e to vitamin Krest riction, and antibio tics that decreas e intesti nal vitamin syn thesis are often added topoultr y diets Phyl loquinone is too expen sive for this purpose, and diff erent forms of mena-dione ha ve been used M enadione itself possess es high biological acti vity in a defic ient ch ick,but its effecti veness dep ends on the presence of lipids in the diet to pro mote absorpt ion Thereare also prob lems of its stability in feed pro ducts, an d because of this , water- soluble forms areused Menad ione form s a water-solub le sodium bisulfit e ad dition produ ct, menadi one sodiumbisul fite (MSB ) (Figure 3.3), which has been used co mmercial ly but which is also somewhat

O

O O

O

O

O O

O des Me-phylloquinone 2 ⬘,3⬘Dihydro-phylloquinone

unstabl e in mixe d feeds In the presen ce of excess sodium bisul fite, MSB crystall izes as acomplex with an add itional mole of sodium bisulfite; this complex , known as menad ionesodium bisul fite co mplex (MSBC ), has increa sed stabi lity, and is wi dely used in the poultr yindust ry A thir d wat er-soluble compoun d is a salt form ed by the addition of dimethylpyr -idinol to M SB; it is called menadi one pyridi nol bisul fite (MP B) [24] Comparis ons of therelative biopotency of these compou nds have often been made on the basis of the weigh t ofthe salt s rather than on the basis of men adione co ntent, and this ha s caused some con fusion inasses sing their value in animal feeds.

The clinical use of vitamin K is large ly lim ited to various preparat ions of phy lloqui none

A water- solubl e form of menadi one, menadi ol sod ium diphosph ate, whi ch was sold asKappad ione or Syn kayvite, was once used to prevent he morrha gic diseas e of the newbor n,but the danger of hyperbil irubin emia associ ated with menadi one usage (see section Effi cacy

the desir ed form of the vita min Phylloq uinone (USP phytonadi one ) is sold as Aqua PHYTO N, Konakion , Mephyton, an d Mo no-Kay These pr eparations are deterg ent stabil-ized pr eparations of phy lloquin one and are used as intramuscul ar injec tions to preventhemorr hagic disease of the newbor n In some co untries, oral pro phylaxis of vitamin K hasbeen promot ed, and these preparat ions are not well absorbed A leci thin and bile salt mixe dmice lle prep aration, Kon akion MM , is now available an d has been sho wn [25] to be effecti vewhen admini stered orally Althou gh not current ly used in the Unit ed State s or Weste rnEuro pe, pha rmacol ogical doses of MK-4 , men atetrenon e, are used as a treatment forosteopor osis in Japan an d other Asi an c ountries (see secti on Hemor rhagic Dis ease of the

Antagon ists of Vitam in Action

The histo ry of the discove ry of the first antagoni sts of vita min K, the co umarin deriva tives,has been docu mented an d discussed by Lin k [26] A hemorr hagic disease of cattle, trace d tothe consu mption of improp erly cu red sweet clover hay, was describ ed in Canada an d theUnited States Midwest in the 1920s The compou nd present in spo iled swe et clover that wasresponsi ble for this diseas e had bee n studied by a numbe r of invest igators but was finallyisolated and ch aracterize d as 30 ,30 -methylbi s-(4-hydr oxycou marin) by Link’s group during theperiod from 1933 to 1941 and was called dicuma rol (F igure 3.4) Dicumarol was success fullyused as a clinical agent for antic oagulant therapy in some early studi es, and a large number of

SO3Na +

SO – Na +

SO –+ H 3

3

FIGURE 3.3 Forms of vitamin K used in animal feeds

substi tuted 4-hydrox yco umarin s were synthes ized both in Link’s laborato ry and elsewhere The most success ful of these, both clinically for lon g-term lowering of the vitaminK-depe nde nt clotting factors an d sub sequently as a rodenti cide, has been war farin,3-( a-aceton ylbenzyl )-4-hydroxy cou marin Althou gh warfarin is the most extens ivel y use ddrug worldw ide for ora l anticoag ulant therapy , other co umarin deriva tives wi th the sametherape utic mechani sm such as its 40 -nitro analog, acen ocoumarol , an d phe nproco umon havebeen used These drugs differ in the degree to which they are ab sorbed from the intes tine, intheir plasm a half-li fe, and presum ably in their effecti veness as a vita min K antagoni st atthe active site Because of this, their clinical use differs Muc h of the informat ion on thestruc ture–a ctivity relationshi ps of the 4-hydrox ycou marins has be en revie wed by Renkand Stoll [27] The clinical use of these compoun ds and many of their pharmac odynami cinter actions have been revie wed by O’Re illy [28]

W arfarin ha s been widely used as a rode nticide and, as might ha ve been predict ed,con tinual use led to developm ent of an ticoagulant -res istant popul ations [29, 30] More hydro-phobi c derivativ es of 4-hydro xycoumar ins are clear ed from the body much more slowly andare effe ctive rodenticides in warfarin -resistant rat stra ins Com pounds such as difena coumand brod ifacoum are now widely used for rodent co ntrol [31] but should be used with care ascon sumption of carcas ses by birds or ca ts can lead to death

A secon d class of compo unds wi th antic oagulant activit y that can be revers ed byvita min K administr ation [32] are the 2-subs titu ted 1,3-indand iones such as 2-phen yl-1,3-inda ndion e (Figur e 3.5) These compou nds ap pear [33] to act by the same mechani sm asthe 4-hydrox ycoumar ins, and althoug h they were admini stered as clinical anticoagu lants androden ticides at one time, they are current ly seldom used Some structural analogs of the vitaminhave also been sh own to antagon ize its acti on Early studies of the structural requir ement s forvita min K activit y [34] demonst rated that replac ement of the 2-methyl group of phy lloqui nones

by a chlori ne atom to form 2-chl oro-3-p hytyl-1, 4-napht hoquino ne resul ted in a compou nd thatwas a pot ent an tagonist of vitamin K In co ntrast to the co umarin an d indandione de rivative s,chloro- K acts like a true compet itive inhibitor of the vitamin at its active site; and , as it is aneffe ctive antico agulant in coumari n antico agulant -resistant rats [35], it has been suggest ed

as a possible rodenticide Another structurally unrelated compound, pyridinol, has anticoagulant activity [36]; and, on the basis of its action in warfarin-resistantrats [33], it would appear that it is functioning as a direct antagonist of the vitamin Sub-sequent studies have demonstrated [37] that other polychlorinated phenols are also effective

2,3,5,6-tetrachloro-4-O

O

O OH

Warfarin O

FIGURE 3.4 Oral anticoagulants that antagonize vitamin K action

vitamin K antagonists Studies of vitamin K antagonists have more recently been studiedusing in vitro assays and are discussed in thesection Vitamin K-Dependent Carboxylase.

