GASTROENTEROLOGY

1992:102:647-655

Cholesterol Malabsorption in Pancreatic Insufficiency: Effects of Enzyme Substitution MATTI

VUORISTO,

Second Department

HANNU

of Medicine,

University

.. . . VAANANEN,

Defective lipolysis, steatorrhea, and hypocholesterolemia characterize pancreatic insufficiency. Lipid metabolism in pancreatic insufficiency was studied by measuring serum lipoproteins, cholesterol absorption with double labels and serum plant sterols, and bile acid and cholesterol synthesis with fecal and dietary steroid analysis and cholesterol precursor sterols before and during exogenous pancreatic enzyme substitution. Baseline fecal fat, masses, bile acids and neutral steroids, and cholesterol synthesis were increased, whereas cholesterol absorption was markedly reduced. In fact, the present data suggest that sterol absorption may be disturbed more sensitively than fat absorption in pancreatic insufficiency. Enzyme substitution significantly reduced fecal fat, masses, bile acids and neutral steroids, and synthesis of cholesterol and improved cholesterol absorption in relation to serum cholesterol, although normal values were not obtained. Serum level of high-density lipoprotein cholesterol was significantly elevated by exogenous enzymes, whereas levels of cholesterol or triglycerides in other lipoproteins remained unchanged. Improved sterol absorption increased also serum levels of plant sterols and reduced levels of cholesterol precursors and cholesterol synthesis and precursor sterol-plant sterol ratios. Thus, reduced intestinal lipolysis with expanded oil phase appears to be a major reason for impaired cholesterol absorption, causing enhanced cholesterol and, consequently, bile acid synthesis and reduced serum cholesterol level. Exogenous enzyme substitution seems partly to correct these abnormalities, improvements of which can be monitored by the gas-liquid chromatographic determination of serum plant sterols or cholesterol precursor-plant sterol ratios.

xocrine pancreatic insufficiency is characterized by steatorrhea and remarkable hypocholesterolemia. Fat maldigestion appears when the lipolytic activity in duodenal juice is reduced by r90%,’ as a

E

and TATU

A. MIETTINEN

of Helsinki, Helsinki, Finland

result of reduced lipase output or inactivation of lipase and precipitation of bile acids in the acidic milieu of the upper small intestine.2 Enhanced bile acid elimination, often observed both in adults with exocrine pancreatic insufficiency3-5 and in children with cystic fibrosis,6 may also be involved in the lowering of serum cholesterol in these patients. Because cholesterol absorption occurs in the upper small intestine and requires micellar solubilization by bile salts,7 it can be inferred that the absorption of cholesterol may also be impaired and account for hypocholesterolemia in patients with pancreatic insufficiency. In contrast to enhanced fecal bile acids, elimination of cholesterol as fecal neutral steroids is only occasionally increased, suggesting inconsistently reduced cholesterol absorption or decreased biliary output of cholesterol in pancreatic insufficiency.4 However, as far as we know, there are no studies available on cholesterol absorption and its effects on serum lipoproteins and cholesterol metabolism in this condition. Therefore, in the present study we measured serum lipids and lipoprotein distribution, cholesterol absorption, elimination of cholesterol as fecal neutral and acidic steroids, and fecal fat and fecal masses before and during exogenous pancreatic enzyme substitution in patients with exocrine pancreatic insufficiency. In addition, we determined serum and fecal squalene and noncholesterol sterols. Of these variables, plant sterols and cholestanol are positively correlated with the absorption efficiency of cholesterol in various clinical conditions,8-10 whereas serum cholesterol precursor levels parallel changes in the cholesterol synthesis”-‘2 and the activity of hepatic hydroxymethylglutaryl coenzyme A reductase in humans.13J4 Thus, it seemed that their determinations could give additional information about the changes in absorption and synthesis of cholesterol before and during enzyme substitution in the patients with pancreatic insufficiency. 0 1992 by the American Gastroenterological 0016-5065/92/$3.00

Association

646

VUORISTO

ET AL.

