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Human serum cholesterol synthesis
EXPERIMENTAL
AND
MOLECULAR
Human
PATHOLOGY
Serum
C. BRUCE TAYLOR, B. MIKKELSON, Department
4, 480-488
(1965)
Cholesterol
Synthesis’
J. A. ANDERSON, D. T. FORMAN, AND S. S. CHOIR
of Pathology, Evanston Hospital, Evanston, Illinois; Northwestern University School, Chicago, Illinois; and Mayo Clinic, Rochester, Minnesota Received
May
Medical
19, 1965
In 1950 Gould and Taylor reported (Fig. 1) a marked suppressionof newly synthesized cholesterol in the liver and plasma of dogs ingesting a high cholesterol diet
Hours FIG. 1. Effect of dietary cholesterol on in vitro cholesterol synthesis by liver slices in dogs. Estimated milligrams of cholesterol synthesis per 100 gm of liver are plotted on the ordinate; hours incubated in presence of Cl4 acetate are plotted on the abscissa. Control dogs were on cholesterolfree diets (for 1 month) before liver biopsies were taken for incubation. Cholesterol-fed dogs were fed 1 gm of cholesterol in oil 12 hours before liver biopsies were taken for incubation. Estimations into cholesof cholesterol synthesis were determined from quantities of Cl4 acetate incorporated terol molecules isolated from incubated liver slices.
(Gould and Taylor, 1950) ; and others demonstrated a similar homeostatic mechanism in rats (Tomkins et al., 1953). The mechanismof this inhibition of cholesterol synthesis by dietary cholesterol has been studied extensively and shown to possessthe characteristics of a negative “feed-back” mechanismor control system. Thus, in this 1 Supported by the Chicago, Illinois and American Heart Associations; The Life Insurance Medical Fund; the Thomas J. Dee and the George C. Moody Memorial Research Funds of Evanston Hospital; and the National Institutes of Health, Department of Health, Education, and Welfare (Graduate Research Training Grant ZGM-697). 2 Research Trainee supported by Graduate Research Training Grant 2GM-697. 480
HUMAN
SERUM
CHOLESTEROL
SYNTHESIS
481
control system, a single reaction, the reduction of ,&OH ,L?methyl glutaryl CO A to mevalonic acid was shown to be the primary site of depression of cholesterologenesis produced by exogenous (dietary) cholesterol (Siperstein and Guest, 1960). Studies in our laboratories (Fig. 2) have shown an almost complete lack of change in the rate of appearance of newly synthesized plasma cholesterol in man regardless of the type of diet (cholesterol-rich or cholesterol-low) employed (Cox et al., 1963). These
endogenously synFIG. 2. Effect of dietary cholesterol on appearance of Cl4 acetate-labeled thesized cholesterol in plasma of dogs (on left) and man (on right). Clear bars represent amounts appearing when dog and 8 subjects were on cholesterol-free diets; dark bars represent relative quantities appearing while dog and subjects were on cholesterol-rich diet. The 100% for cholesterolfree values was arbitrarily chosen as baseline for this graph. Dog shows marked suppression (to 5%) of rate of appearance of labeled plasma cholesterol, whereas the human subjects showed no compensatory suppression. Value of 162% for group of subjects was probably over 100% because of shift in pathways for use of acetate pool caused by feeding of neutral fat with cholesterol.
findings corroborated earlier studies which indicated that man (unlike the dog and rat) has little demonstrable “feed-back” control of in vitro hepatic cholesterol synthesis (Davis et uZ., 1959). Employing an indirect method for estimating relative contributions of dietary and endogenously synthesized cholesterol to plasma cholesterol, reported by Morris and others in studies on rats (Morris et al., 1957), we fed ring-labeled 4-Cl* cholesterol to dogs and humans (Taylor et al., 1960; Kaplan ef al., 1963). Our studies in the dog were comparable to those reported in the rat by Morris and others (1957) ; these animals both demonstrated nearly complete replacement of endogenous sources of
482
C. BRUCE
TAYLOR
ET
AL.
plasma cholesterol by ring-labeled dietary cholesterol (Fig. 3). The indirect studies were in good agreement with earlier studies demonstrating the dominant homeostatic role played by the liver in the cholesterol metabolism of the rat and the dog. The results from the ring-labeled cholesterol feeding studies in man were very different from those observed in dogs and rats; only about a quarter to a third of labeled dietary cholesterol appeared in plasma with the remaining large fraction of 3/4 to g of plasma cholesterol apparently being derived from endogenous synthesis (Fig. 4). As reported earlier, since most subjects showed an appreciable rise in serum cholesterol 280
1
/DIET
DIET:
01 0
I
.
2
3
.
4
WEEKS
5
.
C-14-CHOL @BY/DAY) IN E66 YOLK
.
6
7
6
.
.
9
IO
ON DIET
FIG. 3. Graph presenting appearance of ring-labeled (4-C14) dietary cholesterol in serum cholesterol of dogs. Radioactivity in disintegrations per min./mg. cholesterol is plotted on abscissa; time in weeks, on ordinate. Dotted line across top plots dietary radioactivity. Note that within 3 weeks the 2 dogs showed that 91 and 94% of their serum cholesterol contained the ring label, indicating that these percentages of serum cholesterol were derived from diet.