SYNTHESIS OFVITAMINK

The methods used in the synthesis of vitamin K by early investigators involved the densation of phytol or its bromide with menadiol or its salt to form the reduced additioncompound, which was then oxidized to the quinone These reactions have been reviewed

con-in considerable detail, as have methods to produce the specific menaqucon-inones rather thanphylloquinone [38,39] The major side reactions in this general scheme are the formation ofthe cis rather than the trans isomer at the D2position and alkylation at the 2-position ratherthan the 3-position to form the 2-methyl-2-phytyl derivative The use of monoesters

of menadiol and newer acid catalysts for the condensation step [40] is the basis for the generalmethod of industrial preparation used at the present time Naruta [41] has described

a new method for the synthesis of compounds of the vitamin K series based on the ing of polyprenyltrimethyltins to menadione This method is a regio- and stereocontrolledsynthesis that gives a high yield of the desired product Analytical methods based on high-performance liquid chromatography (HPLC)=MS or GC=MS have been reported, andmethods for the high-yield synthesis of 18O- or 2H-labeled vitamin K homologs have beendescribed [42–44]

coupl-PHYSICAL ANDCHEMICALPROPERTIES

Compounds with vitamin K activity are substituted 1,4-naphthoquinones and, therefore,have the general chemical properties expected of all quinones The chemistry of quinoidshas been reviewed in a book edited by Patai [45], and much of the data on the special andother physical characteristics of phylloquinone and the menaquinones have been summarized

by Sommer and Kofler [46] and Dunphy and Brodie [47] The oxidized form of the K vitaminsexhibits an ultraviolet (UV) spectrum that is characteristic of the naphthoquinone nucleus,with four distinct peaks between 240 and 280 nm and a less sharp absorption at around320–330 nm The molar extinction value e for both phylloquinone and the various mena-quinones is about 19,000 The absorption spectrum changes drastically on reduction to thehydroquinone, with an enhancement of the 245 nm peak and disappearance of the 270 nmpeak Vitamin K-active compounds also exhibit characteristic infrared and nuclear magneticresonance (NMR) absorption spectra that are largely those of the naphthoquinone ring.NMR analysis of phylloquinone has been used to firmly establish that natural phylloquinone

O

O

O

O Cl 2-Phenyl-1,3-indandione 2,3,5,6-Tetrachloro-4-pyridinol

Chloro-K

Cl Cl

Cl OH

3

FIGURE 3.5 Other vitamin K antagonists

is the trans isom er and can be used to establis h the cis–trans rati o in synthet ic mixt ures of thevita min Mass spectr oscopy has been useful in determ ining the lengt h of the side ch ain andthe de gree of satur ation of vita mins of the menaquino ne seri es isolated from na tural sou rces.Phylloq uinone is an oil at room tempe ratur e; the various menaqu iones can easily be cryst al-lize d from organic solvent s and ha ve melting points from 35 8C to 608C, de pending on thelengt h of the isopre noid chain.

ANALYTICAL PROCEDURES AND VITAMIN K CONTENT OF FOOD

Chem ical react ivity of vitamin K is a functi on of the naphthoq uinone nucleus, and as otherquino nes also react wi th many of the colori metric assays that have been develope d [46, 47],they are of littl e an alytical value The numb er of interfer ing substa nces pre sent in crudeextra cts is also such that a significan t amount of sep aration is req uired before UV absorpt ionspectr a can be used to qua ntitate the vita min These sim ple methods are therefo re notpracti cal in the determinat ion of the smal l amo unt of vita min presen t in natural sou rces.All oral bioa ssay proced ures are complica ted by the effects of different rates and extent s ofabsorp tion of the de sired nutrients from the various produ cts assayed They have be ensuperse ded by HP LC techni que s and hav e littl e us e at the presen t time

Anal ytical method s suitab le for the small amou nts of vitamin K present in tissues andmost food sources have been availab le only recent ly The sep aration of the ex tensive mixtures

of menaquini ones in bacter ia and anima l sources was fir st achieve d wi th various thin- layer orpap er ch romatograp hic syst ems [38, 46–48] All separat ions involving con centrated extra cts ofvita min K should be carried out in subdued light to mini mize UV decomposi tion of thevita min Com pounds with v itamin K acti vity are also sen sitive to alkal i, but they arerelative ly stable to an oxidiz ing atmosph ere and to heat and ca n be vacuu m-distill ed withlittl e de composi tion Inter est in the qua ntitatio n of vita min K in serum and anima ltissues eventual ly led to the use of HP LC as an analytical tool to investiga te vita min Kmeta bolism [49]

Sa tisfactory tables of the vitamin K co ntent of various commonl y co nsumed foods werenot made av ailable until the early 1990s Many of the values previous ly quoted in variouspublica tions have apparent ly been recalculat ed in an unspe cified way from data obtaine d by achick bioass ay that was not intende d to be more than qualitativ e and should not be used tocalcul ate intake Tables of food vitamin K content in va rious older text s and reviews may alsocon tain data from this source, as wel l as consider able amou nts of unpublishe d data

Curr ent methodol ogy uses HPLC analysis of lipid extracts, and has been report ed [16] tohave a within-sam ple co efficie nt of varia tion for different foods in the range of 7%–14% and abetw een-samp le coeff icient of variation of 9%–45% Altho ugh green leafy vegeta bles havebeen know n for some time to be the major source of v itamin K in the diet, it is now apparentthat co oking oils , parti cularly soybean oil and rapesee d oil [50], are major con tributors Human milk contain s abou t 1 ng=ml of phylloqui none [51–54 ], whi ch is only 20%–30 % ofthat found in cow’s mil k Infant formulas are cu rrently supp lemented with vitamin K,providi ng a much higher intake than that pro vided by brea st milk

