Cholesterol absorption, turnover, and excretion rates in hypercholesterolemic rhesus monkeys

EXPERIMENTAL

.iND

Cholesterol

MOLE:CCL.iR

PATHOLOGY

Absorption,

14, 75-89

Turnover,

in Hypercholesterolemic P. J. MANNING~,

(1971)

and Rhesus

Excretion

Rates

Monkeys’

T. B. CLARKSON, AND H. B. LOFLAND

Twelve male rhesus monkep

were randomly

divided into tlvo s&groups

of six

and all monkeys were fed a diet which contained 25 gm of lnrd per 100 grn and cholesterol at 1 mg/kcal. The diet of six monkeys also contained the tuberculostatic drug isonicotinic acid hydlazide at 0.06 gm prr 100 gm of diet. The monkeys ate about 550 mg of cholesterol daily. Isoniazid had no statistically significant effects on serum lipid concentrations. iZverage serum total cholesterol concentrations exceeded 600 mg/lOO ml; 95% bring transported by low-density lipoproteins. Fasting serum triglycerides did not exceed 25 mg/lOO ml. Cholesterol absorption. turnorpr. and excretion rates were measured in three monkeys from each of thr ta-o dietary groups. Cllolcsterol-1,2-RH was incorporated into the diets for 195 days, and during the isotopic steady state 83 to 92% of the strum cholesterol was derived from dietary cholesterol. During the isotopic steady statr, cholesterol-4-V was injected intravenously and data on its rate of disappearance from serum, plotted on a semilogarit,hmic scale. fit a two-pool model. Cholesterol absorption ranged from 127 to 192 mg daily. Dail:: esvrction of neutral and acidic steroids was measured in feces collerted for four days when the disappearance of cholesterol+“C from serum was linear. Total daily endbgenous steroid excretion was 28 to 56% less than values for daily cholesterol turnover as calculated with the two-pool model system. This discrepancy was attributed mainly to accumulation of cholesterol in body pools and possible destruction of the neutral steroid nucleus b.v intestinal bacteria.

Use of nonhuman primates in atherosclerosis research has increased during the past 10 years because many investigators believe that induced atherosclerosis in these animals more closely resembles the disease in man than does the disease induced in nonprimate laboratory animals. Most reports of diet-induced atherosclerosis in monkeys describe serum lipid changes in responseto cholesterol-containing diets, and t’he morphology and distribution of arterial lesions. Further search for suit’able nonhuman primate models of atherosclerosis must include investigations of the physiology and pathophysiology of cholesterol metabolism. Radiolabeled cholesterol has been particularly useful in measuring in viva metabolism of cholesterol, and has been used t’o study the following (Grundy and Ahrens, 1969) : ’ Supported by grants FR ? Present addrrss: Sinclair (‘olumhia, Missouri 65201.

05000, FR 00180. HE 04722, and H-5883 from the U.S.P.H.S. Comparativcl Medirine Research Farm. University of Missouri,

(I ) The importance of dietary cholesterol as a sours of strum cholesterol, by feeding constant amount:: of rwriiolat~elcrl cholrstcrol until an isotopic “steady state” is attained (Morris et (II., 19.57I ; aualyais of sc~rnilogaritlimic (2) Turnover of body cliolcsterol, I)y kiuctic curves that’ describe tliv rate tlisaplwarancc~ of ratliolnheletl cholestcrol~ administered intravenously (Gurpitlc et nl., 1964 1; (3) Excretion rates of acidic and neutral stcroitls &rived from body cholcsterol, which are measured by injecting ik single intravcwous dose of radiolabcletl cholesterol, then measuring the radioactivity of neutral steroids an(1 bile acid.< of feces when their specific activities equal that of serum csholesterol (Hcllman et al., 1957) ; 141 The rate of cholesterol absorption Iluring the isotol)ic steady state, 1,) multiplying tinily cholesterol turnover by the fraction of serum cholesterol tlcrired from dietary cholesterol (\Vilron ant1 I,in(lscy, 1965; Grutdy and Ahrew. 1969). Some of these tccliniqucs liavc Iww used to study ~liolest~rol mc~tubolisni ilr some speciesof nonhuman I)rimatcs. The lwcentagt~ of serum cholcskrol tlcrivccl from dietary cholesterol has lwn studictl in ccbus nioukcys (genus Ceblts’) antI noolly monkeys (genus Layuthri.r‘l by nI:lnn i 19661, baboons (genus Papio ) 1)) 1 bp \Vilson ( 19681 Eggen et ~1. ( 196ii, 1969I , squirrel monkeys ( genw Snhiri and Eggen et nl. (19691, ant1 rhesus n10&cJY ( d/rrcacfl /rl/rluttfl'~ h;v 11:\n11 ( 1966) and Eggcbnet nl. ( 19691. Turnover rates of hotly rholestcrol liavc lwcn estimated in cebus monkeys and scluirrcl monkeys by the two-pool moclcl concept (Lofland et crl., 1968; Clarkson et (II.. 1969). A l~reliminsry description of ~holcsterol excretion rates has been rqwrtcvl for rhrws monkeys, l)abooiih, ant1 qldrrrll monkeys (Eggen et al., 19691. In this paper w report tlic results of our stu(lica uii cliolest~wl :~bsorl~tion, turnover, and excretion ratci in six liyl~ercliolcstcroleiiiic rhesus nionlicys. Tlic~ct aspects of chokderol mc+abolixm were atutlicd I)y isotopic tcchniqucs. The ris animals were part of a groul) of 12 monkeys , kix of n-l:ich wcw feel konicotinic acid hydrazidc in the diet. This drug is used prophyl:~cticxlly to prevent tuberculosis and has been used for this l~urpow in rhcsus nionlw~s in atherosclerosis research studies (Cos et nl., 1958; Scott et (11.,1967). In this atlvillary exlwriment, we wishctl to see whctlir~r this chug :df(ic*ts scwni liljitl conccwtratiolrs and cholesterol metabolism. -1IATl~:KIAI,


