European Journal of Clinical Investigation (1979) 9,55-62

Esterification of cholesterol in human small intestine: the importance of acyl-CoA:cholesterol acyltransferase KAARE R. NORUM, ANNE-CHARLOTTE LILUEQVIST, PER HELGERUD, ELDAR R. NORMA", ARVE MO & BODIL SELBEKK, Institute for Nutrition Research, University of Oslo; Surgical Departments I1 and 111; and Research Laboratory of Gastroenterology, Medical Department IX, Ullevgl University Hospital, Oslo, Norway Received 10 April 1978 and in revised form 14 September 1978

Abstract. Human intestinal mucosa contains acyl-CoA:

cholesterol acyltransferase activity. The enzyme has been studied by using oleylcarnitine, CoA and carnitine palmitoyltransferase as an oleyl-CoA regenerating system. The enzyme was found in the particulate fraction of the cells, it had a pH optimum between 7.2 and 8.2, and was inhibited by taurocholate. The specific enzymic activity in biopsies from intestinal mucosa of normal men was found to be 3.6 k 1.37 nmol cholesteryl ester formed mg protein-' h-', an activity which can account for all cholesteryl esters in intestinal lymph. Low enzymic activity was found in biopsies from patients with small intestinal disorders. Two pancreatectomized patients had values within the normal range. Key words. Acyl-CoA:cholesterol acyltransferase; camitine palmitoyltransferase; cholesterol; cholesteryl ester; CoA; intestinal mucosa; taurocholate. Introduction

Cholesterol metabolism in the intestine plays an important role in the overall metabolism of sterols in the body [l]. In the intestinal lumen, cholesterol derives from at least three sources: the diet, the bile, and desquamated mucosal cells. About 30-50% of the intestinal cholesterol is absorbed [ 2 ] . Most of the cholesterol in intestinal lymph is esterified with long chain fatty acids [3]. The cholesteryl esters in mesenteric lymph have several potential sources. Some plasma lipoprotein cholesteryl esters may enter the lymph via interstitial spaces, some may be formed by the action of the enzyme lecithin: cholesteryl acyltransferase (LCAT)* (EC 2.3.1.43), and some may originate within the mucosal cells by esterifi-

* Abbreviations: lecithin :cholesterol acyltransferase (EC 2.3.1.43), LCAT; acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26), ACAT. Correspondence: Dr Kaare R. Norum, Institute of Nutrition Research, School of Medicine, University of Oslo, P.O. Box 1046, Blindern, Oslo 3, Norway. 0014-2972/79/0200-00ss$02.00 0 1979 Blackwell Scientific Publications

cation of newly synthesized or absorbed chol&terol. The intestinal lymph contains only small amounts of LCAT, which furthermore is inhibited to a large extent by the high concentrations of triglyceride-rich lipoproteins present in the lymph [4]. Thus, most of the cholesteryl esters in intestinal lymph are formed in the mucosal cell. The esterification of cholesterol in the small intestine has been explained by a mucosal cholesterol esterase (EC 3.1.1.13) thought to be derived from pancreas [5]. Recently, an acyl-CoA:cholesterol acyltransferase (ACAT) (EC 2.3.1.26) has been found in mucosal cells from rats [6] and guinea-pigs [7]. In the present study we have been able to demonstrate that ACAT is present in human intestinal mucosa, and that the esterification of cholesterol takes place in intestinal mucosa also from pancreatectomized patients. Materials and Methods