GASTROENTEROLOGY

Materials and Methods

cholesterol, and 0.24 pCi of [3H]sitosterol three times a day with the main meals for 7-10 days. During the first visit, basal data without enzyme substitution were recorded. Thereafter, patients received with the main meals two to three capsules of pancreatic enzyme preparation containinglipase, amylase, and protease, 8000,9000, and 450 European Pharmacopoeia units per capsule (Pankreon; KaliChemie Pharma GmbH, Hannover, Germany). The s-day stool collections were performed from day 1 to day 12 to see the immediate effect of enzyme substitution on fecal fat and masses and then on an average of 82 +- 12 days after the patients had consumed the enzyme therapy.

Patients Eight patients with exocrine pancreatic insufficiency secondary to chronic alcoholic pancreatitis were studied (Table 1). The diagnosis of pancreatic insufficiency was based on an earlier observation on the presence of fecal fat > 7 g/24 h on a fat diet of 100 g/day. In addition, in six patients an abnormally low bicarbonate output was found in secretin test; in one patient chronic pancreatitis was the laparotomy finding; and one patient had a history of recurrent pancreatitis and grossly abnormal pancreas in sonographic and computer tomographic examinations. Analysis of fecal samples after the study was completed showed surprisingly that patient 5 had no steatorrhea in any fecal samples. He had clear chronic pancreatitis, diabetes, and abnormal secretion test results, Therefore, he has been included to formal study population. Overall, five of the eight patients had undergone a distal pancreas resection (cases 1-3, 5, and 8), and cholecystectomy was performed on three patients (cases 1, 3, and 4). Six of the patients were receiving insulin treatment for diabetes mellitus (cases 1-3, 5,6, and 8). Patients with symptoms or signs of liver cirrhosis or with abnormal biochemical liver function test results were excluded. All patients were informed of the nature and design of the studies and volunteered to participate. The research protocol was approved by the Ethics Committee of the hospital.

Chemical

All patients were hospitalized during the studies. During each hospital stay, patients received a standard diet, containing 100 g of fat (polyunsaturated/saturated fatty acid ratio, 0.5), 300 mg of cholesterol, and 20 g of medium chain triglycerides-oil per day. Daily energy content of the diet (30-40 kcal/kg body wt) was adjusted to maintain a constant body weight during each study period. For the measurement of cholesterol absorption,‘5 fecal fat, and fecal sterols, each patient received one capsule containing ZOO mg of chromic oxide, 0.06 pCi of [4-14C]-

In the present were used.

the following

calculations

and Fecal Bile Acids and Neutral

Steroids in

Body wt Fecal fat (g/day)

(kg) IlO.

Sex (F/M)

Height (cm)

1 2 3 4 5 6 7 8 Mean +SEM

51 77 75 75 42 65 54 56 62 f5

F M F F M M M M

170 175 154 153 167 193 171 168 169 r4

ApoE, apolipoprotein values.

3/5

E; Off, before therapy;

ApoE phenotype 3/4 3/3 3/3 3/4 3/3 3/3 3/3 3/3

Off

On

67 62 49 36 72 83 86 62 65 ?6

66 63 55 36 71 81 90 66 66 +6

On, during long-term

Analysis

Calculations

Table 1. Clinical Data, Fecal Fat, Percentage Absorption of Cholesterol, Eight Patients With Exocrine Pancreatic Insufficiency

Age (YU

and Isotope

Serum cholesterol and serum triglyceride levels were measured by the routine methods of the hospital.‘6,‘7 Cholesterol and triglyceride contents in very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were measured as described in the Lipid Research Clinics Program.“’ Apolipoprotein E phenotyping was performed by isoelectric focusing.‘g Serum cholestanol, cholesterol precursor sterols, and serum plant sterols were determined by gas-liquid chromatography (GLC) directly from a 0.5mL plasma sample saponified by a mixture of 10N KOH and 99.5% ethanol (1:9).*’ Fecal fat was measured according to van de Kamer et al.‘l and chromic oxide as described by Bolin et al.” Fecal bile acids, fecal neutral steroids, and fecal noncholesterol sterols were determined on a capillary GLC.23-25 The recovery of [3H]sitosterol showed no marked degradation of the sterol nucleus; thus, the fecal data were calculated in relation to chromic oxide. Fecal radioactivities were measured with a liquid scintillation counter with 0.5% 2.5-diphenyl oxazole in toluene (Wallac Rackbeta Scintillation: Model 1215; LKB Stockholm, Sweden).

