Sterol biosynthesis in vitro of rat renal inner medulla

Biochirnica ,O Elsevier BBA

306 (1973) 237-248 Company, Amsterdam

~ Printed

in The Netherlands

56252

STEROL

EIGIL

et Biophysicn Acta, Scientific Publishing

BIOSYNTHESIS

BOJESEN,

INGE

IN VZTRO OF RAT RENAL

BOJESEN

Institute of‘ Experimental Hormone DK-2100 Copenhagen (Denmvk)

and KIRSTEN Research,

(Received August 18th, 1972) (Revised manuscript received December

13th,

University

INNER

MEDULLA

CAPITO of‘ Copenhagen,

71, Nsrre

All&.

1972)

SUMMARY

In vitro studies of lipid production of rat renal papillae with [2-‘4C]acetate or [2-‘4C]mevalonate have shown that sterologenesis is an important part of the total lipogenesis in this tissue. However, only a very small amount of labelled cholesterol could be detected in the sterol fraction. The lipid extract of tissue slices was directly subjected to thin-layer chromatography or reversed-phase column chromatography. The purified sterols were analyzed by radio gas-liquid chromatography on three different stationary phases, argentation thin-layer chromatography and digitonin thin-layer chromatography. By these techniques it was demonstrated that nearly all the sterol radioactivity could be accounted for by 4,4, r4a-trimethyl-gcc-cholesta-8,24-dien-3/?-ol (lanosterol) and three sterols, which were slightly more hydrophilic than cholesterol. Two of these sterols had the same chromatographic mobilities as cholesta-5,24-dien-j/i-o1 (desmosterol) and gee-cholest-7-en-3/J-01 (lathosterol), respectively. The third compound, which accounted for the largest fraction of the radioactivity in the sterol fraction, could be tentatively identified as 5K-cholesta-7,24-dien-3/I-o1.

INTRODUCTION

The recent interest in medullary lipids is largely due to observations suggesting that specific lipids, produced by the medulla, participate in the control of blood pressure (Hickler et al.‘, Lee et al.’ and Muirhead et ~1.~). It has been shown that the number of lipid droplets of the interstitial cells of the rat renal papilla varies rapidly with the functional state of medulla (Nissen4T5). These lipid droplets, morphologically very similar to those of the steroid hormone-producing cells, are a characteristic feature of the interstitial cells in this tissue (Osvaldo and Latta6). Furthermore, the long-chain fatty acid composition of the triglycerides is different from that of the depot fat and the peripheral plasma lipids (Nissen and Bojesen7). Such evidence suggests that the lipid droplets in the interstitial cells of the rat

238

E.

BOJESEN

et cd.

renal papilla are produced locally. In order to substantiate this, the lipogenesis in this tissue was studied in vitro with 2-i4C-labelled acetate. These studies revealed that a chain elongation of fatty acids in fact took place (unpublished). Furthermore, it was observed that almost half of the lipophilic radioactivity had a chromatographic mobility slightly more hydrophilic than cholesterol. It was therefore decided to purify these compounds for identification. MATERIALS

All radioactive compounds were purchased from The Radiochemical Centre, Amersham, England and they were used without further purification. 5b-Cholestan3cc-01, 5r-cholestane, cholesterol and inactive DL-mevalonic acid were procured from Sigma. Standard sterols lathosterol and desmosterol were procured from Schwarz/ Mann and Applied Science, respectively. Other chemicals used were from Merck (analytical grade). Female SPF-Wistar or Sprague-Dawley rats (200-250 g) were used in this work. METHODS