240 DIS. / YIN. 6
PERCENTAGE
‘60:
OF PLASMA
CHOL.
c---3I%-l-32.4
FROM
DIET
-
WOL
120.
0
2
4
12 WEEKS
0:
14
16
16
DIE:’
FIG. 4. Graph similar to that of Fig. 3 presenting rate of appearance of labeled (4-C’*) dietary cholesterol in serum of 32 year old female subject. At about 6 weeks only 31% of serum cholesterol was derived from labeled dietary cholesterol. Interestingly, changing the diet from 2 gm of cholesterol/day to 10 gm/day resulted in only a minimal rise (to 32.4%) in per cent of serum cholesterol derived from labeled dietary cholesterol.
HUMAN
SERUM
CHOLESTEROL
SYNTHESIS
483
while on the ring-labeled cholesterol regimen, there is suggestive evidence that in the human dietary cholesterol is additive to an essentially quantitatively unaltered endogenoussupply of plasma cholesterol (Kaplan et al., 1963). In order to better understand cholesterol metabolism in man and determine the effects of dietary cholesterol on the rate of synthesis of serum cholesterol, a deuteriumlabeling technique was employed. The rationale and methodology were based on the work of London and Rittenberg (1950) who showed that a single adult male subject on a regular diet (probably containing 0.5 to 1 gm of cholesterol per day) had about 0.5 gm of newly synthesized cholesterol appear in his plasma each day (London and Rittenberg, 1950). Since much of the quantitative estimation of human cholesterol synthesis is basedon this single study, we felt the need for additional similar studies with subjects on cholesterol-free and cholesterol-rich diets. MATERIALS
4ND METHODS
Eight endogenous serum cholesterol synthesis studies were carried out on five healthy human subjects (male and female) (Taylor et al., 1965). The subjects’ age, race, sex, and national origin varied considerably. Each subject served as his or her own control and they were alternately placed on a cholesterol-low diet and a cholesterol-rich diet. The cholesterol-low diet consisted mainly of egg whites, beans, lentils, powdered fat-free milk, and dry cottage cheese. This regimen excluded meat, egg yolks, cream, and butter fat. All subjects were placed on this diet six weeks prior to the initiation of the studies. Baseline levels of normally occurring deuterium in urine and in serum cholesterol were obtained from all subjects. Subsequently, subjects were administered an oral priming dose of heavy water (D?O) in concentrations high enough to raise the body water D-0 content to 0.5 moles per cent in excessof normal baseline DaO. Doses averaged from 140 to 250 gm of DZO. Thereafter, subjects received oral dosesof D,O daily, approximating 10% of their original priming dose in order to compensate for normal turnover of body water. All studies were run for at least 40 to SOdays’ duration, and almost daily urine and serum sampleswere collected from subjects. After completion of this initial study, subjects were allowed 7-10 months for the elimination of DZO of body water, cholesterol and other endogenously synthesized deuterated organic molecules.Mass spectrometric analysis verified the return to baseline levels of D,O contents of body water and serum cholesterol. The cholesterol-rich study was then instituted. This study provided an estimated daily dietary intake of 2.5 to 3.0 gm of cholesterol. The egg was the major cholesterolcarrying vehicle since an egg yolk contains approximately 250 mg cholesterol. Again, urine and serum sampleswere collected almost daily for determination of deuterated body water levels and deuterium concentration in newly synthesized cholesterol. The isolation, combustion and reduction of serum cholesterol were carried out in the following manner. Serum specimenswere hydrolyzed with alcoholic KOH and extracted with petroleum ether and one portion of the extract analyzed for total serum cholesterol by the Abell-Kendall procedure (Abel1 et al., 1952) with a Lieberman color reaction (20-1, acetic anhydride-sulfuric acid). The remainder was converted to cholesterol digitonide. Pyridine was then added in order to split off the digitonide and the cholesterol was extracted with acidified ether, purified and weighed. Specimens so isolated usually yielded from 2 to 4 mg of pure cholesterol.
484
C. BRUCE
TAYLOR
ET
AL.
The isolated cholesterol was then combusted with copper oxide at 600°C under high vacuum conditions in a sealed ampule of Corning No. 1720 ignition glass. The ampule containing the COZ and HZ0 of combustion was then placed in liquid nitrogen in order to condense and freeze the gases formed. Carbon dioxide was removed and water driven over into a reduction ampule containing 0.5 gm zinc. The reduction tube was then heated to SOO’C in order to reduce the HZ0 and DzO to Hz and Dz gas. The deuterium assays were carried out in a Nier Model, RCA isotope ratio mass spectrometer at the Mayo Clinic. The mass spectrometric analyses consisted of measuring the mass 3 to mass 2 ratio. This ratio was then converted to heavy water concentration by reference to a calibration derived from five standards of various concentrations of DzO and HzO. The magnitude of error in the reduction and mass spectrometer analyses was of the order of 5 4%. RESULTS
AND
DISCUSSION
The principal advantage of using deuterium is its negligible radioactivity which makes it possible to carry out long-term studies on humans without radiation as a prime hazard. By enriching each subject’s body water deuterium with the initial priming dose
SUBJECT O+
NO. 1
CHOL.
