Lack of degradation of dietary and endogenous sterols in gastrointestinal tract of man

Lack of Degradation of Dietary and Endogenous Sterols in Gastrointestinal Tract of Man By B. J. Kudchodkar,

H. S. Sodhi,

Nine hyperlipidemic subjects consuming their habitual solid diets were investigated by sterol balance techniques to see if there was any degradation of the steroid nucleus by the intestinal microflora. After the subjects had been equilibrated on their controlled habitual diets, 3-day fecal pools were collected first in a steady state for 9-12 days and then for another 12-15 days during the treatment with clofibrate and nicotinic acid. The amounts of cholesterol, P-sitosterol, and their microbial metabolites were estimated from each fecal pool. They were corrected for fecal flow and methodological losses. There was a modest variation in the recovery of the dietary ,Q-sitosterol from pool to pool within a given subject. The mean variation was 9 * 7%. The mean recovery of dietary P-sitosterol in nine subjects

and L. Horlick

was 94 +- 5O/o with a range between 89 and 104O/0. If the absorption of /3-sitosterol is assumed to be 5O/o, these results rule out significant degradation of the steroid nucleus in the gastrointestinal tract. This was true even when the subjects were given clofibrate or nicotinic acid. The mean recovery of ,&sitosterol during this period was 96 2 6O/o. The proportions of microbial conversion products (stanone and stanol) of cholesterol and ,&sitosterol were similar. Clofibrate treatment did not significantly influence these proportions. However, in response to the treatment with nicotinic acid, significant increases in the stanol derivatives of both cholesterol and ,&sitosterol were seen, but the changes were identical for the two sterols.

I

T HAS LONG BEEN RECOGNIZED that unabsorbed dietary or endogenous cholesterol is transformed to coprostanol and other steroid ketones by the action of intestinal microorganisms. lv2 It has also been shown that the plant sterols (e.g., P-sitosterol) undergo identical conversions to those of cholesterol, suggesting that the intestinal microorganisms do not distinguish between P-sitosterol and cholesterol.3 In 1968, Grundy et a1.4 presented evidence suggesting that there were considerable losses of dietary cholesterol and ,8-sitosterol despite very careful and meticulous methodology employed for their recoveries in the feces. They postulated, therefore, that the losses were due to the degradation of the sterol nucleus and not to other possible causes for incomplete recovery of these sterols. Such losses were noted in a number of subjects investigated by the above authors, although Borgstrom reported it in

From the Deparfmenf of Medicine, University of Saskatchewan, Saskatoon, Canada. Received for pubrication September 17, 1971. Supported by Grants MA-Z%ZI and MA-3580 from the Medical Research Council of Canada, from the Canadian and Saskatchewan Hearf Foundations, and from Ayerst Laboratories, Montreal, Canada. 8. J. Kudchodkar, Ph.D.: Postdoctoral Fellow, Department of Medicine, University of Saskafckewan, Saskatoon, Canada. H. S. Sodhi, M.D., Ph.D.: Director of Medical Research, Department of Medicine, University of Saskatchewan, Saskatoon, Canada. L. Horlick, M.D., F.R.C.P.(C.): Professor and Head, Department of Medicine, University of Saskatckewan, Saskafoon, Canada. Metabolism,

Vol. 21, No. 4 (April), 1972

343

KUDCHODKAR,

344 Table 1. Clinical

Subject Age

Sex

Weight (kg)

48 32

F M

61 81

37

F

62

51

M

88

46

M

74

44

M

114

36

M

71

54

F

64

42

M

77

SODHI, AND HORLICK

Data Cholesterol Triglycerides (mg/lOO ml) (mg/lOO ml)

Clinical Diagnosis

Coronary heart disease Agina pectoris; family history of coronary heart disease Xanthelesma; family history of coronary heart disease Coronary heart disease; maycoardial infarction Coronary heart disease; angina pectoris Pulmonary infarction; obesity Family history of coronary heart disease Angina pectoris; family history of coronary heart disease Family history of coronary heart disease