Procedures

Case

Vol. 102. No. 2

Off 98.8 59.2 80.0 35.7 4.8 44.9 40.4 106.0 58.7 2 12.1

enzyme substitution

On 87.0 12.5 27.6 15.1 6.8 22.7 19.9 76.6 33.8 k 11.0"

study

Cholesterol absorption (%) Off 0.3 0.1 0.9 5.4 24.0 6.5 9.6 4.1 6.4 f 2.8

On 1.4 25.5 15.8 18.7 39.6 19.6 7.6 22.6 18.9 * 4.1b

Fecal bile acids (mg/doY)

Fecal neutral steroids (mg/doY)

Off

On

Off

On

1571 603 603 260 1199 656 867 552 789 f147

1561 323 436 239 138 386 574 387 506 +156"

1454 992 642 641 1214 884 1769 1347 1118 t142

1291 677 743 614 451 1211 1510 1207 963 +136

(82+ 12 days, mean + SEM). “P < 0.05, bP < 0.01from pretreatment

CHOLESTEROL

February 1992

Cholesterol

Absorption

(%) fecal 14C/3H 14C/3H in Capsules

1x

=

Influx (mg/day)

Fecal Neutral Steroids (mg/day) X 100 100 - Percentage of Cholesterol Absorption

40 : 0

r = 0.693 p < 0.001

0

x. 20

a

2.” 0 . 0

yxY @

0 r = 0.545 40 - p < 0.01 ‘X

20

0

Data

fat was markedly increased in all but one patient, indicating a severe impairement of pancreatic exocrine function in the present series (Table 1). The mean absorption efficiency of cholesterol, 6.4% t- 2.8%, was very low in the patients and almost zero in some cases. The absorption efficiency of cholesterol was actually negatively correlated with fecal fat, and already a moderate increase in fecal fat was associated with a markedly impaired absorption of cholesterol (Figure 1). It is also seen in Figure 1 that the patients with most severe steatorrhea tended to have the highest values for fecal bile acids, fecal neutral steroids, and cholesterol synthesis. In fact, the bile acid and cholesterol synthesis values were roughly twice of those in a normal male population3’ Correspondingly, serum levels of cholesterol precursor sterols (Table 2) were higher and those of plant sterols lower than in the random male population.31 Fecal fat was positively correlated with fecal mass (r = 0.781; P < 0.05) and fecal dry weight (r = 0.947; P < O.OOl), showing that the high fecal masses closely paralleled the severity of fat malabsorption in the patients. fecal

f

9

2

a

E

B

x

X

0 x X

0

X

o~x.

.

x.

o

X

* 8O

Results

Basal

649

’

Intestinal cholesterol influx, consisting of dietary, biliary, and mucosal cholesterol, was calculated by an indirect method’” assuming that exogenous and endogenous cholesterol were absorbed similarlyz7 and that the normally small mucosal cholesterol loss” was equal during each study period. The intestinal influx of endogenous cholesterol (biliary cholesterol secretion) was calculated as follows: Biliary Cholesterol Secretion (mg/day) = Intestinal Influx of Cholesterol (mg/day) - dietary cholesterol (mg/day) and Absorbed Cholesterol (mg/day) = Cholesterol Absorption (%) X Intestinal Cholesterol influx (mg/day):lOO. Statistical analysis was performed using Student’s paired t test. The multiple correlations of cholesterol absorption with various variables were calculated using analysis of the stepwise regression 2R on a VAX-8600 computer with BMDP Software.”

Baseline

INSUFFICIENCY

The short-term enzyme substitution (9-12 days) reduced markedly fecal fat and fecal masses in the patients (Figure 2). The long-term enzyme substitution (82 + 12 days, mean 4 SEM) reduced fecal fat, masses, bile acids, neutral steroids, and cholesterol synthesis and increased the relative and absolute absorption of cholesterol (Tables 1 and 3). The substitution increased significantly the serum HDL cholesterol level and insignificantly the serum levels of total and lipoprotein cholesterol, whereas levels of

100.