Getled Consitlemtioks. Tw-c~lvc niul~~rhesus iiioiik~~ys n-tw I)urcliasc(l frolii a commercial dealer:’ a8 young adults cdimatc~l to 1~13.5 to 4 yc’ars 01~1on tli(> basis of body n-eight and dental patterns (Humle, 1960; Schultz and &Iarshull, 1933‘1. All monkeys were twtcvl for tubcrculo.~i~ ul)on nrriral awl at nioutlil~ int’errala by intrapalpebral injection of 0.1 nil of undilut,etl mammalian tubcrculin4. They were hou~cl inclividuall\- in stainless steel cagw 46 cm w.j(k, 61 ~111 deep, ancl 81 cm high in :t winclow-lwa room artificially lighted from 7:00 AM to ” Prirn:ltcl Imllorts. Inc.. Srw Y0rk. SW Tork. ’ J~w.cn-,Snlshrry I,nhor:ltorirs. IGu~sns (‘it?-. iLIkwl1ri.

CHOLESTEROL

METSBOLISM

IN

RHESUS

MONKEYS

77

4:00 PM and maintained at 24 t 1°C. Drinking water was provided at all times. Electrocardiograms were recorded as described by Wolf et al. (1969) at the onset of the study and 11 months later. Diets and Isotopes. Diets were prepared in IO-kg batches and frozen until used. For 3 weeks after arrival the monkeys were fed the control diet, which consisted of (gm per 100 gm of diet) lard 25.0, flour 20.0, applesauce 7.8, complete vitamin mix5 2.2, dry milk solids 30.0, casein 13.0, and USP XIV salt mixture 2.0. After 3 weeks they were divided without conscious bias into two subgroups of six. One group was fed the basic diet with 0.5% added cholesterol and 0.06% isonicotinic acid hydraaide and the other group was fed the basic diet with 0.5% added cholesterol. Each monkey was given 160 gm of diet daily. The amount of diet wasted was estimated twice by weighing the uneaten portion for 2-day periods, during months 5 and 11 of the study. During month 5 of the experiment the relative contributions of exogenous and endogenous cholesterol to the overall sterol balance were studied by incorporation of cholesterol-l ,2-3H solution6 into the diet. The radiolabeled diet was fed to six monkeys (three from each subgroup) for 195 days. Specific activities (dpm/mg) of dietary cholesterol were 31,664 * 281 and 32,190 * 280 (mean * SD) for the isoniazid-cholesterol and cholesterol diets, respectively. Cholesterol concentration and radioactivity were measured on blood taken weekly and the results were expressed as the percentage of dietary cholesterol specific activity measured in serum. Cholesterol-4J4C solution was prepared by adding 0.2 ml of stock cholesterol solution (5 mg/ml) to 250 PC of cholesterol in benzene. This mixture was evaporated under nitrogen and the residue was dissolved in 1.6 ml of warm ethanol and 0.3 ml of Tween 2Oj. The mixture was diluted to 8 ml with 0.9% sodium chloride solution. * After the monkeys had eaten the radiolabeled diet for 120 days, the specific activity of serum cholesterol was essentially constant. At’ this time 30 PC of the cholesterol-4J4C solution6 was injected intravenously into the six monkeys to study cholesterol turnover and excretion rates, and blood samples were taken at 1, 5, 10, 15, 28, 42, 48, and 58 days for determination of cholesterol concentration and radioactivity. The decline in specific activity of serum cholesterol with time was plotted on a semilogarithmic scale and the best-fitting curve was calculated (Worsley and Lax, 1962). An IBM 1620 computer was used to analyze the double exponential curves as described by Gurpide et al. (1964). When the specific activity of serum cholesterol-4-14C had declined to its final single exponential rate, feces were collected from the six monkeys for 4 days (days 54-57 after injection of cholesterol-4J4C). The feces of each monkey were pooled in tared glass jars, homogenized, and weighed. Forty to 55 gm of homogenized feces was lyophilized7 in a tared flask for 18 hours and 2 to 4 gm of 5 Nutritional Biochemicals Corporation, Cleveland, Ohio. o New England Nuclear. Boston, Massachusetts. ’ The Virtis Company. Incorporated, Gardiner. New York.

