Proc. Nati. Acad. Sci. USAVol. 87, pp. 6009-6013, August 1990Biochemistry

25-Hydroxyvitamin D3-la-hydroxylase in porcine hepatic tissue:Subcellular localization to both mitochondria and microsomes

(vitamin D/cytochrome P-450/liver)

BRUCE W. HOLLISDepartments of Pediatrics and Biochemistry and Molecular Biology, School of Medicine, Medical University of South Carolina, Charleston, SC 29425

Communicated by Robert Wasserman, Mfay 29, 1990 (received for review February 22, 1990)

ABSTRACT In vitro studies were performed to assess theability of hepatic homogenates, mitochondria, and microsomesto la-hydroxylate 25-hydroxyvitamin D3 (25(OH)D3]. Additionof 25(OH)D3 to either hepatic mitochondria or microsomescaused a concentration-dependent increase in the production of1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Hepatic homogenatesalso produced purported 1,25(OH)2D3, although at a muchreduced efficiency as compared with hepatic mitochondria ormicrosomes. Purported 1,25(OH)2D3 synthesized by hepaticmitochondria or microsomes was idenffiled by its mobility onseveral high-performance liquid chromatographic systems and,ultimately, by its ability to interact with the bovine thymus1,25(OH)2D3 receptor protein. Production of 1,25(OH)2D3 byhepatic mitochondria and microsomes was dependent on time ofincubation, protein content, and pH of incubation medium, andit required an adequate source of reducing equivalents. Gener-ation of 1,25(OH)2D3 by these organelles could be totally blockedby the cytochrome P-450 inhibitor ketoconazole. The microso-mal la-hydroxylase could not be saturated even at the highestconcentration (240 iAM) of 25(OH)D3 used. The mitochondrialla-hydroxylase, however, displayed saturation at -40 IAM25(OH)D3. Eadie-Hofstee reciprocal plot analysis of the hepaticmitochondrial la-hydroxylase gave a Km of 17 ,IM 25(OH)D3and a Vm, of 481 pg of 1,25(OH)2D3 per min per mg of protein.Because of its inability to achieve substrate saturation, mean-ingful kinetic parameters could not be calculated for the hepaticmicrosomal la-hydroxylase. These data demonstrate the liver tobe an even more dynamic organ than was previously believedwith respect to vitamin D metabolism in that the liver has thepotential to produce 1,25(OH)2D3 in situ by at least two separatemechanisms.

It is well established that various side chain and/or A-ringhydroxylation reactions are essential for the metabolicactivation of vitamin D3 (1, 2). Circulating vitamin D3 firstundergoes a hepatic conversion to 25-hydroxyvitamin D3[25(OH)D3] (3). The hydroxylase enzymes responsible for thisconversion are known to be cytochrome P-450 mixed-functionoxidases that are NADPH dependent and are located in bothhepatic mitochondria (4-6) and microsomes (7, 8). Finalactivation of 25(OH)D3 into 1,25-dihydroxyvitamin D3[1,25(OH)2D3] takes place primarily in the kidney (9). Therenal enzyme responsible for this activation, 25(OH)D3 la-hydroxylase, is also known to be a cytochrome P450 mixed-function oxidase that is located solely in the inner mitochon-drial membrane (10, 11). In recent years, other tissues havealso been shown to be capable of synthesizing 1,25(OH)2D3,including placenta (12), bone cells (13), lymphatic tissue (14),and keratinocytes (15). Compared with the renal 25(OH)D3la-hydroxylase, very little information is known with respectto the metabolic control of these extrarenal 25(OH)D3 la-

hydroxylases and essentially no information is available con-cerning their subcellular distribution.Because of the important role of 1,25(OH)2D3 in various

aspects of hepatic function (16-24), the possibility that theliver could synthesize in situ this hormonal form of vitaminD3 was investigated. It is reported here that both hepaticmitochondria and microsomes possess a 25(OH)D3 la-hydroxylase in significant amounts. These enzymes differ insome characteristics, but both appear to behave as cy-tochrome P-450 mixed-function oxidases.