(mg/day). Cholesterol

IN PANCREATIC

Enzyme Substitution

Cholesterol synthesis was calculated as follows: Cholesterol Synthesis (mg/day) = Sum of Fecal Bile Acids and Fecal Neutral Steroids (mg/day) - Dietary Cholesterol

Intestinal

MALABSORPTION

Oc

0

20

x&o ‘0

xx

,”

oz. .uo 10

xx

.o X

x

r = 0.589 p c 0.01

LL 0 30 . r = 0.828 p = 0.001

O 0

20

x

10 .

B

5

0

0

CD

30

60

90

1 0

FECAL FAT, g/day Figure 1. Correlations of absorption efficiency of cholesterol, fecal bile acids, fecal neutral steroids, and cholesterol synthesis with fecal fat in eight patients before (X), during short-term (912 days; 0) and long-term (82 + 12 days: ?? ) enzyme substitution.

650

VUORISTO ET AL.

GASTROENTEROLOGY Vol. 102, No. 2

Table 2. Serum Lipids and Lipoproteins and Serum and Fecal Noncholesterol Sterois Before and During Long-Term Enzyme Therapy in Eight Patients Parameter

Off

On

Correlations

Serum (mmol/L)

Cholesterol VLDL IDL LDL HDL Total Triglycerides VLDL Total

0.33 0.15 1.95 1.17 3.92

+ + f + +

sterol outputs were decreased, whereas fecal cholestanol and plant sterol excretions were uninfluenced by the enzyme substitution.

0.07 0.01 0.26 0.07 0.24

0.83 2 0.13 1.56 +_ 0.15

0.29 0.13 2.23 1.41 4.42

f + + + +

0.06 0.02 0.24 0.05O 0.26

0.69 f 1.30 f

0.26 0.28

Correlations of cholesterol absorption with different variables were usually opposite to those of cholesterol and bile acids synthesis, as shown in Table 4. Thus, the absorption efficiency of cholesterol was negatively correlated with fecal fat, masses, sitosterol, neutral steroids and bile acids, serum cholesterol precursors, or their ratio to plant sterols and cholesterol synthesis and positively correlated with

(pg/lOO mg of cholesterol)

Cholestanol Squalene Lathosterol Aa-cholestanol Desmosterol Campesterol Sitosterol Lathosterol/campesterol Lathosterol/sitosterol As-cholestanol/campesterol A”-cholestanol/sitosterol Campesterol/sitosterol

93.5 20.1 238.2 64.2 69.3 58.0 53.2 5.59 5.42 1.64 1.53 1.07

+ 16.5 + 2.5 f 41.3 + 12.8 + 7.1 + 9.7 + 6.6 + 1.72 + 1.61 k 0.53 + 0.51 + 0.10

87.1 24.3 227.0 42.3 72.9 99.1 80.0 3.50 3.41 0.65 0.62 1.21

f 8.8 k 3.6 rt 48.4 + 10.6” * 7.9 f 18.5’ + 8.8a f 1.33 f 0.95 + 0.30” k 0.18a k 0.13

FECAL FAT

0

1-3

4-6

7-9

lo-12

DAYS

7-9

lo-12

DAYS

lo-12

DAYS

Feces (mg - kg-’ - day-‘)

Cholestanol Squalene Lathosterol Campesterol Sitosterol

0.14 0.22 0.25 0.7 3.6

+ f f + f

0.03 0.03 0.04 0.1 0.5

0.14 0.15 0.16 0.6 3.1

+ * f + +

FECAL MASS

0.02 0.02O 0.03b 0.1 0.4

NOTE. Results are expressed as means * SEM. Length of longterm therapy, 82 f 12 days (mean f SEM). “P < 0.05, bP < 0.01 from pretreatment values. Off, before; on, during long-term enzyme therapy.