Incubations Unanaesthetized animals were decapitated and the renal papillae cut out as described by Nissen and Bojesen’. Longitudinal slices, 0.25-0.35 mm thick, made by a Stadie-Riggs tissue slicer were incubated in Krebs-Ringer phosphate medium (pH 7.4) (95.0 mM NaCl, 4.5 mM KC], 1.0 mM CaCl,, 1.0 mM KH,PO,, 1.0 mM MgS04. 7 H,O, 3.6 mM NaHCO,. Phosphate buffer: 2.6 mM NaH,PO, and I 1.2 mM Na,HPO,, IO mM glucose). The substrates used were sodium [2-14C]acetate and ot_-[2-‘4C]mevalonic acid lactone hydrolyzed in a bicarbonate solution (more than 99 % according to liquid/liquid partition assays). Four different types of incubations were used. (I) 2-h incubations with an acetate concn of 4.5 . 10~~ M and spec. act. 55 Ci/mole, (II) 2-h and (III) 7-h incubations both with a mevalonate concn of 150 * IO-~ M and spec. act. 0.83 Ci/mole and finally (IV) 2-h incubations with a mevalonate concn of 24 1 I op6 M, spec. act. I 0.3 Ci/mole. All incubations were carried out at 37 “C with air as the gas phase in stoppered flasks, supplied with a centre-cup containing 40 $2 M NaOH as CO, absorber. Incubation of 250 mg papillary tissue (wet weight) was carried out in 16 ml buffer. The pH of the medium decreased during 3.5 h incubations to 6.8-7.0. Therefore, in the case of 7-h incubations the medium was renewed after 3.5 h. The slices were removed after incubations, rinsed and homogenized in phosphate buffer. The homogenate (I ml) was extracted once with I ml dichloromethane-methanol (2 : I, by vol.) and twice with I ml dichloromethane, the pooled extracts were dried with Na,SO, and the solvent evaporated in a stream of N,. The lipids were separated in classes according to the methods of Skipski et al.8 for qualitative investigations or subjected to the purification procedure. Pur$cation Reversed-phase column chromatography. filled with a suspension of kieselguhr (silanisiert

The column (5 mm x 260 mm) was 0.2-0.3 mm fur die Gaschromatografie,

STEROL BIOSYNTHESIS

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239

Merck) in heptane. After placing a piece of filter paper in the stop cock bore, a displacement of the heptane with the mobile phase by suction (retention volume 1.5 ml) was possible. The free sterols were eluted from the column with the mobile phase: acetonitrile-acetic acid-water (75 :20:7, by vol.) saturated with heptane. Fractions I-IO (I ml) and I 1-20 (2 ml) were collected. Recovery of added radioactive sterols was 97-100

%.

Argentation-column chromatography. The free sterols were eluted from a AI,O,AgNO, (4.5 : I. I, by wt) column (5 mm x 260 mm) with chloroform-dimethyl ketone (98:2, by vol.) in Fractions I-IO (I ml) and 11-20 (2 ml). Recovery of added radioactive sterols was 77-80 %. Argentation thin-layer chromatography. AgNO, thin-layer chromatography made according to the procedure of Tu et aZ.’ with the modification that all the matograms were run at 4 “C. The spots, which were removed from the thin-layer were eluted with 3 x 0.5 ml dichloromethane-methanol (2: I, by vol.) after the tion of 100 111I % KCN in water and dried with Na,SO,, a procedure which tained LOOy/, recovery.

was chroplate, addiascer-

Digitonin-silica thin-layer chromatography. A modification of the micromethod described by Taylor” was used in this work. The plates activated for I h at I IO “C were developed for 4 h in the solvent system : 2-methylpropan-2-ol-water-ethyl acetate (50: 10:50. by vol.) saturated with digitonin. Radio gas-liquid chromatography. The sterols were converted to trimethylsilyl ethers (Brooks et al.“), and injected dissolved in pure hexane on a Pye gas chromatograph equipped with the detector assembly for radio chromatography manufactured by Panax Equipment Ltd (Simpson”). The ethers were analyzed on 5-ft glass columns of 4-mm internal diameter with liquid phase I ‘A OV-I on 100-120 mesh Gas Chrom Q (operating conditions: carrier gas flow rate 60 ml/min, column temperature 250 “C) with liquid phase I y! EGSS-X on 80-100 mesh Diatomite CQ (operating conditions: carrier gas flow rate 60 ml/min, column temperature 205 “C) and with liquid phase 5 y/, PEGA on 80-100 mesh celite JJ’CQ (operating conditions: carrier gas flow rate 120 ml/min and column temperature 200 “C). In all experiments the detector heater and injection heater was set 20 “C higher than the oven temperature. Scintillation counting. Radioactivity was measured in a Packard liquid scintillation counter, Model 3315 with an efficiency of 57.5 % for 14C and 29 % for 3H under the conditions employed for counting. 5 ml of a liquid scintillation solvent ( IOO g naphthalene, 6.8 g PPO and 0.30 g POPOP per 1 dioxane) was used. RESULTS

Class thin-layer chromatography according to Skipski Type 1 incubations (2-h with [2-14C]acetate) showed on an average that 5 nmoles acetate are incorporated in lipids per g tissue (wet weight) per 2 h. The distribution in lipid classes, presented in Table I, first column, revealed a considerable synthesis of

E. BOJESEN

240 TABLE

ef ol.

