Studies of the In Vivo Metabolism of

Mevalonic Acid in the Normal Rat

KjELL H. HmLTSmoM,MARVIND. SIPERSTEIN, LEE A. BmICEER, andLYNNEJ. LUBY

From the Department of Internal Medicine, The University of Texas South-western Medical School at Dallas, Dallas, Texas 75235

A B S T R A C T Studies were performed to examine syn-thesis, tissue localization, and metabolism of mevalonicacid in normal rats. Circulating mevalonate was foundto have a rapid turnover phase of 5 min and a slowerphase of 40-50 min. Under in vitro conditions the syn-thesis of mevalonate is carried out most actively by theliver and only to a minor extent by the other tissuesstudied. The most unexpected finding of this study wasthat both in vivo and in vitro the kidneys rather thanthe liver are the primary site of the metabolism of cir-culating mevalonate. Whereas mevalonate in the liver israpidly transformed to cholesterol, the major productsof mevalonate metabolism in the renal tissues during thesame time period are squalene and lanosterol. Exogenousin contrast to circulating mevalonate is metabolized pri-marily in the intestine.

INTRODUCTIONThere is now abundant evidence that mevalonic acid is anessential intermediate in the synthesis of all animal andplant sterols (1, 2). In addition, previous studies fromthis laboratory have established that the reaction re-sponsible for the synthesis of mevalonate represents theprimary site of feedback control of cholesterol biosyn-thesis (3, 4), a finding that has been confirmed by anumber of subsequent reports (5-7). Finally, this feed-back regulation of mevalonate has been shown by both in

Dr. Hellstrom's present address is Department of Medi-cine, Serafimerlasarettet, 112 83 Stockholm, Sweden, andDr. Bricker's present address is The University of MiamiSchool of Medicine, P. 0. Box 875, Biscayne Annex, Miami,Fla.

During these studies, Dr. Hellstrom was supported byU. S. Public Health Service Research Fellowship AwardTW891, and Dr. Bricker was supported by U. S. PublicHealth Service Training Grant CA 05200. Dr. Siperstein isthe recipient of a U. S. Public Health Service ResearchCareer Award HE 1958.

Received for publication 21 December 1971 and in re-vised form 29 January 1973.

vitro (8-11) and in vivo studies (12) to be regularlylost following malignant change in liver. This lesion,moreover, remains the single known example of thie con-stant loss of a normal feedback system in cancerous tis-sue (13).

The finding that mevalonic acid plays so key a regu-latory function in sterol synthesis serves to emphasizethe need for a better knowledge of the metabolic fate ofthis compound than is currently available. While nu-merous studies have examined this problem in isolatedin vitro systems (1, 2, 14-16), relatively few studieshave been directed at the more physiologic question ofthe metabolic fate of mevalonic acid in the intact animal.

Gould and Popjak (17) first observed in 1957 that in-jected DL-[14C]mevalonic acid tended to concentrate inthe liver of the rat. Subsequently, Goodman, Avigan, andSteinberg (18) confirmed and expanded this observationby demonstrating that in the rat intravenously adminis-tered DL-[2-14C]mevalonate was rapidly taken up by theliver and there converted into the nonsaponifiable inter-mediates of cholesterol synthesis. Finally, Garattini,Paoletti, and Paoletti (19) also noted that 2 h after theadministration of [14C]mevalonate to rats the label wasfound primarily in the liver.

It is noteworthy that, while each of these earlier stud-ies indicated that intravenously administered mevalonateis metabolized by the liver, only the study of Garat-tini et al. (19) attempted to assess the relative im-portance of the other tissues of the body in the overallmetabolism of mevalonate.

It was the purpose of the present study to determinewhether organs other than the liver play a role in themetabolism of endogenous mevalonate. The unexpectedresult of these studies is that the major portion of circu-lating mevalonate is metabolized not by the liver but bykidneys.

METHODSAnimals. Unless otherwise indicated the rats employed in

this study were 180-200 g adult females of the Sprague-

The Journal of Clinical Investigation Volume 52 June 1973 -1303-1313 1303

Dawley strain. Both before and during the experiment theywere fed ad lib. ground B & D rat cubes1 and water. Atthe completion of the studies the animals were killed by de-capitation, and the organs to be analyzed were immediatelyexcised and prepared as described below.

The labeled mevalonate was dissolved in saline and ad-ministered to the animals, as noted, by intravenous or in-traperitoneal injection or by a gastric tube. When the me-valonate was given parenterally, the animals were lightlyanesthesized with ethyl ether just prior to the study.

Materials. DL- [2-`4C]mevalonate, specific activity 5.03mCi/mmol, DL-[1-`4C]mevalonate, specific activity of 1.54mCi/mmol, and DL-[5-3H]mevalonate with a specific ac-tivity of 50 mCi/mmol were purchased from the NuclearChicago Corp.2 and were used as their potassium salts. [2-"4C]acetate with a specific activity of 2 mCi/mmol wasobtained from New England Nuclear. After treatment withacid both labeled mevalonate preparations were shown tocontain at least 95% of their label in the mevalonolactonepeak isolated by gas-liquid chromatography (21).