FREE
TIME IN DAYS
FIG. 5. Serum cholesterol-deuterium enrichment curves of Subject 1 while on cholesterol-free and cholesterol-rich diets. The middle curve is derived from corrected data of cholesterol-rich curve to correct for dilution of deutero-cholesterol by unlabeled dietary cholesterol (see text for discussion of this correction). Note again lack of effect of dietary cholesterol on rate of appearance of endogenously synthesized, labeled cholesterol.
HUMAN
SERUM
CHOLESTEROL
485
SYNTHESIS
and daily doses of deuterium oxide, we were able to elevate and maintain the subject’s labeled body water between 0.5 and 0.6 moles per cent of DzO. The labeling of newly synthesized cholesterol was adequate to provide meaningful data when D20 enrichment was at 0.5 moles per cent of DzO in body water. Maximum enrichment of 0.1% of D20 in body water was shown to be experimentally unsatisfactory. The maximal deutero-cholesterol concentrations were reached by 38-44 days in all eight studies. (Fig. 5 is a graph of data from two studies on one subject.) These times are comparable to the study reported by London and Rittenberg in which maximum deutero-cholesterol-digitonide concentrations were reached on the 36th day. Exogenous cholesterol feeding markedly increased the serum cholesterol levels. There was a marked increase in serum cholesterol of from 20 to 56 per cent. Since our studies showed simultaneous marked reduction of absolute deutero-cholesterol concentrations in the cholesterol-rich subjects, we felt this lower specific activity was due to the dilution effect of exogenous cholesterol on endogenously synthesized deuterocholesterol. The most striking example was Subject 2 (Fig. 6) whose mean serum SUBJECT
NO,
2 0-0 M
0
IO
20
30
40 TIME
50
60
70
CHOL.
FREE
CHOL. RICH (uncorrected)
00
90
IN DAYS
Serum cholesterol-deuterium enrichment curves of Subject
2. As discussed in the text, this subject’s serum cholesterol increased 5670, while he was on the cholesterol-rich study, resulting in a decreased maximum deuterium enrichment from over .16 to about .06. When corrections for exogenous dilution were made, the lower (cholesterol-rich) curve became very similar to the cholesterol-free curve.
486
C.
BRUCE
TAYLOR
ET
AL.
cholesterol increased 567& while on the cholesterol-rich diet. His absolute deuterocholesterol concentration, however, fell from 0.1640 moles per cent (low-cholesterol diet) to 0.0650 moles per cent of DzO on the cholesterol-rich diet. We corrected for the dilution of endogenously synthesized deutero-cholesterol by dietary cholesterol by determining the degree of dilution by exogenous cholesterol and increasing the moles per cent of D,O absolute by a corresponding factor. The effectiveness of this correction is demonstrated by the corrected curve obtained when the subject was on the cholesterol-rich diet. This effect was seen in all corrected curves. By plotting the data on semi-log paper (in the manner of London and Rittenberg) one can obtain the half-turnover’time of serum cholesterol. This half-turnover time is the time required to reach 50% of maximum deutero-cholesterol concentration. It also represents the time required for half the serum cholesterol molecules present in circulation to be synthesized. After obtaining the t$ (estimated half-turnover time in days), one can calculate the turnover rate of endogenously synthesized plasma cholesterol. This turnover is the mean survival time of serum cholesterol molecules and is related to the rate at which serum cholesterol molecules are synthesized. It is interesting to note the relatively unchanged t$ and T (estimated turnover time in days) in both low-cholesterol and cholesterol-rich groups (Table I). ESTIMATED
TURNOVER
TABLE I AND HALF-TURNOVER TIME VALUES FOR SUBJECTS AND CHOLESTEROL-RICH DIETS
Subjecta Subject of London and Rittenberg No. 1 No. 1 No. 2 No. 2 No. 3 No. 3 No. 4
Diet ( ?) Chol.-rich Chol.-free Chol.-rich Chol.-free Chol.-rich Chol.-free Chol.-rich Chol.-free
t%
ON CHOLESTEROL-FREE
(daysjb 8 10.5 10.0
8.0 8.5 12.5 12.0 9.5
r (davs)b 12 15.0 14.4 11.6 12.3 18.0 17.3 13.7
a A fifth subject receiving a much lower labeling dose of D.,C) is not listed. b t% z estimated half-turnover time in days; T = estimated turnover in days.