Treatment

280 299

94 159

Clofibrate Clofibrate

434

112

Clofibrate

284

183

Clofibrate

223

283

Clofibrate

358

388

Clofibrate

257

284

Clofibrate

480

118

Nicotinic

Acid

309

120

Nicotinic

acid

only one out of 20 subjects and Connor et al6 noted it in only one of six subjects they investigated. When choIestero1 IabeIed with r4C in the ring structure was administered to man, no 14C02 was detected in the expired air,7s and it would appear that except for the aromatization of the ring there is no degradation of the cyclopentanophenanthrene nucleus in the body of man.g It is conceivable, however, that the intestinal bacteria can degrade the ring. The hypothesis of degradation of the steroid nucleus in the intestinal lumen has been advanced to account for the lack of complete recovery of dietary plant sterols. This hypothesis underscores the necessity of monitoring the intestinal losses of cholesterol by measuring the fecal recovery of dietary /3-sitosterol in those subjects who consistently show incomplete recoveries. The relative importance of this phenomenon has yet to be assessed. Our detailed studies on the recoveries of dietary /3-sitosterol in nine subjects failed to show any evidence for its losses in the gastrointestinal tract. Our studies also confirmed the findings of Grundy et a1.,4 suggesting that the proportions of various products of cholesterol and /3-sitosterol produced by the intestinal microorganisms were identical. MATERIALS

AND

METHODS

All subjects had either elevated plasma cholesterol (greater than 250 mg/loo ml) or plasma triglyceride (greater than ISO mg/loo ml) concentrations or both when first seen in the University Hospital. The values given in Table I are those obtained at the time of their participation in these studies. The subjects consumed a diet of constant composition closely resembling their habitual food. Plant sterols including p-sitosterol as well as cholesterol were inherent components of the diets. The diets were prepared from single pools of each of the various dietary constituents. Sufficient servings were prepared and

DIETARY

AND ENDOGENOUS

345

STEROLS

frozen to last for the entire period of the study. One week’s trial on the diet was allowed for the subjects to become accustomed to the diet and to adjust the intake to maintain body weight. The subjects were also given chromic oxide 100 mg three times a day with meals to correct for the variations in the fecal flow. The feces were collected in s-day pools for 9 or 12 days after the subjects had been consuming the experimental diet. The subjects were then given either clofibrate 500 mg four times a day or nicotinic acid 1 g three times a day (Table l), and collections of feces were continued for 12-15 days more. The fecal neutral streoids derived from the cholesterol and plant sterols were extracted, various metabolites isolated by thin-layer chromatography, and the metabolites of cholesterol (coprostanol and coprostanone) and of 6-sitosterol (coprositostanol and coprositostanone) were then estimated by gas-liquid chromatography by the procedures developed by Miettinen et al.10 The recoveries of dietary 6-sitosterol in the feces were corrected for the fecal flow from the data on chromic oxide.11 Any losses in extraction and recovery procedures were corrected by the use of an external standard of radioactive cholesterol. RESULTS Recovery

of

Dietary P-Sitosterol in Feces

The intake of /3-sitosterol varied between 171 and 4.~4 mg/day. For estimation of the recovery of P-sitosterol in the feces, /3-sitosterol and its stanone and stanol derivatives were estimated and their amounts added (Table 2). Three or four fecal pools before treatment and four or five fecal pools after starting the treatment were examined. There was a modest variation in the recovery of dietary P-sitosterol from pool to pool within a given subject during the control period. The range of variations was 3-25% with a mean + SD of 9 + 7%. The per cent recoveries of /3-sitosterol in nine subjects during the steady state conditions of the control period were between 89 and 104 with a mean of 94 * 5 (Table 2). The subjects continued to eat the same diet when treatment with clofibrate of nicotinic acid was started so that the intake of dietary P-sitosterol was the same as during the control period. The fecal recoveries during the treatment period were 96 + 6% indicating that clofibrate or nicotinic acid did not have any significant effect on the fecal recoveries of dietary ,&itosterol (Table 2). The effect of treatment on plasma lipids, however, was significant. There were Table 2. Recovery

of Dietary

p-Sitosterol

Excretion (mQ/day)Mean 2 SD Subject

Intake (mQ/daY)

1 2 3 4 5 6 7 6 9

361 257 255 454 171 290 394 351 404

Pretreatment

399 221 235 436 156 277 360 323 369

Mean + SD *Number of fecal pools. tNS: nonsignificant.