L-[1-14C]`mevalonate was prepared from DL-[1-4C] mevalo-nate as follows: 20 ,uCi (2 mg) of DL-[1-'4C]mevalonate wasdissolved in 3.7 ml of water containing 500 /Amol of potas-sium phosphate, pH 6.7, 50 /Amol of ATP, and 5 mg ofyeast L-mevalonate kinase, prepared as described by themethod of Tchen (22) through the protamine sulfate pre-cipitation step. Following incubation for 1 h at 37°C thecrude mixture was subjected to thin-layer chromatographyon silica gel G employing butanol: H20: ammonium hy-droxide, 60: 30: 10, as the developing system. The D-mevalo-nate and unreacted L-mevalonate moved to the front of thethin layer chromatograph in this system. The location ofL-[1-'4C]mevalonate-P04 Rf of 0.72 was then detected byautoradiography on nonscreen monopak film. The identityof the mevalonate phosphate was confirmed by use of astandard L-mevalonic acid-1-'PO4 synthesized by the L-mevalonate kinase reaction with ATP--y-3P and nonradio-active DL-mevalonate.

The band of silica gel containing the L-[1-14C]mevalonate-P04 was scraped from the thin-layer chromatograph, andthe radioactivity was eluted with 50% ethanol. The L-[1-4C]mevalonate-P04 was next incubated with alkaline phos-

phatase3 2.4 U for 4 h at pH 7.6 to remove the phosphate.The free L-[1-'4Cmevalonate was then rechromatographedin the same solvent system, and the free mevalonate at thefront was recovered by elution of the silica gel with Nano-grade methanol. Analysis of the product by gas-liquid chro-matography demonstrated that at least 90% of the recovered4C was present in mevalonic acid.

Analytical methods. The various tissues to be studiedwere cut into small pieces and extracted for 2 h by re-fluxing repeatedly with chloroform-methanol (1: 1 by vol-ume). A portion of this extract, one-fourth to one-third,was then transferred into a 50 ml screw tube and evapo-rated to dryness under a stream of nitrogen. 2 ml of 1 Npotassium hydroxide in 35% ethanol were added per gramof tissue represented in the evaporated extract. The tubeswere then flushed with nitrogen, closed with a screw capand placed in a waterbath (70°C) for 1 h. The lipids werenext separated into the nonsaponifiable fraction, the allylpyrophosphates and the prenoic acids by the method de-scribed by Goodman and Popjak (16). After extraction of

1B & D Mills, Inc., Grapevine, Tex.2 Nuclear Chicago Corp., Des Plaines, Ill.

Calf intestine alkaline phosphatase obtained from Worth-ington Biochemical Corp., Freehold, N. J.

the nonsaponifiable material with petroleum ether, the waterphase was acidified to pH 1 and left at room temperatureovernight. On the next day the aqueous phase was againmade alkaline, and the hydrolized prenoic alcohols were ex-tracted with petroleum ether. The prenoic acids were ob-tained after a final acidification of the aqueous phase andrepeated extractions with petroleum ether. Each petroleumether fraction was washed twice with a small volume ofwater, which after backwashing with petroleum ether wasadded to the original water phase. The petroleum ether ex-tracts were then combined and evaporated under nitrogen.Since the alcohols contained negligible amounts of radio-activity, the acids and alcohols were extracted together inmost of the experiments. The nonsaponifiable material wasnext redissolved in a small volume of chloroform and frac-tionated by thin-layer chromatography on 20 X 20 cm platescoated with silica gel G (plate thickness 250 gm). Thechromatograms were developed for 1 h in heptane-benzene(97: 3). Squalene' was identified as a distinct spot at Rf

0.94, nerolidol (Rf 0.72), lanosterol and dihydrolanosterol(Rf 0.63), farnesol and methostenol (Rf 0.48-0.54), geranioland 27 carbon sterols (Rf 0.4). It was assumed that the 27carbon sterol band represented cholesterol and no furtherpurification of this compound was carried out. The variousbands were visualized by spraying with 0.05% solution ofrhodamine G in 50% ethanol and scraped from the platesdirectly into the scintillation fluid consisting of 0.3% di-phenyloxazole and 0.015% p-bis-phenyloxazolyl-benzene intoluene. Radioactivity was determined with either Packard'or Beckman Liquid Scintillation Counters.6

The turnover times of DL- and L-mevalonate were deter-mined as follows: Cannulae of P-10 polyethylene tubingwere inserted into the femoral arteries and the tail veinsof lightly anesthetized rats. The rats were then placed inrestraining cages and allowed to recover for at least 1 h.The labeled mevalonate was then injected through the venouscannulae, and at regular intervals 50 ,ul of blood was col-lected from the femoral artery and added to 1.0 ml of 0.9%osaline. A 0.6 ml portion of the diluted blood was then di-rectly assayed for 14C using Beckman Bio-Solve 38 as thescintillation solution.

Mevalonate synthesis by tissue slices was determinedexactly as described in a previous publication (3). Thefurther details of the individual experiments are describedunder Results.