The data in Table 3.1 are taken from a survey of literatu re [55], which con sidered mo st

of the reported HPL C-deriv ed values for v arious food item s and from analyses of the FDAtotal diet study An e xtensive USDA databas e contai ning the vitamin K content of a largenumb er of foods can be accessed at http: == www nal.usda gov =fnic =foodcomp In g eneral,green and leaf y vegeta bles are the be st so urces of the vita min, an d coo king oils are the nextmajor sources In addition to the data from the United States, there are databases publishedfrom a number of other countries [56–58] as well as reports of the vitamin K content of fastfoods [59], mixed dishes [60], and baby food products [61] The major source of vitamin K

in foods, and the source usually reported, is phylloquinone Significant amounts of MK-4 are

found in poultry meat and egg yolk as poultry rations are commonly supplemented withmenadione, and some cheeses can have appreciable amounts of long-chain menaquinones[62] due to the bacterial action during aging Using the available food composition data andfood consumption data, it is possible to calculate average daily intakes of phylloquinione.Based on the Third National Health and Nutrition Examination Survey (NHANES III)data [63], the adult U.S male and female intakes were about 115 and 100 mg=day, respect-ively This is somewhat higher than some previous estimates [64,65] Mean phylloquinoneintakes in Ireland for adult men and women have been reported to be 84 and 74 mg=day [56],

in Scotland 72 and 64 mg=day [66], in Britain 70 and 61 mg=day [67], and in The Netherlands

257 and 244 mg=day [58] The high consumption of cheese in The Netherlands also provides

an intake of about 20 mg=day of long-chain menaquinones As different databases are used,variations in the assumed vitamin K content of those few foods that contribute the mostvitamin to the diet can result in large differences In a study where four metabolic ward dietswere directly analyzed to contain about 100 mg=day, the amount calculated by two differentdatabases ranged from 84 to 160 mg=day [68] Use of the current database information has,however, made it possible to formulate nutritionally adequate diets that contain only

10 mg=day of phylloquinone [69]

The conversion of liquid oils to solid margarines by commercial hydrogenation results inthe formation of substantial amounts of 20,30-dihydrophylloquinone, which in the case of

TABLE 3.1

Vitamin K Content of Ordinary Foods

mg Phylloquinone=100 g of Edible Portion

beef

0.5

Endive 231 Walnut oil 15 Bananas 0.5 Wheat flour 0.6 Turkey <0.1 Green

Lettuce 122 Dry kidney

beans

beans

some of the harder margarines can exceed the amount of unmodified phylloquinone [70,71].Because of the large contribution of high phylloquinone vegetable oils to many diets, theamount of the hydrogenated form represents around 20% of the total vitamin K in Americandiets [72] Although this form of the vitamin has some biological activity, the degree of thisresponse has not been well established in either human subjects or experimental animals.

METABOLISM

ABSORPTION AND TRANSPORT OFVITAMINK

The absorption of nonpolar lipids, such as vitamin K, into the lymphatic system requiresincorporation into mixed micelles, and early studies [73] demonstrated that these phylloquinone-containing micellar structures required the presence of both bile and pancreatic juice Using

an in vitro recirculating perfused isolated rat intestine preparation [74], it was found that theabsorption of radiolabeled phylloquinone was energy-dependent and saturable Normalhuman subjects were found [75] to excrete less than 20% of a large (1 mg) dose of phyllo-quinone in the feces, but as much as 70%–80% of the ingested phylloquinone was excretedunaltered in the feces of patients with impaired fat absorption caused by obstructive jaundice,pancreatic insufficiency, or adult celiac disease

The bioavailability of phylloquinone from different food sources has not been extensivelystudied, and the results reported are somewhat variable Phylloquinone in spinach was found

to be absorbed only about 15% as well as from a detergent-solubilized preparation(Konakion) when it was consumed with 25 g of butter [76], and less than 2% as well when butterwas omitted A second similar study [77] indicated that phylloquinone in broccoli, spinach, orromaine lettuce, consumed with a diet containing 30% fat, was only about 15%–20% as bio-available as added phylloquinone A comparison [78] of the absorption of about 300 mg=day

of phylloquinone in the form of broccoli or 300 mg=day added to corn oil indicated thatbioavailability from the food source was only about 50% that of phylloquinone in corn oil.Some cheeses and a fermented soybean product, natto, consumed mainly in the Japanesemarket, do contain substantial amounts of long-chain menaquinones, and there are indica-tions [62] that these forms may be more bioavailable than phylloquinone from vegetablesources The limited available data would suggest that bioavailability of vitamin K from food

is rather low and variable and very dependent on both the individual food sources and totaldiet composition

Substantial amounts of vitamin K are present in the human gut in the form of long-chainmenaquinones Relatively few of the bacteria that comprise the normal intestinal flora aremajor producers of menaquinones, but obligate anaerobes of the Bacteroide fragilis, Eubac-terium, Propionibacterium, and Arachnia groups are, as are facultatively anaerobic organismssuch as Escherichia coli The amount of vitamin K in the gut can be quite large, and theamounts found in total intestinal tract contents from five colonoscopy patients have beenreported [79] to range from 0.3 to 5.1 mg, with MK-9 and MK-10 as the major contributors.The total amount of long-chain menaquinones, mainly MK-6, MK-7, MK-10, and MK-11,present in human liver also greatly exceed the phylloquinone concentration, which representsonly about 10% of the total [80] There is some evidence [81] that the hepatic turnover of long-chain menaquinones is slower than that of phylloquinone, which would account for theincreased concentration observed, but a major question remaining is how these verylipophylic compounds that are present as constituents of bacterial membranes are absorbedfrom the lower bowel Absorption of menaquinones from rat colonic gut sacs has beenreported [82], but in the absence of bile no uptake of MK-9 from the rat colon to lymph

or blood occurred within 6 h [83] In the presence of bile, MK-9 is absorbed via the lymphaticpathway from rat jejunum [83] The oral administration of 1 mg mixed long-chain

menaquinones to anticoagulated human subjects [84] effectively decreased the extent of theacquired hypoprothrombinemia, demonstrating that the human digestive tract can absorbthese forms of the vitamin from the small intestine but does not address their absorption fromthe large bowel However, a small but nutritionally significant portion of the intestinalcontent of the vitamin is located not in the large bowel but in a region where bile acid-mediated absorption could occur [79].