Chemicals. 7a-3H-Cholesterol (1 2.6 Ci/mmol) was purchased from the Radiochemical Centre, Amersham, England, and purified by thin-layer chromatography [8] before use. Bovine serum albumin, stigmasterol and glutathione were supplied by Sigma Chemical Company, St Louis, Miss., U.S.A. Sodium taurocholate was obtained from Koch-Light Laboratories Ltd, Colnbrooke, England. Camitine palmitoyltransferase (EC 2.3.1.2 1) with a specific activity of about 0.2 U/g of protein was prepared from calf liver mitochondria [9]. 1-Oleylcarnitine and ''C-palmitoyl-l-carnitine were kindly donated by Professor Jon Bremer, Institute of Medical Biochemistry, University of Oslo, Norway. Human material. Biopsies of intestinal mucosa were taken in the morning, usually between 09.00 and 10.00 hours, after an overnight fast, either from normal men, 19-24 years old, or from patients with different diseases, of either sex and different age groups (see Table 4). Informed consent was obtained from all individuals. The biopsies were taken from the transition between duodenum and jejunum corresponding to the ligament of Treitz. We used a modified Crosby capsule [lo] with

55

56

KAARE R. NORUM et al.

which several biopsies could be obtained. The biopsies were rinsed in ice-cold isotonic saline or potassium phosphate buffer (0.2 mol/l, pH 7.2), and were homogenized in the phosphate buffer using a tight-fitted Dounce homogenizer. Unbroken cells and debris were removed by centrifugation in a Sorvall SS34 Rotor, 120 g for 2 min.

Mucosal material was also taken from resected parts of duodenum and jejunum from patients operated for peptic ulcer or carcinoma of the stomach. The unaffected mucosa from these specimens was scraped off with a glass microscopic slide and homogenized as described for the biopsies. In most of these cases we could obtain enough material to isolate a ‘microsomal fraction’. The homogenate was then centrifuged in a Sorvall RC 2B centrifuge for 15 min at 6800 g in a SS34 Rotor at 4OC, in order to sediment cellular debris, nuclei, mitochondria and most of the lysosomes. The ‘microsomal fraction’ was obtained by centrifuging the 6800 g supernatant for 30 min in a Beckman L.2.65 ultracentrifuge at 140,000 g at 4°C using a Ti 60 rotor. The pellet was suspended in the potassium phosphate buffer (0.2 mol/l, pH 7.2). Protein content was analysed according to Lowry et al. [ 111, using bovine serum albumin as standard.

Results

Preliminary experiments revealed that a CoA-dependent cholesterol esterifying enzyme system is present in mucosa from human small intestine. Based on experience from a similar enzyme system in rat [6] and guineapig intestine [7] , optimal incubation conditions for the human enzyme were established. The pH optimum was rather broad, with a plateau between 7.2 and 8.2. A preincubation time of 2 h was enough to obtain a satisfactory isotopic equilibrium between added trace amounts of radioactive cholesterol and endogenous mucosal cholesterol. The esterification was started by adding the acyl-CoA generating system (acylcarnitine, carnitinepalmitoyl-transferase and CoA). The reaction was rectilinear for about 30 min, but we usually incubated for only 15 min to ensure linearity. The amounts esterified, however, were calculated and given in quantities per hour. Cholesterol esterification did not take place without CoA. Fig. 1 shows that cholesterol esterification in total homogenate and microsomal fraction from human jejunal mucosa is dependent on the CoA concentration. A maximum rate of esterification was obtained when CoA concentration was about 8 X mol/l. The apparent K , for CoA in the combined enzyme system is approximately 5 X mol/l (Fig. 1).

Unesterified cholesterol and cholesteryl esters were extracted with chloroform:methanol (2: I), separated by thin layer chromatography, and assayed by gas liquid chromatography [8]. Radioactive unesterified cholesterol and cholesterol esters were counted in a Packard TRI-CARB liquid scintillation spectrometer using Permablend (Packard Instruments Co., Ill., U.S.A.) as liquid scintillator. Counting efficiency for single and double isotope counting was determined by the external standard or channels ratio method. Enzyme assays. The incubations were carried out in plastic vials shaken continuously in a waterbath at 37°C. The incubation volume was 0.5 ml. Endogenous cholesterol was used as substrate, but was labelled by addition of trace amounts of 3H-cholesterol. The homogenate or micrxomal fraction was preincubated for 2 h with an albumin-stabilized emulsion of tritiated cholesterol at 37°C [ 121. Each incubation vial contained approximately 200,000 dpm radioactive cholesterol. The esterification of cholesterol was initiated by adding oleylcarnitine, glutathione, carnitine palmitoyltransferase, and finally CoA. More detailed descriptions of the incubation conditions are given in the Result section or in legends to Figures and Tables. The esterification rate of cholesterol is either given as the fractional esterification, i.e. per cent of labelled cholesterol esterified within a given time period, or as specific activity, i.e. nmoles cholesterol esterified per mg of protein per hour, assuming isotopic equilibrium between endogenous and exogenous free cholesterol. AU values given are means of duplicate determinations.