MATERIALS AND METHODSReagents. 25(OH)D3 and 1,25(OH)2D3 were generously

provided by Hoffman-LaRoche. [26(27)-methyl-3H]25-(OH)D3 (90 Ci/mmol; 1 Ci = 37 GBq) and [26(27)-methyl-3H]1,25(OH)2D3 (90 Ci/mmol) were obtained from T. A.Reinhardt (Ames, IA). C18 Sep-Pak cartridges and silicaBond-Elut cartridges were purchased from Waters and An-alytichem International (Harbor City, CA), respectively.Ketoconazole was a gift from J. L. Napoli (Buffalo, NY).Bovine 1,25(OH)2D3 thymus receptor was obtained fromINCSTAR (Stillwater, MN). Coomassie blue protein assayreagent was purchased from Pierce. Nicotinamide adeninedinucleotide phosphate, isocitrate, N,N'-diphenylethylene-diamine (DPED), dithiothreitol, and ethylenediaminetetra-acetic acid were purchased from Sigma. All solvents were ofHPLC grade and were obtained from Fisher.

Livers. Livers from 3-month-old castrated male pigs, main-tained on stock diet, were obtained at the time of slaughter.The livers were perfused with ice-cold 0.15 M NaCl, minced,and placed in an appropriate volume of ice-cold 50 mMNa2HPO4 (pH 7.4) buffer containing 0.25 M sucrose and 0.5mM EDTA to provide a 20% (wt/vol) homogenate andhomogenized in a Waring blender and stored at -70'C forfurther use.

Preparation of Subcellular Fractions. All procedures wereperformed at 40C unless otherwise stated. Liver homogenateswere removed from -70'C storage and thawed. Cytosol-freehomogenate was obtained by the method of Engstrom et al.(25) with the following modification. Crude 20% homogenatewas centrifuged at 100,000 x g for 1 hr instead of 48,400 Xg for 10 min. The resulting precipitate was resuspended byhand homogenization to the original volume in a buffercontaining 33 mM Na2HPO4, 0.25 M sucrose, and 10mM KCI(pH 7.4) just prior to enzyme assay. The mitochondria andmicrosomes were isolated by the method of Simpson andMiller (26). The washed mitochondrial and microsomal pel-lets were suspended individually in a buffer containing 33 mMNa2HPO4, 0.25 M sucrose, and 10 mM KCl (pH 7.4) by handhomogenization and adjusted to a protein concentration of

Abbreviations: 25(OH)D3, 25-hydroxyvitamin D3; 1,25(OH)2D3,1,25-dihydroxyvitamin D3; DPED, N,N'-diphenylethylenediamine;DPPD, N,N'-diphenyl-p-phenylenediamine; RRA, radioreceptor as-say.

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1-4 mg/ml just before the enzyme assay. All protein con-centrations were determined by using Coomassie blue withbovine serum albumin as a standard (27).

Incubations. Liver homogenates, cytosol-free homoge-nates, mitochondria, or microsomes were incubated for var-ious periods of time at 370C in a shaking water bath. The finalassay incubation vol was 2 ml, and it was performed incapped borosilicate glass culture tubes (13 x 100 mm). Liverhomogenate and cytosol-free homogenate incubation tubescontained a final concentration of 50 mM Na2HPO4, pH7.4/125 mM sucrose/5 mM KCl/2 mM MgCl2/2 mM isoci-trate/3 mM EDTA/1 mM dithiothreitol/1 mM NADPH/10,uM DPED. Mitochondrial incubation tubes possessing aNADPH-generating system contained a final concentrationof 50 mM Na2HPO4, pH 7.4/125 mM sucrose/5 mM KCI/2mM MgCl2/2 mM isocitrate/3 mM EDTA/1 mM dithiothrei-tol/10 ,uM DPED. Microsomal incubation tubes contained afinal concentration of 50 mM Na2HPO4, pH 7.4/125 mMsucrose/5 mM KCI/3 mM EDTA/1 mM dithiothreitol/1 mMNADPH/10 AM DPED. In some incubation tubes ketocon-azole was added in <10 ,ul of ethanol. The approximateprotein content of the incubation tubes was 15-33 mg for thehomogenates and 0.1-3.5 mg for subcellular organelles. Be-fore addition to the incubation tubes, each protein componentwas gassed with a direct flow of 95% 02/5% C02. Thereaction was initiated by the addition of the appropriateamount of 25(OH)D3, which had been prepurified on silicaBond-Elut cartridges, in 40 ,l of ethanol.The incubation was terminated by adding 2 ml of acetoni-