Menadione is widely used in poultry, swine, and laboratory animal diets as a source ofvitamin K It can be absorbed from both the small intestine and the colon by a passive process[85] Menadione itself does not have biological activity, but after absorption it can bealkylated to MK-4, a biologically active form of the vitamin

Absorption of phylloquinone from the intestine is via the lymphatic system [75] and isdecreased in individuals with biliary insufficiency or various malabsorption syndromes.Phylloquinone in plasma is predominantly carried by the triglyceride-rich lipoprotein fractioncontaining very low density lipoproteins (VLDL) and chylomicrons, although significantamounts are located in the low-density lipoprotein (LDL) fraction [86,87] In a study [88]comparing the transport of different forms of vitamin K, significant amounts of MK-4 werefound in the high-density lipoprotein (HDL) fraction, and the half-life of MK-9 was found to

be substantially greater than that of either phylloquinone or MK-4 As expected fromlipoprotein transport, plasma phylloquinone concentrations are strongly correlated withplasma lipid levels [89,90] The major route of entry of phylloquinone into tissues appears

to be via clearance of chylomicron remnants by apolipoprotein E (apoE) receptors Thepolymorphism of apoE has been found to influence the fasting plasma phylloquinone con-centrations in patients undergoing hemodialysis therapy [89], and plasma phylloquinoneconcentrations have been shown to decrease according to apoE genotype: apoE2 > apoE3 >apoE4 This response is correlated to the hepatic clearance of chylomicron remnants fromthe circulation, with apoE2 that has the slowest rate of removal Removal of circulatingphylloquinone by osteoblasts has also been shown [91] to be modulated by the apoEgenotype Details of the secretion of phylloquinone from liver and the movement of thevitamin between organs are not available The total human body pool of phylloquinone isvery small, and early studies [75] using pharmacologic doses of radioactive phylloquinoneindicated that the turnover is rapid There are only limited available data assessing thedisappearance of small amounts (<1 mg) of infused3H-phylloquinone from human subjects,and these [92] are consistent with a body pool turnover of about 1.5 days and a body pool size

of about 100 mg Other data, based on liver biopsies of patients fed diets very low in vitamin Kbefore surgery [93], indicated that about two-thirds of hepatic phylloquinone was lost in

3 days These findings are also consistent with a small pool size of phylloquinone that turnsover very rapidly

PLASMA ANDTISSUECONCENTRATIONS OFVITAMINK

Measurements of endogenous plasma phylloquinone concentrations have been available onlysince the early 1980s The early history of the development of HPLC techniques to quantitateplasma phylloquinone concentrations has been reviewed [49] These methods require apreliminary semipreparative column to rid the sample of contaminating lipids followed by

an analytical column The chief alterations and improvement in methodology in recent yearshave been associated with the use of different methods of detection Early methods used UVdetectors, which lack sensitivity, and electrochemical detection or fluorescence detection ofthe vitamin following chemical or electrochemical reduction have replaced this methodology.Comprehensive reviews of the procedures used to determine plasma phylloquinone concen-trations by both detection methodologies are available [94,95] The most commonly usedmethodology at present involves fluorescence detection following zinc postcolumn reduction

Cont inued modificat ion of this techn ique [96] has great ly increa sed its sensi tivity and ducibility Although earli er report s of plasm a phy lloquinone concen trations wer e somewhathigher, it now appears that normal fasting values are aro und 0.5 ng=ml (1.1 nmo l=L) There is

repro-a strong positive correl repro-ation between plrepro-asmrepro-a triglyce rides repro-and plrepro-asm repro-a phy lloquin one [97] , repro-andthe varia tion betwe en samples measur ed at differen t da ys from the same subject is muchhigher than that for other fat-sol uble vita mins [98] Because of this , extre me cauti on should beused in attempts to determine vita min K status of an indivi dual from a single day’s sampl e ofplasm a Ci rculating phylloqui none con centrations do respond to daily ch anges in intake andfall rather rapidl y when intake is restrict ed [93, 99,100 ] Although there are very few foodscon taining appreci able amounts of long-chain men aquinones, they are de tectable in plasma,and in some cases have been repo rted to present at substa ntial levels [101–1 03]

T ISSUE DISTRIBUTION AND S TORAGE OF V ITAMIN K

The dist ribut ion of vitamin K in various body organs of the rat was fir st studi ed with acti ve form s of the vitamin, using bot h mass ive [104] an d more phy siological [105] amounts ofphy lloquino ne The live r was fou nd to retain the majorit y of the vita min at early time points,but as the half-li fe in the live r app ears to be in the range of 10–15 h [105,106] , it is rapidly lost.Studies using radioac tive phylloqui none [107] indica ted that more than 50% of the live rradioac tivity was recovered in the microsomal fract ion, and substan tial amoun ts were found

radio-in the mito chond ria and cellular debris fractions The specific activit y (picom oles of vitamradio-in

K =mg pro tein) of injec ted radioac tive phylloqui none has been assessed [108], and only themito chondrial and micro somal fract ions ha d a specific activit y that was en riched over that ofthe e ntire hom ogenate, with the highest activit y in the microso mal fraction A more de tailedstudy [109] foun d the highe st spec ific activit y of radioac tive phy lloquin one to be in the Golgiand smoot h micro somal membran e fractions Only limite d data on the distribut ion of mena-quino nes are available, an d M K-9 has be en repo rted [110] to be prefer entia lly local ized in amito chondrial rather than a microso mal subcell ular fraction Fac tors influen cing intr acellulardist ribution of the vitamin are not well unde rstood, an d only pr eliminar y evidence of anintr acellular vitamin K-bi nding protei n that might faci litate intraorganel le movem ent hasbeen present ed [111]

Bec ause of the smal l amou nts of vita min K in anima l tissu es, it is difficul t to de terminewhi ch of the vitamers are presen t in tissue from diff erent specie s Only limited data areavail able, and they have been compiled and reviewed by Duello and Matschiner [112] Thesedata, obtained largely by thin-layer chromatography, indicate that phylloquinone is found inthe liver of those species ingesting plant material and that, in addition to this, menaquinonescontaining 6–13 prenyl units in the alkyl chain are found in the liver of most specie s Morerecent ly, ana lysis of a limit ed number of human live r spec imens has shown that phy lloquin onerepres ents onl y ab out 10% of the total vita min K pool and that a broad mixt ure (Tab le 3.2) ofmenaq uinones is present The predo minant forms appear to be MK-7, MK-8, MK-10, andMK-11 Kayata et al [113] have reported that the hepatic menaquinone content of five

24 month old infants was approximately sixfold higher than that of three infants less than

2 weeks of age, and another study [114] failed to find menaquinones in neonatal livers Althoughthe long-chain menaquinones are potential sources of vitamin K activity in liver, the extent

to which they are used is not known A study conducted in rats [110] has demonstrated thatthe utilization of MK-9 as a substrate for the vitamin K-dependent carboxylase is only about20% as extensive as phylloquinone when the two compounds are present in the liver in equalconcentrations Recent data have suggested that MK-4 may play a role in satisfying a uniquevitamin K requirement of some tissues Most analyses of liver from various species have notdetected significant amounts of MK-4 As commercially raised chickens are fed menadione as asource of vitamin K, chicken liver has been shown [112,115,116] to contain more MK-4 than

phylloquinone, and some nonhepatic tissues of the rat have also been shown [117] to containmuch more MK-4 than phylloquinone.