Ob

I

1

I

I

0

2

4

6

8

co A ( 1 0 - ~ m o 1 / 1 ) Figure 1. Formation of cholesteryl ester and concentration of

CoA. Total homogenate ( 0 ) and microsomal fraction ( 0 ) of mucosa from jejunum were obtained by scraping a piece of intestine resected during a Roux Y operation because of a prepyloric ulcer. The incubation mixture contained total homogenate or microsomal fraction corresponding to a cholesterol concentration of 14.3 and 31.7 mmol/l, respectively, 1%bovine serum albumine coated with trace amounts of radioactive cholesterol, 10 mmol/l glutathione, 0.1 mmol/l 1-oleylcarnitine, 18 U/l of carnitine palmitoyltransferase,and 0.2 mol/l potassium phosphate buffer, pH 7.2. The concentration of CoA varied as shown in the Figure. The total homogenate and the microsomal fraction were preincubated with the labelled cholesterol and the potassium phosphate buffer for 2 h, before the esterification was initiated with the addition of oleylcarnitine, carnitine palmitoyltransferase and CoA. Incubation time was 15 min.

MUCOSAL ESTERIFICATION OF CHOLESTEROL The formation of cholesteryl esters was stimulated by addition of small amounts of long chain acylcamitine. Fig. 2 reveals that the optimal concentration of oleylcamitine had a narrow concentration range which might probably be explained by the fact that the compound is both a substrate and a detergent, which in higher concentration will inhibit the enzyme system. Since the cholesteryl ester hydrolase is stimulated by taurocholate [5], we were interested in testing the effect of taurocholate on the CoA dependent enzyme system, When the usual incubation conditions were used, more than 50% of the microsomal cholesterol was esterified per hour (Table 1). Addition of 5 mmol/l taurocholate, which is the optimal concentration for esterification by the cholesteryl ester hydrolase [5], led t o a marked inhibition of the cholesterol esterification. The concentration of taurocholate influences the formation

57

of cholesteryl esters (Fig. 3). A significant inhibition occurred at 1 mmol/l, and the esterification was almost completely inhibited at 20 mmol/l. Table 1 further reveals that taurocholate cannot substitute for oleylcamitine in the reaction, and that taurocholate neither alone nor in the presence of CoA stimulates the microsoma1 cholesteryl ester formation at pH 7.2. No esterification of labelled cholesterol took place in the absence of CoA, and the reaction was greatly stimulated by oleylcamitine. These findings strongly indicate that the esterification proceeds via the following reactions: acylcarnitine + CoA + acylCoA carnitine, acylCoA cholesterol -+ cholesteryl ester CoA and that the mucosal enzyme is an acy1CoA:cholesteryl acyltransferase (ACAT). In order t o test this we designed an experiment in which we used ''C-palmitoylcarnitine and 3H-cholesterol in the same incubation, The obligate role of CoA was confirmed since no esterification took place in the absence of this compound (Table 2). When calculating the amount of cholesteryl esters formed, using either the specific activity of radioactive cholesterol or palmitoylcarnitine, we obtained the same results, strongly supporting the reaction scheme outlined above. However, these calculations were based upon the assumption of isotopic equilibrium between endogenous cholesterol and added radioactive cholesterol, and that the specific activity of the palmitate was the same in palmitoylcarnitine as in cholesteryl palmitate. The fractional esterification of added radioactive cholesterol was dependent on the amounts of tissue homogenate used in the incubation (Fig. 4). The concentration of homogenate is given as nmol mucosal cholesterol/ml incubation mixture. The optimal concentration

