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Studies on the biosynthesis of cholesterol. VIII. Tissue distribution of cholesterol and presence of precursors in intact animals
Studies on the Biosynthesis of Cholesterol. VIII. Tissue Distribution of Cholesterol and Presence of Precursors in Intact Animals’ Erwin From
Schwenk, George J. Alexander and Charles A. Fish
the Worcester
Foundation Received
for Experimental Massachusetts December
Biology,
Shrewsbury,
23, 1954
Substances of a higher specific activity than the simultaneously biosynthesized cholesterol-Cl4 were found in the neutral nonsaponifiable lipides of pig livers perfused with acetate-1-CY4. Digitonin precipitates a part of these higher-counting companions (HCC) along with cholesterol (1). Fractions obtained by chromatography from these neutral nonsaponifiable lipides were partially converted, after feeding to rats, into cholesterol-CY4, and therefore include precursors in the biosynthesis of this compound (2)In intact rats, injected intraperitoneally with acetate-1-CY4, HCC could only be found in the digitonin-cholesterol when the animals were killed within an hour after the injection but not 4 hr. later (3). In these experiments the rats were worked up as a whole and consequently the isolated cholesterol was a mixture from all the tissues in the body of the animals. It seemed interesting to study the distribution of cholesterol and HCC among different tissues and in various species and to ascertain whether the HCC from intact animals contain precursors of cholesterol as was found for the HCC from liver perfusions (2) and from yeast (4).2 1 This work has been supported by the Damon Runyon Fund; the U. S. Public Health Service (Grant No. C321); an institutional grant from the American Cancer Society; and the Schering Corporation, Blootield, N. J. 2 In reviewing this manuscript one of the referees stressed the necessity of a more precise definition for HCC. The symbol HCC (higher-counting companions) is used to designate the total amount of radioactive substances accompanying cholesterol-C” in the nonsaponifiable lipides in biosynthetic experiments. Digitonin precipitates only a portion of these HCC together with the digitonide of
37
38
SCHWENK,
ALEXANDER
AND FISH
EXPERIMENTAL
A. Cholesteroland Digitonin-Precipitable HCC in Tissuesof Rats, Guinea ,Pigs and Fish Experiment 1. Nineteen male and two female rats8 (170-180 g. each) were injected intraperitoneally with 0.1 mc./IOO g. body weight of an aqueous solution of sodium acetate-l-C 14. Ten male rats were killed after 30 min., nine male rats after 240 min., and the two female rats 48 hr. after the injection. The animals were bled and the blood collected separately. Livers, gastrointestinal tracts (G.I. tracts) (including the contents), skins, lungs, spleens, adrenals, and kidneys were isolated, and all remaining tissues were combined as carcass. They were weighed and hydrolyzed with 30% KOH for 15-20 hr. on the steam bath. Without filtering from the bones, the hydrolyzates were acidified with concentrated hydrochloric acid. Ice was added to avoid overheating. The acidified mixtures were left in the refrigerator overnight, to allow the fatty acids to solidify. After addition of diatomaceous earth (Celite) the mixture was filtered with suction. The wet filter cake was boiled three times with pentane and the solution was filtered or decanted off the Celite. The acidified hydrolyzates of the smaller organs were extracted directly with pentane. The pentane solutions were extracted twice with 10% KOH with addition of ethanol for better separation of the soap solution, which was subsequently acidified to obtain the fatty acids. After twice washing with water the pentane extracts were evaporated to dryness under reduced pressure. If the residue contained water some benzene was added and again evaporated in vacua. Precipitation with digitonin and bromination, etc. of the cholesterol, as well as plancheting and counting on an end-window counter in this and in all following experiments were carried out as described before (5) (Table I). The most interesting fact in Expt. 1 is that the liver alone of all the tissues was free of HCC, even half an hour after the injection. After 4 hr., the G.I. tract and the blood had lost their HCC while the other tissues still contained some. However, after 48 hr., these substances had discholesterol-C14. These digitonin-precipitable HCC are a useful tool for the study of the biosynthesis of cholesterol in intact animals (or other biological systems) as shown in the present paper. Some of these digitonin-precipitable HCC may be precursors (E. Schwenk, Biochemical Symposium, Federation Meeting at San Francisco, April, 1955). That the part of the HCC which is not precipitated by digitonin also contains precursors is shown in this paper. 3 Charles River Strain of Sprague-Dawley rats were used in all experiments.
BIOSYNTHESIS
OF
CHOLESTEROL.
TABLE
Distribution
I
of Cholesterol-C14 and Digitonin-Precipitable Injected with CH&1400Na
= I
ChoIesteroLC’4, counts/min./mg.
--
272 428 28 37 29 48 23 15 25
I
after
.-
.-
320 270 6 20 5 0 0 0 7
9 male rats killed 240 min. Cholesterol-C”, counts/min./mg.
HCC’
I Xbrom.
Digit.
Liver G.I. tract Carcass Skin Lung Spleen Adrenal Kidney Blood
HCC
=
10 male rats killed 30 min.
Digit.
39
VIII
in Tissues
of Rats
= after
I.
EIcca
Dibrom.
2 female
Tqagt;;Ued
after
_Cholesterol-W, counts/min./mg.
-
HCC?
Digit.
Dibrom.
345 676 281
656 711 322
-
% 37 78 46 83 100 100 100 72
227 491 39 89 73 91 0 26 156
246 497 28 40 27 68 0 17 161
% 28 55 63 25 -
% -
34 -
-aHCC
y.
=
digit.
digit.
dibrom.
.I *n,-, A luu.
appeared from carcass, liver, and G.I. tract. Very important also was the absenceof any radioactivity in the cholesterol of the adrenals. Even after 240 min. when other organs of low weight like lung, spleen, or kidney contained cholesterol-C i4, the adrenal was free of it. The absence of HCC in the liver cholesterol made it desirable to investigate the change of HCC in time more thoroughly. The next experiment was therefore carried out using six time intervals. &periment d. Eighteen male rats, weighing from 170 to 190 g. were injected as previously and groups of three rats were killed at 10, 20, 40, 80, and 240 min. and at 1 week after injection. The animals were bled and the blood for each time interval was collected as were the livers, G.I. tract, skins, and remaining parts of the carcass. They were worked up as before (Fig. 1). Again no HCC were found in the liver during the experiment. The rapid incorporation of Cl4 into cholesterol in liver and G.I. tract is remarkable. Within 20 and 40 min. after injection, the cholesterol count of these tissues reached a maximum. However, while the count in the G.I.tract cholesterol remained almost stationary through most of the experi-
40
SCHWENK,
Liver
4000
r > .E 5In E a 0
ALEXANDER
40
80
AND
FISH
4000 c
240
1 wkll0
Blood
40
80
1 WI
240 Skin
G&Tract
. Carcass
.-+. P----b-- ------.o.4.D-.-.---.-.D, ;p 40
80
240
1wklO
40
Minutes
80
240
1 WI
Minutes
FIG. 1. Distribution of radioactivity in rat tissues. Specific activity of cholesterol-Cl4 and fatty acids plotted against time (Expt. 2). + - - - - - - - - - + cholesterol from digitonide. 0 0 cholesterol purified via dibromide. q m..-.-. - 0 fatty acids. One-week points for skin and carcass are drawn to the same scale as the points for the other tissues.
ment and dropped only after 1 week, the count of the liver cholesterol decreasedimmediately after its maximum was reached and was considerably reduced after 1 week. In skin, carcass, and blood, maxima were attained slowly in the first 4 hr., but after a week the count in these tissues had become nearly the same as in the liver and G.I. tract. In all tissues the fatty acids showed a maximum, almost simultaneously with the cholesterol after 2040 min. There appeared a lesswell-defined second maximum in the count of the fatty acids at 240 min., similar to observations of Beeckmanns et al. (6): 4 The
attainment
of the first
maximum
in the count
of the fatty
acids
at about
BIOSYNTHESIS
Distribution
OF
CHOLESTEROL.
