Biochem. J. (1968) 109, 457Printed in Great Britain

Enzyme Studies on the Esterification of Vitamin D in Rat Tissues

By D. R. FRASER AND E. KODICEKDunn Nutritional Laboratory, University of Cambridge and Medical Re8earch Council, Cambridge

(Received 23 April 1968)

1. The mechanism of vitamin D esterification in the rat was studied with liver,small-intestinal mucosa, pancreatic juice and blood plasma as enzyme sources and[1-3H]cholecalciferol, [U-14C]ergocalciferol and [4-14C]cholesterol as substrates.2. No esterification of vitamin D could be detected with liver preparations nor withhomogenates or acetone-dried powder extracts of intestinal mucosa. 3. Pancreaticjuice esterified [1-3H]cholecalciferol with oleic acid, and specificity studies indi-cated that a cholesterol-esterifying enzyme was using vitamin D as substrate.4. Plasma cholesterol-esterifying enzyme also esterified vitamin D. 5. The speci-ficity of the esterification reaction is discussed in relation to (a) the molecularstructure of the substrates and (b) their availability, in a micellar solution, to theenzyme. 6. It is concluded that cholesterol-esterifying enzymes esterify vitamin Din vivo during absorption from the small intestine and while it is transported inblood.

After the oral administration to rats of vitamin Das [1-3H]cholecalciferol, some was found esterifiedwith fatty acids in liver, kidney, intestinal tissue,plasma and intestinal lymph (Fraser & Kodicek,1968a,b). A number of studies have indicated thatesterification takes place during transport of vit-amin D from the intestinal lumen to the intestinallymph (Schachter, Finkelstein & Kowarski, 1964;Bell, 1966; Blomstrand & Forsgren, 1967; Fraser &Kodicek, 1968b). No other site of synthesis has yetbeen described and it was therefore decided toinvestigate the general enzymology of vitamin Desterification; this could be mediated by either an

enzyme or enzymes specific for vitaminD or one thatesterifies some other monohydroxylic compoundsuch as cholesterol or retinol.At least three different enzyme mechanisms ap-

pear to be present in the rat for the esterification ofcholesterol. The first is a direct union of free fattyacid and cholesterol with no external energy source.

Such an enzyme occurs in intestinal tissue andpancreatic juice (Swell, Byron & Treadwell, 1950).A blood-plasma enzyme transfers a fatty acid fromthe ,8-position of lecithin to esterify it with choles-terol, again without the introduction of externalenergy (Glomset, 1962). A third mechanism requiresactivated fatty acids that are esterified with choles-terol under the influence of a fatty acyl-CoA-cholesterol acyltransferase; this mechanism is foundin liver (Goodman, Deykin & Shiratori, 1964) andadrenal gland (Longcope & Williams, 1963).

Retinol is esterified by intestinal and pancreaticenzymes in a similar way to cholesterol; however,

separate and specific enzymes are claimed for thesetwo substrates (Murthy & Ganguly, 1962). Anenzyme in liver esterifies retinol, but, in contrastwith cholesterol esterification in that organ, requiresno activation of the fatty acids (Futterman &Andrews, 1964).

Liver, small-intestinal mucosa, pancreatic juiceand plasma were chosen as material for assay of'vitamin D-esterifying activity'. The conditionsin vitro were based on published methods forcholesterol and retinol esterification so that any

action on vitamin D by these enzymes would belikely to be observed. No vitamin D esters could befound by using preparations of liver or small in-testine as enzyme sources in vitro, but pancreaticjuice did synthesize cholecalciferol esters, and thespecificity of the reaction was studied. Plasma alsoesterified some cholecalciferol, and this reaction as

well as that in pancreatic juice is considered to becatalysed by cholesterol-esterifying enzymes.

EXPERIMENTAL

Materials. Substrate vitamin D was either [1-3H]chole-calciferol or [U-14C]ergocalciferol prepared from their 3,5-dinitrobenzoate esters by the method of Wilson, Lawson &Kodicek (1967). Unlabelled crystalline cholecalciferol(Koch-Light Laboratories Ltd., Colnbrook, Bucks.) andergocalciferol (Glaxo Laboratories Ltd., Greenford, Middx.)were used to dilute the labelled forms to appropriate specificradioactivities. [4-14C]Cholesterol (24-8mc/m-mole) (TheRadiochemical Centre, Amersham, Bucks.) was diluted withunlabelled cholesterol thathad been recrystallized once from

457

D. R. FRASER AND E. KODICEKacetone and twice from ethanol. Radioactivity was meas-ured by liquid-scintillation spectrometry with a countingefficiency for 3H of 20% and for 14C of58%.

Oleic acid, ATP and CoA were purchased from SigmaChemical Co. (St Louis, Mo., U.S.A.), and sodium tauro-cholate (A grade) was from Calbiochem (Los Angeles, Calif.,U.S.A.), sodium deoxycholate from Koch-Light Labora-tories Ltd., p-chloromercuribenzoic acid from British DrugHouses Ltd. (Poole, Dorset) and bovine plasma albuminfrom Armour Pharmaceutical Co. Ltd. (Eastbourne,Sussex). Retinol was prepared immediately before use fromretinyl acetate (Roche Products Ltd., London, W. 1) bysaponification in the presence of 0-25% pyrogallol. Freeretinol was extracted with diethyl ether, its purity checkedby thin-layer chromatography and its concentration meas-ured by ultraviolet spectrophotometry.