+ +

+

60 "0

2

L

I-OLEYLCARNITINE

6

8

10

(lO-Lrnol/l)

Figure 2. Formation of cholesteryl ester and concentration of

1-oleylcarnitine. Total homogenate (0)and microsomal fraction from the same preparation as described in legend to Fig. 1, were incubated. The concentration of CoA was 8 X mol/l. The concentration of 1-oleylcarnitine varied as shown in the Figure. The conditions were otherwise as stated in legend to Fig. 1. (0)

LT W CL

I-

In

Table 1. Influence of taurocholate concentration on the esterifi-

cation of cholesterol by microsomes from human jejunal mucosa

u

: \ W

201

The incubation mixture contained CoA

Taurocholate

Oleylcarnitine

0.8 0.8 0 0.8

0

0.1 0.1

5 5 5

0 0

% Cholesterol esterified per hour 52.8

7.2 0.3 0.6

The incubation mixture contained microsomal fraction in the same concentration as described in legend to Fig. 1. The final concentrations, in mmol/l of CoA, taurocholate and I-oleylcarnitine are tabulated, otherwise the conditions were as described in legend to Fig. 1.

0

I,,,,,\-, 0

5

10

TAUROCHOLATE

15

20

( 1 0 ' ~ rnol/l)

Figure 3. Inhibition of the formation of cholesteryl ester by taurocholate. The incubation mixture contained microsomal fraction from the same source and in the same concentration as described in legend t o Fig. 1. The concentration of CoA was 8 X mol/l and the concentration of sodium taurocholate varied as shown in the Figure. The conditions were otherwise as stated in legend to Fig. 1.

58

KAARE R. NORUM et al. Table 2. Formation of double labelled cholesteryl ester from 3H~7acholesterol and ''C-l-palmitoylcarnitine

Radioactivity (cpm) in: Isotope

Concentration of CoA (mmol/l)

1 4 c

0.8 0

'H

0.8 0

Cholesteryl ester

Cholesterol

Nanomoles cholesteryl ester formed

1265 15

0 0

1.1* 0

2440 90

54950 5 24 00

0

0.95tt

Microsomal fraction of mucosa from jejunum was obtained by scraping pieces of intestine taken from a patient during radical surgery for carcinoma of the stomach. The incubation mixture contained microsomal fraction corresponding to 56 nmol cholesterol/ml, 1% bovine serum albumine coated with 3H~holesterol(approx. 50,000 cpm), 0.1 mmol/l of 14C-palmitoylcarnitine(approx. 67,000 cpm), and CoA, as indicated. The conditions were otherwise as stated in legend to Fig. 1. * Assuming isotopic equilibrium between endogenous cholesterol and added 3H~holesteroltracer. tt Assuming that the radioactive palmitoyl group of cholesterylpalmitate and palmitoylcarnitine has the same specific activity.

'for the human enzyme was a mucosal homogenate corresponding to about 50 nmol cholesterol/ml. This amount of mucosal cholesterol equals about 1 mg of mucosal protein (see Table 4).

Localization of the enzyme system. From a patient operated with a Billroth I1 resection for chronic peptic ulcer, we obtained biopsies from both duodenum proximal to the papilla of Vater, and the jejunum. The enzyme activities in total homogenate and 'microsomal fraction' from these biopsies are given in Table 3, which shows that the specific activity of the enzyme in jejunum is 3 times higher than in proximal duodenum. Furthermore, the activity is higher in the microsomal fraction than in the total homogenate, by a factor of approximately 2.5. Stability of the enzyme activity. Intestinal biopsies or material obtained from gastrointestinal surgery were kept icecold and usually assayed within 3 h. In some instances this was impossible and the homogenate or microsomal fraction were stored at -20°C. The enzyme activity was quite stable for a few days under this condition, but prolonged storage at -20°C reduced the enzyme activity considerably, especially in the total homogenate. This is shown in Fig. 5 , whxh reveals that storage for about 18 days results in retention of only 50% of the original enzyme activity in the total homogenate. Based on preliminary experiments, we have found that the enzyme activity is much more stable when the material is kept at -70°C. Enzyme activity in intestinal biopsies. Biopsies were taken by a modified Crosby capsule or by scraping jejunal mucosa from specimens removed during gastrointestinal surgery. Table 4 shows the results obtained in total homogenates from nine normal men, and from sixteen patients with different diseases. The content of mucosal cholesterol in the normal men had a mean value of 45.5 nmol/mg protein. Values quite similar to this were found in most of the patients. The lowest values were observed in one who had been