total and VLDL triglycerides tended to be decreased (Table 3). Of the cholesterol precursors, the serum levels of squalene and lathosterol decreased insignificantly and the level of A’-cholestanol decreased significantly, whereas the serum contents of campesterol and sitosterol were significantly increased and that of desmosterol remained unchanged (Table 2). The campesterol-sitosterol ratio, strongly related to cholesterol malabsorption in celiac disease,g was only insignificantly increased. However, the cholesterol precursor sterol-plant sterol ratios were reduced because of the simultaneous decrease in cholesterol synthesis and increase in sterol absorption (Table 4). In fact, the precursor sterol-plant sterol ratios seemed to be suitable for monitoring changes in fat and cholesterol absorption during exogenous pancreatic enzyme substitution. Fecal squalene and latho-

0

1-3

g/day

4-6

FECAL DRY WEIGHT

200 r

0

l-3

OFF

4-6

7-9

ON ENZYMES

Figure 2. Effects of short-term enzyme therapy (9-12 days) on fecal fat and fecal masses in eight patients. *P < 0.05, **P < 0.01 from the respective pretreatment values.

CHOLESTEROL MALABSORPTION

February

1992

Table

Metabolism, Fecal Fat, and Fecal Masses in Eight Patients Before and During Long-Term Pancreatic Enzyme Substitution

3. Cholesterol

Parameter Fecal steroids (mg *kg-’ *day-‘) Bile acids Neutral steroids Dietary Endogenous Total Total endogenous steroids Intestinal influx of cholesterol (mg *kg-’ *day-‘) Dietary Endogenous Total Cholesterol absorption (mg . kg-’ *day-‘) Dietary Endogenous Total Cholesterol synthesis (mg *kg-’ . day-‘) Fecal fat (g. kg-’ . day-‘) Fecal mass (g . kg-’ . day-‘) Fecal dry wt (g *kg-’ - day-‘) Whole intestine transit time (h)

Off 12.0 f 1.9

On

7.8k 2.3a

4.7 + 0.6 12.5f 1.5 17.3f_1.4 24.6f 2.9

3.9+ 10.7* 14.6k 18.5+

0.5b 1.4 1.5' 3.3

5.0f 0.6 13.6ic1.8 18.6f 1.6

4.9+ 0.5 12.9+ 1.5 17.7f 1.4

0.3+ 0.1 1.0+ 0.5 1.3+ 0.6

0.9k 0.2b 2.2+ 0.4 3.1IL0.6"

24.3+ 2.9 1.0zk0.2 10.1+ 1.8 2.9f 0.5 22.9+ 3.1

17.5AI3.5a 0.5f 0.2b 5.7+ 1.3b 1.4+ 0.3b 28.3+ 5.7

NOTE. Results are expressed as mean + SEM. Length of long-term therapy, 82 f 12 days (mean f SEM). Off, before; on, after long-term pancreatic enzyme substitution. “P < 0.05, bP < 0.01 from pretreatment values.

serum cholesterol and plant sterols, but not with serum cholestanol. A stepwise multiple regression analysis using cholesterol absorption as a dependent factor and body mass index, dietary and endogenous intestinal influx of cholesterol, cholesterol synthesis, and fecal bile acids, fat, dry weight, campesterol, and sitosterol as independent variables showed that the absorption efficiency of cholesterol was determined by fecal fat (r = 0.662) and fecal sitosterol (r = -0.480).

Discussion The present data are in agreement with earlier reports showing that, in pancreatic insufficiency, dietary substitution of pancreatic enzymes improves fat absorption but does not normalize it.2*32The findings showed for the first time that in patients with untreated pancreatic insufficiency, the absorption of cholesterol is low, resulting in low serum cholesterol and plant sterol levels, high cholesterol synthesis, and cholesterol precursor levels and that malabsorption and other variables of cholesterol metabolism and serum plant sterol contents can also be improved by exogenous pancreatic enzyme substitution. Enhanced cholesterol synthesis, more than twice the level of that found in a normal male population,30