trile followed by Vortex mixing. Each incubation tube re-ceived 1000 cpm of [3H]1,25(0H)2D3 to estimate recoverylosses during the extensive extraction and purificationscheme. The 1,25(OH)2D3 was extracted from the incubationmedium by C18 solid-phase extraction as described (28).Sample Purification and Quantitation of 1,25(OH)2D3. The

samples were evaporated to dryness under N2 and dissolvedin 2 ml of methylene chloride. The samples were then appliedto silica Bond-Elut cartridges and the 1,25(OH)2D3-containing fraction was separated and collected as described(29). The individual fractions containing 1,25(OH)2D3 werethen subjected to normal-phase HPLC with a Beckmanmodel 344 liquid chromatography system (Beckman Instru-ments, Palo Alto, CA). Normal-phase HPLC was performedwith a Zorbax-Sil column (0.4 x 25 cm) (DuPont) developedin and eluted with methylene chloride/isopropanol (96:4,vol/vol) and a flow rate of 2 ml/min. The 1,25(OH)2D3-containing region from this system was quantitated by directUV absorbance at 265 nm and/or integrated peak area,collected, and dried under N2, and resuspended in absoluteethanol for confirmatory quantitation by radioreceptor assay(RRA) (28).The purported 1,25(OH)2D3 generated from four incuba-

tion tubes of either liver mitochondria or microsomes wassubjected to three additional HPLC systems to confirm theidentity of the purported product. These systems, in order ofuse, were normal-phase [hexane/methylene chloride/isopropanol/methanol, 79.5:14.5:5.5:1.0 (vol/vol)] reversephase (Vydac-ODS 0.4 x 25 cm in 100% acetonitrile and aflow rate of 1 ml/min), and, finally, normal-phase [hexane/isopropanol, 92:8 (vol/vol)]. Flow rates were 2 ml/min unlessotherwise noted. The eluting 1,25(OH)2D3 peak from the finalnormal-phase system was quantitated by direct UV absor-bance at 265 nm after its elution from HPLC. This fractionwas collected, dried under N2, suspended in absolute etha-nol, and subjected to further quantitation by RRA using thebovine thymus 1,25(OH)2D3 receptor (28).

Other Analytical Methods. Liver microsomal and mito-chondrial cytochrome P-450 was solubilized as described(30). Spectral measurements of cytochrome P-450 contentwere made with a Schimadzu UV 165 spectrophotometer.

Cytochrome P-450 content was determined from the CO-reduced difference spectrum after reduction with dithioniteas described by Omura and Sato (31).

RESULTSIdentification and Quantitation of 1,25(OH)2D3 Produced by

Hepatic Mitochondria and Microsomes. Pig liver mitochondriaand microsomes incubated with 60 ,uM 25(OH)D3 produced acompound that comigrated with authentic 1,25(OH)2D3 onfour successive HPLC systems, including in order of use,normal-phase (96% methylene chloride/4% isopropanol), nor-mal-phase (79.5% hexane/14.0%o methylene chloride/5.5%isopropanol/1.0% methanol), nonaqueous reverse phase(100% acetonitrile), and finally, normal phase (92% hexane/8% isopropanol) (data not shown). The total amount of puta-tive 1,25(OH)2D3 produced from four pooled incubation tubesof liver mitochondria or microsomes was 133.7 and 136.1 ng,respectively, as quantitated by direct UV detection afterelution from the final HPLC system. Further analysis of thesegenerated compounds by subsequent RRA with the bovinethymus 1,25(OH)2D3 receptor demonstrated both putative1,25(OH)2D3 compounds to give identical binding affinity ascrystalline 1,25(OH)2D3 toward this specific 1,25(OH)2D3 re-ceptor molecule. The amount of compound produced in thisexperiment represents a production rate and turnover numberof 413 pg of 1,25(OH)2D3 per min per mg of protein and 4.5pmol of 1,25(OH)2D3 per min per nmol of cytochrome P-450,respectively, for hepatic mitochondria, and 667 pg of1,25(OH)2D3 per min per mg of protein and 2.1 pmol of1,25(OH)2D3 per min per nmol of cytochrome P-450, respec-tively, for hepatic microsomes.For subsequent experiments, it was determined that quan-