I

DISTRIBUTION INCUBATIONS

OF 14C IN LIPIDS EXTRACTED FROM PAPILLARY WITH [z-14C]ACETATE AND [z-“C]MEVALONATE

Approximately 25 mg papillary slices were incubated Substrate concns:t IO-* M glucose and 4.5. IO-~ 24. IO-” M [2-‘?Z]mevalonate (spec. act. IO Ci,‘mole). layer chromatography, localized by spraying, scraped counter. Results given as “j<; S.E. Lipid cltrrs,~

Phospholipids Unidentified sterols Lanosterols Free fatty acids Triglycerides Sterolesters Squalene

[r-‘JC],4ccttrfc imuhario~~ with air as gas /JhLI.W (type I, II = 14) 22.1

I.5

33.5 -7 1.3 13.0 :: 0.7 6.1

21.6 0.3 2.6

Jo 0.5

+ 1.1

SLICES

AFTER

for 2 h in 2 ml phosphate buffer (pH 7.4). M [z-‘JC]acetate (spec. act. 55 Ci/mole) or Lipids were extracted and separated by thinoff the plate and counted in a scintillation

[r-‘4C]Acernte

itlcubtrtion with nitrogen as gas phase itype I, II = 41

[z-‘“C]Meralontrte incubatiun with air as gas phase itype IV, II : 4)

26.3 ?I 5.7 3.5 I~_0.2 5.2 :r 3.5

6.0 1 2.2 56.8 _I~0.3 22.1 1 0.7

I.9

1: 0.2

2.8

d_ 0.8

11.8t 3.3

4.5 I

45.6 -

7.9 k 1.3

I.0

LO.1

0.4

1.6

a compound running as lanosterol and of one or several compounds slightly more hydrophilic than cholesterol. The exclusive sterol nature of these compounds was ascertained by gas-liquid chromatography and anaerobic incubations (Table I, second column) as well as incubations with mevalonate (Table I, third column). After acetate as well as mevalonate incubations the hydrophobic part of the cholesterol spot contained very little 14C , indicating that at most 12 “/, and probably a smaller fraction of the total sterol 14C was present as cholesterol. A more quantitative estimation could not be achieved by this crude analysis which nevertheless clearly revealed a striking contrast to the results of incubations of whole kidney slices reported by Dietschy and Siperstein13. For this reason papillae slices were incubated in KrebsRinger bicarbonate medium and also liver slices in the phosphate medium. Neither of these control experiments indicated that the medium was important, since cholesterol was also poorly labelled compared with other sterols when bicarbonate medium was used, while liver slices in the phosphate medium gave the expected high yields of cholesterol according to thin-layer chromatography and gas-liquid chromatography analyses (90 f/i of the total sterol 14C). The fact that we were using plain atmosphere as the gas phase could not be of any importance, since the Q,, of papillae slices is as low as 4.5 ~1 O,/mg dry tissue per h. The rute

of sterologenesis

When labelled unidentified sterols were produced for identification purposes, we noticed that the rate of sterol accumulation was dependent upon incubation time as well as substrate concentration. An evaluation of such quantitative aspects was possible, since the yield of the lipid extraction procedure was maximized and the chromatographic losses small. Only about I .5-2 times more of the labelled sterols, including lanosterol, could be extracted after 7-h, than after 2-h incubations. As shown in Table II, the rate of accumulation of radioactive unidentified sterols was increasing during the initial 3 h