pancreatectomized (J.N.), and in one with gluten enteropathy (M.B.), while the highest value was found in a patient with the Zollinger-Ellison syndrome (A.P.). The mean fractional esterification in normal men was 8.0%/h. Most of the fractional cholesterol esterification in the patients had values within the normal range. It is noteworthy that the two pancreatectomized patients (J.N. and T.G.) both had normal values. Very low fractional esterification was found in a patient with gluten enteropathy in an active phase (P.B.), in a patient with ulcerative colitis (S.H.) and in a woman with malabsorption and an atrophic jejunal mucosa (I.S.). Two patients with stomach ulcer (W.B., S.S.) had remarkably high fractional cholesterol esterification. The specific activity of the esterification in the normal men had a mean value of 3.6 nmol cholesteryl ester formed per mg protein per hour. Patients with malabsorption and diseases of the small intestine had the lowest values, while high values were generally found in the patients with stomach ulcer (except B.O., who had had gastric retention for several days). Discussion

The mechanism of cholesterol esterification in the mucosal cell has been poorly understood. Treadwell & Vahouny have suggested that the esterification does not require activated fatty acids, and that it is catalysed by a bile salt stimulated cholesterol esterase, which is similar to the esterase of pancreatic origin [5] . hversion of pancreatic juice leads to a reduction in mucosal cholesterol esterase activity, suggesting that the enzyme is secreted from the pancreas and absorbed from the gut lumen into the mucosal cells [13]. Gallo et al. [14] recently found that when isolated mucosal cells were incubated with purified cholesterol esterase, there was an increase in the cellular formation of cholesteryl esters. The esterification of cholesterol by this enzyme requires a concentration of bile acid which is in far excess of the level thought to be present in the mucosal cell. The esterification of cholesterol in the mucosal preparations described in the present study is most

MUCOSAL ESTERIFICATION OF CHOLESTEROL

59

Table 3.Esterification of cholesterol in total homogenate and microsomal fraction of mucosa from proximal duodenum and jejunum

Cholesterol (nmoles/mg protein)

Enzyme source Proximal duodenum Total homogenate Microsomal fraction Jejunum Total homogenate Microsomal fraction

Specific activity (nmoles cholesteryl ester formed mg protein-' h-')

Fractional cholesterol esterification

54 106

3.9 4.9

2.1 5.2

44 91

14.4 18.6

6.3 16.9

The mucosa was obtained by scraping pieces of intestine taken from surgical specimens from a patient who had a Billroth I1 resection carried out due to chronic peptic ulcer. The duodenum specimen was taken approximately 2 cm distal to the pylorus, and the jejunum specimen was taken approximately 20 cm distal to the ligament of Treitz. The incubation mixtures contained the same amounts of endogenous cholesterol (approx. 25 mmol/l), the concentration of CoA was 0.8 mmol/l, otherwise the conditions were as described in legend to Fig. 1

I

0

0

10

30

50

70

ENDOGENOUS CHOLESTEROL Inmoleslrnl I

Figure 4. Esterification of cholesterol and the concentration of homogenate in the incubation mixture. Total homogenates of jejunal mucosa were obtained from biopsies taken from two normal men ( 0 and &). The concentration of CoA was 8 X mol/l and the amounts of homogenate varied as shown in the Figure. Otherwise the incubation conditions were as stated in legend to Fig. 1. The ratios of endogenous cholesterol to protein in the two homogenates were 49 nmole/mg in that marked with 0,and 47 nmole/mg in that marked with A.