titation of hepatic-generated 1,25(OH)2D3 could be achievedadequately following a single normal-phase HPLC system in96% methylene chloride/4% isopropanol. This determinationwas achieved by comparison of direct quantitation versuscombined HPLC-RRA quantitation of purported 1,25(OH)2D3generated by hepatic mitochondria and microsomes. Thesecomparisons yielded significant linear relationships ofy = 0.45ng + 1.05(x) (r = 0.99), and y = 0.42 ng + 0.97(x) (r = 0.99)for mitochondria and microsomes, respectively. Thus, thedirect UV quantitation technique of 1,25(OH)2D3 was adaptedfor subsequent experiments with the exception of experimentsin which low substrate concentration gave rise to low productformation. In these cases, RRA was used as the final quanti-tation step for 1,25(OH)2D3.Comparison of the Relative la-Hydroxylase Activities of

Liver Homogenates, Mitochondria, and Microsomes. Pig liverhomogenates, cytosol-free homogenates, mitochondria, andmicrosomes incubated with 60 pM 25(OH)D3 exhibited var-ious abilities, on a protein basis, to generate 1,25(OH)2D3(Table 1). Crude homogenate and cytosol-free homogenateproduced 0.09 and 0.63 ng of 1,25(OH)2D3 per mg of proteinper 20 min, respectively. This was much less than observedfor isolated mitochondria and microsomes, which generated8.26 and 16.1 ng of 1,25(OH)2D3 per mg of protein per 20 min,respectively.

Kinetic Properties of Hepatic Mitochondrial and Microso-mal 25(OH)D3 la-Hydroxylase. The time course, proteindependence, and substrate concentration curves for theconversion of 25(OH)D3 to 1,25(OH)2D3 in hepatic mitochon-dria and microsomes are shown in Figs. 1 and 2A, respec-tively. The reaction is linear for up to 30 min for mitochondriaand for up to 45 min for microsomes and is dependent onprotein concentration, which ranged from 0.23 to 3.7 mg pertube for mitochondria and from 0.12 to 1.8 mg per tube formicrosomes (Fig. 1B). With respect to substrate saturation,only the reaction with mitochondria reached saturation andthis occurred at =40.uM 25(OH)D3 (Fig. 2A). Production of

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Table 1. Distribution of 25(OH)D3 la-hydroxylase activity in porcine liver

Total 1,25(OH)2D3 Relative 1,25(OH)2D3 RelativeProtein content, produced,* produced, ng per mg la-hydroxylase

Fraction mg per incubation ng per incubation of protein per 20 min activitytHomogenate 32.4 2.96 0.09 1.0Homogenate

precipitate: 15.8 9.91 0.63 7.0Mitochondria 2.7 22.3 8.26 92Microsomes 1.5 24.1 16.1 179

Tissue preparation, incubation conditions, and quantitation of 1,25(OH)2D3 were performed as described in the text. Eachincubation tube contained 60 juM 25(OH)D3 as substrate.*Values represent the mean of four incubation tubes.tinitial value of 1.0 based on 20% liver homogenate.tResuspension of 100,000 X g 20% liver homogenate precipitate.

1,25(OH)2D3 by hepatic mitochondria was thus dependent onsubstrate concentration as shown by the reciprocal plot of thedata in Fig. 2B. As previously reported by Vieth and Fraser(32) using renal mitochondria, 1,25(OH)2D3 production is notlinear at low substrate concentrations but was linear at highersubstrate concentrations. Thus, using the linear portion ofthegraph, a significant inverse relationship was achieved: y =481 pg of 1,25(OH)2D3 per min per mg of protein - 17 (v/[S])(r = 0.95) (v, Velocity; [S], substrate concentration). Forhepatic mitochondria this relationship yielded a Km and Vmaxof 17 ,uM 25(OH)D3 and 481 pg of 1,25(OH)2D3 per min permg of protein, respectively, for this hepatic 25(OH)D3 la-hydroxylase (Fig. 2B). Because of the inability of the mi-crosomal 25(OH)D3 la-hydroxylase to saturate at the highestsubstrate concentration tested, Km and Vma could not becalculated.