STEROL

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in contrast to the decreasing rate of accumulation of other lipids. The rate lation of labelted squalene and Ianosterol was found to be highest after the maximal value is probably reached before. The influence of substrate concentration on the sterol accumulation from the data in Table III. For the purpose of comparison the calculations upon the production of a C,, sterol, which per molecule contains I 5 14C TABLE

mg

glucose ~-

is apparent are based atoms from

11

INCORPORATION OF [z-“%]ACETATE fnmoles/g SES AFTER DIFFERENT TIMES OF INCUBATtON 25

of accumu0.5 h, while

per 2 h) INTO

THE

MAJOR

LIPID

papillary slices were incubated for 2 h in 2 ml phosphate buffer (pH 7.4) contaming and 4.5 IO-~ M [z-“%]acetate (spec. act. 55 Ci/mole). Results expressed as nmoles/g _--_. - .__ 0.5 h

I h

2h

.: h

3.5 2.4 3.0 1.0 3.6 0.6

2.5 3.0 2.5 0.9 3.4 0.5

1.7 3.4 1.4

1.7 3.4

0.4 2.4 0.4

0.3 1.4 0.2

14.1

12.8

9-7

8.0

CLASIO mM per 2 h.

_______.

Pllospholipids Unidentified Lanosterol

sterols

Free fatty acids Triglycerides Squalene and sterol Total incorporation of [2-‘Vlacetate

TABLE

esters

I .o

111

ACCUMULATED DE NOVO-PRODUCED STEROLS (nmoleslg per z h) CALCULATED ON INCORPORATED FROM [z-t4C]ACETATE AND 5 ‘“C THE BASIS OF 15 14C ATOMS ATOMS FROM [2-‘4~]MEVALONATE Renal papillary cose, containing were separated Time qf incubntiatz ih) 2 2

slices were incubated for 2 or 7 h in phosphate buffer fpH 7.4) with IO mM glu[2-‘VJacetate or [2-‘4C]mevalonate at different concentrations. Extracted lipids either by thin-layer chromatography or reversed-phase column chromatography. -..-. _-...______ COflC?l Substrate Rate of sterolI I . 1om6 M) accumulation (nrnoles/g per 2 h & S.E.) ~_~_______._______.__ -. [2-“V]Acetate 4.5 0.15 i 0.013 (n = 14) 30.0 0.68 + 0.047 fn =: II)

2 2 2 7 7 7

50.0 [2-‘4C]Mevalonate

1.3

24.0 I 24.0 f 24.0 300.0

0.60 t 5.6 2.4 6.8

300.0

9.4

0. I 3

(n = 4)

disregarding the small differ[2-14C]acetate or 5 14C atoms from [2-‘4C]mevalonate, ence between lanosterol and the other sterols. High mevalonate concentration (3 * 10~~ M) ascertained a high rate of sterologenesis during 7-h incubations. Separation and pur$ication qf unident$ed sterols The labelled sterols were purified by the different

kinds of liquid chromatog-

E. BOJESEN

242

er tll.

raphies described and column fractions were assayed by radio gas-liquid chromatography analyses recording simultaneously, the mass and the 14C activity. The major problem was to eliminate completely the large amount of unlabelled cholesterol, which was present in an amount IOO times greater than any of the labelled sterols accumulated in the tissue, even after 7-h incubations. However, this problem was solved by the following procedure in which column chromatographies were the major tools. Extensive use of thin-layer chromatography was abandoned because of incomplete re-

L-G-

h Fraction

I.

number

Fig. I. Chromatographic separation of the free unidentified sterols, cholesterol and lanosterol on a \-mmi , column (5 mm x 260 mm) of silanisiert Kieselguhr (0.2-0.3 mm). 0-0, [3H]cholestero1; r4C-labelled sterol I, II and 111, cholesterol and lanosterol synthetized by renal medulla. The solvent was a mixture of acetonitrile-acetic acid-water (75: 20:7, by vol.) saturated with heptane. Fractions of I ml and 2 ml were collected. The flow rate was 0.2 mlimin.

_Solvent ‘Front

Fig. 2. Thin-layer chromatogram of free sterols on AgNO,-impregnated silica-gel plates. The solvent was chloroform-dimethyl ketone (95: 5, by vol.). The spots were detected by spraying. I, lanosterol; 3, sterol III, da, sterol II; qb, sterol I; ga, cholesta-5,7-dien-38-01; gb, cholesterol; 6. 2. lathosterol; desmosterol.