SYNTHESIS OFMENAQUINONE-4

Long-chain menaquinones are synthesized by bacteria via pathways that have been wellestablished [20] It is now well established that MK-4 is not a major product of bacterialmenaquinone biosynthesis, but that tissue MK-4 is formed by an alternate pathway Mena-dione can be converted to MK-4 by in vitro incubation of rat or chick liver homogenates withgeranylgeranyl pyrophosphate [118], and it has been demonstrated [119] that other isoprenoidpyrophosphates can serve as alkyl donors for menaquinone synthesis Early animal studies[120] also suggested that both phylloquinone and other long-chain menaquiniones could beconverted to MK-4 It was originally believed that the dealkylation and subsequent realkyla-tion with a geranylgeranyl side chain occurred in the liver, but it was subsequently concludedthat phylloquinone was not efficiently converted to MK-4 unless it was administered orally.This suggested that intestinal bacterial action was required for the dealkylation step Morerecent studies [115–117,121] have demonstrated that the phylloquinone-to-MK-4 conversion

is very extensive in tissues such as brain, pancreas, and salivary gland and that its trations in those tissues exceed that of phylloquinone Similar distributions of MK-4 havebeen observed in human tissues [122], and it has been established that high tissue concentra-tions of MK-4 are more readily obtained in rats by phylloquinone supplementation than byadministering MK-4 [123] Gut bacteria are not needed for this conversion [124,125], andcultures of kidney cells are able to convert phylloquinone to MK-4 in a sterile incubationmedium [124] As both phylloquinone and MK-4 are effective substrates for the only knownfunction of vitamin K, the vitamin K-dependent g-glutamyl carboxylase, the metabolicsignificance of this conversion is not yet apparent

concen-METABOLICDEGRADATION ANDEXCRETION

The conversion of phylloquinone or long-chain menaquiniones to MK-4 is a major metabolicpathway of vitamin K utilization in some tissues, but does not indicate ultimate excretion

TABLE 3.2Vitamin K Content of Human Liver

pathways Evidence for metabolism of the naphthoquinone ring is lacking, and thephosphate, sulfate, and glucuronide of administered menadiol have been identified[126,127] in urine and bile Studies with hepatectomized rats [128] have indicated thatextrahepatic metabolism is also significant Early studies of phylloquinone metabolism[104] demonstrated that the major route of excretion was in the feces and that very littleunmetabolized phylloquinone was present The side chains of phylloquinone and MK-4are shortened by the rat to seven carbon atoms, yielding a terminal carboxylic acid groupthat cyclized to form a g-lactone [129] This lactone is excreted in the urine, presumably as

a glucuronic acid conjugate Studies [75] of the metabolism of radioactive phylloquinone inhumans indicated that about 20% of an injected dose of either 1 or 45 mg of vitamin K wasexcreted in the urine in three days, and that 40%–50% was excreted in the feces via the bile.Two different aglycones of phylloquinone were tentatively identified as the 5- and 7-carbonside-chain carboxylic acid derivatives (Figure 3.6) These studies concluded that the g-lactonepreviously identified was an artifact formed by the acidic extraction conditions used

in previous studies

The most abundant metabolite of phylloquinone is its 2,3-epoxide (Figure 3.6) formed as

a product of the action of the vitamin K-dependent g-glutamyl carboxylation This bolite was discovered by Matschiner et al [130] who was investigating an observation [107]that warfarin treatment caused a buildup of radioactive vitamin K in the liver This increasewas shown to be due to the presence of a significant amount of a metabolite more polar thanphylloquinone that was isolated and characterized as phylloquinone 2,3-epoxide Furtherstudies of this compound [131] revealed that about 10% of the vitamin K in the liver of anormal rat is present as the epoxide and that this can become the predominant form of thevitamin following treatment with coumarin anticoagulants Warfarin administration alsogreatly increases urinary excretion and decreases fecal excretion of phylloquinone [132].The distribution of the various urinary metabolites of phylloquinone is also substantiallyaltered by warfarin administration The 7-carbon and 5-carbon side-chain major urinaryglucuronides (Figure 3.6) are decreased [133], and other uncharacterized metabolites, pre-sumably arising from the epoxide, are increased The major degradation products of vitamin

meta-K metabolism appear to have been identified and they are apparently formed from eitherphylloquinone or menaquinones, but there may be a number of urinary and biliary productsnot yet characterized Methodology useful for the routine analysis of the two major urinary

O

O Phylloquinone-2,3-epoxide

COOH COOH

1,4-naphthoquinone

aglycone s of vitamin K has been de veloped [134] , and it has be en suggest ed that qua ntitatio n

of these meta bolites might be a useful nonin vasive marker of vita min K status

VITAMIN K-DEPENDENT PROTEINS

P LASMA-CLOTTING FACTORS

Soon afte r Dam’ s discove ry of a he morrha gic con dition in chicks that could be cured bycerta in plant extra cts, it was demonst rated that the plasma of these c hicks co ntained adecreas ed con centration of prothrom bin This protein (also called c lotting factor II) wasthe fir st plasma protein -clotting facto r to be discove red It is the most ab undan t of theseprotei ns and was also the first protei n demonst rated to contai n g-carbox yglut amic acid (Gla)resid ues Plasm a-cl otting facto r VII, fact or IX, and fact or X were all init ially identifi edbecause their activit y was decreas ed in the plasm a of a patient with a hereditary bleedi ngdisorde r [135] and were subsequen tly shown to depend on vita min K for their synthes is Untilthe mid- 1970s these four vita min K-depe nden t clotting fact ors wer e the only pro teins known

to require this vita min for their synthes is

The pro cess of blood coagu lation is essent ial for hemo stasis and , along with plateletactivati on, invo lves a comp lex series of events (F igure 3.7) which lead to the gen eration ofthrom bin by pr oteolytic activatio n of proteas e zymogens [136,137] The vitamin K-dep endentclotting factors are involv ed in these activati on an d prop agation events through membr ane-associ ated complex es with each other and with access ory protei ns These proteins arecharact erized by an amino term inal domain which co ntains a number of glutamic acidresid ues which have been posttransl ationa lly con verted to g-carbox yglut amyl resi dues (see

procoagulants is very homologous, and the 10–13 Gla residues in each are in essentially thesame position as in prothrombin