TABLE
II
41
VIII
of Cholesterol-C 14) Digitonin-Precipitable HCC and Fatty Acids in Tissues of Rate S Min. after Injection with CH&““OONa Counts/min./mg. Cholesterol
HCC’
C’” Fatty
Digit.
Dibrom.
260 159 6
128 14 0
acids
%
Liver G.I. tract Carcass ~HCC~~ b Not
=
digit.
-
dibrom.
digit.
x
1058 243 b
50 91 100
100.
counted.
The liver was free of HCC in Expt. 2 even as early as 10 min. after injection A still shorter reaction time was therefore tried. Experiment S. Two male rats, 170 f 10 g. each, were injected as before and killed exactly 3 min. after the injection (Table II). In this experiment considerable HCC were found in the liver, and the radioactivity in the G.I. tract and carcass digitonides was almost entirely due to HCC. Only small amounts of cholesterol-C? had been formed. The fatty acids in the liver showed a high count. The following experiments demonstrate that species other than the rat have a different pattern of biosynthesis of precursors and cholesterol. Experiment !+. Four guinea pigs, each weighing about 800 g. were injected with 0.2 mc. of carboxyl-labeled sodium acetate-C4 each. They were killed after 15,30,60, and 180 min. Livers, G.I. tracts, and carcasses were separately hydrolyzed and worked up as usual (Fig. 2). Unlike in experiments with rats, livers as well as G.I. tract contained considerable amounts of HCC, but incorporation of Cl4 into cholesterol was much smaller in both tissuesthan in the rat experiments, even when the smaller amount of radioactive acetate which was administered is taken into the time when the cholesterol reaches a maximal count suggests a correlation between the first-formed acids and cholesterol. They may include precursors of cholesterol. Accordingly, some of the acid material obtained from the livers in Expt. 2 was fed to two rats, but the cholesterol isolated from the livers and G.I. tract of these animals contained only traces of radioactivity; a more thorough investigation of these acid fractions seems advised.
42
SCHWENK,
ALEXANDER
AND
FISH
G.I.-Tract \ \
+. ‘\\
Carcass & Blood
10 -
,+--- -*------------+ +- A 1530 60 Minutes
FIG. 2. Distribution of radioactivity cholesterol-04 and fatty acids plotted cholesterol from digitonide. 0 El -.-.-.-.0 fatty acids.
I I I :.-.
‘\
‘\
‘\
‘\
‘\
‘+
0
o--9
‘I ‘., 1, 0 J-0 180 15 30
‘\
.D---.-60
-.-.-0 180
Minutes
in guinea-pig tissues. Specific activity of against time (Expt. 4). + - - - - - - - - + 0 cholesterol purified via dibromide.
consideration. There was more cholesterol-Cl4 and especially HCC in the G.I. tract than in the liver. The maximum counts were reached in the first hour after injection as in the rats but HCC disappeared faster from the liver than from the other tissues.The fatty acids had low counts with well-defined maxima in the first 30 min. Experiment 5. A male rainbow trout, 2 years old and weighing 492.5 g. was injected intraperitoneally with 0.5 mc. of carboxyl-labeled acetateCl4 in water of 8°C. After 1 hr. the animal was killed and dissected into liver, G.I. tract, and carcass and worked up as usual. Experiment 6. Eight dogfish (Mustelus cams) pups, each weighing about 110 g., were injected intraperitoneally in water of 6°C. with 0.09 mc. of carboxyl-labeled acetate-C4 and killed 1 hr. later. For reasons of transport the tissues had to be kept for 3 days in 95 % ethanol and were then hydrolyzed and worked up as usual (Table III). The liver in these fish incorporated more Cl* in cholesterol-Cl* than the G.I. tracts or carcasses,but the values for the specific counts in all fractions were very low; HCC were present in all tissues.A large amount of activity is not precipitable with digitonin and appears in the crude nonsaponifiable or in the mother liquor from the cholesterol digitonides.
BIOSYNTHESIS
Distribution
OF
CHOLESTEROL.
TABLE
III
43
VIII
HCC in Tissues of Fishes
of Cholesterol-C14 and Digitonin-Precipitable Injected with CH#dOONa Trout COUlltS/ min./mg.
Dog!ish I-m?
Countsl min./mg.
EIcc*
-~ %
Nonsaponifiable Liver
Cholesterol-W
816” Digit. Dibrom.
%
351
5 1
80
57 29
49
--Fatty acids
11,370
Nonsaponifiable G.I. tract
Cholesterol-Cl”
122a Digit. Dibrom.
Fatty acids
26
Nonsaponifiable Carcass and blood
Cholesterol-04
6 2
136” Digit. Dibrom.
-es-
66 --------
14
6 Traces
100
630 62
-----
Fatty acids
82
---
2 2
35
-
11 2
81
149
* In these experiments the mother liquor material from the digitonide precipitation of cholesterol-C’* was counted and is reported in the cohunn for nonsaponifiable material. digit. - dibrom. bHCC% = x 100. digit.
The mother liquor residue from all digitonin precipitations in Expt. 6 was isolated and had a specific count of 338 counts/min./mg. It was chromatographed on alumina. The pentane fraction was radioactive. After another chromatogram on alumina the colorless, radioactive oil was treated with thiourea (7). Of the resulting colorless oil (26 mg.), an infrared spectrum was obtained which showed the main bands of squalene, but its fingerprint region did not allow complete identification with this hydrocarbon.