RESULTS

Kinetic studies of mevalonate in vivo

Turnover time of mevalonate in normal rats. Theinitial experiments of this study were designed to deter-mine the rate at which circulating mevalonate is metabo-lized in the normal rat. The turnover time of a relativelysmall dose of mevalonate was estimated by injecting 7.2iug (2 ACi) of DL[5-3H]mevalonate intravenously into a

'The methostenol was a gift from Dr. Ivan Franz; thedihydrolanosterol a gift from Dr. Jean D. Wilson. Farnesoland geraniol were obtained from Aldrich Chemical Co., Inc.,Milwaukee, Wisc., and lanosterol was purchased from MannResearch Laboratories, New York. The squalene and choles-terol were obtained from Eastman Kodak Co., Rochester,N.Y.

Packard Instrument Co., Inc., Downers Grove, Ill.6 Beckman Instruments, Inc., Fullerton, Calif.

1304 K. H. Hellstrom, M. D. Siperstein, L. A. Bricker, and L. J. Luby

L [I- _4C] - Mevolonate

,[ t 1/2 ' 5.2 minatz 7.3min

bhet1/ 24.6 minbt z 42.6 min

10 20 30 40 50 60 70 80 90TIME (MINUTES)

FIGURE 1 Turnover study of DL-[5-'H]mevalonate in a nor-

mal rat. The rapid phase of mevalonate turnover was calcu-lated using the method of Gurpide et al. (20).

200 g Sprague-Dawley rat. The resulting die-awaycurves were analyzed according to the two-pool modeldescribed by Gurpide, Mann, and Sandberg (20). Afteran initial rapid decrease in blood radioactivity with a

corrected turnover time that can be approximated at 7min (Fig. 1), a somewhat slower turnover rate with a

half-time of 28 min and a turnover time of 40 min was

observed.Both because of the possibility of the incorporation

of ['H]mevalonate into circulating labeled compoundsother than mevalonate and because of the complicatingproblem of the nonphysiologic D-mevalonate influencingthe turnover kinetics of administered DL-mevalonate,these turnover studies were repeated using the physio-logically active L-isomer of mevalonate, prepared as de-scribed under Methods. In addition, in this experimentthe mevalonate was labeled in the carboxyl carbon. Themetabolism of mevalonate to isopentenyl pyrophosphateresults in the loss of its first carbon as 'CO2, which inturn is diluted by the large body pool of '2CO2 and ex-

pired; as a result it would be highly unlikely that sig-nificant amounts of "C present in the blood of the rat

injected with L-[1-"C]mevalonate would be present incompounds other than mevalonate itself. As shown inFig. 2, studies utilizing L-[1-"C]mevalonate indicatea rapid turnover phase of 7 min and a slower componentwith a turnover time of 52 min, figures that do not differgreatly from the turnover times observed with DL-[5-'H]mevalonate.

TIME (MINUTES)

FIGURE 2 Turnover study of L-[1-14C]mevalonate in a nor-mal rat.

Metabolism of endogenous mevalonateTissue distribution of circulating mevalonate. Studies

were next carried out to determine where within the bodythe metabolism of this circulating endogenous pool ofmevalonate takes place. The initial experiments were de-signed to determine the overall tissue distribution andrelative affinities of various organs for endogenousmevalonic acid. Accordingly, DL- [2-14C]mevalonate (150t'g or 75 lyg of L-mevalonate) were injected intravenouslyinto rats, the animals were killed 2 h later, and the total14C recovered in the various tissues following chloroformand methanol extraction was determined. The resultsshown in Fig. 3 indicate that, contrary to expectation,

0

_Jc20 AlW

w

z

5-_

a~~~~~~

FIGURE 3 Total organ distribution of ["C] mevalonate andlabeled metabolites 2 h following intravenous administrationof DL-[2-"C]mevalonate.

Mevalonic Acid Metabolism In Vivo 1305

la00roI

2U6

la0.20E 1,000S.2a.0

a)U)

i, W< 10 ;>

Co. 8

2 6

0w4 a D

tabolites per gram of tissue 2 h following intravenous ad-ministration of DL- [2-4C] mevalonate.

the kidneys, rather than the liver, represent the majorsite of uptake of the "C, with four times as much of thelabel being present in the kidney (16%) as was foundin the liver (3.9%). The entire gastrointestinal tractand its contents accumulated a total of only 2.4% of therecovered radioactivity with lesser amounts of radio-activity being found in the other tissues examined. Itwas assumed that the remainder of the mevalonate andits metabolites were either distributed throughout thecarcass, which was not examined, or else excreted inthe urine.

The ability of the kidneys to accumulate mevalonateand its metabolites is even more strikingly demonstratedwhen the distribution of the label is calculated on thebasis of the weight of the tissues studied. As is shownin Fig. 4, the kidneys per gram of tissue concentrate

KIDNEY

18

a 16 '

W 14

12a1-41 8

LA> 0

Ia.

['C] mevalonate to an extent at least 20 times that ob-served in the liver.

Tissue localization of mevalonate metabolites at vari-ous time intervals. While observations of the previousexperiments strongly suggest that the kidneys play an

important role in the metabolism of circulating mevalo-nate, it was conceivable that the localization of 14C inthe kidneys represented simply the renal concentrationof mevalonate itself, particularly of the unmetabolizedD-mevalonate, rather than the actual metabolism ofmevalonate by this organ. To resolve this question the"C present within the kidney, liver, and intestine was

fractionated four separate times as described underMethods, into total nonsaponifiable lipids, allyl pyro-phosphates, and prenoic acids.