A compilation of the data on all subjects and comparison with the data of London and Rittenberg indicate that the absence of cholesterol or the presence of 2.5 to 3.0 gm per day in the diet results in no significant change in the t$ or r of plasma cholesterol. The estimated quantities of newly synthesized cholesterol appearing in the plasma each day are also reasonably comparable to the quantity reported by London and Rittenberg (Table II). Again, the essentially complete lack of an effect of dietary cholesterol on estimated daily plasma synthesis is striking. The above studies back” mechanism in suggest a continuous plasma regardless of
CONCLUSION and our earlier studies indicate an essential absence of a “feedman for compensation for dietary cholesterol. These studies unaltered rate of delivery of endogenous cholesterol to the diet. They further suggest that absorbed dietary cholesterol is
HUMAN
DATA USED
FOR
SERUM
ESTIMATION
CHOLESTEROL
OF GRAMS
TABLE II OF PLASMA
Plasma Subject Subject of London and Rittenberg
Diet
T
(days)n
vol. (ml)
487
SYNTHESIS
CHOLESTEROL
SYNTHESIZED
PER DAY
Mean serum cholesterol
Mb = total plasma
mC:gm plasma cholesterol
(mg
cho1. (gm)
syn ./day
percent)
( ?) Chol.-rich
12
3750
175
6.56
,546
No.
1”
Chol.-free Chol.-rich
15.0 14.4
2990 2990
218 261
6.5 (7.8-1.3)
.43 .45
No.
2d
Chol.-free Chol.-rich
11.6 12.3
2330 2330
175 273
4.1 (6.4-2.3)
.3S .33
No.
3”
Chol.-free Chol.-rich
18.0 17.3
2891 2891
150 193
4.3 (5.6-1.3)
.24 .25
4
Chol.-free
13.7
3180
110
No.
-
3.5
.26
a T = estimated turnover time in days. b M = total quantity of plasma cholesterol derived from plasma volume and mean serum cholesterol. c m = quantity of plasma cholesterol synthesized per day derived from dividing M by !?, in gram. d In subjects 1, 2, and 3 the total plasma cholesterol in grams during cholesterol-rich studies has been corrected for estimated dilution by unlabeled dietary cholesterol.
added to endogenous serum cholesterol, resulting in a net higher serum cholesterol concentration. There still remains another important, incompletely studied facet of human cholesterol metabolism; this concerns the ability of man to alter his rate of conversion of cholesterol to bile acids and alter fecal excretion of both bile acids and cholesterol to compensate for variable quantities of dietary cholesterol. Studies of this nature have been reported in dogs (,4bell et aE., 19.561,rats (Wilson, 19641, and are being actively studied in man (Grundy et al., in press; Miettinen et al., in press). On the basis of our present knowledge it would appear that man’s principal protection from hypercholesteremia of dietary origin is his rather limited capacity for intestinal absorption of cholesterol (Kaplan et al., 1963). REFERENCES ABELL, L. L., LEVY, B. B., BRODIE, B. B., and KENDALL, F. E. (1952). A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Ckem. 195, 357. ABELL, L. L., MOSBACH, E. H., and KENDALL, F. E. (1956). Cholesterol metabolism in the dog, J. Biol. Chem. 220, 527-536. Cox, G. E., TAYLOR, C. B., PATTON, D., DAVIS, JR,, E. B., and BLANDIN, IV. (1963). Origin of plasma cholesterol in man. Arch. Pathol. 76, 60-88. DAVIS, JR., E. B., Cox, G. E., TAYLOR, C. B., and CROSS, S. L. (1959). Cholesterol synthesis in human liver. Surgical Forum 9, 486-489. GOULD, R. G., and TAYLOR, C. B. (1950). Effect of dietary cholesterol on hepatic cholesterol synthesis. Federation Pvoc. 9, 179. GRUNDY, S. M., AHRENS, JR., E. H. and MIETTINER, T. A. (1965). Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids J. Lipid Res, 6, 397-410. KAPLAN, J. A., Cox, G. E., and TAYLOR, C. B. (1963). Cholesterol metabolism in man. Arch. Pathol. 76, 359-368.
488
C. BRUCE
TAYLOR
ET
AL.
LONDON, I. M., and RITTENBERG, D. (1950). Deuterium studies in normal man. I. The rate of synthesis of serum cholesterol. II. Measurement of total body water and water absorption. J. Bid. Chem. 184, 687-691. MIETTINEN, T. A., AHRENS, JR., E. H., and GRUNDY, S. M. (1965). Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J. Lipid Res. 6, 411-424. MORRIS, M. D., CEAIKOFF, I. L., FELTS, J. M., ABRAHAM, S., and FANSAH, N. D. (1957). The origin of serum cholesterol in the rat: diet versus synthesis. 1. Biol. Chem. 224, 1039. SIPERSTEIN, M. D., and GUEST, M. J. (1960). Studies on the site of feedback control of cholesterol synthesis. /. Clin. Invest. 99, 462. TAXZOR, C. B., MIKKELSON, B., ANDERSON, J. A., and FORMAN, D. T. (1965). Human Serum Cholesterol Synthesis Measured with the Deuterium Label. Arch. Pathol. (to be published). TA~OR, C. B., PATTON, D., YOGI, N., and Cox, G. E. (1960). Diet as source of serum cholesterol in man. Proc. Sot. Ezptl. Bid. Med. 103, 768-772. TOMKINS, G. M., SHEPPARD, H., and CHAIKOFF, I. L. (1953). Cholesterol synthesis by liver. III. Its regulation by ingested cholesterol. J. Biol. Chem. 201, 137. WILSON, JEAN D. (1964). The quantification of cholesterol excretion and degradation in the isotopic steady state in rat: The influence of dietary cholesterol. 1. Lipid Res. 6, 409-417.