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covery, low capacity and lack of reproducibility. [3H]Cholesterol was generally used as an internal reference standard. The initial purification step was reversed-phase column chromatography. The distribution of radioactivity on different fractions is presented in Fig. I. According to radio gas-liquid chromatography, the first peak (Fractions 4-10) contained two labelled sterols, named sterol I and II. Sterol I, being the more hydrophilic compound, was mainly present in Fractions 4-7. After repeating this column twice with Fractions 4-10, an effective separation of these two sterols could be achieved by argentation thin-layer chromatography (Fig. 2). The second peak (Fig. I) partly coinciding with the [3H]cholesterol peak was found to contain one major i4C-labelled sterol, named sterol III, which was not

Fig. 3. Chromatographic separation of free sterol III and cholesterol on a column (5 mm x 260 mm) of neutral A1203-AgN03. 0-0, [3H]cholesterol; C-O, sterol I11 and cholesterol synthetized by renal medulla. The solvent was a mixture of chloroform-dimethyl ketone (98 : 2, by vol.). Fractions of I ml and 2 ml were collected. The flow rate was 0.2 mlimin.

cholesterol. However, sterol III could be completely separated from cholesterol by two chromatographic runs on AgNO,-Al,O, columns (Fig. 3). Radio gas-liquid chromatography of the third peak (Fig. I) showed only one labelled compound with a retention time coinciding with that of lanosterol. Quantification

qf cholesterol synthesis

By the argentation-column chromatography (Fig. 3) a small fraction of i4C activity remained associated with the [3H]cholesterol marker. In order to decide whether this r4C activity represents incorporation into cholesterol, two different incubations (Types II and III) were performed. Middle fractions of the [3H]cholesterol peak from a reversed-phase column were rechromatographed by the same system and the new middle peak fractions subjected to argentation-column chromatography. Nearly all the r4C activity ran ahead of cholesterol as sterol III, but a small fraction gave an illdefined peak coinciding quite well with the 3H peak. In the case of the short incubation study, the total i4C counts were too few to permit a more rigorous identification and only a maximal figure of cholesterol synthesis could be calculated to 2.5 % of the sterol 14C, on the basis of the 14C/3H ratio of the peak samples. Increased incorpora-

E. BOJESEN

244

et

lrl.

tion of 14C in the sterol fraction was achieved by a 7-h incubation. Contrary to the 2h incubation, this experiment now resulted in as much as 7.4% of the sterol activity coinciding with the [3H]cholesterol peak. Two observations showed unequivocally in cholesterol. Firstly, the expected isotope that this r4C was indeed incorporated fractionation gave a variation of the “C/3H ratio through the peak, which was exactly cholesterol on the same the same after running a sample of authentic “C/3H-labelled column. Secondly, crystallization of the labelled compound of the peak with unlabelled cholesterol before and after acetylation showed that no other compound than cholesterol contained 14C (Table IV). These results showed therefore that labelled cholesterol could indeed be synthetized from acetate and mevalonate by the tissue in vitro although at a very low rate compared with other sterols. Radio gas-liquid

chromatography

The results from radio gas-liquid chromatography (Table V) show that the relative retention times of sterol 111and lathosterol trimethylsilyl ethers are identical on TABLE

IV

SPECIFIC ACTIVITIES CHOLESTEROL AND METHANOL-ETHANOL

(dpm/mg), ISOTOPE RATIOS (3H/‘4C) AND MELTING POINTS OF CHOLESTERYL ACETATE AFTER RECRYSTALLIZATION FROM MIXTURES Compound Cholestrryl

Cholesterol

Pool 1st crystallization Mother liquor Melting

point

TABLE

V

(“C)

‘1C

-‘Hl’“C

20.0 20.0

51.6 51.6

18.5

51.9

3H/‘4C

18.2

50.9

‘7.5 18.6

51.2 52. I

116-117

147-148

GAS-LIQUID CHROMATOGRAPHY SILYL ETHERS ON COLUMNS OF CHOLESTANE

acetate

‘T

RETENTION TIMES OF I x, OV-I, I “/, EGSS-X AND

STEROL TRIMETHYL5:/, PEGA RELATIVE

Retention times were measured from the solvent front. Retention times for cholestane liquid phases used were 6.2, 2.5 and I I min on I :i, OV-I, I % EGSS-X and 5 “A PEGA, .~ ~~~~ ~~~~~-~ Stud trimrthylsiiyl ethers Relative retention time on liquid phases

Lanosterol Ergosterol Cholesta-5,7-dien-3D-ol Cholesterol Lathosterol Desmosterol Cholest-8( 14)-en-3b-ol 5cc-Cholestan-3P-ol Sterol I Sterol II Sterol III

I “A ov-I

I % ECSS-X

5 ‘4 PEGA

2.88

3.52 3.44 3.05

3.69

2.14 I .96 2.21

2.06

2.15

2.65 3.02 2.13

3.25 2.28 2.96 3.42

I .02 2.03 2.20

3.70 3.08 2.63

4.18 3.39 2.89

TO

on the three respectively.