44 B. Precursors
SCHWENK,
ALEXANDER
AND
FISH
of Cholesterol in the Dig&w&-Nonprecipitable Rat and Guinea Pig
HCC from
The mother liquors of the digitonin precipitation of cholesterol from 18 rats in Expt. 2 were combined and reduced to dryness in vacua. Digitonin was eliminated as usual and the residue, 3100 mg. of brown oil, 150 counts/min./mg., a total of 465,000 counts, was chromatographed on alumina (Alcoa, grade I). Elution with pentane gave 650 mg. of a white oil which solidified partly on cooling, 79 counts/min./mg., a total of 51,000 counts. The eluates with benzene, ether, and methanol were combined and gave 2500 mg., 166 counts/min./mg., a total of 414,000 counts. Of the pentane fraction, 165 mg. was fed to a rat (Expt. 7) and the remainder purified twice through the thiourea compound. The white oil had a count of 363 counts/min./mg. However, while the infrared spectrum of this substance contained the main absorption bands of squalene, the fingerprint region did not permit positive identification with squalene. The other fractions from the chromatogram were submitted to a washout procedure as described before (2) using 3 g. of especially purified cholesterol. A quantity of 2680 mg. of cholesterol, 13 counts/min./mg., was obtained; this count remained unchanged when the substance was purified through the dibromide. The mother liquors from the washout, 146 counts/min./mg., were precipitated with digitonin and gave 13.8% of cholesterol, which after bromination had 15 counts/min./mg., in agreement with the figure above. An amount of 520 mg. of the mother liquor from the digitonin precipitation, 144 counts/min./mg., a total of 75,000 counts, was fed to two rats (Expt. 8). A male guinea pig of 935 g. was injected with 0.94 mc. of acetate-Cl4 and killed after 30 min. The neutral nonsaponifiables were obtained from liver and G.I. tract as usual and precipitated with digitonin. The mother liquor from this precipitation was worked up as described in the preceding paragraphs. A total of 257 mg. of a brown oil, 184 counts/min./mg., was obtained which was chromatographed on alumina (Alcoa, grade I). The pentane fraction gave 25 mg., 216 counts/min./mg., a total of 54,000 counts of a white oil which partly solidified on standing. It was fed to one rat (Expt. 9). The column was stripped with benzene, ether, and methanol, and the combined material, 185 mg., 127 counts/min./mg., a total of 23,400 counts, was reprecipitated with digitonin. The digitonide after decomposition gave 5 mg. of cholesterol, 93 counts/min./mg. The mother liquor from this digitonin precipitation gave 120 mg. of a brown-
BIOSYNTHESIS
OF CHOLESTEROL.
VIII
45
ish oil, 188 counts/min./mg., a total of 22,000 counts which was fed to one rat (Expt. 10). Feeding Experiments. Experiment 7. Two female rats, 185 g. each, received 165 mg. of pentane fraction, 79 counts/min./mg., a total of 13,000 counts, dissolved in 4 ml. olive oil in four feedings 6 hr. apart, two on the first day and two on the second day. They were killed 48 hr. after the last feeding. Weights: livers 20.4 g., G.I. tract 40.0 g., carcass and blood 314.6 g. Experiment 8. One female rat (185 g.) recieved 390 mg. of the substance after precipitation with digitonin 144 counts/min./mg., a total of 56,000 counts, and a second female rat (185 g.) received 130 mg., a total of 18,800 counts, in four feedings as before. Weights: liver and G.I. tract 26.3 g., 25.6 g. ; carcass and blood 146.0 g., 132.6 g. Experiment 9. One male rat (180 g.) received 25 mg. of the pentane fraction 216 counts/min./mg., from the guinea-pig experiment a total of 5400 counts, in four feedings as before. Weights: liver and G.I. tract, 40.7 g.; carcass and blood, 182.6 g. Experiment 10. One male rat received 115 mg. of the mother liquor from the guinea-pig experiment 188 counts/min./mg., a total of 22,000 counts, in four feedings as above. Weights: liver and G.I. tract, 31.6 g.; carcassand blood, 187.9 g. (Table IV). DISCUSSIONS
The experiments of this investigation show that the main part of the biosynthesis of cholesterol-Cl4 both in rats and in guinea pigs occurs in the liver and the gastrointestinal tract in the first 30-60 min. after injection of labeled acetate. This result agrees with findings of Gould et al. (8) who observed that synthesis ceasesin rats and rabbits after 4 hr., with Van Bruggen et al. (9) who showed the rapid uptake of Cl4 into cholesterol in the first hours after injection in rats and with Busch et al. (10) who noticed complete disappearance of CY4-labeledacetic acid from the blood of rats in the first minutes after intravenous injection. Considerable incorporation of Cl4 from acetate into squalene, presumably a precursor of cholesterol, was observed by Langdon et aE. (11) 30 min. after the injection. Half an hour after the injection of acetate into rats, the count in liver 6 This discussion is based on the consideration of the specific counts of cholesterol-Cl4 because calculation of the data of this investigation in the form of efficiency factors as used by Werthessen et al. (5) or by Van Bruggen et al. (9) have not given a better or different interpretation of the experimental results.
SCHWENK,
ALEXANDER
I -I
M
B 8 d
2 -
-
-
+ca
-
-
. sf$ -
-
-
co
-1 -
AND FISH
rti
co
d
-
-
-
-
-
-
-
7-l
m
5-i
rl
-
w
-
-
-
-
-
-
Q ”
Fa
-
-
-
I
BIOSYNTHESIS
OF CHOLESTEROL.
VIII
47
TABLE V Weights in Grams and Total Counts for Tissues of Rats Znjected with CH&l4OONa Taken from Experiment 1
i
Time
Liver
.----------
min.
30 Weight count/ min. x
G’l’ tract
CZWXSS
Skin
Lung
Spleen
tt$
*ttf-
Blood
Total
77.3 177.0 1126.4 314.0 11.9 11.7 15.8 0.26 59.51793.9 57.6 50.7 7.3 13.7 0.2 0 0 0 0.2 129.7
10-s
Per cent of total count 240 Weight count/ min. X
44.4
39.1
5.6
10.5
0.1
-
-
-
0.1 100.0
---------74.4 150.6 1003.2 290 11.9 13.8 15.6 0.24 60.51620.2 44.3 76.5 36.9 28.3 4.0 4.6 1.2 0 9.0 204.8
10-a
Per cent of total count
21.6
37.4
18.0
13.8
2.0
2.2
0.6
-
4.4
100.0
and G.I. tract together amounts to more than 80% of the total incorporated CY4,and 240 min. after injection these tissues still account for 60% of the total. The skin contains another 10 and 14%. This contribution of liver and G.I. tract becomes even more impressive when the weights of the tissues are taken into consideration. The contribution of tissues other than the above mentioned is very small (Table V). Whether the slow increase with time of the specific activities in these tissues is due to a sluggish conversion of precursors formed in these tissues or to the transport of precursors and/or cholesterol from liver and G.I. tract must be further investigated. Liver and G.I. tract account also in the guinea pig for the larger part of Cl4 incorporated into cholesterol-CY4,but the G.I. tract contains much more newly synthesized cholesterol than the liver. Very striking are the dissimilar patterns of biosynthesis in the different tissues. Except in the first 3 min. of the experiment the liver cholesterol of the rat (Fig. 1) doesnot contain HCC, while the cholesterol of the G.I. tract holds considerable amounts of HCC during the first hour after injection. These disappear in the course of 4 hr. In the liver the cholesterol reaches a maximum count between 20 and 30 min. after injection. This
48
SCHWENK,
ALEXANDER
AND
FISH
count decreases possibly by conversion of cholesterol to other substances and stabilizes itself after 40 min. on a much lower level which persists then for a long time. In the G.I. tract cholesterol reaches a lower maximum than in the liver at 40 min. and holds it for at least 4 hr. The other tissues here investigated, including the blood, contain HCC for the main part of the experiment although the level of specific activity of their cholesterol is considerably lower than in the liver or the G.I. tract. HCC are retained in these tissues for some time. However, 1 week after the injection, as Fig. 1 shows, HCC have almost disappeared from digitonin cholesterol and the nearly identical counts of purified cholesterol in different tissues demonstrate that a redistribution of the biosynthesized cholesterol has taken place. The gastrointestinal tract appears to retain it longer than any other tissue. It is possible that other specific patterns would be found in tissues not considered in this investigation if larger amounts of acetate-C’* could be used. In the guinea pig (Fig. 2) as in the rat, liver and G. I. tract incorporate much more C?* into cholesterol than the carcass, but the specific counts reached in all tissues of the guinea pig are considerably lower than in the rat. HCC are present not only in the G.I. tract but, in contradistinction to the rat experiments, also in the liver throughout the duration of the experiment. Evidently the reactions which convert acetate into cholesterol and this into other products are much slower than in the rat. The guinea pig does not synthesize cholesterol as easily and it cannot cope with it as well as the rat. This suggests a possible correlation to the fact that the guinea pig shows atherosclerotic lesions when fed cholesterol for a time, while the rat is not so affected. Gould et al. (12) have observed that cholesterol metabolism is slower in the rabbit, another animal susceptible to atherosclerosis after cholesterol feeding, than in other species. Compare also Duff (13) and Pilgeramt et al. (14). The few experiments in fish here reported imply a very low level of synthesis of cholesterol, but the large amount of radioactivity not precipitated by digitonin from the nonsaponifiable material shows a pattern different from the rat and the guinea pig. HCC are found in liver and in the G.I. tract. Observations of dissimilar patterns of cholesterol synthesis for different tissues of animals other than rats, guinea pigs, and fish were also made by Heard et al. (15) who claim in the laying hen and in the pregnant mare “that independent routes of synthesis of cholesterol operate in different tissues.”