f 11 (3)' -e 65(4) f 32(3) f 24 (4) ?I lS(3) f 14(3) + 23(4) f 33(4) f 12(4)

Treatment

363 232 267 459 162 277 354 315 404

f 66 (5)' +- 21 (4) + 15(4) -c SS(4) f 10(5) + 32(5) + Q(5) r 51 (4) r 14(5)

Recovery (%) Pretreatment

104a 3 69 * 25 92 ?z 13 96k 6 92 f 10 95k 5 Qlk 5 90 -c 14 962 3 94-c 5

Treatment

103 A 20 93k 6 104a 6 101 2 14 99 k 15 95 rt 11 90' 2 94k 5 lOOr. 3 96* 6

P

NSt NS NS NS NS NS NS NS NS NS

Subject

Nit acid

Nit acid

Clofibrate

Clofibrate

Clofibrate

Clofibrate

Clofibrate

Clofibrate

Clofibtate

Treatment

Cholesterol @-Sitester Cholesterol p-Sitesterol Cholesterol p-Sitesterol Cholesterol p-Sitesterol Cholesterol pSitesterol Cholesterol p-Siiesterol Cholesterol p-Sitesterol Cholesterol @Sitester Cholesterol @-Sitester

Sterol

407r 3992 803 f 221 k 682 f 235 f 1110 k 436 k 791 r 158 jI 1307 ” 277 k 1241 f 360 2 1113 -’ 323 f 735 k 389 -e

9 11 48 65 200 32 50 24 67 16 33 14 148 23 157 33 96 12

Total Steroids (m&day) Pretreatment

553 EY 53 3831 66 1005 A 48 232 -t 21 1043 1. 102 267 2 15 1438 r?:331 459 f 66 747 r 90 162 f 10 1253 -+ 112 277 +- 32 1199 -c 103 354 -e 9 1091-+ 71 315 -+ 51 893 z!z 57 4042 14 2 2

2 2 1 0.5 5 5 0.5 0.4 3 3 l-cl 1 1 1 0.5 -il -cl

21 k.0 a0 f 0 -

It1 fl 20 r 0 fl k2 f 0 f 0 FO *o

2 2

-c2 -cl

4 -t2 4 %2 1 +o 0.5 f 0 5 -cl 6 -cl I -c 0 0.3 * 0 2 20 2 %O 121 2 +I 2 21 2 -co 1 t 1

Stanone Pretreatment Treatment

of Dietary Sterols (% of Total)

Treatment

Table 3. Fecal Metabolites

1

942 1 822 4 81 2 4 85-c 7 045 1 982 2 98-c 2 762 3 7.5-c 3 66-c 4 69r 3 952 4 952 2 65 k 13 67 2 12 77k 6 772 6

952

Pretreatment

92 91 71 70 88 88 98 98 77 75 72 71 91 91 95 93 88 86

+. 2 + 5 + 4 -c 6 f 3 +- 4 -c 4 -c 2 2 7 -c 5 f 4 f 5 + 2 f 3 f 4 -c 2 f 8 k 7

Stan01 Treatment

1 1

28 r 4 29*5 8-c3 a*3 l-F1 Ikl 21 2 5 22 r 5 27 -c 5 28 2 5 623 7r3 323 723 11 28 12 r 4

422 4-12

Sterol Treatment

162 4 182 4 llf 1 115 1 2f 1 2* I 21 k 3 222 3 321 4 30-c 3 4-1: 2 6% 2 35 1 12 33 r 12 22-c 6 21k 3