Following the discovery of Gla residues in vitamin K-dependent proteins, three more Glacontaining plasma proteins with similar homology were discovered Protein C [138,139] andprotein S [140] are involved in a thrombin-initiated inactivation of factor Va, a clotting factorwhich is not vitamin K-dependent, and therefore plays an anticoagulant rather than procoa-gulant role in normal hemostasis [141] In addition to the approximately 40 residue Gladomain, the vitamin K-dependent proteins have other common features The Gla domain ofprothrombin is followed by two kringle domains, which are also found in plasminogen, and aserine protease domain Factor VII, factor IX, factor X, and protein C contain two epidermalgrowth factor domains and a serine protease domain, whereas protein S contains fourepidermal growth factor domains, but is not a serine protease The function of protein Z[142], the seventh Gla-containing plasma protein which is also not a protease zymogen, wasnot known for some time, but has now been shown [143] to have an anticoagulant functionunder some conditions As these proteins play a critical role in hemostasis, they have beenextensively studied, the cDNA and genomic organization of each of them is well-documented[144], and a large number of genetic variants of these proteins have been identified as riskfactors in coagulation disorders [145]

CALCIFIEDTISSUEPROTEINS

The first vitamin K-dependent protein discovered that was not located in plasma was isolatedfrom bone [146,147] This 49 residue protein contained 3 Gla residues, was called osteocalcin(OC) or bone Gla protein (BGP), and had little structural homology to the vitaminK-dependent plasma proteins Although it is the second most abundant protein in bone,

its function has been very difficult to define Production of the biologically active Gla form ofosteocalcin can be blocked when rats are fed a diet containing the anticoagulant warfarin andalso given large amounts of vitamin K to maintain plasma vitamin K-dependent protein

Protein S Prothrombin

Factor VII

Factor IX

Factor X Factor X

Thrombin

TM Protein C

Protein Ca(Inactive)

(Inactive) V

IIaXI

XIa

IXa

IIaXIIa

VIIaTissue factor

Xa

FIGURE 3.7 Vitamin K-dependent clotting factors The vitamin K-dependent procoagulants (grayovals) are zymogens of the serine proteases; prothrombin, factor VII, factor IX, and factor X Coagu-lation is initiated when they are converted to their active (subscript a) forms This process can beinitiated by an extrinsic pathway when vascular injury exposes tissue factor to blood The product of theactivation of one factor can activate a second zymogen, and this cascade effect results in the rapidactivation of prothrombin to thrombin and the subsequent conversion of soluble fibrinogen to theinsoluble fibrin clot A number of steps in this series of activations involve an active protease, a secondvitamin K-dependent protein substrate, and an additional plasma protein cofactor (circles) to form a

occur through an intrinsic pathway involving thrombin activation of factor XI and subsequently factor

IX Two other vitamin K-dependent proteins participate in hemostatic control as anticoagulants, not

called thrombomodulin (TM) Protein C is then able to function in a complex with protein S to

production Using this protocol, no defects in bone were seen when bone osteocalcin wasdecreased to about 2% of normal after 2 months, and fusion of the proximal tibia growthplate was observed after 8 months [148,149] These observations indicate that osteocalcin isinvolved in some manner in the control of tissue mineralization or skeletal turnover However,osteocalcin gene knockout mice have been shown [150] to produce more dense bone rather than

a defect in bone formation Some of the osteocalcin produced in bone does appear in plasma atconcentrations that are high in young children and approach adult levels at puberty

A second low-molecular-weight (79 residue) protein with five Gla residues was also firstisolated from bone [151] and called matrix Gla protein (MGP) This protein is structurallyrelated to osteocalcin but is also present in other tissues and has been shown to be synthesized

in cartilage and many other soft tissues [152] MGP has been difficult to study because of itshydrophobic nature, relative insolubility, and tendency to aggregate The details of thisphysiological role are unclear, but it has been shown that MGP knockout mice die fromspontaneous calcification of arteries and cartilage [153], and arterial calcification has beendemonstrated in a warfarin-treated rat model [154] Although evidence to support a specificfunction in calcified tissues is lacking, the plasma protein, protein S, which is produced in theliver, is also synthesized by bone cells

OTHERVITAMINK-DEPENDENTPROTEINS

A relatively small number of other mammalian proteins have now been shown to contain Glaresidues and are therefore dependent on vitamin K for their synthesis The most extensivelystudied is Gas 6, a ligand for the tyrosine kinase Ax1 [155], which appears to be a growth factor formesangial and epithelial cells The physiological function of the protein is not clearly defined, butthere are indications of its possible role in nervous system function [156], vascular cell function[157], and platelet activation [158] Two proline-rich Gla proteins (PRGP-1, PRGP-2) werediscovered [159] as integral membrane proteins with an extracellular amino terminal domainthat is rich in Gla residues Subsequently, two other members of this transmembrane Gla proteinfamily (TMG-3 and TMG-4) have been cloned [160] The specifics of the role of these cell-surfacereceptors are not yet known Vitamin K deficiency has been reported to alter brain sphingolipidsynthesis [121,161], but the mechanism of the response or the role that it plays in neural functionhas not been clearly identified [162] There have also been reports of other peptide-bound Glaresidues in mammalian tissues, but no specific proteins have been identified