Precholecalciferol was prepared from cholecalciferol bycreating equilibrium conditions for the previtamin D=evitamin D reaction at which 20% of previtamin is present(Velluz, Amiard & Petit, 1949). Cholecalciferol, dissolved inbenzene, was heated for 16hr. at 600. The mixture wasapplied to a preparative thin-layer chromatography plateand developed in chloroform, on which cholecalciferol(RpO 37) separated from precholecalciferol (RpO-48). Thelatter was eluted with diethyl ether and collected at 00. Theeluted material was checked by repeating the chromato-graphy, when most ran as previtamin D. A small region ofcholecalciferol could be observed, but this may have formedduring the chromatography. Labelled precholecalciferolwas prepared in the same way from [1-3H]cholecalciferol andcontained 91% of the radioactivity as precholecalciferol.The preparations were stored at -20° before use.

Rachitic vitamin D-deficient hooded rats were raised aspreviously described (Fraser & Kodicek, 1968a), and theseor vitamin D-supplemented rats on the rachitogenic dietsupplied all tissues for enzymic assay.

All other materials were as described by Fraser & Kodicek(1968a,b).

Presentation of substrate. Vitamin D is unstable in anaqueous environment (Chen, Terepka, Lane & Marsh, 1965)unless protected by high concentrations of organic solventsor adsorbed on protein molecules. For short-term experi-ments in vitro it was decided either to emulsify vitamin Ddirectly in water or to present it bound to plasma albumin.The procedure of Hernandez & Chaikoff (1957) for theemulsification of cholesterol was used to emulsify vitaminD, oleic acid and taurocholate in buffer without any organicsolvent in the final solution. An alternative method was theaddition of vitamin D and oleic acid in ethanol to bufferedtaurocholate to give an ethanol concentration of5-10%; thesuspension was vigorously agitated for 30 sec. on a vibratingrubber disk to produce a clear or opalescent solution.To prepare a stable aqueous solution of vitamin D a

method was devised to bind it to plasma albumin with theminimum of destruction. Aq. 4% (w/v) bovine plasmaalbumin, pH7-3 (5ml.), was placed in a 150ml. round-bottomed ground-glass-necked flask provided with a mag-netic stirrer and set up in a CaCl2-ice freezing mixture. Thetemperature was maintained at -4° to- 5, and 0-2 ml. ofethanol containing 2501&g. of [1-3H]cholecalciferol was runin over a 10min. period from a micrometer syringe pipette,the tip of which dipped beneath the surface of the albuminsolution. The water and ethanol were then removed byfreeze-drying, and the albumin was redissolved in lOml. of

water at 00 to give a clear solution containing 20mg. ofbovine plasma albumin and 25,tg. of cholecalciferol/ml.This was stored at -15°, and the stability was confirmed byextracting the vitamin with 25 vol. of acetone-ethanol(1:1, v/v) and chromatographing the extract on thin layerswith chloroform. Only one radioactive spot was found andthis ran adjacent to marker vitamin D (RFO-35). Aqueoussolutions of [4-14C]cholesterol were also prepared by theabove methods.

Studie8 of e8teriftcation in vitro

Liver. In general 4g. of liver was homogenized with 12 ml.of 0-44m-sucrose at 40 in a Potter-Elvehjem homogenizer.The disrupted tissue was fractionated by differential centri-fugation at 40 into debris, mitochondria, microsome andsoluble-supernatant fractions, and in some experimentsnuclei were purified from the debris fraction (Hogeboom,1955). Pellets were resuspended in 5ml. of 0 44M-sucrose.The interval between killing and the beginning of incubationwas 2-3 hr.

Incubation mixtures contained: 1-4ml. of 0-2M-sodiumphosphate buffer, pH7-3; 01ml. of 015M-NaF; 0-2ml. of75mM-ATP, pH 7-3; 0-2ml. of0-6mM-CoA; 2-5 ,g. of[1-3H]-cholecalciferol or [4-14C]cholesterol on albumin (0-Iml.);2ml. of liver cell fraction. The total volume was 4ml.Flasks were shaken at 370 for 2 hr., after which the contentswere extracted with chloroform-methanol (2:1, v/v).Extracts were chromatographed by a method of Deykin &Goodman (1962), and radioactivity was measured in'esterified' and 'non-esterified' substrate fractions.

Inte8tinal Muco0a. Methods of assaying esterifyingactivity were modified from procedures published previously(Mahadevan, Murthy, Krishnamurthy & Ganguly, 1961;Murthy, Mahadevan, Seshadri Sastry & Ganguly, 1961b;Murthy & Ganguly, 1962; Lossow, Migliorini, Brot &Chaikoff, 1964). Small intestine was freed from pancreatictissue and mesenteric adipose tissue and placed in 500ml. ofaq. 0-9% NaCl soln. at 4°. The lumen was washed throughwith 20-30ml. of NaCl soln., the intestine was slit open andblotted on a porcelain slab, and the mucosa was scraped offwith the edge of a microscope slide. Some mucoid materialwas removed by washing the scrapings in 150ml. of NaClsoln., and the cells were recovered by centrifuging at 10OOgfor 5min. The pellet (1g.) was homogenized in a Potter-Elvehjem homogenizer with 10ml. of 0-15M-KC1.

Acetone-dried powders were also prepared from themucosa of groups of 16 rats. In one procedure the washedmucosal scraping was homogenized in 120ml. of water in aWaring Blendor for 30sec. at 00. The homogenate wasstirred rapidly into 11. of acetone at - 15°, filtered andwashed with acetone and peroxide-free diethyl ether in aBuchner funnel, and finally dried in a desiccator at lowpressure. Other batches of mucosal powder were preparedby the method of Mahadevan et al. (1961) and both types ofpowder were extracted with water at 00 as described by thoseauthors.