3-c 32

Pretreatment

Y

DIETARY

AND ENDOGENOUS

decreases of 23 + s?& and 35 +- 12% concentrations. Bacterial Transformation

347

STEROLS

in plasma cholesterol

of Cholesterol and &Sitosterol

and triglyceride

in lnfestinal Lumen

Stanone and stanol derivatives were the major compounds resulting from the metabolism of intestinal bacteria and they were estimated individually for both cholesterol and ,&sitosterol. Their amounts were expressed as per cent of the total constituted by the stanone, stanol, and sterols in the feces (Table 3). The results computed from different pools showed little variation from pool to pool (Table 3). The proportions of stanone, stanol, and sterol derivatives of cholesterol and ,f3-sitosterol were remarkably similar in all subjects. Stan01 derivatives constituted anywhere from 66-95% of the total neutral steroids and stanone derivatives accounted for only OS-~%. The remainder was made up by the parent sterol molecule. In four subjects clofibrate increased the quantities of fecal total neutral steroids derived from cholesterol, and in three others it caused a decrease. However, the treatment with clofibrate changed neither the total nor the proportions of the various derivatives of dietary /3-sitosterol produced by the action of intestinal flora. In response to treatment with nicotinic acid, the fecal excretion of total neutral steroids derived from cholesterol showed no change in one and a significant increase in the other. There were significant increases in the stanol derivatives for both cholesterol and /3-sitosterol and corresponding reductions in the amounts of parent sterols present in the feces in each of the two subjects. DISCUSSION

A number of reports have indicated that the absorption of dietary P-sitosterol in man is up to 5%. 12*13 The fecal recoveries of dietary /3-sitosterol in our studies were 94 * 4% during the control and 96 + 6% during the treatment periods indicating that the losses of dietary &sitosterol during its passage through the intestinal tract could not have been significant in these subjects. Borgstrom investigated 20 subjects, and unexplained losses of radioactive /3-sitosterol were observed in only one subject. Connor et al.6 investigated six subjects and found significant losses also in only one subject. Further work on this subject by DenBesten et al. l4 showed that the losses of dietary sterols occurred when the patient was given a liquid formula and not when he was fed solid food of the same composition as that of the liquid formula. They also made an interesting observation that the addition of solids such as cellulose and lactose to the liquid formula in this patient prevented the losses observed when the patient consumed only the liquid formula. Most of the subjects in whom such losses were observed by Grundy et a1.4 had been given liquidformula diets and so the suggestion that the sterol balance studies should be corrected for the losses of neutral sterols15 may be applicable to the subjects given liquid formulae. Lack of any losses in our subjects may possibly be related to the consumption of solid-food diets. These data suggest that the losses of dietary sterols in the intestinal lumen

340

KIJDCHODKAR, SODHI, AND HORLICK

may not be an important phnomenon in subjects who consume solid-food diets. However, the one subject reported by Borgstrom in his study of 20 individuals did consume a solid-food diet. The nature of these losses observed in only an occasional subject must remain speculative at the moment. Until the end-products of bacterial action on sterols accounting for losses are identified, the degradation of the steroid nucleus remains only one of the many possibilities explaining the incomplete recoveries of dietary sitosterol. The word degradation or destruction implies breaking of the steroid nucleus producing fragments which are lost by the methods of fecal extraction.rO Rosenfeld and Hellman16 reported destruction of sterol during in vitro incubations of labeled /3-sitosterol and cholesterol with human stools. However, the extent of conversion of these sterols to materials soluble in 70% ethanol rather than in petroleum ether was not measured. It has been shown by Grundy et a1.4 that after its incubation with fecal cultures, 1540% of the radioactive cholesterol is not extracted by petroleum ether from the alkali aqueous phase. The radioactivity remaining behind in the aqueous phase was attributed to the possible conversion of cholesterol to cholesterol sulphate.4 In view of these findings, it is questionable if the radioactive sterols not extracted by petroleum ether from the aqueous phase could be equated to destruction.ie In similar in vitro studies, Wood and Hatoffr’ were able to recover all the radioactivity from the fecal cultures to which radioactive cholesterol had been added. Since there was significant bacterial conversions of cholesterol to coprostanol and other derivatives, it demonstrated that their fecal cultures were metabolically active. In absence of the knowledge of the degradative products of cholesterol, it may be better to view its losses as an open question. However, it is worth emphasizing that this appears to be an uncommon phenomenon in subjects given solid-food diets. Grundy et a1.4 gave radioactive cholesterol and ,&itosterol to their subjects and were the first to show that the relative amounts of stanone and stanol derivatives of the two sterols produced by the intestinal bacteria were identical. The results of our studies on the amounts of various products of cholesterol and P-sitosterol corroborate the work of these authors. The treatment with clofibrate had significant effects on plasma lipid levels and on the fecal excretion of total steroids in most, but it had no effect on the bacterial conversion of the sterol to stanone and stanol derivatives. Nicotinic acid also showed significant effects on plasma lipids in both subjects. It increased the fecal neutral steroids in only one of the two but had a significant effect on the bacterial conversion of the parent sterols to stanols in both subjects. The effect was identical on both sterols indicating that cholesterol and P-sitosterol are metabolized identically by the intestinal microorganisms. These data also support the suggestion of Kellog and Wostmann18 that changes in the gut microflora could not be a major factor in the changes in the fecal excretion of dietary or endogenous cholesterol. ACKNOWLEDGMENT The clofibrate Canada.