BIOSYNTHESIS
OF
CHOLESTEROL.
VIII
49
The adrenal of the rat seems especially interesting because of the absence of cholesterol-C4 even after 240 min. as shown in Table I, although some HCC are found in this organ after 30 min. All other tissues of the rat contain cholesterol-Cl4 within 240 min. This must not necessarily mean that this substance was not synthesized in the adrenal. It is possible that the rapid decrease of the maximum count in the liver and the nonappearance of cholesterol-U4 in the adrenal have a common background. If synthesis of cholesterol as well as its conversion (into bile acids in the liver and into cortical hormones in the adrenal) occur simultaneously and in the same sites, one would expect the count in these tissues to rise first and soon to decrease, as seen in the liver in the present experiments. In the adrenal the amounts of cholesterol-Cl4 may be too small to be registered by available methods. Once cholesterol leaves these reaction sites it becomes diluted by the nonradioactive cholesterol originally present in the tissues. It appears then quite inert and the specific count may remain comparatively constant for a long time. This was found in the hen, where cholesterol-C4 persisted for 4 months after the injection of acetate-C4 (16). The assumption of specific reaction sites for the biosynthesis of cholesterol and its further catabolism is not contradictory to the fact that all tissues which have been investigated, either as slices or in perfusion experiments, synthesize cholesterol. Isolated tissues surviving under artificial conditions need not necessarily behave as in the living body. The idea that cholesterol synthesis may be a general property of all growing cells but that biosynthesis in the mature animal may take place to a higher degree in reticulum cells and histiocytes, especially the reticulum cells of the liver, has been mentioned by Thannhauser (17) ; and Landon et al. (18) conclude from their data that liver, intestine, and perhaps skin are important sites of biosynthesis, and that large-scale catabolism may be restricted to the same tissues and the adrenals. The feeding experiments reported here show that the digitonin nonprecipitable HCC from rats and guinea pigs contain substances similar in their chromatographic behavior to the HCC found in perfusions of pig livers (2) and in yeast (4). Like these they generate cholesterol-Cl4 after feeding to rats. From the pentane fraction of the chromatogram of HCC from rats and guinea pigs a hydrocarbon fraction was obtained which after feeding to ra,ts was converted in good yields into cholesterol-C14, as in the fundamental experiments of Langdon et al. (11). However, this fraction did not give, even after purification through the thiourea adduct, an infra-
50
SCHWENK,
ALEXANDER
AND FISH
red spectrum completely identical with that of squalene.6 In spite of its high conversion to cholesterol-Cl4 no claim therefore can be made that this hydrocarbon is identical with squalene-C14.It may contain this substance, but at this time there is nowayof proving this contention. This argument is also valid for the earlier reports from this laboratory on hydrocarbon fractions from HCC in liver perfusions or yeast, although these fractions undoubtedly represented squaleneof high purity as shown by infrared spectra, analysis, and conversion into hydrochloride. It must be considered possible that the radioactivity in these hydrocarbon fractions is due not only to squalene-Cl4but to someother substance, similar enough to squalene, but present in such small amounts that it does not change the infrared spectrum or modify the properties of the main constituent enough to be recognizable as a separate entity. Mondon’s “isosqualene” (19) could be such an admixture. For the same reasons the elegant elucidation by Bloch’s group (20) and by Cornforth et al. (21) of the distribution of Cl4 in cholesterol-C4 and recent similar experiments with squalene by Cornforth et al. (22) do not allow a decision on the squalenehypothesis. From a study of turnover rates PopjBk (23) has concluded recently that squalene is not an obligatory precursor of cholesterol. ACKNOWLEDGMENTS The authors are grateful to Mr. Paul Skogstrom for infrared spectra; to Mrs. Joan Fitzgerald and Miss Lucy Oulohojian for help in the biological experiments; and to Mr. Dean Stevens for Liebermann-Burchard determinations. SUMMARY
The biosynthesis of cholesterol-C4 and of radioactive companion substances has been studied in time experiments and in different tissues of rats, guinea pigs, and fish injected intraperitoneally with acetate-1-CY4. Varying and specific patterns have been found for the biosynthesis of these substances in different tissues of the animals. Radioactive companion substances of rats and of the guinea pig were fed to rats and yielded cholesterol-CY4; they contain therefore precursors of this substance. REFERENCES 1. SCJXWENK,
E., AND WERTHESSEN,
N. T., Arch.
Biochem. and Biophys.
40, 334
(1952). 6 Langdon
el al. (11) added carrier
squalene in their experiments
with rats.
BIOSYNTHESIS
OF
CHOLESTEROL.