Incubation mixtures contained: 1-3 ml. of0-14 M-barbital-acetate buffer, pH6-4; 0-lmI. of aq. 80mM-sodium tauro-cholate; 0-Iml. of 40,UM-oleic acid (in ethanol or as albumin-stabilized aq. potassium oleate); 0-Iml. of 20iM-[1-3H]-cholecalciferol (approx. 280000counts/min.); 0-4ml. ofenzyme preparation. The total volume was 2ml. After a

1968458

ENZYMIC SYNTHESIS OF VITAMIN D ESTERS2 hr. incubation at 370 the contents of each flask were addedto 50ml. of acetone-ethanol (1: 1, v/v) and shaken for 4hr.After being filtered and dried under reduced pressure, eachextract was fractionated into 'free' and 'esterified' vitaminD by standard thin-layer chromatography with chloroformas ascending solvent (Fraser & Kodicek, 1968a).When a soluble protein extract was assayed for activity a

simpler extraction procedure was adopted in which 20,p. ofincubation mixture was removed in a disposable pipette andadded to 20,ul. of acetone-ethanol on a spotting tile. Thisentire mixture was applied with 10,ug. each of standardcholecalciferol and cholecalciferol palmitate to micro-thin-layer plates (Vahouny, Borja & Weersing, 1963), whichwere developed with hexane-diethyl ether-acetic acid(20:4:1, by vol.). The micro-plates were stained withiodine vapour to locate free and esterified fractions and de-stained by heating at 500, and the silica gel was scrapedquantitatively into two vials for measurement of the radio-activity in each area by liquid-scintillation spectrometry.

Pancreatic juice. Bile and pancreatic juice were collectedseparately in tubes at 00 from cannulae implanted in thehepatopancreatic duct. The bile-collecting cannula wasinserted where the duct ran free in the lesser omentumbetween the porta hepatis and the pancreas, and thesecond cannula collected pancreatic juice from the pointwhere the duct entered the duodenal wall. The rat wasrestrained after surgery in a cage described by Bollman(1948) and was supplied with a commercial cube diet andwater containing 5% glucose. Pancreatic juice, a clearcolourless liquid, was stored in lml. volumes at - 150. Themean daily output over 16 days was 9ml.The assay of esterifying activity was similar to that des-

cribed by Shah, Lossow & Chaikoff (1965), except that Iml.volumes were used in 12 cm. x 1-5 cm. test tubes closed withaluminium foil. Incubation was performed with continuousshaking of 135 oscillations/min. in a water bath at 37°. Theincubation mixture contained: 0-86ml. of 0-1m-sodiumphosphate buffer, pH6-2; 0-05ml. of 80mM-sodium tauro-cholate; 0-4,.tmole of cholecalciferol or cholesterol plus0-8pmole of oleic acid in 0-02 ml. of ethanol; 0-7mnjumole of[1-3H]cholecalciferol (45000counts/min.) or 1-5m,umoles of[4-14C]cholesterol (43 000 counts/min.) in 0-02 ml. of ethanol.Dispersion of the substrate was aided by agitation on avibrating rubber disk for 30sec. Pancreatic juice (0-05ml.)was added 15sec. before the tube was placed in the waterbath. At the end of the incubation, lml. of acetone-ethanolwas added to each tube and approx. 0-Iml. of this mixturewas directly chromatographed on micro-thin-layer plates,and radioactivity in the free sterol and ester fractions wasestimated.Plasma. Rat blood was collected into heparinized syringes

by cardiac puncture and plasma was obtained immediatelyby centrifuging at 4°. Incubation flasks contained 2ml. ofplasma, to which was added [1-3H]cholecalciferol or[4-14C]cholesterol as albumin-bound substrate. A blankflask contained plasma heated at 600 for 30min. Incubationwas carried out with shaking at 370 for 24hr. bya method forthe determination of cholesterol esterification by plasma(Glomset, Parker, Tjaden & Williams, 1962). At the end ofthe incubation period the plasma was extracted with 5ml.of acetone-ethanol, and the extract was filtered and driedunder reduced pressure. By means of floridin-earth chro-matography (Fraser & Kodicek, 1968a) ester fractions wereobtained from the extract.

RESULTS

Esterification studies with tissues

Esterification studies with liver. The overall re-sults indicated that vitamin D was not esterified byliver under the conditions provided. A small amountof radioactivity in ester fractions from incubationswas sometimes higher than from correspondingfractions from boiled-enzyme controls. However,this radioactivity was so low (0-4% of total) that itsidentity as ester could not be established. The con-trol experiments with [4-140]cholesterol showedclearly that esterification of this substrate tookplace in a 2hr. incubation, when 50-70% of theradioactivity was found in the ester fraction.To exclude the possibility that unfavourable con-

ditions were the cause of the failure to synthesizevitamin D esters a range of variables was intro-duced: incubation time, the presence or absence ofMg2+, concentration of protein, and concentrationand method of presentation of substrates. None ofthese succeeded in revealing esterifying activity.Kidney homogenates were also used in one experi-ment and again no evidence of cholecalciferol esteri-fication was found.

It is concluded that there is no positive proof thatvitamin D is esterified by liver or kidney, and thatthis supports the suggestion that the small amountof vitamin D ester found in these sites in vivo isprobably synthesized elsewhere (Fraser & Kodicek,1968b).