AND
MOLECULAR
Human
PATHOLOGY
Serum
C. BRUCE TAYLOR, B. MIKKELSON, Department
4, 480-488
(1965)
Cholesterol
Synthesis’
J. A. ANDERSON, D. T. FORMAN, AND S. S. CHOIR
of Pathology, Evanston Hospital, Evanston, Illinois; Northwestern University School, Chicago, Illinois; and Mayo Clinic, Rochester, Minnesota Received
May
Medical
19, 1965
In 1950 Gould and Taylor reported (Fig. 1) a marked suppressionof newly synthesized cholesterol in the liver and plasma of dogs ingesting a high cholesterol diet
Hours FIG. 1. Effect of dietary cholesterol on in vitro cholesterol synthesis by liver slices in dogs. Estimated milligrams of cholesterol synthesis per 100 gm of liver are plotted on the ordinate; hours incubated in presence of Cl4 acetate are plotted on the abscissa. Control dogs were on cholesterolfree diets (for 1 month) before liver biopsies were taken for incubation. Cholesterol-fed dogs were fed 1 gm of cholesterol in oil 12 hours before liver biopsies were taken for incubation. Estimations into cholesof cholesterol synthesis were determined from quantities of Cl4 acetate incorporated terol molecules isolated from incubated liver slices.
(Gould and Taylor, 1950) ; and others demonstrated a similar homeostatic mechanism in rats (Tomkins et al., 1953). The mechanismof this inhibition of cholesterol synthesis by dietary cholesterol has been studied extensively and shown to possessthe characteristics of a negative “feed-back” mechanismor control system. Thus, in this 1 Supported by the Chicago, Illinois and American Heart Associations; The Life Insurance Medical Fund; the Thomas J. Dee and the George C. Moody Memorial Research Funds of Evanston Hospital; and the National Institutes of Health, Department of Health, Education, and Welfare (Graduate Research Training Grant ZGM-697). 2 Research Trainee supported by Graduate Research Training Grant 2GM-697. 480
HUMAN
SERUM
CHOLESTEROL
SYNTHESIS
481
control system, a single reaction, the reduction of ,&OH ,L?methyl glutaryl CO A to mevalonic acid was shown to be the primary site of depression of cholesterologenesis produced by exogenous (dietary) cholesterol (Siperstein and Guest, 1960). Studies in our laboratories (Fig. 2) have shown an almost complete lack of change in the rate of appearance of newly synthesized plasma cholesterol in man regardless of the type of diet (cholesterol-rich or cholesterol-low) employed (Cox et al., 1963). These
endogenously synFIG. 2. Effect of dietary cholesterol on appearance of Cl4 acetate-labeled thesized cholesterol in plasma of dogs (on left) and man (on right). Clear bars represent amounts appearing when dog and 8 subjects were on cholesterol-free diets; dark bars represent relative quantities appearing while dog and subjects were on cholesterol-rich diet. The 100% for cholesterolfree values was arbitrarily chosen as baseline for this graph. Dog shows marked suppression (to 5%) of rate of appearance of labeled plasma cholesterol, whereas the human subjects showed no compensatory suppression. Value of 162% for group of subjects was probably over 100% because of shift in pathways for use of acetate pool caused by feeding of neutral fat with cholesterol.
findings corroborated earlier studies which indicated that man (unlike the dog and rat) has little demonstrable “feed-back” control of in vitro hepatic cholesterol synthesis (Davis et uZ., 1959). Employing an indirect method for estimating relative contributions of dietary and endogenously synthesized cholesterol to plasma cholesterol, reported by Morris and others in studies on rats (Morris et al., 1957), we fed ring-labeled 4-Cl* cholesterol to dogs and humans (Taylor et al., 1960; Kaplan ef al., 1963). Our studies in the dog were comparable to those reported in the rat by Morris and others (1957) ; these animals both demonstrated nearly complete replacement of endogenous sources of
482
C. BRUCE
TAYLOR
ET
AL.
plasma cholesterol by ring-labeled dietary cholesterol (Fig. 3). The indirect studies were in good agreement with earlier studies demonstrating the dominant homeostatic role played by the liver in the cholesterol metabolism of the rat and the dog. The results from the ring-labeled cholesterol feeding studies in man were very different from those observed in dogs and rats; only about a quarter to a third of labeled dietary cholesterol appeared in plasma with the remaining large fraction of 3/4 to g of plasma cholesterol apparently being derived from endogenous synthesis (Fig. 4). As reported earlier, since most subjects showed an appreciable rise in serum cholesterol 280
1
/DIET
DIET:
01 0
I
.
2
3
.
4
WEEKS
5
.
C-14-CHOL @BY/DAY) IN E66 YOLK
.
6
7
6
.
.
9
IO
ON DIET
FIG. 3. Graph presenting appearance of ring-labeled (4-C14) dietary cholesterol in serum cholesterol of dogs. Radioactivity in disintegrations per min./mg. cholesterol is plotted on abscissa; time in weeks, on ordinate. Dotted line across top plots dietary radioactivity. Note that within 3 weeks the 2 dogs showed that 91 and 94% of their serum cholesterol contained the ring label, indicating that these percentages of serum cholesterol were derived from diet.