VIII
51
2. SCHWENK, E., TODD, D., AND FISH, C. A., Arch. Biochem. and Biophys. 49,187 (1954.) 3. SCHWENK, E., AND WERTHESSEN, N. T., Arch. Biochem. and Biophys. 42, 91 (1953). 4. SCHWENK, E., ALEXANDER, G. J., AND FISH, C. A., Arch. Biochem. and Biophys. 63, 274 (1955). 5. WERTHESSEN, N. T., AND SCHWENK, E., Am. J. Physiol. 171, 55 (1952). 6. BEECBIMANNS, L. M., AND DE ELLIOTT, G., Nature 167, 200 (1951). 7. RABOTJRN, W. J., QUACKENBUSH, F. W., AND PORTER, J. W., Arch. Biochem. and Biophys. 48, 267 (1954). 8. GOULD, G. R., Circulation 2, 460 (1950); GOULD, G. R., SINE, F. M., ROSENBERQ, I. N., SOLOMON, A. K., AND HASTINGS, A. B., J. Biol. Chem. 177,295 (1949). 9. VAN BRUQQEN, J. T., HUMHENS, T. T., CLAYCOMB, C. K., AND WEST, E. S., J. Biol. Chem. 200,31 (1953). 10. BUSCH, H., AND BALTRUSH, H. A., Cancer Research 14, 448 (1954). 11. L~NODON, R. G., AND BLOCH, K., J. Biol. Chem. 200, 129, 135 (1953). 12. GOULD, G. R., AND TAYLOR, C. B., Federation Proc. 9, 179 (1950); TAYLOR, C. B., AND GOULD, G. R., Circulation 2, 467 (1950). 13. DUFF, G. L., Arch. Pathol. 20, 109 (1935). 14. PILOERAMT, L. O., AND GREENBERG, D. H., Science 120, 760 (1954). 15. HEARD, R. D. H., JACOBS, R., O’DONNELL, V. J., AND WILLOUOHBY, H., Abstr. Communs. XIX Intern. Physiol. Congr., p. 922. Montreal, 1953. 16. SCH~ENK, E., AND BAKER, C. F., Arch. Biochem. and Biophys. 46, 341 (1953). 17. THANNHAUSER, S. J., New Engl. J. Med. 246, 695 (1952). 18. LANDON, E. J., AND GREENBERQ, D. M., J. Biol. Chem. 209, 493 (1954). 19. MONDON, A., Angew. Chem. 66, 333 (1953); Ber. 88,724 (1955). 20. LITTLE, H. N., AND BLOCH, K., J. Biol. Chem. 183, 33 (1950); WUERSCH, J., HUANQ, R. L., AND BLOCH, K., J. Biol. Chem. 196, 439 (1952); BLOCH, K., Helv. Chim. Acta 36, 1611 (1953). 21. CORNFORTH, J. W., HUNTER, G. D., AND POPJAK, G., Biochem. J. 64, 597 (1953). 22. CORNFORTH, J. W., AND POPJ~K, G., Biochem. J. 68,403 (1954). 23. POP,& G., Arch. Biochem. and Biophys. 48, 102 (1954).
Schwenk, George J. Alexander and Charles A. Fish
the Worcester
Foundation Received
for Experimental Massachusetts December
Biology,
Shrewsbury,
23, 1954
Substances of a higher specific activity than the simultaneously biosynthesized cholesterol-Cl4 were found in the neutral nonsaponifiable lipides of pig livers perfused with acetate-1-CY4. Digitonin precipitates a part of these higher-counting companions (HCC) along with cholesterol (1). Fractions obtained by chromatography from these neutral nonsaponifiable lipides were partially converted, after feeding to rats, into cholesterol-CY4, and therefore include precursors in the biosynthesis of this compound (2)In intact rats, injected intraperitoneally with acetate-1-CY4, HCC could only be found in the digitonin-cholesterol when the animals were killed within an hour after the injection but not 4 hr. later (3). In these experiments the rats were worked up as a whole and consequently the isolated cholesterol was a mixture from all the tissues in the body of the animals. It seemed interesting to study the distribution of cholesterol and HCC among different tissues and in various species and to ascertain whether the HCC from intact animals contain precursors of cholesterol as was found for the HCC from liver perfusions (2) and from yeast (4).2 1 This work has been supported by the Damon Runyon Fund; the U. S. Public Health Service (Grant No. C321); an institutional grant from the American Cancer Society; and the Schering Corporation, Blootield, N. J. 2 In reviewing this manuscript one of the referees stressed the necessity of a more precise definition for HCC. The symbol HCC (higher-counting companions) is used to designate the total amount of radioactive substances accompanying cholesterol-C” in the nonsaponifiable lipides in biosynthetic experiments. Digitonin precipitates only a portion of these HCC together with the digitonide of
37
38
SCHWENK,
ALEXANDER
AND FISH
EXPERIMENTAL
A. Cholesteroland Digitonin-Precipitable HCC in Tissuesof Rats, Guinea ,Pigs and Fish Experiment 1. Nineteen male and two female rats8 (170-180 g. each) were injected intraperitoneally with 0.1 mc./IOO g. body weight of an aqueous solution of sodium acetate-l-C 14. Ten male rats were killed after 30 min., nine male rats after 240 min., and the two female rats 48 hr. after the injection. The animals were bled and the blood collected separately. Livers, gastrointestinal tracts (G.I. tracts) (including the contents), skins, lungs, spleens, adrenals, and kidneys were isolated, and all remaining tissues were combined as carcass. They were weighed and hydrolyzed with 30% KOH for 15-20 hr. on the steam bath. Without filtering from the bones, the hydrolyzates were acidified with concentrated hydrochloric acid. Ice was added to avoid overheating. The acidified mixtures were left in the refrigerator overnight, to allow the fatty acids to solidify. After addition of diatomaceous earth (Celite) the mixture was filtered with suction. The wet filter cake was boiled three times with pentane and the solution was filtered or decanted off the Celite. The acidified hydrolyzates of the smaller organs were extracted directly with pentane. The pentane solutions were extracted twice with 10% KOH with addition of ethanol for better separation of the soap solution, which was subsequently acidified to obtain the fatty acids. After twice washing with water the pentane extracts were evaporated to dryness under reduced pressure. If the residue contained water some benzene was added and again evaporated in vacua. Precipitation with digitonin and bromination, etc. of the cholesterol, as well as plancheting and counting on an end-window counter in this and in all following experiments were carried out as described before (5) (Table I). The most interesting fact in Expt. 1 is that the liver alone of all the tissues was free of HCC, even half an hour after the injection. After 4 hr., the G.I. tract and the blood had lost their HCC while the other tissues still contained some. However, after 48 hr., these substances had discholesterol-C14. These digitonin-precipitable HCC are a useful tool for the study of the biosynthesis of cholesterol in intact animals (or other biological systems) as shown in the present paper. Some of these digitonin-precipitable HCC may be precursors (E. Schwenk, Biochemical Symposium, Federation Meeting at San Francisco, April, 1955). That the part of the HCC which is not precipitated by digitonin also contains precursors is shown in this paper. 3 Charles River Strain of Sprague-Dawley rats were used in all experiments.
BIOSYNTHESIS
OF
CHOLESTEROL.
TABLE
Distribution
I
of Cholesterol-C14 and Digitonin-Precipitable Injected with CH&1400Na
= I
ChoIesteroLC’4, counts/min./mg.
--
272 428 28 37 29 48 23 15 25
I
after
.-
.-
320 270 6 20 5 0 0 0 7
9 male rats killed 240 min. Cholesterol-C”, counts/min./mg.
HCC’
I Xbrom.
Digit.
Liver G.I. tract Carcass Skin Lung Spleen Adrenal Kidney Blood
HCC
=
10 male rats killed 30 min.
Digit.
39
VIII
in Tissues
of Rats
= after
I.
EIcca
Dibrom.
2 female
Tqagt;;Ued
after
_Cholesterol-W, counts/min./mg.
-
HCC?
Digit.
Dibrom.
345 676 281
656 711 322
-
% 37 78 46 83 100 100 100 72
227 491 39 89 73 91 0 26 156
246 497 28 40 27 68 0 17 161
% 28 55 63 25 -
% -
34 -
-aHCC
y.
=
digit.
digit.
dibrom.