Esterification studies with small-intestinal mucosa.In preliminary experiments with whole-mucosalhomogenates, no esterification of vitamin D couldbe measured, and with [4-14C]cholesterol under thesame conditions only 2-3% of the recovered radio-activity was esterified. Acetone-dried powders ofmucosa, prepared by both methods described above,were assayed in a similar manner to that describedby Murthy & Ganguly (1962); however, in no casewas more than 2-7% of cholesterol ester formed. Itwas concluded that the poor esterification of choles-terol by these preparations was due either to thepresence of an active hydrolytic enzyme or to thevirtual absence of the esterifying enzyme. Since thepH optimum for cholesterol-ester hydrolysis isreported to be pH8-6 (Murthy & Ganguly, 1962),the pH of 6-4 used in this study should not befavourable for this reverse reaction. Inactivation ofthe enzyme was again unlikely as a 10% homogenateof rat pancreas in water in the same assay systemesterified as much as 30-40% of added cholesterol.Hence the mucosal cholesterol-esterifying enzymewas considered to be present in very low amounts.

Variation in the activity of intestinal cholesterol-esterifying enzyme was observed by Nieft & Deuel(1949), who reported that activity was present only

Vol. 109 459

D. R. FRASER AND E. KODICEKwhen rats had been given a diet containing 1% oflanolin or 1% ofcholesterol. Murthy, Mahadevan &Ganguly (1961a), by feeding a diet with 1% ofcholesterol to young rats for 5 weeks, showed thatmucosal acetone-dried powders from rats on thehigh-cholesterol diet had a 2-5-fold greater choles-terol-esterifying activity and a fourfold greaterretinol-esterifying activity than control rats. Sincethe rachitogenic diet of Steenbock & Black (1925)contains no cholesterol, the rats in the present ex-periments had not received dietary cholesterol fromweaning. Therefore weanling male or female ratswere raised for 5 weeks on the 1% cholesterol diet ofMurthy et al. (1961a) supplemented with vitaminsA, D, E and K; over this period the rats increasedin weight from 35g. to 200g. Acetone-dried powderof intestinal mucosa was prepared and extracted bythe method of Mahadevan et al. (1961), and assayedfor cholesterol-esterifying activity.

In several assays, these extracts only esterified3.1-3.9% of the [4-14C]cholesterol in 2hr. In thesimilar experiments by Murthy et al. (1961a) thespecific activity of the enzyme was increased from52-4 to 130-6m,umoles of cholesterol esterified/mg.of protein/hr., whereas the present enzyme extracthad a specific activity of only 0 4-1 7m,umoles ofcholesterol esterified/mg. of protein/hr.

It was concluded that the hooded piebald rats ofthis Laboratory have a very low intestinal choles-terol-esterifying activity and it was not possible totest in vitro the specificity ofits action for vitamin D.If, however, this enzyme were responsible for thevitamin D ester appearing in thoracic-duct lymphthe much smaller amounts found in the lymph ofour rats (Fraser & Kodicek, 1968b) compared withAmerican reports on albino rats (Schachter et al.1964; Bell, 1966) could be explained by a differencein this enzyme's activity between the two strains ofrats.

E8teriftcation studies with pancreatic juiceBecause the pancreas had high cholesterol-

esterifying activity and because the reactionmechanisms of the pancreatic and intestinal en-zymes were very similar (Murthy & Ganguly, 1962),we compared the specificity of cholesterol esterifi-cation catalysed by pancreatic juice with a similarreaction for vitamin D. The methods used fordefining the specificity of one enzyme towards twosubstrates were based on criteria listed by Dixon &Webb (1960), the main ones being: (1) failure toseparate the two activities by all available frac-tionation procedures; (2) maintenance of a constantratio between the two activities as the enzymeundergoes inactivation; (3) the 'mixed-substratephenomenon': equal quantities of two substratesadded together produce a lower total rate of reac-tion than the sum of the rates measured separately.

Preliminary experiments, with cholesterol assubstrate, revealed that maximum esterificationoccurred by 30min. and that the rate in the first5min. was much faster than subsequently (Fig. la).There are three possible explanations for this non-linearity of reaction: the enzyme is inactivatedduring incubation, or a secondary reverse reactioncomes into play as ester concentration rises, or themicellar state of the substrate changes.

Proteolytic inactivation of the enzyme may havedecreased its effective concentration, although theenzyme is said to be protected by taurocholate(Vahouny, Weersing & Treadwell, 1965). However,the addition of soya-bean trypsin inhibitor (50,ug./assay) did not prevent the tailing off of the re-action.The second possibility, that the ester was being

hydrolysed as well as synthesized, seemed morelikely. When incubation was continued for 2hr. theamount of ester sometimes dropped (Fig. la, curvesi and ii) and the use of more enzyme and less sub-strate decreased the overall synthetic reaction (Fig.la, curve iii). This, along with a marked decline inspecific activity as the protein concentration in-creased (Fig. lb), suggested that the hydrolyticcholesterol esterase was active. Although the pan-creatic hydrolysing enzyme has a pH optimum of8-6, its activity is only halved at pH 6-2 (Murthy &Ganguly, 1962). To overcome this effect, it wasconsidered that, if the enzyme concentration weredecreased to give a lower reaction rate, linearesterification could be observed before esterhydrolysis became significant. This proved to bethe case, for when pancreatic juice was diluted 1: 3with O-lM-phosphate buffer, pH6-2 (giving 0-27mg.of protein/assay), a linear rate of esterification wasmeasured over 5-30min. (Fig. lc).