MAPL’KIKG,

78

CLARKSON,

AND

LOFLAND

lyophilizcd feces was extracted for 12 hours with 250 ml of 95:; ethanol in a Soxhlet cstraction apparatus. Duplicat’e 2.0 ml portions of the ethanol extract of fete:: Tvere cvapornted to dryness by air. The residue was dissolred in a solution of 6 gm of 2,5-tliphcnyloxazole per liter of toluenc, and 0.04 ml of a 10% solution of benzoyl peroxide in toluene was added to oxidize pigments. After 2 days the samplc~ wew nearly colorless. Fifteen drops of Rio-SoW were added to solubilize the bile acids and radioactivity was counted after the vials had been in darkness for 2 hours. Neutral steroids and bile acids were separated from the remaining ethanol extract by ion-exchange cllronlatograpll~ (hlitchell and Diver, 1967). Total radioactivity in the ethanol extract was compared with the sum of the radioactivity in the neutral steroid and bile acid fractions clutetl separat’ely from the ion-exchange resins. Four samples of feces that contained no radioactive material were lyophilizcd and extracted by ethanol as described above after a known amount of cholesterol-“C and cholic :~citl-l-‘C was added. Recovery of the added radioactivity from the ion-exchange resins ranged from 94 to 99% for cholic acid-1K and 97 to lOOF& for cholestcrol-l”C. Biochemicnl Techniques and illea.surenrmzt of Rndioactivit!/. Serum lipids and radioactivity wcrc measured in blood t)aken from t)he femoral artery or vein after the monkeys were fasted for 16 to 18 hours. Blood was allowcd to clot at’ room temperature for 2 hourti and serum was collcctcd after the tubes wrc centrifuged were measured by at 1500 rpm for 5 minutw. Strum cholesterol am1 triglyccridw semiautomated procetlures (Block et al.. 1966; Lofland, 1964). Cholesterol content of serum lipoproteins watt mca~ured by a technique in which low-density lipoproteins are pn~cipitatcd with dcxtran sulfak (Kritehcwky ef al., 1963). Dietary cholesterol was extracted I)y mixing 1-2 gm of diet with 20 volumes of hot chloroforn-methanol 2: 1. After 16 hours thcb mixture war filtered through Whatman number I filter paper and the residue and filter paper were washed three times with hot chloroform-methanol 2:l. The volume of the extract was reduced to 10 ml by evaporation with air; a portion was put into a glass Trial and dried. The residue was dissolved in isopropanol, and cholesterol was measured ar; described previously (Block et al., 1966). Radioactivity was counted on isopropanol extracts of diet, or serum which were air-dried and the residue, dissolved in 10 ml of a solution of 6 gm of 2,5-diphenyloxazole per liter of toluenc, was counted with a Beckman DPM-100 liquid scintillation spcctromete@ to a statistical error of 25. Quench correction was made by a channels-ratio system, wit’11 l”‘Ce as the external standard. Cholesterol Absorption. Daily cholesterol a,bsorption was calculated in the isotopic et)eady &ate by the method developed by Wilson ancl Lindsey (1965) and modified by Grundy and Ahrens (1969). The equation is Dietary

cholesterol

daily cholesterol

Cholesterol

absorbed daily (mg) =

turnover

Turnoue~.

(mg’) X fraction of serum cholesterol derived from absorbed dietary cholesterol during isotopic steady state.

The clccrcase in specific

’ Beckman In>tr~~nwnt~, Fullerton.