Vitamin K-dependent proteins are not confined to vertebrates, and a large number of thetoxic venom peptides secreted by marine Conus snails are rich in Gla residues [163] VitaminK-dependent proteins have also been found in snake venom [164,165], and the carboxylasehas been cloned from a number of vertebrates, the Conus snail, a tunicate, zebrafish, anddrosophila [166–168], and has been identified in the genome of bacteria and archaea [169].The strong sequence homology of the enzyme from these phyllogenetic systems suggests thatthis posttranslational modification of glutamic acid is of ancient evolutionary origin, and thatnumerous vitamin K-dependent proteins are yet to be discovered within the wide range oforganisms capable of synthesizing this modified amino acid

BIOCHEMICAL ROLE OF VITAMIN K

DISCOVERY OFg-CARBOXYGLUTAMICACID

A period of approximately 40 years elapsed between the discovery of vitamin K and thedetermination of its metabolic role Beginning in the early 1960s, studies of prothrombinproduction in humans and experimental animals eventually led to an understanding of themetabolic role of vitamin K Early theories that vitamin K controlled the production of

specific proteins at a transcriptional level could not be proven, and alternate hypotheses wereconsidered Involvement of an intracellular precursor in the biosynthesis of prothrombin wasfirst clearly stated by Hemker et al [170] who postulated that an abnormal clotting time inanticoagulant-treated patients was due to a circulating inactive form of plasma prothrombin.

It was subsequently demonstrated [171] that the plasma of patients treated with coumarinanticoagulants contained a protein that was antigenically similar to prothrombin but lackedbiological activity A circulating inactive form of prothrombin was first demonstrated inbovine plasma by Stenflo [172], but it appears [173] to be present in low concentrations oraltogether absent in many other species Other observations [174] were consistent with thepresence of a hepatic precursor protein pool in the hypoprothrombinemic rat that was rapidlysynthesized and that could be converted to prothrombin in a step that did not require proteinsynthesis

Studies of the inactive abnormal prothrombin [175] demonstrated that it containednormal thrombin, had the same molecular weight and amino acid composition, but didnot adsorb to insoluble barium salts as did normal prothrombin This difference, and thealtered calcium-dependent electrophoretic and immunochemical properties, suggested adifference in calcium-binding properties of these two proteins that was subsequentlydemonstrated by direct calcium-binding measurements The critical difference in the two proteinswas the inability of the abnormal protein to bind to calcium ions, which are needed for thephospholipid-stimulated activation of prothrombin by factor Xa[176] Acidic, Ca2þ-binding,peptides could be isolated from a tryptic digest of the amino terminal domain of normalbovine prothrombin but could not be obtained when similar isolation procedures wereapplied to preparations of abnormal prothrombin Stenflo et al [177] succeeded in isolat-ing an acidic tetrapeptide (residues 6–9 of prothrombin) and demonstrated that the glutamicacid residues of this peptide were modified so that they were present as g-carboxyglutamicacid (3-amino-1,1,3-propanetricarboxylic acid) residues (Figure 3.8) Nelsestuen et al [178]independently characterized g-carboxyglutamic acid (Gla) from a dipeptide (residues 33 and

34 of prothrombin), and these characterizations of the modified glutamic acid residues inprothrombin were confirmed by Magnusson et al [179], who demonstrated that all of the 10Glu residues in the first 33 residues of prothrombin are modified in this fashion

O R

CH3O

CH3R OH

CH2

H NH O

generates vitamin K-2,3-epoxide

V ITAMIN K-DEPENDENT CARBOXYLASE

The discove ry of Gla residu es in prothrom bin led to the de monstrati on [180] that crud e ratliver microsomal prep arations c ontaine d an enzymat ic activit y (the vitamin K-depe nde ntcarboxyl ase) that pro moted a vitamin K-depe ndent incorpora tion of H 14CO3 

into end ous precurs ors of vitamin K-depe nden t pro teins present in these prep arations The fixe d

ogen-14

CO2 was present in Gla residu es, and subsequen t studi es [181] establ ished that de solubi lized microsomal prep arations retained this carbo xylase activity The same microsomalpreparations and incubation conditions that fixed CO2would convert vitamin K to its 2,3-epoxide [182] (Figure 3.8) In the solubi lized preparat ion, smal l pep tides con taining adjacentGlu–Glu sequences such as Phe–Leu–Glu–Glu–Val were substrates for the enzyme [183], andthey were used to study the properties of this unique carboxylase The rough microsomalfraction of liver was found to be highly enriched in carboxylase activity, and lower butsignificant activity was found in smooth microsomes These initial studies were consistentwith the hypothesis that the carboxylation event occurs on the lumenal side of the roughendoplasmic reticulum [184]

tergent-A general understanding of the properties of the vitamin K-dependent carboxylase wasgained from studies using this crude detergent-solubilized enzyme preparation, and these datahave been adequately reviewed [185–189] The vitamin K-dependent carboxylation reactiondoes not require ATP, and the energy to drive this carboxylation reaction is derived from theoxidation of the reduced, hydronaphthoquinone, form of vitamin K (vitamin KH2) by O2toform vitamin K-2,3-epoxide (Figure 3.8) The lack of a requirement for biotin and studies ofthe CO2or HCO3requirement indicate that carbon dioxide rather than HCO3 is the activespecies in the carboxylation reaction Studies of substrate specificity at the vitamin K-bindingsite of the enzyme have shown that active substrates are 2-methyl-1,4-naphthoquinonessubstituted at the 3-position with a rather hydrophobic group Although some differences

in carboxylase activity can be measured, phylloquinone, MK-4, and the predominant tinal forms of the vitamin, MK-6 and MK-8, are all effective substrates The 2-ethyl and des-methyl analogs of the vitamin have little activity, and methyl substitution of the benzenoidring has little effect, or decreases substrate binding The vitamin K antagonist, 2-chloro-3-phytyl-1,4-naphthoquinone, is an antagonist of the enzyme, and the reduced form has beenshown to be a competitive inhibitor Synthesis and assay of a large number of rather high Km

intes-low-molecular-weight peptide substrates of the enzyme have failed to reveal any uniquesequences surrounding the Glu residue that are needed as a signal for carboxylation.Normal functioning of the vitamin K-dependent carboxylase poses an interestingquestion in terms of enzyme–substrate recognition This microsomal enzyme recognizes asmall fraction of the total hepatic secretory protein pool and then carboxylates 9–12 Glu sites

in the first 45 residues of these proteins Cloning of the vitamin K-dependent proteins hasrevealed that the primary gene products contain a very homologous propeptide between theamino terminus of the mature protein and the signal peptide [144] This region appears to

be a docking or recognition site for the enzyme [190] and has also been shown [191] to be amodulator of the activity of the enzyme by decreasing the apparent Km of the Glu sitesubstrate Although the carboxylase-binding affinities of the propeptides for differentproteins differ significantly [192], propeptides are required for efficient carboxylation, andglutamate-containing peptides with no homology to vitamin K-dependent proteins are sub-strates for the carboxylase if a propeptide is attached [193,194]