Because cholesterol and oleic acid are almost in-soluble in water and are presented to the enzymeas a micellar dispersion, it is difficult to know theexact substrate concentration. Also the preferredsubstrate state is found to vary for different en-zymes acting on lipids. The cholesterol-esterifyingenzyme requires a micellar substrate (Vahouny,Weersing & Treadwell, 1964), in contrast withpancreatic lipase, which acts on the water-oil inter-face of triglyceride emulsions (Desnuelle & Rovery,1961). Hence, when the substrate concentration isgiven, it is a measure of the total in the assay ratherthan that which is always exposed to the enzyme.Nevertheless, when the cholesterol concentrationwas raised to 0-6,umole/ml. with a constant oleicacid concentration of 1-6,moles/ml., a linear in-crease in activity was obtained (Fig. ld). Thisagrees with the results ofMurthy & Ganguly (1962),who found that the activity increased linearly withup to 1,umole of cholesterol/ml. The rate of in-crease was much greater with dilute (0-27mg. of

460 1968

ENZYMIC SYNTHESIS OF VITAMIN D ESTERS

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Fig. 1. (a) Time-course of cholesterol-oleic acid esterification with pancreatic juice: (i) and (ii), 0-6ytmole ofcholesterol and 0-05ml. of pancreatic juice; (iii), 0-3,mole of cholesterol and 0-1 ml. of pancreatic juice. (b) Effectof concentration of pancreatic juice on esterification (0-3,mole of cholesterol/assay). (c) Time-course of esterifica-tion: 0-05 ml. of pancreatic juice diluted 1:3 with 0-1 M-phosphate buffer, pH 6-2 (0-61,mole/assay). (d) Substrate-concentration curves for cholesterol. Oleic acid was constant at 1-6,umoles/ml.: (i) 0-05ml. of whole pancreaticjuice; (ii) 0-05ml. of pancreatic juice diluted 1:3 with 0-I M-phosphate buffer, pH 6-2.

protein/assay) rather than whole (1-08mg. ofprotein/assay) pancreatic juice.

It was not possible to have cholesterol concentra-tions above 0-6,umole/ml- unless the ethanol con-

centration was also increased. Thus a balance hadto be reached between the maximum amount ofsubstrate and the minimum amount of ethanol toproduce an efficient reaction rate. As a standardprocedure, giving repeatable results, 0-4,umole of

cholesterol and 4% of ethanol in 1 ml. of incubationmiXtures were used.Blank enzyme reactions with pancreatic juice

previously heated at 600 for 30min. gave no radio-activity in the ester fraction.

Clompari8on of cholesterol and cholecalciferol e.steri-fication by pancreaticjuice. Cholesterol and cholecal-ciferol (0-4 tmole ofeach) were incubated separatelyas substrates in the esterification assay. Vitamin D

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radioactivity was provided by 0-7m,umole of[1-3H]cholecalciferol (45 000 counts/min.). After15min. 16% of the cholesterol was esterified, andabout 2% of the cholecalciferol radioactivity chro-matographed as ester. This was concentrated byfloridin-earth chromatography and was run withstandard vitamin D stearate, oleate, linoleate andlinolenate on a 20cm. x 5cm. silver nitrate-impreg-nated plate, as described for the first dimension ofester separation by Fraser & Kodicek (1968a). Theesters were observed under ultraviolet light afterbeing sprayed with dichlorofluorescein (0.2%, w/v,in ethanol), and radioactivity in the spots wasmeasured after elution with diethyl ether. Over90% of this radioactivity was in the monounsatur-ated vitamin D ester, thus confirming that vitaminD oleate had been synthesized.To identify the enzyme that was esterifying vit-

amin D, an attempt was made to fractionate whole,freeze-dried, pancreatic juice by DEAE-cellulosecolumn chromatography (Marchis-Mouren, Charles,Ben Abdeljlil & Desnuelle, 1961). Although goodresolution into about 12 peaks of protein wasachieved, the cholesterol-esterifying activity wasnot associated with any of these peaks. It wasdecided therefore to examine the specificity ofesterification indirectly with unfractionated pan-creatic juice.

Mixed-8ub8trate e8terification. If cholecalciferoland cholesterol are esterified by the same enzymethen the rate of reaction for either substrate shouldbe less when both are present, because of competi-tion for the one active enzyme centre. A 15min.

incubation was performed in duplicate with0-2pmole of cholesterol and of cholecalciferol/ml.,separately and together. This short time-intervaldecreased the effect of the hydrolytic reaction bykeeping the ester concentration at a low value.Cholesterol esterification was hardly altered by thepresence of cholecalciferol and the esterification rateof the latter fell by 19% with cholesterol present(Table 1). Ideally, both rates should have droppedif the same enzyme were involved, but as the choles-terol reaction was over 10 times faster, competitionby cholecalciferol may not have been observed.A similar experiment was carried out on 0-2 ,umole

of retinol/ml. incubated with cholesterol and withcholecalciferol. Retinol esterification was not meas-ured, but the fact that it occurred was confirmedvisually by retinol fluorescence in ultraviolet lighton the micro-thin-layer plates (free retinol RFO-1;ester RpO-6). Cholesterol and cholecalciferol esteri-fication rates rose by 16% and 17% respectively inthe presence of retinol. The cause of this rise couldnot be determined; however, as there appears to becompetition for esterification in the presence ofcholesterol but not in the presence of retinol, theinvolvement of the cholesterol-esterifying enzymeis favoured in the synthesis of vitamin D ester.Moreover, the equal stimulatory effect of retinol onboth cholesterol and cholecalciferol esterificationsuggests that again the same enzyme mechanismwas affected.

Cofactor requirements. Studies on bile-salt require-ments supported the concept that the cholesterol-esterifying enzyme was acting on cholecalciferol.