Cnlifornin.

activit,y

of intravenously

(1 )

in-

CHOLESTEROL

METABOLISM

IX

RHESUS

79

MONKEYS

jetted cholesterol-4-l% was plotted on a semilogarithmic scale versus time. The curves were analyzed according to a two-pool model (Fig. 1). As shown by Gurpide et al. (1964) when a radiolabeled compound is injected into pool A and only that pool can be sampled, t,he characteristics that can be calculated are: 1) volume and mass of pool A; 2) rate constants describing exchange of the radiolabeled compound between pools A and B; 3) metabolic clearance rate of the radiolabeled compound from pool A. The calculations are

PR,

= daily cholesterol

turnover

R, = dpm of radiolabeled

cholesterol

O( = slope of first exponential

injected into pool -2;

0.693 T+ of first exponential

=

in clays ’

0.693 = T; of second exponential

fi = slope of second exponential

c, =

R.44 CYCB+ pc, ;

(mg) =

dpm

in days ’

at, zero time of first exponential;

mg of serum total cholesterol

at zero time of second exponent,ial; CB = mg of serum dpm total cholesterol &I,

= mass of cholesterol

in pool A =

R.4 CA + CB ;

K AA = rate constant for removal of cholesterol = atJf*C*

K nB = rate constant

- HfACB RA

from pool A ;

for removal of cholesterol = -(a

from pool B

+ P + KA.4).

If cholesterol is assumed not to be irreversibly excreted from pool B, as appears to be the case in man (Goodman and Noble, 1968), then K, = 0 and

K BA

=

-KBB

K,

=

&/‘KB.~

, ,

and

K,n

= -A-,,

- K, .

Cholesterol Excretion. Daily excretion of fecal endogenous neutral steroids and bile acids was calculated by the isotopic balance technique developed by Hellman et al. (1957). The principle is described in detail by Gruncly and Ahrens (1969). The calculation is Daily excretion of body = Daily radioact,ivit,y in fecal neutral steroids and bile acids cholesterol (mg) Specific act)ivity (dpm/mg) of serum total (2) cholesterol 3 days previously for neutral st,eroids and 6 days previously for bile acids

80

MANNING,

CLARISON,

AND

LOFLAND

Synfhesls

‘X

wlthin

\

FIG. 1. Turnover of plasma cholesterol in a system composed of two kinetically distinguishable open pools. Two-pool model is presented schematically as pools A and B. Radiolabled cholesterol, injected intravenously, exchanges with a pool of readily miscible cholesterol (pool A), which at the same time exchanges more slowly with a second pool of cholesterol (pool B). Pool A repwsents blood, liver, and other unidentified tissurs. Cholesterol fsnicw pool A from diet. from synthesis within pool A. and from pool B. Cholesterol leaves pool A by exchange with pool B, and by excretion as bile acids and neutral steroids into the intestine. Pool B is presumed to be muscle and other tissues. Cholesterol enters pool B b> exchange with pool A and by synt.hesis within pool B. It is assumed that cholesterol leaves pool B only by exchange with pool A. Rat? constants are denoted by K values. Semilogarithmic graph depicts theoretical rate of disappearance of substances from a two-pool system. Circles connectrd by solid line are observed cspcrimental values. Line with slope p is extrapolated to lrertical axis to derive CB. Values on this extrapolated line are subtracted from corresponding experimental values to obtain line with elolw a and intercept Ca. Modified from Gurpide et ~1. (1964) and Goodman and Nohlc (1969).

RESTiLTS General Considerations. The monkeys ate the diets readily and each animal gained an average of 0.94 kg during the 11 months of study. Monthly tests for tuberculosis were negative. Electrocardiograms had no changes indicative of either myoc.ardial i&emia or conduction wbnormalitic -8 thought to be associat,ed with vascular lesions. Premature vent’ricular contractions during p&othal ancethesia were seen commonly. The characteristics of the electrocardiograms of these monkeys were similar to those reported by Atta and Vanace (1960) for anesthetized rhesus monkeys. Sernm Lipids and Lipoprotein Cholesterol. Each monkey ate an average of 110 gm of diet daily (about 550 mg of cholesterol). The concentrations of serum lipids for the cholesterol group and the cholesterol-iaoniazid group respectively were (mean * SD) total cholesterol 701 -C 169 mg/lOO ml and 775 * 182 mg/lOO ml (0.1 > P > 0.05), triglycerides 21.9 * 12.4 mg/lOO ml and 21.2 + 6.6 mg/lOO ml (0.9 > P > 0.7), and average percent’age of serum total cholesterol transported by low-density lipoprot,eina, 94.9 and 95.0%. Since theye differences

CHOLESTEROL

METABOLISM TABLE

SERUM

LIPIDS

.&ND

LIPOPROTEIN

IN RHESUS

Serum

CHOLESTEROL RHESUS MONKEYS

cholesterol (mg/lOO ml) percentage of serum total cholester01 t,ransported by @ lipoproteins Serum total triglycerides (mg/lOO ml) total

a Mean

f

81

I IN

Baseline

Average

MOSKEYS

12 HYPERCHOLESTEROLEMIC

5 months

11 months

205 f 78.0

9.5”