The data in Fig. 5 demonstrate that within 30 minand at each time interval thereafter a large percentageof the total radioactivity present in the kidney can beaccounted for by the metabolites of mevalonate. Thisconclusion has been confirmed by the observation thatonly traces of free ['C]mevalonate can be recovered inthe kidneys by gas chromatography (21) 2 h after in-jection of the labeled mevalonate. After 2 h over 75% ofthe remaining ["C]mevalonate can be recovered in theurine. Moreover, analysis of the urine by gas-liquidchromatography (21) has demonstrated that within thelimits of the method, 100% of the label in the urine ispresent as free mevalonic acid. It is likely that, as sug-gested by Gould and Popjak (17), this radioactivityrepresents primarily the D-isomer of ["C] mevalonate.The relative importance of the kidneys in the metabolismof mevalonate is emphasized by comparison of the totalmetabolites of mevalonate present in the kidney, with

LIVER INTESTINE

EOTOTAL 1C METABOLITES_ NONSAPONIFIABLE LIPID,1M PRENOIC ACID 1

E ALLYL PYROPHOSPHATE

2TIME (HOURS)

FIGURE 5 Fractionation of mevalonate metabolites following intravenous administration ofDL- [2-"C] mevalonate.

1306 K. H. Hellstrom, M. D. Siperstein, L. A. Bricker, and L. J. Luby

2 24 48 1 2TIME (HOURS)

FIGuRE 6 Fractionation of mevalonate metabolites follow-ing intraperitoneal administration of DL-[2-14C]mevalonate.

those of the liver and intestine (hatched bars, Fig. 5).By 30 min the kidneys were found to contain 15% of theinjected [14C]mevalonate as mevalonate metabolites.Since only the L form of mevalonate is metabolized inthe body, actually 30% of the "metabolizable" mevalo-nate is present in the kidneys at this time interval. Bycontrast, the liver, 30 min after injection of mevalonate,contained only 3% of the administered 14C as metabolicproducts of mevalonate, while the intestine accumulatedonly 0.5% of the label during this 30 min period. More-over, this predominance of the kidneys in mevalonatemetabolism persists throughout the 1st and 2nd h afteradministration of the [1'C]mevalonate, and in fact therelative concentration of the label in the kidneys is moremarked at the 4th h of the study, at which time over 20times the amount of 14C is found in the kidneys than ispresent in the liver.

The data in Fig. 5 also document that the majority,averaging over 90%, of these renal metabolites of mev-alonate is recoverable in the nonsaponifiable lipid frac-tion with only a few percent noted in the acidic or allylpyrophosphate fractions. This distribution of mevalo-nate metabolites in the kidney was observed as early as30 min after injecting the [14C]mevalonate and persistedthroughout the entire 4 h of observation. A slightlyhigher percentage of the 14C was noted in the acidicfraction of the liver; however, even in this tissue it isclear that the nonsaponifiable fraction contains the ma-jority of mevalonate metabolites. Finally, in the intestinethe predominant mevalonate metabolites were againrecovered in the nonsaponifiable fraction.

Subsequent to the above studies it was recognized that,while the amounts of mevalonate employed are much less

tabolism, these levels of mevalonate probably far exceedthan those used in previous studies of mevalonate me-the extracellular mevalonate pool; however, studies em-ploying tracer amounts of DL-mevalonate (0.5 lug or 0.25Ag of L-mevalonate) have given very similar results, i.e.in 2 h 12% of the injected DL-[2-UC]mevalonate was me-tabolized by kidneys and 3% in the liver.

As indicated in Fig. 6, when the labeled mevalonatewas administered by the intraperitoneal route, an identi-cal tissue distribution of 14C was observed even after 48h. Moreover, the pattern of mevalonate metabolism, withthe predominance of label in the nonsaponifiable fractionof the kidneys, which was observed following the intra-venous administration of mevalonate, was confirmedwhen the labeled mevalonate was given by the intra-peritoneal route.

Taken together these studies therefore provide strongevidence not only that the kidney serves as the majortissue of mevalonate uptake but that in addition the kid-ney may well be the major site of metabolism of circu-lating endogenous mevalonate.

Subfractionation of nonmaponifiable lipids derived from[2-"C]mevalonate. To establish whether the kidneysplay a role in sterol metabolism, it was necessary todemonstrate that there is a renal conversion of circulatingmevalonate to precursors of cholesterol or to cholesterolitself. Consequently the nonsaponifiable 14C recoveredfollowing the intravenous injection of 0.5 ug of [2YGC]mevalonate was subfractionated by thin-layer chromatog-raphy, as described under Methods, and the bands cor-responding to squalene, lanosterol, and cholesterol wereassayed for 14C. The results (Fig. 7) indicate a markedqualitative difference between kidney and liver in themetabolism of circulating mevalonate. Within 30 min65% of the [14C]mevalonate present in the nonsaponi-fiable fraction of the kidney has been incorporated intosqualene with 21% in lanosterol and only 10% in cho-

_

80KIDNEY

C0

, 60

o40

0

0~

£0 60-LIVER

40

20f-F

= SqualeneLanosterol

1 Cholesterol

0.5 2TIME (HOURS)

FIGURE 7 Subfractionation of nonsaponifiable metabolites ofDL-[2-14C]mevalonate following intravenous administration.