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all three stationary phases and that sterol II trimethylsilyl ether is eluted as desmosterol trimethylsilyl ether from the EGSS-X and the PEGA phases. Gas-liquid chromatography data suggest that sterol I is 5!x-cholesta-7,24-dien-3P-o1. A retention time for cholesta-7,24-dienol trimethylsilyl ether of 3.72 was calculated as the product of the measured retention time of the trimethylsilyl ether of lathosterol (2.65) and the retention factor (I .405) for the A24 bond estimated from the cholesterol and desmosterol gas-liquid chromatography data. This calculated value of 3.72 is identical with the retention time of 3.70 actually found for sterol I trimethylsilyl ether. It should be mentioned here that small amounts were found of a sixth r4Clabelled sterol, which may be cholesta-5,7,24-trien-3P-01. The trimethylsilyl derivative of this sterol has a retention time of 4.26 on EGSS-X columns, which is undistinguishable from 4.28, the retention time which can be calculated from the cholesta-5,7-dien30-01 data corrected for the retention factor for the A24 bond. Argentation thin-layer chromatography of this sterol supports this suggestion since the R, (0-0.07) of the compound is that expected for a sterol with three double bonds. The radio gas-liquid chromatography permitted an approximate estimation of specific activities of sterol I, II and III extracted from the tissue after incubation with [2-14C]mevalonate (spec. act. 0.83 Ci/mole). Triangulation was carried out of correspondingactivity and mass peak together with a [‘4C]cholesterol sample for calibration (spec. act. 4.2 Ci/mole, corresponding to that of the substrate). The specific activities were found to increase in the order sterol II (0.8 Ci/mole), sterol III (2.2 Ci/mole) and sterol I (4.3 Ci/mole). Since the specific activity of sterol I is approximately the same as that of the cholesterol reference, it was concluded that very little of this compound is prevalent in the tissue, whereas the two others probably are preformed compounds. Also, lanosterol had the high specific activity of a & nova-synthetized sterol. Digitonin-silica chromatography The classical digitonin precipitation of 3p-hydroxysterols is not directly applicable to the pg scale of substances available in this study. In this case, the digitonin silica thin-layer chromatography yields the same kind of information. This technique was therefore applied to sterol I, II and III and the results shown in Table VI indicate that they are all retained close to the application spot as expected for 3p-hydroxy compounds. TABLE

VI

RF OF 14C-LABELLED STEROLS ON PLATES WITH AND IMPREGNATION RELATIVE TO SD-CHOLESTAN-3a-OL

WITHOUT

DIGITONIN

In the case of digitonin thin-layer chromatography the plates contained 0.5 g digitonin per 20 g silica-gel H (Fluka) and the developing solution (z-methylpropan-z-ol-water-ethyl acetate (~o:Io:~o, by vol.)) was saturated with digitonin. The RF of 5/I-cholestan-3cc-ol was in both cases 0.88 (0.84~092).

Sterols

Relative

RF

With digitonin [26-“%Z]Cholesterol [26-‘4C]Desmosterol 14C-Labelled sterol 14C-Labelled sterol “C-Labelled sterol

0.07

Without 1.00 1.10

I II III

0.07 0.03 0.07

1.10 I.10

1.03

digitonin

7-46

E. BOJESEN

et al.