728 A 95.3

39

699 f 95.4

52

14.2

l.la

22.5

1.5

20.1

2.2

+

f

f

8EM.

were not statistically significant the monkeys were treated as a single group and the data were combined. The concentration of serum total cholesterol for all the monkeys averaged 205 mg/lOO ml at the start and 663 mg/lOO ml within 1 month after adding 0.5% cholesterol to the diet. When the monkeys were fed the control diet without added cholesterol an average 78% of serum cholesterol was transported by low-density lipoproteins. With 0.5 gm cholesterol per 100 gm of diet, about 95% of serum cholesterol was transported by low-density lipoproteins. The average concentration of serum triglycerides did not, exceed 22.5 mg/ 100 ml. Serum cholesterol and triglyceride concentrations and the percentage of serum cholesterol transported by low-density lipoproteins are tabulated in Table I. Attainment of Isotopic Stendg State nnd Amount of Serum Cholesterol Derived fror~ Diet. After cholesterol-1,2-3H was added to the diets the specific activity of serum cholesterol increased during the first 40 days. At this time and for the next t,wo weeks the monkeys were inadvertently fed the same diets without radiolabeled cholesterol (Fig. 2). When the radiolabeled cholesterol was fed again, the specific activity of serum cholesterol increased up to clays 120-140; t’hen it increased very slowly, and it continued to do so even aft’er day 195. The average percentage of serum cholesterol derived from dietary cholesterol was 89.9% for the monkeys fed the isoniazid-cholesterol diet and 87.8% for the group without isoniazid (n = 3 for each group). Analysis of Turnover Curves of Serum Cholesterol. In all monkeys the semilogarithmic plot of the decline in specific activity of intravenously injected cholesterol-4J4C described a double exponential curve. Individual curves from each monkey were analyzed to estimate the number of kinetically distinguishable pools involved in plasma cholesterol turnover (Goodman and Noble, 1968) ; for each, the plot was consistent with a two-pool model (Gurpide et al., 1964). Values of the characteristics derived from analysis of the curves are tabulated in Table II. In most instances corresponding values among the animals were similar. Differences in the mass of cholesterol in pool A were mainly the result of differences in body weight. When this mass was calculated per 5 kg body weight it ranged from 3100 to 3800 mg. The rate constant .K, multiplied by the size of pool A is an estimate of the amount of cholesterol removed from this pool daily. This constant indicated that 4.9 to 6.5% of the total mass of cholesterol in pool A was turned over daily.

Percentage Specific

of Dietary Cholesterol Activity Measured in Serum Percentage Specific

of Activity

.

. .

. .

.

. .

Die fury Cholesterol Measured tn Serum 0

. .

.

.

”

”

” .

C’

.

g

..

”

ET

.

.

.

’

0

Dietary Cholesterol Measured insSerum

fage of Activity A E 0

Percen Spectftc

CHOLESTEROL

METABOLISM

IOOr

0

20

40

60 Days

100

120

_

.

140

160

83

180

200

in Diet

59

l * .

.

l

.

.

l

l

.

09

l

.

.

60--

. .

>: +. 40z g” ,D - 20-

.

.

$2 u

11IIIIIIII

$3 ?$

MONKEYS

Choles+erol-l,2-3H

.

32

.$S QY

58

Monkey

80-

uz

Monkey

80 Fed

F ‘0s L k IOO-

i?Q 3s C”b

IN RHESUS

11

20

0

40

60 Days

80 Fed

111

120

100

Cholesterol-

l.2-3H

Monkey

IOO-

IllI

140

180

I

200

In Diet

61 ***

80-

l

I60

l *

.

0.

. .

60-

*a.

l

* .

l

. .

.

40-

l -

.

20i I 0

I 20

I

I

40

I

I

I

60 Days

II

80 Fed Fm.

I

100

Cholesterol2-continued

I

I

120

I

I

140 I, 2-3H

I

I

I

160 in Diet

I

I80

I

I

200

J

a 6 c d

-

C C c C&I C&I C&I

785 GO7 635 1050 663 741

f f f & zk *

18 26 39 36 32 44

11 17 12 21 17 12

955 083 848 723 457 525

-

3382 6274 4305 4581 5808 3185

C B (dpm md

For explanation of symbols, see test. C = cholesterol diet; C&I = cholesterol and Mean f SEM for the 9 weeks of this portion Calculated by assumiq KR = 0.

51 52 53 58 59 61

Diet*

PlasmaC cholesterol (m&!/100 ml)

PLASM.~

I

isouiaeid diet. of the study.