240 DIS. / YIN. 6
PERCENTAGE
‘60:
OF PLASMA
CHOL.
c---3I%-l-32.4
FROM
DIET
-
WOL
120.
0
2
4
12 WEEKS
0:
14
16
16
DIE:’
FIG. 4. Graph similar to that of Fig. 3 presenting rate of appearance of labeled (4-C’*) dietary cholesterol in serum of 32 year old female subject. At about 6 weeks only 31% of serum cholesterol was derived from labeled dietary cholesterol. Interestingly, changing the diet from 2 gm of cholesterol/day to 10 gm/day resulted in only a minimal rise (to 32.4%) in per cent of serum cholesterol derived from labeled dietary cholesterol.
HUMAN
SERUM
CHOLESTEROL
SYNTHESIS
483
while on the ring-labeled cholesterol regimen, there is suggestive evidence that in the human dietary cholesterol is additive to an essentially quantitatively unaltered endogenoussupply of plasma cholesterol (Kaplan et al., 1963). In order to better understand cholesterol metabolism in man and determine the effects of dietary cholesterol on the rate of synthesis of serum cholesterol, a deuteriumlabeling technique was employed. The rationale and methodology were based on the work of London and Rittenberg (1950) who showed that a single adult male subject on a regular diet (probably containing 0.5 to 1 gm of cholesterol per day) had about 0.5 gm of newly synthesized cholesterol appear in his plasma each day (London and Rittenberg, 1950). Since much of the quantitative estimation of human cholesterol synthesis is basedon this single study, we felt the need for additional similar studies with subjects on cholesterol-free and cholesterol-rich diets. MATERIALS
4ND METHODS
Eight endogenous serum cholesterol synthesis studies were carried out on five healthy human subjects (male and female) (Taylor et al., 1965). The subjects’ age, race, sex, and national origin varied considerably. Each subject served as his or her own control and they were alternately placed on a cholesterol-low diet and a cholesterol-rich diet. The cholesterol-low diet consisted mainly of egg whites, beans, lentils, powdered fat-free milk, and dry cottage cheese. This regimen excluded meat, egg yolks, cream, and butter fat. All subjects were placed on this diet six weeks prior to the initiation of the studies. Baseline levels of normally occurring deuterium in urine and in serum cholesterol were obtained from all subjects. Subsequently, subjects were administered an oral priming dose of heavy water (D?O) in concentrations high enough to raise the body water D-0 content to 0.5 moles per cent in excessof normal baseline DaO. Doses averaged from 140 to 250 gm of DZO. Thereafter, subjects received oral dosesof D,O daily, approximating 10% of their original priming dose in order to compensate for normal turnover of body water. All studies were run for at least 40 to SOdays’ duration, and almost daily urine and serum sampleswere collected from subjects. After completion of this initial study, subjects were allowed 7-10 months for the elimination of DZO of body water, cholesterol and other endogenously synthesized deuterated organic molecules.Mass spectrometric analysis verified the return to baseline levels of D,O contents of body water and serum cholesterol. The cholesterol-rich study was then instituted. This study provided an estimated daily dietary intake of 2.5 to 3.0 gm of cholesterol. The egg was the major cholesterolcarrying vehicle since an egg yolk contains approximately 250 mg cholesterol. Again, urine and serum sampleswere collected almost daily for determination of deuterated body water levels and deuterium concentration in newly synthesized cholesterol. The isolation, combustion and reduction of serum cholesterol were carried out in the following manner. Serum specimenswere hydrolyzed with alcoholic KOH and extracted with petroleum ether and one portion of the extract analyzed for total serum cholesterol by the Abell-Kendall procedure (Abel1 et al., 1952) with a Lieberman color reaction (20-1, acetic anhydride-sulfuric acid). The remainder was converted to cholesterol digitonide. Pyridine was then added in order to split off the digitonide and the cholesterol was extracted with acidified ether, purified and weighed. Specimens so isolated usually yielded from 2 to 4 mg of pure cholesterol.
484
C. BRUCE
TAYLOR
ET
AL.
The isolated cholesterol was then combusted with copper oxide at 600°C under high vacuum conditions in a sealed ampule of Corning No. 1720 ignition glass. The ampule containing the COZ and HZ0 of combustion was then placed in liquid nitrogen in order to condense and freeze the gases formed. Carbon dioxide was removed and water driven over into a reduction ampule containing 0.5 gm zinc. The reduction tube was then heated to SOO’C in order to reduce the HZ0 and DzO to Hz and Dz gas. The deuterium assays were carried out in a Nier Model, RCA isotope ratio mass spectrometer at the Mayo Clinic. The mass spectrometric analyses consisted of measuring the mass 3 to mass 2 ratio. This ratio was then converted to heavy water concentration by reference to a calibration derived from five standards of various concentrations of DzO and HzO. The magnitude of error in the reduction and mass spectrometer analyses was of the order of 5 4%. RESULTS
AND
DISCUSSION
The principal advantage of using deuterium is its negligible radioactivity which makes it possible to carry out long-term studies on humans without radiation as a prime hazard. By enriching each subject’s body water deuterium with the initial priming dose
SUBJECT O+
NO. 1
CHOL.