IN PANCREATIC

INSUFFICIENCY

651

was caused by fecal loss of both bile acids and cholesterol itself. In fact, high cholesterol synthesis may have activated also bile acid synthesis, although both fecal bile acids and fecal neutral sterols were related to fat malabsorption (Table 4; Figure 1). Thus, fat maldigestion (the reason for enhanced fecal fat) may have contributed to the patients’ fecal losses probably through solubilization of cholesterol by large oil phase to unabsorbable form33*34and precipitation of cholesterol and bile acids during impaired of unabsorblipolysis at low pH. 2,35Administration able sucrose polyester, which forms an indigestable large oil phase in intestinal lumen, in fact reduces cholesterol absorption and enhances slightly fecal bile acid output.36 The absorption efficiency of cholesterol was already quite low in the patients with only modest steatorrhea, whereas this slight impairment of lipolysis disturbed bile acid output only modestly (see Figure 1). The reduced pancreatic lipolysis is most likely mainly caused by the low pancreatic enzyme output,’ possibly contributed by reduced duodenal pH2 and dietary fiber-induced inhibition of pancreatic lipase.37-3g The stepwise regression analysis suggested that, in addition to fat maldigestion, the dietary sitosterol intake (fecal sitosterol) reduced the absorption efficiency of cholesterol in the patients. These findings suggest that at low cholesterol absorption level even an ordinary dietary sitosterol intake may markedly disturb the absorption of cholesterol and activate cholesterol synthesis. However, a similar association has been found between cholesterol absorption and dietary plant sterols in a normal male population.30 Furthermore, the cholesterol absorption and fecal bile acids were closely coupled. The intraluminal dilution and precipitation of bile acis at low duodenal pH may markedly reduce effective micellar bile acid concentrations.‘*35 Resulting impaired micelle formation could contribute to the observed cholesterol malabsorption in this condition. Interestingly, fecal fat was not increased in patient 5, but like the other patients with high fecal fat, this patient also had reduced fractional absorption of cholesterol and increased fecal sterol excretion (corrected by enzyme substitution), suggesting that the sterol absorption may be disturbed more sensitively than the fat absorption in this patient. It is possible that the limited action of pancreatic lipase on intestinal oil phase may continue through the whole small intestinal transit in the early stage of pancreatic insufficiency, resulting in fatty acid absorption also from the lower small intestine.40 However, cholesterol absorption may be impaired by large oil phase in the upper part of the small intestine, where cholesterol absorption takes place normally.41 Thus,

652

VUORISTO ET AL.

GASTROENTEROLOGY Vol. 102, No. z

Table 4. Correlations of Absorption and Synthesis of Cholesterol and Fecal Bile Acids With Serum Cholesterol Levels and Noncholesterol Sterols, Parameters ofCholesterol Metabolism, Fecal Variables, and Whole Intestinal Transit Time Cholesterol

absorption Total

Parameter Serum cholesterol (mmoI/L) Total VLDL IDL LDL HDL Noncholesterol sterols (pg/mg cholesterol) Lathosterol A’-Cholestanol Campesterol Sitosterol Lathosterol/campesterol Lathosterol/sitosterol Aa-Cholestanol/campesterol A”-Cholestanol/sitosterol Campesterol/sitosterol Intestinal influx of cholesterol (mg . kg-’ - day+) Dietary Endogenous Total Fecal steroids (mg - kg-’ - day-‘) Neutral steroids Dietary Endogenous Total Bile acids Campesterol Sitosterol Cholesterol synthesis (mg. kg-‘. day-‘) Fecal fat (g. kg-’ . day-‘) Fecal mass (g - kg-’ - day-‘) Fecal dry wt (g +kg-’ - day-‘) Whole intestine transit time (h)

(mg - kg-’ * day-‘)

%

Cholesterol synthesis (mg - kg-‘. day-‘)

Fecal bile acids

(mg. kg-’ *day-‘)

0.414O 0.001 -0.378 0.225 0.554b

0.278 -0.169 -0.413' 0.129 0.567b

-0.128 -0.409 -0.158 0.045 0.020

-0.605b -0.615b 0.696' o.74gc -0.690' -0.694' -0.677' -0.636" 0.290

-0.58ab -0.594b 0.512a 0.638' -0.652' -0.660C -0.659' -0.619' 0.163

0.768" 0.793c -0.478' -0.463a 0.720' o.700c o.730c 0.732' -0.289

0.870' -0.373 -0.369 0.701= 0.697' 0.765' 0.769' -0.220

-0.104 -0.113

-0.017 0.206 0.224

-0.257 0.563b 0.563b

-0.084 0.241 0.236

-0.384 -o.451a -0.595b -0.584b -0.036 -0.408'

-0.376 -0.175 -0.312 -0.512'= 0.053 -0.201

0.017 o.754c 0.772' 0.921C 0.052 0.540b

-0.653' -0.682' -0.618b -o.707c -0.024

-0.485'= -0.610b -0.576b -o.666c 0.080

0.527b 0.644' 0.630' 0.020

0.010

NOTE. Values before and during short-term “P < 0.05, bP < 0.01, cP < 0.001.