Metabolic Requirements and Effect of Ketoconazole onHepatic 25(OH)D3 la-Hydroxylase(s). la-Hydroxylation of25(OH)D3 by both hepatic mitochondria and microsomes wasgreatly influenced by the pH of the incubation medium (datanot shown). Production of 1,25(OH)2D3 by both organelleswas sharply reduced at pH 7.0 and was essentially abolishedat pH 6.4. Fig. 3A displays the dependence of the la-

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hydroxylation of 25(OH)D3 in hepatic mitochondria andmicrosomes on an adequate source of reducing equivalents.These reducing equivalents were supplied to mitochondria asisocitrate and to microsomes directly as NADPH. Without asource of reducing equivalents, the production of1,25(OH)2D3 did not proceed in either organelle. Supplying aslittle as 0.1 ,uM isocitrate to mitochondria or 0.1 /iM NADPHto microsomes initiated the production of 1,25(OH)2D3 andwith the appropriate reducing source at a concentration of-0.5 ,uM, the la-hydroxylation of25(OH)D3 was saturated inboth organelles (Fig. 3A). It should be noted that supplyinghepatic microsomes with 4 mM isocitrate in place ofNADPHresulted in a total lack of production of 1,25(OH)2D3 from25(OH)D3 (data not shown). Fig. 3B depicts the effects of anincreasing concentration of ketoconazole on the la-hydroxylation of 25(OH)D3 by hepatic mitochondria and

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protein concentration (B) on the generation of 1,25(OH)2D3 byhepatic mitochondria (A) and microsomes (e). The concentration ofsubstrate 25(OH)D3 was 60 ,uM in all incubation tubes.

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FIG. 2. Substrate curve for the hepatic mitochondrial (A) andmicrosomal (e) 25(OH)D3 la-hydroxylase during a 20-min incuba-tion (A). Eadie-Hofstee plot for hepatic mitochondrial 25(OH)D3-la-hydroxylase of the dependence of 1,25(OH)2D3 production onsubstrate concentration (v versus v/S) (B). This relationship wasderived from the data in A. Because of nonlinearity at the lowestsubstrate concentrations (o), the regression line includes only pointsat the higher substrate concentrations [>6 AM 25(OH)D3] (solidsymbols) (B). r = 0.95, Vmax = 481 pg per min per mg of protein, Km= 17.0,M.

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FIG. 3. The effect of isocitrate (mitochondria) (v), NADPH(microsomes) (e) (A), and ketoconazole (B) concentrations on theproduction of 1,25(OH)2D3 by hepatic mitochondria and mi-crosomes. While not depicted on the figure, it should be noted thatsupplying hepatic microsomes with only isocitrate results in a totallack of 1,25(OH)2D3 production by this organelle. Substrate concen-tration of 25(OH)D3 was 60 /iM in all incubation tubes.

microsomes. In both cases, inhibition of la-hydroxylationcommenced at as little as 0.1 ,uM ketoconazole and was

completely inhibited at 2.0 /iM.

DISCUSSION

It has been demonstrated here that porcine hepatic tissuecontains enzyme(s) that are capable of la-hydroxylating25(OH)D3. This observation is at odds with earlier studies(33, 34) and it is important to attempt to reconcile some of thediscrepancies. Fraser and Kodicek (33) reported that chickenliver homogenate failed to produce radiolabeled 1,25(OH)2D3from radiolabeled 25(OH)D3. It is possible that the tech-niques used in this earlier study simply could not detect anypossible metabolism that had occurred. A more likely pos-sibility, however, would be that the small amount ofsubstrateused, 17 ng, in the earlier study was totally sequestered by thehomogenate and was simply made unavailable to the en-

zyme(s) for subsequent metabolism. In fact, data from thepresent study show crude liver homogenate to be a poorsynthesizer of 1,25(OH)2D3, although the efficiency of la-hydroxylation by hepatic tissue rapidly increases as tissuefractionation progresses (Table 1). The increase in efficiencycould result from several factors that remain beyond thescope of this report. It is also important to note that extra-renal production of 1,25(OH)2D3 has been seriously ques-tioned (34), although it is now known to occur under phar-macological conditions and to contribute to circulating1,25(OH)2D3 (35, 36). It is also possible that liver serves toproduce 1,25(OH)2D3 in a paracrine, autocrine, and/or in-tracrine mode and not an endocrine mode. Thus, undernormal physiological conditions possible 1,25(OH)2D3 pro-duction by hepatic tissue would be internally utilized andwould not significantly contribute to circulating levels.