The medium The medium from the incubations has been extracted on several occasions. The medium extract contains in general about I ,7 % of the radioactivity found in the tissue extract and no “C-labelled compounds, other than those found in the tissue extract, were observed. Damage of the tissue when it is sliced cannot be avoided, thus the radioactivity extracted from the medium is believed to come from the debris, which can sometimes be seen and is present in the medium after isolation of the papillary slices. DISCUSSION

This work differs in regard to techniques from most previous studies on sterol biosynthesis in renal and other tissues partly because a considerable sterologenesis was an unexpected observation. In the present experiments the tissue concentration was low, glucose was included in the medium at substrate concentration (IO mM) and acetate was present in lower concentrations approximating the tracer level. In other studies on sterol biosynthesis of tissue slices, (Daly14, Dietschy and Siperstein13, Ockner and Laster”) the concentration of tissue was much higher and labelled substrate present at the mM level. Only Daly14, working with arterial tissue and Ockner and Laster ” working with intestinal mucosa have included glucose in the incubation medium. Glucose is required by the renal papilla, since the energy expenditure is furnished to a large extent by the phosphogluconate oxidative pathway. At least this route plays a dominating part in the metabolism of glucose in the renal medulla (Bernanke and Epstein16, Sternberg et al.“) and histochemical methods have failed to show the presence of key enzymes of the Krebs cycle (Nissen and Andersen18). These two observations are in agreement with the observation that the renal papilla exhibits a Crabtree effect (Rosenthal et al.‘“) Lipogenic substrates of high specific activity were used at the beginning of this work to obtain maximal sensitivity of detection. Later on, unlabelled substrates were added to increase the yield of sterols (Table III). In all other studies cited above, the sterols produced have been precipitated with digitonin after saponification of the tissue, whereas in this study the sterols were extracted directly together with the other lipids. This procedure was applied to prevent loss by saponification of specific unknown lipids which we were looking for. This has two consequences for our data on sterols. One is that the esterified sterols have been grouped together in the sterolester class, but since this never contained significant fractions of the radioactivity (Table I) no quantitative important sterol remained undetected. The second consequence is that alkali-labile sterols missed or modified in other studies may appear in ours. However, the papillary sterols, prevalent and synthetized in vitro, are probably identical with sterols obtained from other tissues after saponification. The choice of buffer and gas phase has been commented on earlier and is of little, if any, concern. The chromatographic data, including the retention on digitonin plates, strongly suggest that sterol I, II and III are identical with the cholesterol precursors (jcccholesta-7,24-dien-$-01, desmosterol and lathosterol) known from studies on other tissues, particularly from triparanol(l-[p-(~-diethylaminoethoxy)-phenyl]-l-(p-tolyl)-~-

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(p-chlorophenyl)ethanol)-treated animals (Horlick and Avigan*‘, Frantz and Mobof these berley2’, Clayton et ~1.~~). The calculated values for rates of accumulation sterols shown in Table III are minimal values for several reasons, such as incomplete recovery, decreasing rate of production and suboptimal substrate concentration. Finally, dilution of labelled substrates and intermediates with endogenous metabolites may imply underestimation to such an extent that the results could be postulated to be completely misleading. However, the specific activity of sterol 1 corresponds approximately to that of the substrate and therefore this dilution factor was found to play no important part at least when 24 . IO -6 M mevalonate was the substrate. On the other hand, it is quite possible that the calculated rate of accumulations with an acetate concentration of 4.5 . IO-~ M is too low. In contrast to liver tissue, the renal papilla in vitro accumulates mainly a series of labelled sterols, which have been tentatively identified as recognized cholesterol precursors and very little labelled cholesterol. A similar phenomenon was noticed by Daly I4 working with rat aorta and by Ockner and Laster” working with mucosal scrapings. Since the presumptive cholesterol precursors were highly labelled, the low rate of accumulation of cholesterol indicates that the cholesterol production is greatly impeded at the latest stages. Considering the rather low substrate concentrations used in the present work (less than 3 . IO-~ M) the observed magnitude of sterologenic capacity of this tissue (between I and IO nmoles C,, sterols produced per g tissue wet weight per 2 h) is quite impressive compared with other extrahepatic tissues according to data in the literature. When the capacity is recalculated to the above-mentioned unit from the original data the values of Dietschy and Siperstein13 for the intestinal tract tissue (ileum: 7.6, transverse colon: 3.8 and stomach: 2.4) are a little higher than found in this work for the renal papilla with the same substrate (acetate), whereas they found a value for skin (0.33) which is lower. With mevalonate the value found by DalyI for rat aorta (0.1-0.2) indicates a capacity much lower than that of the renal papilla with this substrate (~-IO), whereas the intestinal mucosa (studied by Ockner and Laster’“) had a similar capacity. In spite of such a considerable capacity for sterologenesis with a low rate of cholesterol production, the free sterol fraction of lipid extracts of papillary tissue has a quantitative and qualitative composition which is roughly similar to that of most other tissues of the rat according to the data of D’Hollander and Chevallier23. These authors found that the free sterol fraction only from the skin tissues contains more than a few per cent of sterols different from cholesterol. The renal papilla contains about I mg of free cholesterol per g tissue wet weight, determined spectrophotometritally and only about to pg of desmosterol and 3 pg of lathosterol according to gasliquid chromatography data. From a physiological point of view the results were disappointing. The lipids produced in vitro from the two radioactive substrates are probably unspecific compounds widely distributed in all rat tissues. Therefore, the results lent no support to the hypothesis that the interstitial cells of the renal medulla produce lipid hormones of some kind related to the blood pressure regulation. Preliminary investigations with renal cortical slices have revealed the same phenomenon (unpublished), in contrast to the data of Dietschy and Siperstein13 who found that renal tissue has a low production of sterols among which cholesterol con-