FREE
TIME IN DAYS
FIG. 5. Serum cholesterol-deuterium enrichment curves of Subject 1 while on cholesterol-free and cholesterol-rich diets. The middle curve is derived from corrected data of cholesterol-rich curve to correct for dilution of deutero-cholesterol by unlabeled dietary cholesterol (see text for discussion of this correction). Note again lack of effect of dietary cholesterol on rate of appearance of endogenously synthesized, labeled cholesterol.
HUMAN
SERUM
CHOLESTEROL
485
SYNTHESIS
and daily doses of deuterium oxide, we were able to elevate and maintain the subject’s labeled body water between 0.5 and 0.6 moles per cent of DzO. The labeling of newly synthesized cholesterol was adequate to provide meaningful data when D20 enrichment was at 0.5 moles per cent of DzO in body water. Maximum enrichment of 0.1% of D20 in body water was shown to be experimentally unsatisfactory. The maximal deutero-cholesterol concentrations were reached by 38-44 days in all eight studies. (Fig. 5 is a graph of data from two studies on one subject.) These times are comparable to the study reported by London and Rittenberg in which maximum deutero-cholesterol-digitonide concentrations were reached on the 36th day. Exogenous cholesterol feeding markedly increased the serum cholesterol levels. There was a marked increase in serum cholesterol of from 20 to 56 per cent. Since our studies showed simultaneous marked reduction of absolute deutero-cholesterol concentrations in the cholesterol-rich subjects, we felt this lower specific activity was due to the dilution effect of exogenous cholesterol on endogenously synthesized deuterocholesterol. The most striking example was Subject 2 (Fig. 6) whose mean serum SUBJECT
NO,
2 0-0 M
0
IO
20
30
40 TIME
50
60
70
CHOL.
FREE
CHOL. RICH (uncorrected)
00
90
IN DAYS
Serum cholesterol-deuterium enrichment curves of Subject
2. As discussed in the text, this subject’s serum cholesterol increased 5670, while he was on the cholesterol-rich study, resulting in a decreased maximum deuterium enrichment from over .16 to about .06. When corrections for exogenous dilution were made, the lower (cholesterol-rich) curve became very similar to the cholesterol-free curve.
486
C.
BRUCE
TAYLOR
ET
AL.
cholesterol increased 567& while on the cholesterol-rich diet. His absolute deuterocholesterol concentration, however, fell from 0.1640 moles per cent (low-cholesterol diet) to 0.0650 moles per cent of DzO on the cholesterol-rich diet. We corrected for the dilution of endogenously synthesized deutero-cholesterol by dietary cholesterol by determining the degree of dilution by exogenous cholesterol and increasing the moles per cent of D,O absolute by a corresponding factor. The effectiveness of this correction is demonstrated by the corrected curve obtained when the subject was on the cholesterol-rich diet. This effect was seen in all corrected curves. By plotting the data on semi-log paper (in the manner of London and Rittenberg) one can obtain the half-turnover’time of serum cholesterol. This half-turnover time is the time required to reach 50% of maximum deutero-cholesterol concentration. It also represents the time required for half the serum cholesterol molecules present in circulation to be synthesized. After obtaining the t$ (estimated half-turnover time in days), one can calculate the turnover rate of endogenously synthesized plasma cholesterol. This turnover is the mean survival time of serum cholesterol molecules and is related to the rate at which serum cholesterol molecules are synthesized. It is interesting to note the relatively unchanged t$ and T (estimated turnover time in days) in both low-cholesterol and cholesterol-rich groups (Table I). ESTIMATED
TURNOVER
TABLE I AND HALF-TURNOVER TIME VALUES FOR SUBJECTS AND CHOLESTEROL-RICH DIETS
Subjecta Subject of London and Rittenberg No. 1 No. 1 No. 2 No. 2 No. 3 No. 3 No. 4
Diet ( ?) Chol.-rich Chol.-free Chol.-rich Chol.-free Chol.-rich Chol.-free Chol.-rich Chol.-free
t%
ON CHOLESTEROL-FREE
(daysjb 8 10.5 10.0
8.0 8.5 12.5 12.0 9.5
r (davs)b 12 15.0 14.4 11.6 12.3 18.0 17.3 13.7
a A fifth subject receiving a much lower labeling dose of D.,C) is not listed. b t% z estimated half-turnover time in days; T = estimated turnover in days.
A compilation of the data on all subjects and comparison with the data of London and Rittenberg indicate that the absence of cholesterol or the presence of 2.5 to 3.0 gm per day in the diet results in no significant change in the t$ or r of plasma cholesterol. The estimated quantities of newly synthesized cholesterol appearing in the plasma each day are also reasonably comparable to the quantity reported by London and Rittenberg (Table II). Again, the essentially complete lack of an effect of dietary cholesterol on estimated daily plasma synthesis is striking. The above studies back” mechanism in suggest a continuous plasma regardless of
CONCLUSION and our earlier studies indicate an essential absence of a “feedman for compensation for dietary cholesterol. These studies unaltered rate of delivery of endogenous cholesterol to the diet. They further suggest that absorbed dietary cholesterol is
HUMAN
DATA USED
FOR
SERUM
ESTIMATION
CHOLESTEROL
OF GRAMS
TABLE II OF PLASMA
Plasma Subject Subject of London and Rittenberg
Diet
T
(days)n
vol. (ml)
487
SYNTHESIS
CHOLESTEROL
SYNTHESIZED
PER DAY
Mean serum cholesterol
Mb = total plasma
mC:gm plasma cholesterol
(mg
cho1. (gm)
syn ./day
percent)
( ?) Chol.-rich
12
3750
175
6.56
,546
No.