probably catalysed by an acy1CoA:cholesterol acyltransferase (ACAT). The esterification is totally dependent on CoA, stimulated by an acylCoA-regenerating system, and the pH optimum and intracellular localization are in accordance with ACAT from other tissues [3]. In the experiment where both the cholesterol and the acyl moiety were labelled with radioactive isotopes we obtained double labelled cholesteryl esters. The rate of esterification was in the same order of magnitude based on calculations of specific activities of either labelled substrate, showing that the esterification requires activated fatty acids. The enzymic reaction described in the present study

2ol 0

0

5

1 15

25

TIME (days) Figure 5. Stability of the mucosal acyl-CoA:cholesterol acyltransferase. Total homogenate ( 0 ) and microsomal (m) fraction from jejunum (same preparation as described in Table 3) were tested freshly prepared and after some days stored in a potassium phosphate buffer (0.2 mol/l, pH 7.2) at -20°C. The incubation conditions for the enzyme assay were as described in Table 3. The cholesteryl ester formed in the fresh total homogenate and the microsomal fraction were 6.3 and 16.9 nmol mg protein-' h-I, respectively.

could not be explained by the activity of cholesterol esterase. The pH optimum was higher than that of the cholesterol esterase, the enzyme was located in a particulate fraction of the cell and the enzyme activity was well retained in two patients who were pancreatectomized. Furthermore, in the incubation system used, the esterification of cholesterol was inhibited by taurocholate (Table 2 and Fig. 3). This inhibition could probably be due to an inhibition of carnitine palmitoyltransferase by the bile acid [15]. However, we have in unpublished experiments [16] found that 5 mmol/l of taurocholate

f

m

f

f

m

f

f f

M.A.

K.A.

RG

M.B.

P.B.

S.H.

I.S. E.B.

M.B.

55

m

m

m

A.P.

J.N.

T.G.

C

C

Stomach ulcer Stomach ulcer Carcinoma of the stomach Carcinoma of the stomach Gluten enteropathy Gluten enteropathy Gluten enteropathy Ulcerous colitis Malabsorp tion Chronic diarrhoea Carcinoma of the colon; resected ileum ZollingerEllison's syndrome Pancreatectomized; carcinoma of the pancreas Pancreatectomized; chronic pancreatitis

ulcer

Normal Stomach ulcer Stomach

Diagnosis

Normal

Normal

Normal

Slight chronic inflammation Atrophy of mucosa Normal

-

Small inflammatory changes

Normal

31.1

65.7

49.6

40.8 50.0

45.4

25.0

54.9

45.3

42.3

44.0

57.5

44.0

45.5 48.8 ?

5.53

nmoles cholesterol/ mg protein

2.8

5.2

2.4

3.6

2.0 3 .O

1.6

1.6

4.8

4.4

4.4

11.0

40.0

20.0

14.4

8.0 3 .O

* 2.97

Fractional cholesterol esterification

* 1.37

1.6

1.6

1.8

0.82 1.5

0.77

0.73

1.7

2.4

2.0

4.7

17.6

11.5

6.3

3.6 1.5

Specific activity nmoles cholesteryl ester formed mg protein-' h-'

Protein in homogenate not determined due to laboratory error

Weight reduction

Weight reduction

Weight reduction; diarrhoea Weight reduction

Marked weight reduction; Marked diarrhoea weight reduction

Gastrectomized ad modum Billroth I1 18 years ago

Gastrectomized ad modum Billroth I1 3 years ago

Fasted several days before biopsy were taken due to retension of gastric content

Remarks

* C = Crosby capsule; S = scraping of surgical specimens. All biopsies were taken from jenunum or duodenum distal to papilla Vaten. t - = Not examined.

42

54

C

62

f

C

C C

C

C

C

c

S

69 32

18

48

29

45

61

S

S

36

m

S.S.