Table 1. Compari8on of reaction rate8 of esterification with pancreatic juice and mixed 8ub8trate8

The reaction rate (m,umoles of substrate esterified/mg. of protein/hr.) was calculated on 15 min. assays. Expt. 1:cholesterol and cholecalciferol as mixed substrates; Expt. 2: cholesterol and cholecalciferol as mixed substrateswith retinol. Oleic acid concentration was 0-8timole/assay; substrate concentration was 0-2,mole of eachsubstrate/assay.

Cholesterol esterification Cholecalciferol esterification

Substrate Reaction rate Mean

Cholesterol

Cholecalciferol

Cholesterol + cholecalciferol

Cholesterol

Cholecalciferol

Cholesterol + retinol

Cholecalciferol+ retinol

4684711

5014561

546534f

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Change (%) Reaction rate Mean Change (%)

470 0

479 + 2

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35-6 -19

540 0

35-632-71 34-2 0

624 +16

} 40-0 +17

Expt. 1

Expt. 2

462 1968

ENZYMIC SYNTHESIS OF VITAMIN D ESTERS

When taurocholate was replaced by deoxycholate,the cholesterol and cholecalciferol reaction rates de-clined by 78% and 32% respectively (Table 2). Theab3ence of bile salt gave similar decreases in thereaction rates. Bile salts are not required for theesterification of retinol by pancreas acetone-driedpowder extracts (Pollard & Bieri, 1960) or small-intestine slices (Olson, 1964); indeed, their presenceinhibits the reaction. Hence the opposite effectfound with both cholesterol and cholecalciferolmakes it less likely that the retinol enzyme isinvolved.

Inhibitorstudies. Heavy-metal ions andp-chloro-mercuribenzoic acid are potent inhibitors of thepancreatic cholesterol-esterifying enzyme (Hernan'-dez & Chaikoff, 1957; Murthy & Ganguly, 1962).The effect of these inhibitors on the present pan-creatic-juice enzyme is given in Table 3. With10 [LM-p-chloromercuribenzoic acid and 1O/tM-CU2+(as cupric acetate), instead of inhibition there wasactivation of both cholesterol and cholecalciferolesterification. When the concentration of p-chloro-mercuribenzoic acid was raised to 0 1 mM, the degreeof activation of the cholesterol reaction was dce-

creased, and cholecalciferol esterification was, in-hibited by 14%. Both reactions approached thecontrol value when 1mM-cysteine was added with0-1 mM-p-chloromercuribenzoic acid. Increasing theCu2+ ionl concentration to 0-1 mm caused inhibitionof cholesterol and cholecalciferol esterification by13% and 59O/% respectively.Activation of the reaction at the low inhibitor

concentration is interpreted as a greater inhibitionof hydrolysis than of esterification. With inerease(dconcentration, both reactions were inhibited,although cholecalciferol esterification was moresensitive to the effect of the inhibitors.

Relative esterification rates. A careful measurewas made of the relative esterification rates ofcholesterol, cholecalciferol and precholecalciferol bypancreatic juice. Precholecalciferol was ineltuded asit was known to be present in equilibrium withcholecalciferol in the previous assays. It also hasbiological activity, explainable by its conversion intocholecalciferol (Hanewald, Rappoldt & Roborgh,1961), and, as it is the immediate precursor ofcholecalciferol (Velltuz, Amiard & Goffinet, 1955), it

Table 2. Effect of bile salts on esterification typancreatic juice

The reaction rate (mMumoles of substrate esterified/mg. ofprotein/hr.) was calculated on 30min. assays. Oleic acidconcentration was 0.8/,mole/assay, the substrate concentra-tion 0.4,umole/assay, and the bile-salt concantration4 ,tmoles/assay.

Bile salt

Cholesterol TaurocholateDeoxycholateNone

Cholecalciferol TaurocholateD)eoxycholateNone

Reactionrate Change (%)825 0182 -78142 -8372 049 -3248 -33

Table 4. Comparative rates of esteriftcation bypancreatic juice for cholesterol, cholecalciferol and

precholecalciferol

Reaction rate (m,umoles of substrate esterified/mg. ofprotein/hr.) was calculated on 15min. assays. Oleic acidlconcentration was 0-8,umole/assay; substrate concentrationwas 0 4/tmole/assay.

Substrate

Cholesterol

Cholecalciferol

Precholecalciferol

Reactionrate

12441273103113315348f

Rel.Mean reaction

rate

1258 100

108 9

332 26

Table 3. Inhibition of pancreatic-juice esterifying enzyme

The reaction rate (mHtmoles of substrate esterified/mg. of protein/hr.) was calculated on 30min. assays. Oleicacid concentration was 0-8umole/assay, and substrate concentration 04p,mole/assay. PCMB, p-chloromercuri-benzoic acid.

Substrate for esterification ... ...

Inhibitor

None10oM-PCMB0-1 mM-PCMB0lmM-PCMB+ 1 mM-cysteine1JOjM-CU2+0 1 mM.-Cu2+

Cholesterol

Reaction rate Change (0o)662 0793 + 20735 +11713 + 8698 + 5578 -13

Cholecalciferol

Reaction rate Change (%)98116849510940

0

+18-14-3+11-59

Substrate

Vol. 1()9 463

Table 5. Comparative rates of e8terification of cholecalciferol and ergocalciferol catalysed by pancreatic juicein vitro

Reaction rate (m,umoles of substrate esterified/mg. of protein/hr.) was calculated on 15min. assays. Oleicacid concentration was 0 8,umole/assay.