48.0 32.3 46.5 40.0 41.5 49.5

2nd exp

T: (days)

CHOLESTEROL

TABLE

0.1535 0.1802 0.2350 0.1568 0.1538 0. IGO9

a

'~URNWER

-

-

0.0144 0.0215 0.0148 0.0173 0.0167 0.0140

B

OF SIX

II

4342 2852 3882 2532 2863 4239

MA (mg)

~~HESUS

213 172 193 165 146 218

PRA w/day KAA

-0.123 -0.138 -0.180 -0.132 -0.120 -0.131

R/~ONKEYS~

-0.045 -0.064 -0.070 -0.042 -0.051 -0.044

KBA~

0.074 0.077 0.130 0.067 0.069 0.079

KAd

i5 0.051

5 d

s 3

s5 x $ 3 0.000 0.049 0.065 0.050

0.049

is zE K*‘”“0 Q

CHOLESTEROL

METABOLISM TABLE

CHOLESTEROL

EXCRETION

IX

Monkey

number

51 52 53 5s 59 61 u Calculated before separation * Calculated

cholesterol in fecesa (mg/day)

85

MONKEYS

III OF SIX Fecal

Endogenous

RHESUS

RHESUS

MONKEYS

endogenous

neutral and acidic steroids eluted from ion-exchange resins* (mg/day)

Daily excretion of cholesterol as bile acids (yO)

111 109 119 95 91 110

38 26 34 47 45 26

139 119 135 106 98 158 from 14C radioact,ivity into neutral and acid by Eq. (2).

in ethanol extract steroid fractions.

of feces

Fecal Escretion of Endogenous Neutral and Acidic Steroids. Excretion rates for body cholesterol are presented in Table III. Since the lyophilized feces collected over 4 clays weighed 26.0 to 31.0 gm, differences in total excreted steroids could not be accounted for simply on the basis of differences in fecal weight. The percentage of fecal total steroids in the form of bile acids varied: ranging from 26 to 47% of the total endogenous cholesterol excreted daily. Radioactivity in the ethanol extract’ of feces was, in all instances, greater than the sum of the radioac.tivity in the bile acid and neutral steroid fractions eluted from the ion-exchange resins. This discrepancy may have resulted from degradat,ion by intestinal bacteria of neutral steroids to compounds that were not eluted from the ion-exchange resin. On this basis, an estimat’ed 7 to 30% of neutral steroids were degraded. Cholesterol Absorption Bwing the Isotopic Steady State. Table IV compares daily cholesterol absorption with daily production and excretion rates. In all monkeys cholesterol absorption exceeded total endogenous steroid excretion by 25 to 46 mg daily. Similarly, the production rate of pool A (PR,) exceeded values of cholesterol absorption and values of tot’al daily excretion of endogenous neutral and acidic steroids. DISCUSSION Data from two of the monkeys were largely responsible for wide standard deviations of mean serum total cholesterol concentration. In one, the serum total cholesterol concentration was 385 * 64 mg/lOO ml (mean * SD of 11 determinations done once monthly) and in the other it was 1088 t 128 mg/lOO ml. The mean concentration for the remaining 10 monkeys was 736 -C 94 mg/lOO ml (range 517 to 992 mg/lOO ml). Other investigators have also reported large variations of serum cholesterol concentration in rhesus monkeys fed atherogenic diets (Cox et al., 1958; Scott et al., 1967; Younger et al., 1969). These investigators fed much more cholesterol in the diet and in some instances also suppressed t,hyroid function (Scott et al.? 1967; Younger et al., 1969) to induce hypercholes-

86

MANNING,

CLARKSON,

AND

TABLE V;\LUES

FUR

??I~A~,

CHOLESTEROL CRETIOK

Monke) number 51

52 53 58 59 61

IV ABSORPTION,

OF SIX

LOFLAND

HHESUS

Estimated cholesterol intake h/da?)

Cholesterol absorptiod (w/day)

550 550 550 550 550 550

183 157

AND

CHUI~ESTEROL

Ex-

MONKEYS

Total dail? endogenous steroid escretionc (mg/day)

PRa”

(w/da?)

-___-

-

139

213

119 135

172

lti0

152

106

165 146 318

127

193

!I8

158

103

a PK.4 = productiou rate of pool A or amount of ne\v cholesterol rntering pool A daily escltlsive of recirculat,ed cholesterol. b Calculated by Eq. (1). c Vahles in this column are same as values in col~unn 3 of Table III. terolemia. contained