.I *n,-, A luu.
appeared from carcass, liver, and G.I. tract. Very important also was the absenceof any radioactivity in the cholesterol of the adrenals. Even after 240 min. when other organs of low weight like lung, spleen, or kidney contained cholesterol-C i4, the adrenal was free of it. The absence of HCC in the liver cholesterol made it desirable to investigate the change of HCC in time more thoroughly. The next experiment was therefore carried out using six time intervals. &periment d. Eighteen male rats, weighing from 170 to 190 g. were injected as previously and groups of three rats were killed at 10, 20, 40, 80, and 240 min. and at 1 week after injection. The animals were bled and the blood for each time interval was collected as were the livers, G.I. tract, skins, and remaining parts of the carcass. They were worked up as before (Fig. 1). Again no HCC were found in the liver during the experiment. The rapid incorporation of Cl4 into cholesterol in liver and G.I. tract is remarkable. Within 20 and 40 min. after injection, the cholesterol count of these tissues reached a maximum. However, while the count in the G.I.tract cholesterol remained almost stationary through most of the experi-
40
SCHWENK,
Liver
4000
r > .E 5In E a 0
ALEXANDER
40
80
AND
FISH
4000 c
240
1 wkll0
Blood
40
80
1 WI
240 Skin
G&Tract
. Carcass
.-+. P----b-- ------.o.4.D-.-.---.-.D, ;p 40
80
240
1wklO
40
Minutes
80
240
1 WI
Minutes
FIG. 1. Distribution of radioactivity in rat tissues. Specific activity of cholesterol-Cl4 and fatty acids plotted against time (Expt. 2). + - - - - - - - - - + cholesterol from digitonide. 0 0 cholesterol purified via dibromide. q m..-.-. - 0 fatty acids. One-week points for skin and carcass are drawn to the same scale as the points for the other tissues.
ment and dropped only after 1 week, the count of the liver cholesterol decreasedimmediately after its maximum was reached and was considerably reduced after 1 week. In skin, carcass, and blood, maxima were attained slowly in the first 4 hr., but after a week the count in these tissues had become nearly the same as in the liver and G.I. tract. In all tissues the fatty acids showed a maximum, almost simultaneously with the cholesterol after 2040 min. There appeared a lesswell-defined second maximum in the count of the fatty acids at 240 min., similar to observations of Beeckmanns et al. (6): 4 The
attainment
of the first
maximum
in the count
of the fatty
acids
at about
BIOSYNTHESIS
Distribution
OF
CHOLESTEROL.
TABLE
II
41
VIII
of Cholesterol-C 14) Digitonin-Precipitable HCC and Fatty Acids in Tissues of Rate S Min. after Injection with CH&““OONa Counts/min./mg. Cholesterol
HCC’
C’” Fatty
Digit.
Dibrom.
260 159 6
128 14 0
acids
%
Liver G.I. tract Carcass ~HCC~~ b Not
=
digit.
-
dibrom.
digit.
x
1058 243 b
50 91 100
100.
counted.
The liver was free of HCC in Expt. 2 even as early as 10 min. after injection A still shorter reaction time was therefore tried. Experiment S. Two male rats, 170 f 10 g. each, were injected as before and killed exactly 3 min. after the injection (Table II). In this experiment considerable HCC were found in the liver, and the radioactivity in the G.I. tract and carcass digitonides was almost entirely due to HCC. Only small amounts of cholesterol-C? had been formed. The fatty acids in the liver showed a high count. The following experiments demonstrate that species other than the rat have a different pattern of biosynthesis of precursors and cholesterol. Experiment !+. Four guinea pigs, each weighing about 800 g. were injected with 0.2 mc. of carboxyl-labeled sodium acetate-C4 each. They were killed after 15,30,60, and 180 min. Livers, G.I. tracts, and carcasses were separately hydrolyzed and worked up as usual (Fig. 2). Unlike in experiments with rats, livers as well as G.I. tract contained considerable amounts of HCC, but incorporation of Cl4 into cholesterol was much smaller in both tissuesthan in the rat experiments, even when the smaller amount of radioactive acetate which was administered is taken into the time when the cholesterol reaches a maximal count suggests a correlation between the first-formed acids and cholesterol. They may include precursors of cholesterol. Accordingly, some of the acid material obtained from the livers in Expt. 2 was fed to two rats, but the cholesterol isolated from the livers and G.I. tract of these animals contained only traces of radioactivity; a more thorough investigation of these acid fractions seems advised.
42
SCHWENK,
ALEXANDER
AND
FISH
G.I.-Tract \ \
+. ‘\\
Carcass & Blood
10 -
,+--- -*------------+ +- A 1530 60 Minutes
FIG. 2. Distribution of radioactivity cholesterol-04 and fatty acids plotted cholesterol from digitonide. 0 El -.-.-.-.0 fatty acids.
I I I :.-.
‘\
‘\
‘\
‘\
‘\
‘+
0
o--9
‘I ‘., 1, 0 J-0 180 15 30
‘\
.D---.-60
-.-.-0 180
Minutes
in guinea-pig tissues. Specific activity of against time (Expt. 4). + - - - - - - - - + 0 cholesterol purified via dibromide.
consideration. There was more cholesterol-Cl4 and especially HCC in the G.I. tract than in the liver. The maximum counts were reached in the first hour after injection as in the rats but HCC disappeared faster from the liver than from the other tissues.The fatty acids had low counts with well-defined maxima in the first 30 min. Experiment 5. A male rainbow trout, 2 years old and weighing 492.5 g. was injected intraperitoneally with 0.5 mc. of carboxyl-labeled acetateCl4 in water of 8°C. After 1 hr. the animal was killed and dissected into liver, G.I. tract, and carcass and worked up as usual. Experiment 6. Eight dogfish (Mustelus cams) pups, each weighing about 110 g., were injected intraperitoneally in water of 6°C. with 0.09 mc. of carboxyl-labeled acetate-C4 and killed 1 hr. later. For reasons of transport the tissues had to be kept for 3 days in 95 % ethanol and were then hydrolyzed and worked up as usual (Table III). The liver in these fish incorporated more Cl* in cholesterol-Cl* than the G.I. tracts or carcasses,but the values for the specific counts in all fractions were very low; HCC were present in all tissues.A large amount of activity is not precipitable with digitonin and appears in the crude nonsaponifiable or in the mother liquor from the cholesterol digitonides.
BIOSYNTHESIS
Distribution
OF
CHOLESTEROL.
TABLE
III
43
VIII
HCC in Tissues of Fishes
of Cholesterol-C14 and Digitonin-Precipitable Injected with CH#dOONa Trout COUlltS/ min./mg.
Dog!ish I-m?
Countsl min./mg.
EIcc*
-~ %
Nonsaponifiable Liver
Cholesterol-W
816” Digit. Dibrom.
%
351
5 1
80
57 29
49
--Fatty acids
11,370
Nonsaponifiable G.I. tract
Cholesterol-Cl”
122a Digit. Dibrom.
Fatty acids
26
Nonsaponifiable Carcass and blood
Cholesterol-04
6 2
136” Digit. Dibrom.
-es-
66 --------
14
6 Traces
100
630 62
-----
Fatty acids
82
---
2 2
35
-
11 2
81
149
* In these experiments the mother liquor material from the digitonide precipitation of cholesterol-C’* was counted and is reported in the cohunn for nonsaponifiable material. digit. - dibrom. bHCC% = x 100. digit.