Assay 1

Rel. reactionReaction rate rate

18435

5.31

Assay 2

Rel. reactionReaction rate rate

254 5-249 1

Table 6. Recovery of vitamin D after administration of ergocalciferol in vivo

[U-14C]Ergocalciferol (sp. radioactivity OlOmc/m-mole) was given to rats on a rachitogenic diet supplementedwith cholecalciferol: (1) oral administration of1-4mg. of [U-14C]ergocalciferol; (2) intravenous injection of0-54mg.of [U-14C]ergocalciferol.

(1)

% of recovered% of dose vitamin Drecovered found as ester10*40-36

4-80

12-5

(2)1I I

% of recovered% of dose vitamin Drecovered found as ester

25-30

13-7

25-90X8

would be formed in vivo in skin under normal condi-tions. A 15min. incubation enabled a determina-tion ofthe relative reaction rates to be made withoutallowing the previtamin D vitamin D equilibrium

time to shift to the right. At 400 only 2% ofvitaminD is formed from previtaminD in 24min. (Hanewaldet al. 1961). The comparative activities (Table 4)show that precholecalciferol is esterified at a rateabout three times that of cholecalciferol and abouta quarter that of cholesterol. When the esteri-fication of cholecalciferol and ergocalciferol was

compared it was observed that the rate for [1-3H]-cholecalciferol was five times that of [U-14C]ergo-calciferol (Table 5). The lowered specificity of thepancreatic-juice enzyme for ergocalciferol in vitrowas not reflected in the whole animal, where esteri-fied forms of ergocalciferol and cholecalciferol were

found in comparable quantities (Table 6).

E8terification studies with blood pltama

As a preliminary to studies in vitro, an experimentwas performed in vivo to see whether vitamin Desterification occurred without intestinal transport.If vitamin D were given parenterally to rats withhepatic-duct cannulae, then the intestinal enzymewould not be exposed to the vitamin. A rat weigh-ing 90g., prepared with a cannulated bile duct, wasinfused via a jugular vein cannula with 0.34mg. of[1-3H]cholecalciferol (sp. radioactivity 141 mc/

Table 7. Recovery of vitamin D after intravenousinfusion of 0-34mg. of [1-3H]cholecalciferol (8p. radio-activity 141 mc/m-mole) into a rat with bile-duct

cannulation

Tissue 0°LiverKidneysSmall intestineLeft tibia, femur and patellaThyroid glandPlasma

% of recoveredvitamin D

Vo of dose found as ester

70 3-31-7 460-5 00-1 00-004 01-6 7-0

m-mole) bound to bovine plasma albumin (50mg./ml.) in lml. of aq. 0.9% sodium chloride solution.To simulate absorption of vitamin D from the in-testine, continuous infusion with a slow-injectionapparatus was carried out over 18hr. The rate ofinfusion was faster for the first 0-2ml., to give a peakat 2-3hr. resembling the absorption curve fromintestine (Fraser & Kodicek, 1968b). The rat was

killed 24hr. after the start ofthe infusion and tissueswere collected for the estimation of free and esteri-fied vitamin D by thin-layer chromatography.Esterified vitamin D was found in liver, kidneyand plasma, but not in small intestine, bone or thy-roid (Table 7). The absence of ester from intestine

CholecalciferolErgocalciferol

LiverKidneysPlasma

464 D. R. FRASER AND E. KODICEK 1968

ENZYMIC SYNTHESIS OF VITAMIN D ESTERS

Table 8. E8terification of [4-14C]chole8terol and [1-3H]cholecalciferol by rat pla8ma in vitro

Substrate added(m,umoles) Enzyme

3-5 Plasma (2ml.)4-4 Plasma (2ml.)

17-3 Plasma (2ml.)17-3 Plasma (2ml.)4-4 Heated plasma (2 ml.)

(600 for 30min.)

% ofadded substrateesterified in 24hr.

262-21.01.90

Table 9. Compariaon of the fatty acid compo8itions of [4-14C]choleoterol and [1-3H]cholecalciferol e8ter88ynthesized by rat pla8ma in vitro

Two chromatograms (i and ii) were run on each sample.

Composition of fatty acids (%)

Degree of fatty acid unsaturation

SaturatedMonounsaturatedDiunsaturatedTriunsaturatedTetraunsaturated

Cholesterol esters

(i) (ii) Mean10 9 107 7 7

31 28 290-2 2 1

52 54 53

Vitamin D esters

(i) (ii) Mean16 12 145 6 6

23 25 245 3 4

50 54 52

indicated that this had not been the site of synthe-sis, and, as the concentration of ester was higher inplasma than in liver and kidney, it was possible thatit had been synthesized in circulating blood.Experiments in vitro revealed that both chole-

calciferol and cholesterol were esterified by plasma(Table 8). The identity of the vitamin D ester wasconfirmed by thin-layer chromatography withchloroform, when 98% of the radioactivity ran

opposite standard vitamin D ester. The degrees ofunsaturation of the esterified fatty acids of theesters were compared. Silver nitrate-impregnatedthin-layer plates were prepared, spotted with theradioactive esters plus standard cholesterol or vit-amin D stearate, oleate, linoleate and linolenateesters, and developed with diethyl ether-hexane(1: 4, v/v). The separated esters were observedunder ultraviolet light after being sprayed withdichlorofluorescein (0.2% in ethanol) and theirradioactivity was measured after elution with di-ethyl ether. Much of the natural plasma cholesterolester was the tetraunsaturated arachidonate, andcholecalciferol radioactivity in this spot was as-

sumed to be the tetraunsaturated vitamin D ester.The synthesized vitamin D esters were similar incomposition to those of cholesterol with most of theradioactivity in the di- and tetra-unsaturated esters(Table 9).Another rat was prepared as described above and

blood was collected 48hr. after the beginning of an

Table 10. Compo8ition of whole-blood vitamin Dester8 after intravenous infusion of 0-2mg. of [1-3H]-cholecalciferol (8p. radioactivity 141 mnc/rn-mote) into

a rat

Blood was collected 48hr. after the start of the infusion,which lasted 16hr. Tworun on each sample.