Our monkeys became hypercholeeterolemic by eating a clict which cholesterol at 1 mg/kcal. In our monkeys, fasting concentrations of

serum triglycerides were considerably lower than concentrations reported by others for rhesus monkeys fed atherogcnic diets. Cox et al. (1958) reported that the average concent’ration of serum “nwtral fat” wad about 235 mg/lOO ml for monkeys fed either low-fat diets or diets with a high fat, and cholesterol content. Similarly, strum triglyceride concentration of rhesus monkeys used by Scot,t et al. (1967) ranged from about 275 mg/lOO ml in monkeys fed a “stock” diet to over 1000 mg/lOO ml in monkeys fed a diet that cont’ained both 10% cholesterol and propylthiouracil. A probable explanation for their high values is that the monkeys were not, fasted long enough, a conclusion supported b>- the data of Armstrong et nl. (1967 i in which fasting serum triglyceride concent’rat’ions did not exceed 75 mg/lOO ml in hypercholesterolcmic rhesus monkeys. Several aspects of cholesterol metabolism in our monkeys can be usefully compared with similar studies clone by other investigators. In the six monkeys of t,his study that were fed radiolabeled cholesterol, 83 to 92% of their serum cholesterol was derived from dietary cholesterol. These dat#a arc compared with data obtained from other primate speciesin Table V. The extent to which serum cholesterol increases in response to ingesting large amounts of dietary cholesterol appears to vary considerably among primate species. Our monkeys absorbed from 127 to 192 mg of cholesterol daily and their serum cholesterol concentration increased about threefold. Many lvorkers have concluded that man can absorb a maximum of about 350 mg of cholesterol daily regardless of cholesterol intake (Kaplan et al.? 1963; Wilson and Lindsay, 196.5; Grundy et al., 1969). In addition, man’s serum cholesterol concentration seldom increases more than 50 mg/ 100 ml (Kaplan et al.i 1963) in response to a very high dietary intake of cholesterol. The baboon appears to share this characteristic with man, more so, than t,he other monkeys which have been studied (Table V). These varied responsesmight in small part result from differences in diet, but t,here are important differences in cholesterol metabolism among various primate species.

CHOLESTEROL

METABOLISM TABLE

IN

RHESUS

MONKEYS

87

V

PERCENTAGE OFSERUMTOTALCHOLESTEROLDERIVED FROMDIETARY CHOLESTEROLINMAN AND VARIOUS SPECIES OF MONKEYS

Animal

Rhesus monkey Squirrel monkey Cebus monkey Baboon Man

Duration of stud! ’ (days)

195 45-51 42 100 5R-119

No subjects’

G 3 4 3 24

Approx. daily intake chol. (g/day) 0.55 0.40 0.90 0.50-10.4

Serum cholesterol (mg/lOO ml)

(ioo-1000 232-343 168350 < 195 < 240

Serum chol. derived from diet c%)

Reference

83-92 57-84 3462 51-57 10-41

this study Wilson (1968) Mann (196G) Eggen (1965) Kaplan (1963)

The failure of the specific activity of serum cholesterol-1,2-3H to level off could bc the result of at least two main factors: (1) the animals were growing and gaining body weight, and therefore the size of the total miscible cholesterol pool was changing; (2) relatively large fluctuations in serum total cholceterol (average standard deviation for 12 measurements done once monthly in these six monkeys was 108 mg/‘lOO ml). Changes in serum cholesterol derived from dietary cholesterol from days 120 to 195 (Fig. 2) were not considered significant since the slopes of regression lines between these points were not different from zero (0.3 > p > 0.1). The production rate of pool A (PR,) is the amount of cholesterol which enters pool A daily, exclusive of recirculated cholesterol (Gurpide et nl., 1964). In the metabolic steady state, PRA is synonymous with cholesterol turnover rate. If endogenous synthesis of cholesterol were negligible, values of PR,, daily cholesterol absorption, and daily endogenous cholesterol excretion should be nearly equal. In our monkeys PR.1 exceeded cholesterol absorption by 13 to 33 mg daily. Similarly, PRa exceeded cxcretcd total steroids of endogenous origin by 28 to 56%) perhaps because: (1) cholesterol was accumulating in the body, as inclicated by the xanthoderma or tendinous xanthomas seen in seven of the monkeys; (2) the two-pool model may overestimate turnover by 3-11% (Samuel et ~1.~ 1968) ; (3) no correction was made for variation in fecal flow rates and; (4) no correct#ion was made for possible destruction of the neutral steroid nucleus (Grundy and Ahrens, 1969). Steroid-ring degradation may have caused the sum of the radioact’ivity in the neutral steroid and bile acid fractions of feces to be less than the total activity of the unseparated fractions. Eggen and Strong (1969) presented data which also suggested that the steroid nucleus was altered in the intestine of rhesus monkeys. Discrepancies between PRA and daily steroid excretion, as reported in cebus monkeys by Lofland et al. (1968)) ranged from 31 t,o 61% and in 11 human beings PR, exceeded daily steroid excretion by an average of 15% (Grundy and Ahrens, 1969). In our monkeys endogenous synthesis of cholesterol was not completely suppressed by absorption of large amounts of dietary cholesterol. Two observations support, this conclusion: (1) the specific activity of serum cholesterol was less than the specific activity of dietary cholesterol, which indicates that nonlabeled cholesterol of endogenous origin was responsible for the decreased specific act’iv-