Mevalonic Acid Metabolism In Vivo 1307

1,0002

._

%.)

-J0,

%_ 500a

-J

Fe

ENE

-o cpm/min

CHROMATOGRAPH'TRACING

0'AI _

0 6 12 16 24 30 36 42 46MINUTES

FIGURE 8 Gas-liquid chromatographic identification of squalenemevalonate in the kidney.

54 bU 66 r

as the major metabolite of

lesterol. By contrast, even at the earliest interval studiedthe liver had incorporated the injected mevalonate pri-marily into cholesterol. This general pattern of mevalo-nate metabolism was maintained both in the kidneys andliver 2 h after mevalonate administration.

In view of the importance of the identification ofsqualene as the major end-product of mevalonate in thekidneys, the squalene band isolated by thin-layer chro-matography was further identified by gas-liqid chroma-tography using OV-17 as the stationary phase, tempera-ture 2160 C, gas flow 100 ml/min. As shown in Fig. 8,squalene under these conditions has an Rf of 9 min, andas is apparent at least 95% of radioactivity characterizedas squalene on thin-layer chromatography is in factfound in the squalene peak of the gas-liquid chromato-

10 KIDNEY

g8-z § | 1 B ~~LANOSTEROL|6 MI~~~~~~DCHOLESTEROL

ZN

,,e,2 ~ ~ ~ LVE0

1 2 24 48TIME (HOURS)

FIGURE 9 Subfractionation of nonsaponifiable lipids in kid-ney and liver following intraperitoneal administration of DL-[2-"4C] mevalonate.

gram. An incidental finding of this gas-liquid chroma-tographic analysis of squalene is that when analyzedwithout carrier squalene, the natural concentration ofisqualene in renal tissue is approximately 14 ttg per gramof tissue.

Examination of the nonsaponifiable end-products ofmevalonate metabolism was carried out at longer inter-vals, 24 and 48 h, following the administration of themevalonate by the intraperitoneal route (Fig. 9). At theearlier time intervals, i.e. 1 and 2 h, the mevalonatemetabolites in renal tissue conform to those found fol-lowing the intravenous administration of mevalonate inthat the synthesis of squalene and lanosterol by the kid-ney represents the predominant fate of the mevalonate.At 24 and 48 h, however, "4C in these precursors of cho-lesterol has decreased markedly, and the preponderanceof radioactivity is now found in 27 carbon sterols, pre-sumably cholesterol itself.

The metabolism of substrate amounts of circulatingmevalonate. In order to determine whether larger con-centrations of mevalonate would be similarly metabolized,the fate of this compound was examined in animals re-ceiving 1 mg of DL-[2-'4C]mevalonate intravenously.

As indicated in Fig. 10, the distribution and metabo-lism of mevalonate under these conditions is very dif-ferent from that noted with smaller amounts of meva-Ionic acid, in that at the 2 h time interval studied, theliver rather than the kidneys represents the major siteof mevalonate metabolism. In addition, the metabolismof mevalonate in the liver, as expected, results in a pre-ponderance of the mevalonate metabolites being presentin the 27 carbon sterols, presumably cholesterol. The

1308 K. H. Hellstrom, M. D. Siperstein, L. A. Bricker, and L. J. Luby

finding that the metabolism of these large, probably un-physiologic, doses of mevalonate leads to the liver as-suming the major role in mevalonate uptake may serveto reconcile the results of the present studies with thoseof previous investigators (19) in which the distributionof only relatively large doses of mevalonate was studied.

In vitro metabolism of mevalonate by kidney andliverMetabolism of DL-[2-'4C]mevalonate. To rule out the

possibility that the mevalonate metabolites found in thekidneys might be derived via the blood stream from theliver, studies were carried out under in vitro conditionsin an attempt to determine whether in fact the kidneycan, in the absence of liver, actively convert mevalonateinto sterols and sterol precursors. 500 mg of either kid-ney or liver slices were incubated as described underMethods with tracer doses of [2-'C] mevalonate for 2 h;the nonsaponifiable lipid fraction was isolated, assayedfor "C, and then further subfractionated into its sterolcomponents. The results demonstrate first (upper halfof Fig. 11) that kidney slices are capable of significantconversion of mevalonate into nonsaponifiable lipids. Intwo experiments the kidneys incorporated 17 and 19% ofthe added mevalonate to nonsaponifiable sterols whilethe comparable figures for the liver were 20 and 29%.

As expected the conversion of ["C] acetate to non-saponifiable sterols was significantly less than for mev-alonate in each of the two tissues. However, while theliver converted 2% of the added acetate to nonsaponi-fiable lipids, the kidney was capable of incorporating onlyan insignificant 0.03% of the acetate into this fraction.