Degree of fatty acidunsaturation

SaturatedMonounsaturatedDiunsaturatedTriunsaturatedTetraunsaturated }

chromatograms (i and ii) were

Composition of fatty acids invitamin D ester (%)

(i)526

(ii) Mean49 517 7

13 13 13

29 31 30

intravenous infusion of 0-2mg. of [1-3H]cholecalci-ferol. Esterified vitamin D was 2-9% of the totalvitamin D extracted, and its composition, whencompared with vitamin D esters synthesized byplasma in vitro, showed a decrease in the proportionof polyunsaturated esters and a corresponding in-crease in the saturated esters (Table 10).

DISCUSSION

The failure to synthesize vitamin D esters in

vitro with preparations from liver and kidney is

SubstrateCholesterolCholecalciferol

Vol. 109 465

466 D. R. FRASER AND E. KODICEK 1968iinsufficienit evidenice to say that these organs do notesterify vitamin D. However, the findings thatvitamin D ester can be formed by an intestinalenzyme anid by plasma provide explanations for thepresence of ester in liver and kidney. Thus, unless acomplex situation exists where synthesis, hydrolysisand transport of ester are all taking place in liver,-the small amount of esterified vitamin D at that sitewas probably carried there along with vitamin Dalcohol. It may be that vitamin D, rather thanbeing esterified, was catabolized by liver enzymesto material insoluble in chloroform. Indeed, re-covery of radioactivity in chloroform after incuba-tion of [1-3H]cholecalciferol with liver was nevermore than 50%. This loss was not investigated, butanother cause for it might be inability fully to ex-tract vitamin D, which in an aqueous environmentis tightly bound to protein (Chalk & Kodicek, 1961;Chen & Lane, 1965).

Similarly the failure to synthesize vitamin Desters with mucosal preparations throws some doubtoni the ability of the intestine to esterify the vitamin.Nevertheless, the finding of esters in intestinallymph and the esterification of vitamin D in vitroby a pancreatic cholesterol-esterifying enzyme, thereported properties of which are indistinguishablefrom a mucosal enzyme, support the idea of vitaminD esterification by the intestine in vivo. Moreover,the activity of the mucosal cholesterol-esterifyingenzyme was very low in vitro whereas the livercholesterol ester synthetase, which has a quite dif-ferent mechanism, was found to be very active. Thiswould mean that this fatty acyl-CoA-cholesterolacyltransferase in liver was far more specific for itssubstrate than the intestinal esterifying enzymeswere.

Pancreatic cholesterol-esterifying enzyme isknown to have varying activities for other sterols.From published results (Table 11) the essentialsubstrate requirements of the reaction can be de-fined as (1) f-configuration of the C(3) hydroxylgroup, (2) A/B-trans-decalin configuration, and (3)saturation of the C(7)-C(8) carbon-carbon bond.Certain side-chain modifications such as a C(24) ethylgroup, as in f-sitostanol and f-sitosterol, or a C(24)ethyl group and a C(22)-C(23) trans double bond, asin stigmasterol, depress but do not abolish esterifi-cation.Apart from the configuration of the hydroxy

group the integrity of the B ring appears to be themost important factor for esterification. The pres-ence of a C(s)-C(6) double bond is not essential, buta C(6)-C(7) double bond, a 7/3-hydroxy group(Korzenovsky, Rust & Diller, 1955) or alteration inthe size of the B rinlg (e.g. in B-norcholesterol),abolishes activity.The experiments describe(I in the presenit paper

suggest that cholecalciferol is esterified by a chole-

Table 11. Comparison of pancreatic cholesterol-esterifying enzyme activities for different substrates

Values were taken from the literature and are related toan activity for cholesterol of IOe. Oleic acid was co-substratein all experiments. References: (1) Swell, Field & Treadwell(1954) (pig pancreas); (2) Hernandez & Chaikoff (1957) (pigpancreas); (3) Korzenovsky, Diller, Maishall & Auda (1960)(pig pancreas); (4) Murthy & Ganguly (1962) (rat pancreas).

Substrate Rel. activities

References ... (1) (2) (3) (4)Cholestanol 148 109 135Cholesterol 100 100 100 100f-Sitostanol - - 92 -

/-Sitosterol 41 39 69 35Stigmasterol 15 22 - 4Ergosterol 12 3 -

7-Dehydrocholesterol - 0 -

Epicholesterol - 0 5Epicholestanol - - 5Coprostanol - 7B-Norcholesterol - - 0Cholesteryl chloride 0 0Oestradiol - 0 -

Diethylstilboestrol - 0 -

sterol-esterifying enzyme. There is a competitiveeffect when the two substrates are together, thesame cofactor is required and enzyme inhibitorsproduce similar changes in activity. Yet chole-calciferol has no B ring and has a 3-hydroxy groupwhich, although defined from the parent sterol as3fl-, is physically in the 3cx-position. On thesegrounds it should not be esterified at all by theenzyme. However, the use of molecular models hasdemonstrated that there are spacial similaritiesbetween vitamin D and cholesterol, which suggestthat these substances are not as unlike as theirformulae indicate.

We are grateful to Dr P. A. Bell for the supply of [1-3H]-cholecalciferol 3,5-dinitrobenzoate. D.R.F. acknowlcdgesthe assistance of the Commonwealth Scholarship Com-mission.

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