The role of vitamin K in the overall reaction catalyzed by the enzyme is to abstract thehydrogen on the g-carbon of the glutamyl residue to allow attack of CO2at this positioncoupled to conversion of the vitamin to its 2,3-epoxide A number of studies [195–197]that used substrates tritiated at the g-carbon of each Glu residue have defined the actionand the stoichiometry involved The enzyme catalyzes a vitamin KH2and O2-dependent, but

CO2 -independent, release of tritium from the substrate and at saturating concentrations

of CO2 there is an apparent equivalent stoichiometry between vitamin K-2,3-epoxide mation and Gla formation The mechanism by which epoxide formation is coupled tog-hydrogen abstraction is key to a complete understanding of the role of vitamin K Theenzyme has been shown to catalyze a vitamin KH2and O2-dependent exchange of3H from

in Figure 3.9 is consistent with all of the available data, the mechanism remains a hypothesis

at this time

A general understanding of the mechanism of action of the vitamin K-dependent lase was gained through studies of very impure preparations Progress in purifying the enzymewas slow, but the enzyme was eventually purified to near homogeneity and cloned [201]

Gla Vitamin K epoxide

Vitamin KH2

O−

CH3R OH

H NH

NH

O H NH

C

C H

HR

O

O

CH3O R O O

the reduced (hydronaphthoquinone) form of vitamin K, generates intermediates eventually leading to

an oxygenated metabolite that is sufficiently basic to abstract the g-hydrogen of the glutamyl residue

carbanion leads to the formation of a g-carboxyglutamyl residue (Gla) The bracketed peroxy, tane, and alkoxide intermediates have not been identified in the enzyme-catalyzed reaction but arepostulated based on model organic reactions The available data are consistent with their presence

dioxe-The carboxyl ase is a uniq ue 758 amino acid resid ue pr otein with a sequen ce suggest ive of aninteg ral membran e protei n wi th a num ber of membr an e-spannin g domains in the N-term inus,and a C-terminal domain locat ed in the lumen of the endo plasmic reticu lum It has beendemonstrated that the multiple Glu sites on the substrate for this enzyme are carboxylatedprocessively as they are bound to the enzyme via their propeptide [202,203], while the Gladomain undergoes intramolecular movement to reposition each Glu for catalysis, and thatrelea se of the ca rboxyla ted substr ate is the rate- limiting step in the reaction [204]

The membr ane top ology of the ca rboxyla se has not yet been fir mly establis hed Theamino acid sequence of the carboxyl ase indica tes seven hy dropho bic regions in the protei n[205], and it has been proposed that the enzyme has five transmembr an e regions spanning theendo plasmic reticu lum [206] Alternat ive models of the topo logy [207] are also possible, an dadditio nal data are needed To generat e the strong vita min K base needed to gen erate acarbani on on the g-carbon of the Glu resi due woul d req uire depro tonation of the redu cedform of the vitamin so that it could react with O2 For some time, the avail able da ta suggest edthat specific active-s ite Cys resi dues pe rformed this function , but more recent data indica tethat an activated His resi due carri es out this functi on during catal ysis [208] Further de tails ofprogres s in an underst anding of the de tails of the actio n of this unique fat-sol uble vitamin-depen dent react ion are available in recent revie ws [207,209, 210]

V ITAMIN K-E POXIDE R EDUCTASE

The de gradat ion of vita min K-depe nde nt protein s gen erates Gla resi dues whic h are notmeta bolized but are excreted in the urine [211] Human adult Gla excret ion is in the range

of 50 m mol=da y, ind icating that a simila r amount is formed each da y The average dieta ryintake of vita min K is only abo ut 0.2 mmol =day, an d a mo le of vitamin is oxidiz ed for eachmole of Gla form ed It is clear that the vita min K 2,3- epoxide generat ed by the carboxyl asemust be actively recycled , and the he patic ratio of the epo xide relat ive to that of the vita min isincrea sed in anima ls admini stered the 4-hydro xycoumar in antic oagulant warfarin [212] Thissuggest ed that war farin inhibi tion of v itamin K a ction was indirect through an inhibition ofthe enzyme called the vitamin K-epoxide reductase [213] Blocking of the reductase preventsthe reduction of the epoxide to the quinone form of the vitamin and eventually to thecarboxylase substrate, vitamin KH2 Widespread use of warfarin as an anticoagulant roden-ticide led to the appearance of strains of warfarin-resistant rats [214], and the study of theactivity of the epoxide redutcase in livers of these animals was key to an understanding[215, 216] of the details of what is now referred to as the ‘‘vitamin K cycle’’ (Figur e 3.10) Thr eeforms of vitamin K (the quinone (K), the hydronaphthoquinone (KH2), and the 2,3-epoxide(KO)) can feed into this liver vitamin K cycle In normal liver, the ratio of vitaminK-2,3-epoxide to the less oxidized forms of the vitamin is about 1:10 but can increase to amajority of epoxide in an anticoagulated animal The quinone and hydronaphthoquinoneforms of the vitamin can also be interconverted by a number of NAD(P)H-linked reductasesincluding one that appears to be a microsomal-bound form of the extensively studied liverDT-diaphorase activity The epoxide reductase uses a sulfhydryl compound as a reductant

in vitro, but the physiological reductant has not been identified Efforts to purify and terize the protein or proteins responsible for this enzyme activity from liver have not beensuccessful, and a clear understanding of the enzymatic mechanism of the reduction is notavailable Recent identification of the human and rat genes for the vitamin K epoxide reductase[217,218] will aid in efforts to more completely understand this enzyme It is not yet established

charac-if the small, 18 kDa protein expressed by this gene is completely responsible for the observedactivity, or if other as-of-yet-unidentified proteins of the endoplasmic reticulum are needed

to form an active complex [219] The presence of the identified gene in Drosophila and otherinsects [220] suggests that this activity may be as widespread as the carboxylase The importance