TOTALANDNONSAPONIFIABLE

I LIPIDS I ,

10

_ TOTAL T=NOSAPONIFIABLE b 5

w.,

SUFMACTIONATIONOF tNONSAPONIFIABLE

LIPIDS j

10s

=:: SQUALEN

1::LvINOSTENOL °

CHOLESTEROL Z

FIGURE 10 Distribution and metabolism of substrate amountsof DL-[2-"C]mevalonate 2 h following intravenous adminis-tration.

IUNLT~ LIVLFK

COUVEIMSO OF F30 _-SU STRATE TO

NONSAPONIFABLE | 20 _

80 1

SUSBRACTIOATIONOF

NONSAPOIFIALE

LIPIDS4

MDSOVALENE STEROL

MI_ LANOSTEROL

CHOLESROL

0

FIGURE 11 Synthesis of nonsaponifiable lipids from DL-[2-"C] mevalonate by kidney and liver slices in vitro.

The latter finding would suggest that the kidney has onlya limited ability to synthesize mevalonate from acetate,a suggestion that will be confirmed directly in a laterexperiment (see below). On the other hand, the subse-quent steps in sterol synthesis that are responsible forthe conversion of mevalonate to nonsaponifiable cho-lesterol precursors are obviously carried out effectivelyby renal tissue.

The latter conclusion was supported by the subfrac-tionation of the nonsaponifiable lipid synthesized by thekidney, an analysis that led to results (lower half of Fig.11) almost identical to those observed in the in vivo ex-

periments. Specifically, the ability of the kidney to con-

vert mevalonate predominately to squalene and lanosterolrather than to cholesterol is again confirmed; and thissame pattern is demonstrated with [2-14C]acetate as thelabeled substrate. As is well known, liver slices are read-ily capable of incorporating both acetate and mevalonateinto cholesterol, and this finding is likewise confirmedby the results of the studies in Fig. 11.

In vitro metabolism of L-[l-"C]mevalonate. An in-dependent means of determining the relative abilities ofvarious tissues to metabolize mevalonate became avail-able with the synthesis of L- [I-'C]mevalonate (seeMethods). With the use of this label the conversion ofthe mevalonate to isoprenoid units could be simply anddirectly assayed by determining the ability of the kidneyand liver to convert [1-"C]mevalonate to "CO2. Resultsof such an experiment are presented in Table I. Again, itis apparent that per gram of tissue the kidney is ap-

proximately as capable as is the liver of converting thephysiologically active form of mevalonate to isoprenecompounds.

Mevalonic Acid Metabolism In Vivo 1309

TABLE IMetabolism of L-[1-14C]mevalonate to 14CO2

Nanomoles/Percent gram

Organ added 1"C tissue

Liver 28.6 22.9Kidney 25.8 20.6Intestine 2.7 2.2Spleen 1.7 1.4

200 mg of tissue slices incubated with 16 nmol of L-[1-'4C]-mevalonate for 1 h in Krebs' phosphate buffer pH 7.0. 14CO2was collected as previously described (3).

Sites of endogenous mevalonate productionThe observation that plasma mevalonate represents

an actively turning over pool, which is metabolized inthe kidney, raises the question as to the tissue of originof this endogenous mevalonate. Slices from a number oftissues were therefore incubated with [1-14C]acetate inthe presence of an unlabeled pool of mevalonate and theincorporation of the labeled acetate into mevalonate wasdetermined as described in earlier studies (4, 21). Asshown in Table II, of the tissues studied, liver has byfar the greatest ability to synthesize mevalonic acid fromacetate; the kidney and testes in fact had only a barelydetectable rate of mevalonate synthesis, which averagedat most 1/20 of the liver. The finding that the kidneysynthesizes mevalonate at only a minimal rate was ofcourse anticipated by the observation, described in anearlier section of this study, that the conversion of ace-tate to sterols procedes very slowly in renal tissue.

Metabolism of exogenous mevalonateMetabolism of orally administered DL- [2-"C]mev-

alonate. To study the metabolic fate of exogenous mev-alonate, DL-[2-14C]mevalonate in a tracer dose was ad-ministered by stomach tube to 200 g Sprague-Dawley

TOTAL AND 9zIOh LIVERNONSAPONIFIABLEI !

LIPIDS x 5

_TOTAL - °INONSAPONIFIABL W

SUBFRACTIONATIONOFNONSAPONIFIABLE

LIPIDS

EN SQUALENE

m LANOSTEROL

EM CHOLESTEROL

TABLE I IMevalonate Synthesis in Rat Tissue Slices

[2-14C]acetateconverted to

mevalonate/gTissue tissue/h

Liver 14.0Kidney 0.6Testes 0.6Skin 0.2Spleen 0Brain 0

Incubation conditions as described under Methods.

rats, and 2 h later the animals were killed and the dis-tribution of 14C determined as previously described. Theresults presented in Fig. 12 demonstrate a rather strik-ing difference in the sites of metabolism of exogenousmevalonate as compared with that of endogenous origin.While, as emphasized above, systemically administeredmevalonate is metabolized primarily, by the kidneys,the results in the upper half of Fig. 12 demonstrate thatafter 2 h the majority of the orally administered [14C]mevalonate is present in the intestinal wall and in theliver, with significant but still lesser amounts of 1'Cmetabolites being found in the kidneys.