(9-12 days) and long-term

the malabsorption of cholesterol could actually precede that of dietary fats. Furthermore, a possible precipitation of bile acids by low intestinal pH should also disturb the absorption of cholesterol more than that of dietary fats.42,43 Fecal loss of bile acids was actually only modest. For instance, in patients with ileal exclusion,M the mean fecal bile acid loss was 51 vs. 12 mg . kg-’ day-’ in the present study, 6 mg* kg-‘. day-’ in a normal male population,30 and 8 mg - kg-l - day-l in celiac disease.’ At present, the factors involved in enhanced bile acid synthesis in pancreatic insufficiency are still unclear. Bile acid pool size is only modestly reduced in cystic fibrosis.45 The cholesterol malabsorption-induced high cholesterol synthesis may be a factor for ??

-0.080 -0.330 -0.181 0.036 0.095

0.729=

0.149

0.442a 0.502" -0.011 0.472a

-

(82 + 12 days) enzyme substitution

0.921= o.490°

0.714= 0.644' -0.108 are included;

N = 24.

increased bile acid synthesis without actual bile acid malabsorption. Basal cholesterol synthesis of the present series was enhanced more than two times compared with a normal male population3’ but was clearly less than in pure cholesterol, bile acid, or cholesterol plus bile acid malabsorptions.44 Proportion of fecal bile acids of cholesterol synthesis is obviously positively related to the degree of bile acid absorption efficiency. In the present series, the basal proportion of bile acids was 49% of cholesterol synthesis compared with 21% in celiac disease,g 57% in a normal male population,30 88% in bile acid malabsorption, and 67% in bile acid plus cholesterol malabsorption.44 These findings suggest that actual bile acid malabsorption was negligible, with proportionately normal amounts of bile acids be-

February 1992

CHOLESTEROL MALABSORPTION IN PANCREATIC INSUFFICIENCY

ing formed from newly formed cholesterol. The studies on vitamin B,, absorption with intrinsic factor have not actually disclosed any ileal dysfunction for bile acid malabsorption in patients with pancreatic insufficiency.*’ Additional factors for enhanced fecal bile acids may be rapid small intestinal transit time,46 low duodenal pH,lVMand high intestinal nondigestible lipid bulk.47 However, in the present study, the whole intestinal transit time was not changed during the two study periods, and an increase of pH by cimetidine during enzyme therapy reduced further fecal fat but not fecal bile acids in pancreatic insufficiency.5 Fecal fat is also positively correlated with cholesterol malabsorption and fecal bile acids in celiac disease,48 in which intestinal lipolysis is, however, quite intact*’ and the terminal ileum is frequently hypertrophicz8 Thus, an increased bile acid reabsorption may explain the normal bile acid output in celiac disease. It should be born in mind that expansion of oil phase by unabsorbable sucrose polyester enhances slightly also fecal bile acid excretion.36 According to the current knowledge of cholesterol metabolism, the improved absorption of cholesterol should inhibit cholesterol synthesis, downregulate hepatic LDL receptors, and, accordingly, elevate serum LDL cholestero15’ The enzyme substitution actually decreased cholesterol synthesis, which was inversely related to cholesterol absorbed (Table 4). Unexpectedly, the serum total and LDL cholesterol levels were not increased, suggesting that enhanced entry of absorbed cholesterol to the liver was balanced by suppression of hepatic cholesterol and bile acid synthesis with no requirements to downregulate receptor activity for LDL apolipoprotein B. Improved cholesterol and fat absorption markedly increased the serum HDL cholesterol level in the patients with pancreatic insufficiency. The same phenomen is also associated with an increased absorption of cholesterol in celiac disease,51 in vegetarian.s5’ or even in normal populations,30*53 although the mechanism is at present poorly understood. The serum levels of cholesterol precursor sterols, of which those of lathosterol and A’-cholestenol were basally higher than in a normal male population,31 were proportionate to cholesterol synthesis (Table 4) but were only modestly decreased by the enzyme substitution-induced lowering in cholesterol synthesis. In fact, the serum level of desmosterol, reflecting the side chain saturated pathway of cholesterol synthesis, was unchanged. Improvement of cholesterol absorption by gluten-free diet decreased clearly the precursor levels including desmosterol in celiac diseaseeg The relatively small changes in cholesterol precursors may be caused by a relative small improvement in cholesterol malabsorption. The subsequent reduction of cholesterol syn-