used in these studies was provided

by Ayerst

Laboratories,

Montreal,

DIETARY

AND ENDOGENOUS

STEROLS

349

REFERENCES 1. Snog-Kjaer, A., Prange, I., and Dam, H.: Conversion of cholesterol into coprosterol by bacteria in vitro. J. Gen. Microbial. 14~2.56,1956. 2. Rosenfeld, R. S., Fukushima, D. K., Hellman, L., and Gallagher, T. F.: The transformation of cholesterol to coprostanol. J. Biol. Chem. 211:301,1954. 3. Coleman, D. L., Wells, W. W., and Baumann, C. A.: Intestinal sterols. II. Determination of coprostanol and certain related sterofs. Arch. Biochem. 60:412, 1956. 4. Grundy, S. M., Ahrens, E. H., Jr., and Salen, G.: Dietary /3-sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies. J. Lipid Res. 9~374, 1968. 5. Borgstrom, B.: Quantification of cholesterol absorption in man by fecal analysis after the feeding of a single isotope-labeled meal. J. Lipid Res. 10:331, 1969. 6. Connor, W. E., Witiak, D. T., Stone, D. B., and Armstrong, M. L.: Cholesterol balance and fecal neutral steroid and bile acid excretion in normal men fed dietary fats of different fatty acid composition. J. Clin. Invest. 48:1363, 1969. 7. Chaikoff, I. L., et al.: C14-cholesterol. II. Oxidation of carbon 4 and 26 to carbon dioxide by intact rat. J. Bioi. Chem. 194: 413, 1952. 8. Siperstein, M. D., and Chaikoff, I. L.: C14-cholesterol. III. Excretion of carbon 4 and 26 in feces, urine and bile. J. Biol. Chem. 198:93, 1952. 9. Hellman, L., Bradlow, H. L., Adesman, J., Fukushima, D. K., Kulp, J. L., and Gallagher, T. F.: The fate of hydrocortisone4-Cl4 in man. J. CIin. Invest. 33:1106, 1954.

10. Miettinen, T. A., Ahrens, E. H., Jr., and Grundy, S. M.: Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J. Lipid Res. 6 :411,1965. 11. Stanley, M. M., and Cheng, S. H.: Excretion from the gut and gastrointestinal exchange studied by means of the inert indicator method. Amer. J. Dig. Dis. 2 :628, 1957. 12. Gould, R. G., Jones, R. J., LeRoy, V. G., WissIer, R. W., and Taylor, C. B.: Absorbability of P-sitosterol in humans. Metabolism 18 ~652,1969. 13. Salen, G., Ahrens, E. H., Jr., and Grundy, S. M.: Metabolism of /3-sitosterol in man. J. Clin. Invest. 49:952, 1970. 14. DenBesten, L., Connor, W. E., Kent, T. H., and Lin, D.: Effect of cellulose in the diet on the recovery of dietary plant sterols from the feces. J. Lipid Res. 11:341, 1970. 15. Ahrens, E. H., Jr.: A review of the evidence that dependable sterol balance studies require a correction for the losses of neutral sterols that occur during intestinal transit. In Tones, R. J. (Ed.): Atherosclerosis, Proceedings of the Second International Symposium. New York, SpringerVerlag, 1970, p. 248. 16. Rosenfeld, R. S., and Hellman, L.: Reduction and esteriflcation of cholesterol by homogenates of feces. J. Lipid Res. 12: 192, 1971. 17. Wood, P. D. S., and Hatoff, D.: Incubation of human fecal homogenates with 4-C14-cholesterol. Lipids 5 :702,1970. 18. Kellogg, T. F., and Wostmann, B. S.: Fecal neutral steroids and bile acids from germ free rats. J. Lipid Res. 10:495, 1969.