88

MANNING,

CLARKSON,

AND

LOFLAND

ity of serum cholesterol; (2) values of PRa exceeded values of cholesterol absorption by 13 to 33 mg daily, which indicates the endogenous origin of some of the cholesterol entering pool A. These values are only an estimate, since total body mass was increasing slowly. However, values of PR, were calculated from cholesterol-14C die-away curves over 50 days when changes in pool size were presumably small. Different tissues probably continue to synthesize cholesterol under these conditions. Though a marked suppression of hepatic cholesterol synthesis in response to cholesterol-feeding haa been demonstrated in rhesus monkeys (Manalo-Eetrella et al., 1963j, the rak of cholesterol synt’hcsis by other organs is unaffcct,ed by cholesterol-feeding (Wilson, 1968’1. REFERENCES M. L., CONXER. W. E., AND WARSER. E. D. (1967). Xanthomatosis in rhesus monfed a hypercholesterolemic diet. Arch. Pathol. 84,227-237. ATTA, A. G. AND VANACE, P. W. (1960). Electrocardiographic studies in t,he Macaca mzdatta monkey. Ann. N. Y. Acatl. Sci. 85,811X318. BLOCK, W. D., JARRETT. J. Ii.. BND LEVINE, J. B. (1966). An improved automated determination of serum total cholesterol with a single color reagent. Clin. Chena. 12, 681-689. CLARPSOS. T. B., LOFLAND, H. B., AND BULLCOK, B. C. (1969). Effect of type of dietav fat on sterol distribution and metabolism in squirrel monkeys. Circulation 40,111-5. Cos, G. E., TAYLOR, B. C., Cos. L. G.. ASD COUNTS. M. A. (195s). Atherosclerosis in rhesus monkeys: I. Hypcrcholesterolemis induced by diet,ary fat and cholesterol. Arch. Pathol. 66, 32-52. EGGEN, D. A.. NEwnlan-, W. P.. AND STRONG, J. P. (1965). Zn “The Baboon in Medical Research” (H. Vagtborg, ed.). Vol. 2, pp. 55%569. Univ. of Texas Press, Austin, Texas. EGGES. D. A. AND STRONG, J. P. (1969). Cholesterol metabolism in baboon, rhesus and squirrel monkeys on an athrrogenic diet,. Circulntion 40,111-7. GOODMAX. D. S. AKD NOBLE. R. P. (1968). Turnover of plasma cholesterol in man. J. Clin. Invest. 47,231-241. GRUNDY, S. M. AXD AHRENS. E. H.. JR. (1969). Measurements of cholesterol turnover, synthesis. and absorption in man carried out by isotope ltinctic and sterol balance methods. J. Lipid Res. 10, 91-107. GURPIDE, E.. MAKS, J.. AXD SAXDBERG, E. (1964). Detcrminniion of kinetic parameters of a two-pool system by administration of one or more tracers. Biochemistry 3, 1250-1255. HELLMAP;, L.. ROSESFELD. R. S.. IKS:TLI,. TV.. ASD AzmEss. E. H.. JR. (1957). Intestinal excretion of cholesterol: A mrchanism for regulation of plasma levels. J. Clin. Invest. 36, 898. HUR~IE, V. 0. (1960). Estimation of monkey age by dental formula. Ann. N. Iy. Acnd. hi. 85,795-799. KAPLAN, J. A., Cos. G. E.. ASD TATLOR. C. B. (1963). Cholesterol metabolism in man: Studies on absorption. Arch. Pnthol. 76,359-368. KRITCHEVSKY, D.. TEPPER. S. A., ALAUPOVIC, P.. AND Fuanl.4N, R. H. (1963). Cholesterol content, of human swum lipoproteins obtained by dcxtran prccipitntion and by preparative ultracentrifugation. Plot. Sot. Exp. Biol. Xed. 112, 259-262. LOFL.~XD. H. B. (1964). A semiautomated procedure for determination of triglycerides in serum. Annl. Chem. 9, 393-400. LOFLAA-D. H. B.. CLaaKsoX. T. B.. ST. &AIR, R. Tf-.. hHXER. N. L). M.. .\~n EkiLLoc~;. B. C. (1968). Athrrosclcrosic in Cehlls nlbifrons monkeys. I. St,erol metabolism. Exp. Mol. Pathol. 8,302-313. ARMSTROSG.

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