The label in each of these tissues, including the in-testine, is found primarily in the nonsaponifiable lipidsindicating that the intestine itself must have metabolizedthe mevalonate delivered to it from the lumen. Moreover,fractionation of the nonsaponifiable fraction of the mev-alonate metabolites, as demonstrated in the lower half ofFig. 12, demonstrates that the [1'C]mevalonate in theintestine was present primarily as cholesterol. This wasalso the case with the liver. On the other hand the ex-ogenous mevalonate reaching the kidney is metabolized,as was endogenous mevalonate, primarily to squaleneand lanosterol during the time interval studied.

INTESTINE

EI

iF. . . ...-....

FIGURE 12 Distribution of metabolites of [2-TC]mevalonate 2 h after oral administration.

1310 K. H. Hellstrom, M. D. Siperstein, L. A. Bricker, and L. 1. Luby

0sr I

.w4I.ZO

< 2LLO,.)o v

~-Iz NIL

L) -i

wa.

24

22

20

18

16

14

12

10

8

6

4

2

0

FIGURE 13 Distributionintraportal injection.

KIDNEY

LIVER

1 2

TIME (HOURS)of DL- [2-"C] mevalonate following

These data suggest that orally administered mevalonateis largely metabolized by the intestine to nonsaponifiableendproducts that would be delivered, primarily via thelymph, to the systemic circulation and from there takenup preferentially by the liver. Alternately, mevalonatemight be absorbed by the portal circulation and hencedelivered and metabolized by the liver, and only sec-ondarily by the kidney. To examine this question a tracerdose of [2-"C]mevalonate was administered through anintestinal venule into the portal circulation, and the dis-tribution of lipid "C was determined. As shown in Fig.13, at both 1 and 2 h the kidney is the major site ofmevalonate concentration; and as was observed followingadministration of mevalonate by the systemic route, thevast majority of the label present in the kidney has beenconverted to nonsaponifiable compounds. While the ratioof 14C in the kidney to that in the liver is between 3 and4: 1 rather than the 5 and 7: 1 ratio observed followingthe systemic administration of labeled mevalonate, it isstill apparent that most of the mevalonate administeredinto the portal vein must pass through the liver to bedelivered to and metabolized primarily by the kidneys.It is very likely therefore that the exogenous mevalonatethat is adsorbed as such from the intestine must, likeendogenous mevalonate, be chiefly metabolized in thekidneys.

Most exogenous mevalonate, however, is metabolizedby the intestine before it can reach the systemic circula-

tion. This conclusion was further supported by experi-ments in which a lymph cannula was inserted into a ratand the percent of administered mevalonate that was ab-sorbed as nonsaponifiable lipid via the lymph determined.The results shown in Table III demonstrate that over35% of the total administered DL-mevalonate or over70% of the L-mevalonate in the racemic mixture has beenconverted to nonsaponifiable material, in all probabilityby the intestine during the 6 h period of the experi-ment; moreover, of this nonsaponifiable sterol, over 18%had been delivered to the lymph during the 6 h of theexperiment. Finally, it is of interest that subfractionationof nonsaponifiable "C in the lymph reveals that approxi-mately 80% of this lipid is located in the squalene bandon thin-layer chromatography.

It would seem highly likely, therefore, that exogenousmevalonate must be primarily metabolized by the in-testine to cholesterol and squalene during the process ofabsorption. The latter lipid is then delivered to the sys-temic circulation via the lymph and in all probability issubsequently further metabolized by the liver to cho-lesterol.

DISCUSSIONThe primary purpose of the present study was to de-termine the metabolic fate of circulating mevalonic acidin the intact animal.

Clearly, the most unexpected finding to developfrom this investigation is that the kidneys representthe major organ responsible for the metabolism ofcirculating mevalonic acid. Despite the fact that, incontrast to the liver, the kidney has almost no ca-pacity to synthesize mevalonate, 60-70% of the ["C]mevalonate recovered, whether the labeled compound wasgiven intravenously or intraperitoneally, is found in thekidneys while less than 20% is accounted for in the liver.This ability of the kidney to take up mevalonate waseven more clearly illustrated by the fact that per gram oftissue the kidney concentrates ["C] mevalonate to an

TABLE I I IConversion of Orally Administered [2-'4C]mevalonate into

Nonsaponifiable Lipids in a Rat with a LymphFistula for 6 h

Liver Kidney Intestine Lymph

Nonsaponifiable* 1.6 1.1 15.4 18.6Subfractionj

Squalene 5.0 39.0 16.0 80.0Lanosterol 1.0 28.0 3.0 9.0Cholesterol 80.0 27.0 62.0 6.0

* Percent administered [E4C]mevalonate in nonsaponifiablelipids.I Percent of total nonsaponifiable lipids.

Mevalonic Acid Metabolism In Vivo 1311

I

.I

0-

extent 10 times that of the liver. More important, almostall of the mevalonate retained by the kidney was foundto be rapidly converted into the precursors of choles-terol, specifically squalene and lanosterol, a finding thatwould indicate that the kidney represents not only a ma-jor site of mevalonate uptake but is also the primaryorgan in which circulating mevalonate is metabolized inthe body.