The mother liquor residue from all digitonin precipitations in Expt. 6 was isolated and had a specific count of 338 counts/min./mg. It was chromatographed on alumina. The pentane fraction was radioactive. After another chromatogram on alumina the colorless, radioactive oil was treated with thiourea (7). Of the resulting colorless oil (26 mg.), an infrared spectrum was obtained which showed the main bands of squalene, but its fingerprint region did not allow complete identification with this hydrocarbon.
44 B. Precursors
SCHWENK,
ALEXANDER
AND
FISH
of Cholesterol in the Dig&w&-Nonprecipitable Rat and Guinea Pig
HCC from
The mother liquors of the digitonin precipitation of cholesterol from 18 rats in Expt. 2 were combined and reduced to dryness in vacua. Digitonin was eliminated as usual and the residue, 3100 mg. of brown oil, 150 counts/min./mg., a total of 465,000 counts, was chromatographed on alumina (Alcoa, grade I). Elution with pentane gave 650 mg. of a white oil which solidified partly on cooling, 79 counts/min./mg., a total of 51,000 counts. The eluates with benzene, ether, and methanol were combined and gave 2500 mg., 166 counts/min./mg., a total of 414,000 counts. Of the pentane fraction, 165 mg. was fed to a rat (Expt. 7) and the remainder purified twice through the thiourea compound. The white oil had a count of 363 counts/min./mg. However, while the infrared spectrum of this substance contained the main absorption bands of squalene, the fingerprint region did not permit positive identification with squalene. The other fractions from the chromatogram were submitted to a washout procedure as described before (2) using 3 g. of especially purified cholesterol. A quantity of 2680 mg. of cholesterol, 13 counts/min./mg., was obtained; this count remained unchanged when the substance was purified through the dibromide. The mother liquors from the washout, 146 counts/min./mg., were precipitated with digitonin and gave 13.8% of cholesterol, which after bromination had 15 counts/min./mg., in agreement with the figure above. An amount of 520 mg. of the mother liquor from the digitonin precipitation, 144 counts/min./mg., a total of 75,000 counts, was fed to two rats (Expt. 8). A male guinea pig of 935 g. was injected with 0.94 mc. of acetate-Cl4 and killed after 30 min. The neutral nonsaponifiables were obtained from liver and G.I. tract as usual and precipitated with digitonin. The mother liquor from this precipitation was worked up as described in the preceding paragraphs. A total of 257 mg. of a brown oil, 184 counts/min./mg., was obtained which was chromatographed on alumina (Alcoa, grade I). The pentane fraction gave 25 mg., 216 counts/min./mg., a total of 54,000 counts of a white oil which partly solidified on standing. It was fed to one rat (Expt. 9). The column was stripped with benzene, ether, and methanol, and the combined material, 185 mg., 127 counts/min./mg., a total of 23,400 counts, was reprecipitated with digitonin. The digitonide after decomposition gave 5 mg. of cholesterol, 93 counts/min./mg. The mother liquor from this digitonin precipitation gave 120 mg. of a brown-
BIOSYNTHESIS
OF CHOLESTEROL.
VIII
45
ish oil, 188 counts/min./mg., a total of 22,000 counts which was fed to one rat (Expt. 10). Feeding Experiments. Experiment 7. Two female rats, 185 g. each, received 165 mg. of pentane fraction, 79 counts/min./mg., a total of 13,000 counts, dissolved in 4 ml. olive oil in four feedings 6 hr. apart, two on the first day and two on the second day. They were killed 48 hr. after the last feeding. Weights: livers 20.4 g., G.I. tract 40.0 g., carcass and blood 314.6 g. Experiment 8. One female rat (185 g.) recieved 390 mg. of the substance after precipitation with digitonin 144 counts/min./mg., a total of 56,000 counts, and a second female rat (185 g.) received 130 mg., a total of 18,800 counts, in four feedings as before. Weights: liver and G.I. tract 26.3 g., 25.6 g. ; carcass and blood 146.0 g., 132.6 g. Experiment 9. One male rat (180 g.) received 25 mg. of the pentane fraction 216 counts/min./mg., from the guinea-pig experiment a total of 5400 counts, in four feedings as before. Weights: liver and G.I. tract, 40.7 g.; carcass and blood, 182.6 g. Experiment 10. One male rat received 115 mg. of the mother liquor from the guinea-pig experiment 188 counts/min./mg., a total of 22,000 counts, in four feedings as above. Weights: liver and G.I. tract, 31.6 g.; carcassand blood, 187.9 g. (Table IV). DISCUSSIONS
The experiments of this investigation show that the main part of the biosynthesis of cholesterol-Cl4 both in rats and in guinea pigs occurs in the liver and the gastrointestinal tract in the first 30-60 min. after injection of labeled acetate. This result agrees with findings of Gould et al. (8) who observed that synthesis ceasesin rats and rabbits after 4 hr., with Van Bruggen et al. (9) who showed the rapid uptake of Cl4 into cholesterol in the first hours after injection in rats and with Busch et al. (10) who noticed complete disappearance of CY4-labeledacetic acid from the blood of rats in the first minutes after intravenous injection. Considerable incorporation of Cl4 from acetate into squalene, presumably a precursor of cholesterol, was observed by Langdon et aE. (11) 30 min. after the injection. Half an hour after the injection of acetate into rats, the count in liver 6 This discussion is based on the consideration of the specific counts of cholesterol-Cl4 because calculation of the data of this investigation in the form of efficiency factors as used by Werthessen et al. (5) or by Van Bruggen et al. (9) have not given a better or different interpretation of the experimental results.
SCHWENK,
ALEXANDER
I -I
M
B 8 d
2 -
-
-
+ca
-
-
. sf$ -
-
-
co
-1 -
AND FISH
rti
co
d
-
-
-
-
-
-
-
7-l
m
5-i
rl
-
w
-
-
-
-
-
-
Q ”
Fa
-
-
-
I
BIOSYNTHESIS
OF CHOLESTEROL.
VIII
47
TABLE V Weights in Grams and Total Counts for Tissues of Rats Znjected with CH&l4OONa Taken from Experiment 1
i
Time
Liver
.----------
min.