The present study has localized these enzymes to bothhepatic mitochondria and microsomes. These enzymes ap-pear to be distinct from one another and their presence inboth organelles is not due to cross contamination of onesubcellular fraction with another. While both hepatic organ-elles are capable of la-hydroxylating 25(OH)D3, there aredistinct differences in the mitochondrial and microsomalenzymes. First, the time course of 1,25(OH)2D3 productionbetween hepatic mitochondria and microsomes is slightlydifferent (Fig. 1A). The mitochondrial production of1,25(OH)2D3 appears to be linear for only 30 min, whilelinearity of the microsomal enzyme is up to 45 min. Second,a comparison of the substrate saturation curves betweenhepatic mitochondria and microsomes depicts major differ-ences in their relative abilities to la-hydroxylate 25(OH)D3(Fig. 2A). The mitochondrial enzyme demonstrates satura-tion at -40 ,M 25(OH)D3. Conversely, the microsomalla-hydroxylase was not saturated at up to 240 /.M 25(OH)D3.The reason for the nonsaturability of the microsomal enzymeis unknown, but it could be due to either sequestration ofsubstrate and/or the presence of more than one 25(OH)D3la-hydroxylase that exhibit different abilities and rates ofhydroxylation. Because of this nonsaturability, it is virtuallymeaningless to try to calculate further enzyme kinetics on thehepatic microsomal 25(OH)D3 la-hydroxylase(s). However,the Eadie-Hofstee reciprocal plot (37) has been used toestimate the Km and Vma,, of the hepatic mitochondrial25(OH)D3 la-hydroxylase (Fig. 2B). As observed by Veithand Fraser (32), 1,25(OH)2D3 production was not linear at thelowest substrate concentration, but it was linear at highersubstrate concentrations and yielded a Km of 17 AM25(OH)D3 and a Vm. of 481 pg of 1,25(OH)2D3 per min permg of protein. The nonlinearity of low substrate concentra-tions may again be due to either substrate sequestration bythe hepatic mitochondria (32) or the potential mediation of25(OH)D3 transport within the mitochondria as occurs to7-dehydrocholesterol and cholesterol in the liver and adrenalglands, respectively (38, 39). At any rate, the Vma,, obtainedfor the hepatic mitochondrial 25(OH)D3 la-hydroxylase issimilar to that observed for the pig and rat renal mitochon-drial 25(OH)D3 la-hydroxylases (25, 32).However, the Km ofthis hepatic mitochondrial enzyme is a magnitude higher thanthat observed for the pig and rat renal la-hydroxylases (25,32) or the reconstituted 25(OH)D3 la-hydroxylase from pigrenal mitochondria (40). This difference in enzyme Km mayaccount for why serum 1,25(OH)2D3 begins to increase inanephric humans (35), dogs (35), and pigs (36) as circulating25(OH)D3 approaches pharmacological levels. Thus, underpharmacological conditions, it may be possible for the liverto act as an endocrine organ for 1,25(OH)2D3 production. Itis interesting, however, that the hepatic mitochondrial25(OH)D3 la-hydroxylase has a very similar Km, Vmax, andturnover number when compared to the rat hepatic mito-chondrial vitamin D3 25-hydroxylase (6).

Dissimilarity between the hepatic mitochondrial and mi-crosomal la-hydroxylases also exists in their respectiverequirements for reducing equivalents (Fig. 3A). Hepaticmitochondria were able to generate their own reducingequivalents when supplied with isocitrate, as has been pre-viously reported for the rat hepatic mitochondrial vitamin D325-hydroxylase (5). Hepatic microsomes were unable togenerate any 1,25(OH)2D3 when supplied only with isoci-trate, but they readily produced 1,25(OH)2D3 when supplieddirectly with NADPH (Fig. 3A). The requirement forNADPH by the renal mitochondrial 25(OH)D3 la-hydroxy-lase system is well documented (10, 11).