E. BOJESEN

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stituted 79 %. Therefore, we believe that the observation identification of sterol I, I[ and III is in progress.

is important

et al.

and a complete

ACKNOWLEDGEMENTS

This work is supported by grants to Lange Bojesen from Statens Naturvidenskabelige Forskningsrad. Miss Lange Andersen and Mrs Kirsten Olsen are gratefully acknowledged for their conscientious technical assistance. REFERENCES I Hickler, R. B., Lauler, D. P., Saravis, C. A., Vanucci, A. I., Steiner, G. and Thorn, G. W. (1964) Corn. Med. Assoc. J., 90, 28oo287 2 Lee, J. B., Covino, B. G., Takman, B. H. and Smith, E. R. (1965) Circ. Res. 17, 57-77 3 Muirhead, E. E., Brooks, B., Pitcock. J. A. and Stephenson, P. (1972) J. C/in. Invest. 5r. 181-t90 4 Nissen, H. M. (1968) Z. Zellforsclr. Mikrosk. Anat. 85, 483-491 5 Nissen, H. M. (1968) Z. Zel@rsch. Mikrosk. Anut. 92, 52-61 6 Osvaldo, 1. and Latta, H. (1966) J. Ultmstruct. Res. 15, 5899613 7 Nissen, H. M. and Bojesen, I. (1969) Z. Ze/[forsch. Mikrosk. Amt. 97, 274-284 8 Skipski, V. P., Smolowe, A., Sullivan, R. C. and Barclay. M. (1965) Biochim. Biophys. Actn 106, 386-396 9 Tu, C., Powrie, W. D. and Fennema, 0. (1969) Lipids 4, 369-379 I o Taylor, T. ( I 97 I) Anal. Biochrtn. 4 I, 435-445 1 I Brooks, C. J. W., Horning, E. C. and Young, J. S. (1968) Lipids 3, 391-402 12 Simpson, T. H. (1968) J. Chromatogr. 38, 24-34 13 Dietschy, J. M. and Siperstein, M. D. (1967) J. Lipid Res. 8, 977104 14 Daly, M. M. (1971) J. Lipid Res. 12, 367-375 15 Ockner, R. K. and Laster, L. (1966) J. Lipid Res. 7, 750~757 16 Bernanke. D. and Epstein, F. H. (1965) Anl. J. Physiol. 208, 541~545 17 Sternberg, W. H., Farber, E. and Dunlap, C. E. (1956) J. Histochern. Cytochem. 4. 226-283 18 Nissen. H. M. and Andersen, H. (1971) Histochemie 27, 109-118 19 Rosenthal, O., Bowie, M. A. and Wagoner, G. (1940) Science 92. 382-383 20 Horlick, L. and Avigan, J. (1963) J. Lipid Res. 4, 160-165 21 Frantz, I. D. and Mobberley, M. L. (1961) Fed. Proc. 20, 285 22 Clayton, R. B., Nelson, A. N. and Fran& Jr, I. D. (1963) J. Lipid Res. 4. 166-176 23 D’Hollander, F. and Chevallier, F. (1969) Biochim. Biophys. Actn 176, 146-162