47

S

45

m

W.B.

S

C S

Biopsy*

57

19-24 43

Age

f

m f

Sex

9.N.

€3.9.

Normal (mean ? SD, n = 9)

Patients

Jejunal histologyt

Table 4. Cholesterol, fractional and specific activity of cholesterol esterification in intestinal mucosa from normal men and some patients

0

o\

MUCOSAL ESTERIFICATION OF CHOLESTEROL inhibits formation of cholesteryl ester by microsomes from intestinal mucosa when oleyl-CoA is used as substrate. Morin et al. [ 171 have found the same order of inhibition by taurocholate with ACAT from swine arteries. ACAT has been found in several organs and tissues [3], e.g. liver [18, 191, adrenal gland [20], arteries [17], skin [21], and cultivated fibroblasts [22]. The ACAT we have found in intestinal mucosa has many similarities with ACAT in other tissues and organs: it is microsomal, has pH optimum between 7.2 and 8.3 and the preferred acyl group is oleate [3, 6, 18, 19, 221. ACAT i'n cells, grown in tissue culture, shows a remarkable increase in activity when excess cholesterol is taken up by the cells [22]. Animals fed cholesterol and fat show a higher specific activity of ACAT in intestinal mucosa [7] and in aorta [23]. This increase can, to a great extent, be explained by an increased load of cholesterol on the microsomal membranes [24], but it can not explain the increase in cholesterol esterification capacity by fibroblast given excess cholesterol or 25-hydroxycholesterol [22]. The present experiments show that the specific activity of ACAT is higher in the jejunum than in the proximal duodenum, suggesting that lipid absorption may influence the enzyme activity. However, the number of experiment up till now are few, and much more research is needed to define the mechanism underlying the ACAT activation. The activities of ACAT in mucosal biopsies from normal young men varied somewhat. The variability, both in the fractional cholesterol esterification and the specific activity, could probably be explained by contamination of submucosal tissue in the mucosal biopsy. The rather constant ratio of cholesterol to protein, however, does not support this explanation. A variation in the ratio of villi to crypt cell material in the biopsies may also explain the variability in esterification capacity, since cells from the villi differ in many aspects from the crypt cells [25]. The ACAT activity, however, has not been tested in the two cell types. Based on this small patient material it is not possible to draw firm conclusions on what influences different intestinal diseases have on the mucosal ACAT activity. Furthermore, in the biopsies from the patients the specific activity of ACAT varied more than in the normal men. It seems, however, that diseases affecting the mucosa lead to a low intestinal ACAT, and that undernourished patients have a rather low enzyme activity. ACAT was also found in patients with n o pancreatic tissue, an observation which may be of importance when one discusses the role of the pancreas in the esterification of mucosal cholesterol [5, 141. We believe that ACAT and not the cholesteryl esterase is the enzyme which is of importance for making the cholesteryl esters of intestinal origine. The ACAT operates under normal physiological conditions, whereas the cholesteryl esterase has to be carried from the intestine through the unstirred water layer and the mucosal membrane, and needs for its activity a bile acid concentration which does not exist inside the mucosal cell.

61

Cholesteryl esters formed within the mucosal cell will be packed together with triglycerides into the core of the chylomicrons [26] and thereby absorbed, whereas excess unesterified cholesterol may reside on the intracellular membranes, being lost into the gut lumen together with the desquamated mucosal cell. Thus, the esterification of cholesterol has physiological importance and may to some extent regulate the absorption of cholesterol. The ACAT most probably has an important role in this respect. In unpublished experiments [16] we have found that the ACAT activity is rather constant throughout jejunum and ileum. Assuming that a normal man has an estimated 500 g of small intestinal mucosa, or about 75 g of mucosal protein, the data of Table 4 can be used to calculate that the ACAT of normal small intestine has a capacity to esterify about 2.5 g of cholesterol per day. Thus, ACAT most probably has an important role in the production of the cholesteryl esters in normal chyle. Acknowledgments