Similarities do, however, exist in two important aspectsbetween the la-hydroxylases in hepatic mitochondria andmicrosomes. Both enzymes appear to function optimally inthe pH 7.5 range, as has been reported for other; vitamin D3

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hydroxylases (32, 41). Also, both subcellular la-hydroxyl-ases in the present study were very sensitive to the presenceof ketoconazole (Fig. 3B). Ketoconazole, a broad spectrumantifungal drug, is known to specifically inhibit cytochromeP-450 mixed-function oxidases by interacting with the hemeiron of the cytochrome (42). Ketoconazole is also known toinhibit 25(OH)D3 la-hydroxylase activity in renal mitochon-dria (43). Indeed, in the present study suppression of la-hydroxylase activity occurred in the presence of as little as

0.1 AtM ketoconazole and was totally suppressed by thisinhibitor at 2 ,.M (Fig. 3B). Although not definitive, thesedata suggest that the la-hydroxylases in hepatic mitochon-dria and microsomes are of the cytochrome P-450 mixed-function oxidase class of enzyme.Another important aspect of the present work is the

localization of a 25(OH)D3 la-hydroxylase to the microsome.Every definitive study to date has localized the la-hydroxylase to the mitochondria (10, 11, 40). One exceptionto this was a study by Paulson et al. (44), which described a

purported 25(OH)D3 la-hydroxylase to be localized in mi-crosomes of the rat yolk sac. They also reported in the studythat the purported microsomal la-hydroxylase was totallyinhibited by N,N'-diphenyl-p-phenylenediamine (DPPD), a

free radical scavenger that is sometimes used as an antioxi-dant (45). Indeed, Hollis et al. (43) have also described thegeneration of 1,25(OH)2D3 by microsomes isolated fromhuman trophoblastic tissue. However, the production of1,25(OH)2D3 by trophoblastic microsomes was totally abol-ished by DPPD, while production of 1,25(OH)2D3 by renalmitochondria was totally unaffected by the antioxidant (43).It was concluded in this latter study that the 1,25(OH)2D3 was

produced from 25(OH)D3 by free radical chemistry and was

not due to a specific 25(OH)D3 la-hydroxylase in the mi-crosomes. Because of these previous observations, 10 ,uMDPED, a more soluble form of DPPD, was included in allincubation tubes to eliminate this nonspecific production of1,25(OH)2D3.

Physiologically, the observation that the liver has the po-

tential to synthesize 1,25(OH)2D3 in situ is potentially an

important one. Recent studies have demonstrated that hepatictissue has several physiological responses to 1,25(OH)2D3(16-24, 46) and appears to contain high-affinity receptors forthis potent hormone (47). General vitamin D deficiency in therat has been shown to alter hepatic function as manifested bydelayed bromosulfthalein clearance and elevated plasma tran-saminases (16). Furthermore, this study also noted liver his-tology changes consistent with periportal necrosis due tovitamin D deficiency. Hepatic isocitrate lyase and malatesynthase are also known to be decreased in vitamin D defi-ciency (46). In addition to these generalized symptoms ofvitamin D deficiency, 1,25(OH)2D3 is known to specificallyincrease hydroxymethylglutaryl CoA reductase activity (18),decrease liver regeneration time independent of calcium (17),regulate the synthesis of hepatic DNA polymerase a (19, 20),increase [3H]thymidine incorporation into hepatic DNA (17),and enable regenerating liver cells to make functional ribonu-cleotide reductase subunits (21). 1,25(OH)2D3 also increaseshepatocyte cytosolic calcium levers (22) through its potentialaction on phospholipase A activity (23). Given the vast arrayofknown actions of 1,25(OH)2D3 on hepatic function, it is notsurprising that in situ production of the hormone exists in theliver. The exact cell type(s) responsible for the hepatic pro-duction of 1,25(OH)2D3 and the control of the hepatic25(OH)D3 la-hydroxylases remains to be determined by fur-ther study.

I am grateful to Daisy Dominick for her excellent technicalassistance. This work was supported by National Institutes of HealthGrant HD-22542.

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