1”
Chol.-free Chol.-rich
15.0 14.4
2990 2990
218 261
6.5 (7.8-1.3)
.43 .45
No.
2d
Chol.-free Chol.-rich
11.6 12.3
2330 2330
175 273
4.1 (6.4-2.3)
.3S .33
No.
3”
Chol.-free Chol.-rich
18.0 17.3
2891 2891
150 193
4.3 (5.6-1.3)
.24 .25
4
Chol.-free
13.7
3180
110
No.
-
3.5
.26
a T = estimated turnover time in days. b M = total quantity of plasma cholesterol derived from plasma volume and mean serum cholesterol. c m = quantity of plasma cholesterol synthesized per day derived from dividing M by !?, in gram. d In subjects 1, 2, and 3 the total plasma cholesterol in grams during cholesterol-rich studies has been corrected for estimated dilution by unlabeled dietary cholesterol.
added to endogenous serum cholesterol, resulting in a net higher serum cholesterol concentration. There still remains another important, incompletely studied facet of human cholesterol metabolism; this concerns the ability of man to alter his rate of conversion of cholesterol to bile acids and alter fecal excretion of both bile acids and cholesterol to compensate for variable quantities of dietary cholesterol. Studies of this nature have been reported in dogs (,4bell et aE., 19.561,rats (Wilson, 19641, and are being actively studied in man (Grundy et al., in press; Miettinen et al., in press). On the basis of our present knowledge it would appear that man’s principal protection from hypercholesteremia of dietary origin is his rather limited capacity for intestinal absorption of cholesterol (Kaplan et al., 1963). REFERENCES ABELL, L. L., LEVY, B. B., BRODIE, B. B., and KENDALL, F. E. (1952). A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Ckem. 195, 357. ABELL, L. L., MOSBACH, E. H., and KENDALL, F. E. (1956). Cholesterol metabolism in the dog, J. Biol. Chem. 220, 527-536. Cox, G. E., TAYLOR, C. B., PATTON, D., DAVIS, JR,, E. B., and BLANDIN, IV. (1963). Origin of plasma cholesterol in man. Arch. Pathol. 76, 60-88. DAVIS, JR., E. B., Cox, G. E., TAYLOR, C. B., and CROSS, S. L. (1959). Cholesterol synthesis in human liver. Surgical Forum 9, 486-489. GOULD, R. G., and TAYLOR, C. B. (1950). Effect of dietary cholesterol on hepatic cholesterol synthesis. Federation Pvoc. 9, 179. GRUNDY, S. M., AHRENS, JR., E. H. and MIETTINER, T. A. (1965). Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids J. Lipid Res, 6, 397-410. KAPLAN, J. A., Cox, G. E., and TAYLOR, C. B. (1963). Cholesterol metabolism in man. Arch. Pathol. 76, 359-368.
488
C. BRUCE
TAYLOR
ET
AL.
LONDON, I. M., and RITTENBERG, D. (1950). Deuterium studies in normal man. I. The rate of synthesis of serum cholesterol. II. Measurement of total body water and water absorption. J. Bid. Chem. 184, 687-691. MIETTINEN, T. A., AHRENS, JR., E. H., and GRUNDY, S. M. (1965). Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J. Lipid Res. 6, 411-424. MORRIS, M. D., CEAIKOFF, I. L., FELTS, J. M., ABRAHAM, S., and FANSAH, N. D. (1957). The origin of serum cholesterol in the rat: diet versus synthesis. 1. Biol. Chem. 224, 1039. SIPERSTEIN, M. D., and GUEST, M. J. (1960). Studies on the site of feedback control of cholesterol synthesis. /. Clin. Invest. 99, 462. TAXZOR, C. B., MIKKELSON, B., ANDERSON, J. A., and FORMAN, D. T. (1965). Human Serum Cholesterol Synthesis Measured with the Deuterium Label. Arch. Pathol. (to be published). TA~OR, C. B., PATTON, D., YOGI, N., and Cox, G. E. (1960). Diet as source of serum cholesterol in man. Proc. Sot. Ezptl. Bid. Med. 103, 768-772. TOMKINS, G. M., SHEPPARD, H., and CHAIKOFF, I. L. (1953). Cholesterol synthesis by liver. III. Its regulation by ingested cholesterol. J. Biol. Chem. 201, 137. WILSON, JEAN D. (1964). The quantification of cholesterol excretion and degradation in the isotopic steady state in rat: The influence of dietary cholesterol. 1. Lipid Res. 6, 409-417.