30 Weight count/ min. x
G’l’ tract
CZWXSS
Skin
Lung
Spleen
tt$
*ttf-
Blood
Total
77.3 177.0 1126.4 314.0 11.9 11.7 15.8 0.26 59.51793.9 57.6 50.7 7.3 13.7 0.2 0 0 0 0.2 129.7
10-s
Per cent of total count 240 Weight count/ min. X
44.4
39.1
5.6
10.5
0.1
-
-
-
0.1 100.0
---------74.4 150.6 1003.2 290 11.9 13.8 15.6 0.24 60.51620.2 44.3 76.5 36.9 28.3 4.0 4.6 1.2 0 9.0 204.8
10-a
Per cent of total count
21.6
37.4
18.0
13.8
2.0
2.2
0.6
-
4.4
100.0
and G.I. tract together amounts to more than 80% of the total incorporated CY4,and 240 min. after injection these tissues still account for 60% of the total. The skin contains another 10 and 14%. This contribution of liver and G.I. tract becomes even more impressive when the weights of the tissues are taken into consideration. The contribution of tissues other than the above mentioned is very small (Table V). Whether the slow increase with time of the specific activities in these tissues is due to a sluggish conversion of precursors formed in these tissues or to the transport of precursors and/or cholesterol from liver and G.I. tract must be further investigated. Liver and G.I. tract account also in the guinea pig for the larger part of Cl4 incorporated into cholesterol-CY4,but the G.I. tract contains much more newly synthesized cholesterol than the liver. Very striking are the dissimilar patterns of biosynthesis in the different tissues. Except in the first 3 min. of the experiment the liver cholesterol of the rat (Fig. 1) doesnot contain HCC, while the cholesterol of the G.I. tract holds considerable amounts of HCC during the first hour after injection. These disappear in the course of 4 hr. In the liver the cholesterol reaches a maximum count between 20 and 30 min. after injection. This
48
SCHWENK,
ALEXANDER
AND
FISH
count decreases possibly by conversion of cholesterol to other substances and stabilizes itself after 40 min. on a much lower level which persists then for a long time. In the G.I. tract cholesterol reaches a lower maximum than in the liver at 40 min. and holds it for at least 4 hr. The other tissues here investigated, including the blood, contain HCC for the main part of the experiment although the level of specific activity of their cholesterol is considerably lower than in the liver or the G.I. tract. HCC are retained in these tissues for some time. However, 1 week after the injection, as Fig. 1 shows, HCC have almost disappeared from digitonin cholesterol and the nearly identical counts of purified cholesterol in different tissues demonstrate that a redistribution of the biosynthesized cholesterol has taken place. The gastrointestinal tract appears to retain it longer than any other tissue. It is possible that other specific patterns would be found in tissues not considered in this investigation if larger amounts of acetate-C’* could be used. In the guinea pig (Fig. 2) as in the rat, liver and G. I. tract incorporate much more C?* into cholesterol than the carcass, but the specific counts reached in all tissues of the guinea pig are considerably lower than in the rat. HCC are present not only in the G.I. tract but, in contradistinction to the rat experiments, also in the liver throughout the duration of the experiment. Evidently the reactions which convert acetate into cholesterol and this into other products are much slower than in the rat. The guinea pig does not synthesize cholesterol as easily and it cannot cope with it as well as the rat. This suggests a possible correlation to the fact that the guinea pig shows atherosclerotic lesions when fed cholesterol for a time, while the rat is not so affected. Gould et al. (12) have observed that cholesterol metabolism is slower in the rabbit, another animal susceptible to atherosclerosis after cholesterol feeding, than in other species. Compare also Duff (13) and Pilgeramt et al. (14). The few experiments in fish here reported imply a very low level of synthesis of cholesterol, but the large amount of radioactivity not precipitated by digitonin from the nonsaponifiable material shows a pattern different from the rat and the guinea pig. HCC are found in liver and in the G.I. tract. Observations of dissimilar patterns of cholesterol synthesis for different tissues of animals other than rats, guinea pigs, and fish were also made by Heard et al. (15) who claim in the laying hen and in the pregnant mare “that independent routes of synthesis of cholesterol operate in different tissues.”
BIOSYNTHESIS
OF
CHOLESTEROL.
VIII
49
The adrenal of the rat seems especially interesting because of the absence of cholesterol-C4 even after 240 min. as shown in Table I, although some HCC are found in this organ after 30 min. All other tissues of the rat contain cholesterol-Cl4 within 240 min. This must not necessarily mean that this substance was not synthesized in the adrenal. It is possible that the rapid decrease of the maximum count in the liver and the nonappearance of cholesterol-U4 in the adrenal have a common background. If synthesis of cholesterol as well as its conversion (into bile acids in the liver and into cortical hormones in the adrenal) occur simultaneously and in the same sites, one would expect the count in these tissues to rise first and soon to decrease, as seen in the liver in the present experiments. In the adrenal the amounts of cholesterol-Cl4 may be too small to be registered by available methods. Once cholesterol leaves these reaction sites it becomes diluted by the nonradioactive cholesterol originally present in the tissues. It appears then quite inert and the specific count may remain comparatively constant for a long time. This was found in the hen, where cholesterol-C4 persisted for 4 months after the injection of acetate-C4 (16). The assumption of specific reaction sites for the biosynthesis of cholesterol and its further catabolism is not contradictory to the fact that all tissues which have been investigated, either as slices or in perfusion experiments, synthesize cholesterol. Isolated tissues surviving under artificial conditions need not necessarily behave as in the living body. The idea that cholesterol synthesis may be a general property of all growing cells but that biosynthesis in the mature animal may take place to a higher degree in reticulum cells and histiocytes, especially the reticulum cells of the liver, has been mentioned by Thannhauser (17) ; and Landon et al. (18) conclude from their data that liver, intestine, and perhaps skin are important sites of biosynthesis, and that large-scale catabolism may be restricted to the same tissues and the adrenals. The feeding experiments reported here show that the digitonin nonprecipitable HCC from rats and guinea pigs contain substances similar in their chromatographic behavior to the HCC found in perfusions of pig livers (2) and in yeast (4). Like these they generate cholesterol-Cl4 after feeding to rats. From the pentane fraction of the chromatogram of HCC from rats and guinea pigs a hydrocarbon fraction was obtained which after feeding to ra,ts was converted in good yields into cholesterol-C14, as in the fundamental experiments of Langdon et al. (11). However, this fraction did not give, even after purification through the thiourea adduct, an infra-
50
SCHWENK,
ALEXANDER
AND FISH
red spectrum completely identical with that of squalene.6 In spite of its high conversion to cholesterol-Cl4 no claim therefore can be made that this hydrocarbon is identical with squalene-C14.It may contain this substance, but at this time there is nowayof proving this contention. This argument is also valid for the earlier reports from this laboratory on hydrocarbon fractions from HCC in liver perfusions or yeast, although these fractions undoubtedly represented squaleneof high purity as shown by infrared spectra, analysis, and conversion into hydrochloride. It must be considered possible that the radioactivity in these hydrocarbon fractions is due not only to squalene-Cl4but to someother substance, similar enough to squalene, but present in such small amounts that it does not change the infrared spectrum or modify the properties of the main constituent enough to be recognizable as a separate entity. Mondon’s “isosqualene” (19) could be such an admixture. For the same reasons the elegant elucidation by Bloch’s group (20) and by Cornforth et al. (21) of the distribution of Cl4 in cholesterol-C4 and recent similar experiments with squalene by Cornforth et al. (22) do not allow a decision on the squalenehypothesis. From a study of turnover rates PopjBk (23) has concluded recently that squalene is not an obligatory precursor of cholesterol. ACKNOWLEDGMENTS The authors are grateful to Mr. Paul Skogstrom for infrared spectra; to Mrs. Joan Fitzgerald and Miss Lucy Oulohojian for help in the biological experiments; and to Mr. Dean Stevens for Liebermann-Burchard determinations. SUMMARY
The biosynthesis of cholesterol-C4 and of radioactive companion substances has been studied in time experiments and in different tissues of rats, guinea pigs, and fish injected intraperitoneally with acetate-1-CY4. Varying and specific patterns have been found for the biosynthesis of these substances in different tissues of the animals. Radioactive companion substances of rats and of the guinea pig were fed to rats and yielded cholesterol-CY4; they contain therefore precursors of this substance. REFERENCES 1. SCJXWENK,
E., AND WERTHESSEN,
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(1952). 6 Langdon
el al. (11) added carrier
squalene in their experiments
with rats.
BIOSYNTHESIS
OF
CHOLESTEROL.
VIII
51
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