These in vivo studies suggest that while the kidneycontains an active mechanism for converting mevalonateto squalene and lanosterol, the further conversion ofthese intermediates to cholesterol procedes slowly in thistissue. This observation was confirmed in tissue slicesin which it could be demonstrated that mevalonate is in-corporated into squalene at rates per gram of tissue ap-proximately equal to that of liver; however, the kidney,in contrast to liver, converts squalene and lanosterol tocholesterol to an insignificant extent. The studies of Goldand Olson (23) have shown that kidney slices convertmevalonate to Coenzyme Qo at a rate significantly morerapid than that of heart or liver. The synthesis ofCoenzyme Q was not measured in the present study;however, since Gold and Olson observed that the con-version of mevalonate to Coenzyme Q in kidney slicesprogressed at a rate less than 1/10 that of cholesteroland since the present studies in turn demonstrate that theincorporation of mevalonate into cholesterol amounts toonly 1/10 the conversion of mevalonate into nonsaponi-fiable material, Coenzyme Q synthesis could account foronly a very minor fraction, approximately 1%, of themevalonate metabolized by renal tissue.

The findings of this study obviously raise the ques-tion of the role played by the kidneys in the overallmetabolism of cholesterol, a question that can only bepartially resolved at present. In studies to be publishedin detail elsewhere),7 the concentration of mevalonate inthe blood was determined using thin-layer and gas-liquidchromatography and analyzed by mass spectroscopy. Inthe normal rat, blood mevalonate is present at a level ofapproximately 5 Ag/100 ml. At a blood volume of 9.2 mlin a 200 g rat (24) the amount of mevalonate in thecirculation totals about 0.46 stg; assuming an equilibriumthroughout the extracellular space of 24% of body weight(25), the mevalonate present in this volume wouldamount to 2.4 iAg. On the basis of the rapid turnoverphase of the L-[1-14C]mevalonate die-away curve of 5min, which probably represents this rapidly misciblepool, the turnover rate of mevalonate through the extra-cellular space would be 0.7 mg/day.

7Bricker, L. A., K. J. Hellstrom, and M. D. Siperstein. Amethod for measuring tissue levels of mevalonic acid. Inpreparation.

A reasonable estimate of total 24 h sterol productionby a 200 g rat is approximately 6 mg (26), and if, as isgenerally assumed, mevalonate is an obligatory inter-mediate in sterol synthesis it would follow that about 14mg of mevalonate would be required to statisfy thisdaily sterol requirement. If it is assumed that this figureapproximates the total mevalonate produced per day,then the above data would indicate that about 5% ofthe mevalonate escapes, presumably largely from theliver, into the plasma to comprise the circulating misciblemevalonate pool examined in the present study. It is pos-sible that the kidney utilizes this circulating mevalonatepool to synthesis small amounts of cholesterol for struc-tural purposes. On the other hand, both the in vivo andin vitro results demonstrate that the kidney convertssqualene and lanosterol to cholesterol at a slow rate, andthe evidence from the present study demonstrates thatthe kidney does not excrete detectable amounts of thesemevalonate metabolites into the urine. It seems not im-probable, therefore, that the kidney may release a por-tion of these cholesterol precursors into the circulationto be carried to the liver, there finally to be converted tocholesterol. Goodman's finding that significant quanti-ties of squalene are present in the blood of both manand rat lends some support to the likelihood of such anactive circulating pool of squalene in these species (27).

It should be emphasized that, while the above con-clusions are based largely on the tissue distribution ofinjected DL-mevalonate, i.e. the mixture of the metaboli-cally inactive D-isomer and the active L-isomer, it islikely that the findings are physiologically sound. Ex-amination of the tissue distribution of the metabolites ofmevalonate, which could be derived only from theL-isomer, confirms the fact that the kidney is the majorsite of mevalonate metabolism; and secondly, the turn-over studies using the physiologically active L-isomer ofmevalonate confirmed the turnover data obtained initiallywith the racemic mevalonate.

The metabolism of exogenous mevalonate differs fromthat of the endogenously derived compound in that, wheningested in the diet, this sterol precuror is no longermetabolized primarily by the kidneys. Ingested mevalo-nate is largely converted in the intestinal wall to non-saponifiable lipids, which are then absorbed via the lymphinto the systemic circulation. It is likely therefore thatthe intestine rather than the kidneys plays the more im-portant role in the primary metabolism of the smallquantities of mevalonate that may be consumed in thediet.

Finally, the conclusions of this study are restricted atpresent only to the rat, and it remains to be determinedwhether the importance of the kidney in mevalonate me-tabolism, which has been demonstrated in this study, canbe applied to other species as well.

1312 K. H. Hellstrom, M. D. Siperstein, L. A. Bricker, and L. J. Luby

ACKNOWLEDGMENTSWe wish to express our appreciation for invaluable assist-ance in the study to Dr. Millie Wiley, Marianne McKissock,Susie Abright, Judith Hedderman, Judy Johns, VioletFagan, Ann Gyde, Alice Clark, and Carol Jenkins.

This work was supported by U. S. Public Health ServiceGrant CA 08501, and by Damon Runyon Memorial Fundfor Cancer Research Grant 747.

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Mevalonic Acid Metabolism In Vivo 1313