This study was supported by grants from Norwegian Medical Council, Anders Jahre Foundation and Carl Semb Foundation. References 1 McIntyre N. & Isselbacher K.J. (1973) Role of the small intestine in cholesterol metabolism. Am J Clin Nu@ 26, 647656. 2 Grundy S.M. & Mok H.Y.I. (1977) Determination of cholesterol absorption in man by intestinal perfusion. J Lipid Res 18,263-271. 3 Goodman D.S. (1965) Cholesterol ester metabolism. Physiol Rev 45,747-839. 4 Bennett Clark S. & Norum K.R. (1977) The 1ecithin:cholesterol acyl transferase activity of rat intestinal lymph. JLipid Res 18,293'-300. 5 Treadwell C.R. & Vahouny G.V. (1968) In: Handbook of Physiology, Sect. 6, vol. 111, pp. 1407-1438, Am. Physiol. SOC.,Washington D.C. 6 Haugen R. & Norum K.R. (1976) Coenzyme-A-dependent esterification of cholesterol in rat intestinal mucosa. Scand J Gastroent 11,615-621. 7 Norum K.R., Lilljeqvist A-C. & Drev0nC.A. (1977) CoenzymeA-dependent esterification of cholesterol in intestinal mucosa from guinea-pig. Influence of diet on the enzyme activity. Scand JGastroent 12, 281-288. 8 Drevon C.A. & Norum K.R. (1975) Cholesterol esterification and lipids in plasma and liver from newborn and young guinea pigs raised on milk and non-milk diet. Nutr Metabol 18,137-151. 9 Norum K.R. (1964) Palmitoyl-CoA:carnitine palmitoyltransferase. Purification from calf-liver mitochondria and some properties of the enzyme. Biochim Eiophys Acta 89,95-108. 10 Gjone E., Myren J. & Semb L. (1970) Gnstroenterologisk undersdkelsesmetodikk, p. 64. Universitetsforlaget, Oslo. 11 Lowrey O.H., Rosebrough N.J., Farr A. L. & Randall R.J. (1 951) Protein measurement with the Folin phenol reagent. J Biol Chem 193,265-275. 12 Stokke K.T. & Norum K.R. (1971) Determination of lecithin: cholesterol acyltransferase in human blood plasma. Scand J Clin Lab Invest 27, 21-27. 13 Hernandez H.H., Chaikoff I.L. & Kiyasu J.Y. (1955) Role of pancreatic juice in cholesterol absorption. A m J Physioll81, 523-5 26.

62

KAARE K. NORUM et al.

14 Gallo L.L., Newbill T., Vahouny G.V. & Treadwell C.R. (1977) Role of pancreatic cholesterol esterase in the uptake and esterification of cholesterol by isolated intestinal cells. Proc SOCExp Biol Med 156,277-281. 15 Bremer J. & Norum K.R. (1967) The effects of detergents on palmitoyl coenzyme A :carnitine palmitoyltransferase. J Biol Chem 242,1749-1755. 16 Norum K.R. & Lilljeqvist A-C. (1978) Unpublished observations. 17 Morin R.J., Edralin G.G. & Woo J.M. (1974) Estersication of cholesterol by subcellular fractions from swine arteries, and inhibition by amphipathic and polyanionic compounds. A therosclerosis 20, 2 1-39. 18 Goodman D.S., Deykin D. & Shiratori T. (1964) The formation of cholesterol esters with rat liver enzymes. JEiol Chem 239, 1335-1345. 19 Stokke K.T. & Norum K.R. (1970) Subcellular distribution of acyl-CoA:cholesterol acyltransferase in rat liver cells. Biochim Biophys Acta 210,202-204. 20 Longcope C. & Williams R.H. (1963) Cholesterol esterifica-

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Esterification of cholesterol in human small intestine: the importance of acyl-CoA:cholesterol acyltransferase.

European Journal of Clinical Investigation (1979) 9,55-62 Esterification of cholesterol in human small intestine: the importance of acyl-CoA:choleste...
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