653

thesis may also inactivate the postlanosterol steps of cholesterol synthesis pathway, analogously to an activation observed during a neomycin-induced increase of synthesis by cholesterol malabsorption.54 The reduced cholesterol synthesis was apparently also related to reduced fecal output of squalene and lathosterol. The present study showed also for the first time that in pancreatic insufficiency the serum campesterol and p-sitosterol levels are markedly decreased compared with healthy subjects,31 that the plant sterol contents are proportionate to cholesterol absorption efficiency, and that the low serum plant sterol levels are significantly elevated by pancreatic enzyme substitution. Plant sterols are normally poorly absorbed.55 In addition to low absorption efficiency, the low serum plant sterol levels in pancreatic insufficiency may be related to their low dietary intake or increased biliary elimination as acidic or neutral steroids.55*56The dietary plant sterol intakes (shown by fecal plant sterols) were normal in the present study and similar during the two study periods. The biliary influx rate of cholesterol, which probably determines plant sterol secretion,3* was not significantly changed by the enzyme therapy, suggesting that biliary plant sterol secretion was not decreased and that the observed low base-line serum plant sterol contents and their increase during the enzyme treatment were mainly related to the absorption efficiency of cholesterol. Thus, it seems that in the patients with pancreatic insufficiency the defective lipolysis and micellar formation was apparently also the primary reason for the lowered absorption and serum levels of the two plant sterols. Interestingly, the serum campesterol-sitosterol ratio was as low in pancreatic insufficiency as previously observed in celiac disease,g showing that also in the digestive malabsorption the serum level of campesterol is reduced to a higher degree than that of sitosterol. Overall, the close associations of serum plant sterol contents with cholesterol and fat malabsorptions suggest that their serum determination may be valuable even for diagnostic purposes of pancreatic insufficiency and for monitoring effectiveness of pancreatic enzyme therapy. The determination is simple with capillary GLC from nonsaponifiable serum material, when the same run quantitates cholesterol, its precursors, and plant sterols. Accordingly, low serum plant sterol contents associated with a low campesterol-sitosterol ratio, high precursor sterol contents, or high serum lathosterol-plant sterol ratios seem to occur in pancreatic insufficiency, celiac disease,g or ileal dysfunction** with cholesterol malabsorption. High serum lathosterol levels with normal or high plant sterol contents occur in patients with ileal dysfunction without cholesterol malabsorption.**

654

VUORISTO ET AL.

GASTROENTEROLOGY Vol. 102, No. 2

References 1. DiMagno EP, Go VLW, Summerskill

21.

2.

22.

3.

4.

5.

6.

7.

8. 9.

10.

11. 12.

13.

14.

15.

16.

17.

18.

19.

20.

WJH. Relations between pancreatic enzyme outputs and malabsorption in severe pancreatic insufficiency. N Engl J Med 1973;288:813-815. Regan DT, Malagelada JR, Di Magno EP, Go VLW. Reduced intraluminal bile acid concentrations and fat maldigestion in pancreatic insufficiency: correction by treatment. Gastroenterology 1979;77:285-289. Roller RJ, Kern F Jr. Minimum bile acid malabsorption and normal bile acid breath tests in cystic fibrosis and acquired pancreatic insufficiency. Gastroenterology 1977;72:661-665. Pasanen AVD, Tarpila S, Miettinen TA. Relationships between serum lipid and malabsorption of bile acids, neutral sterols and fats in exocrine pancreatic insufficiency. Stand J Gastroenterol 1980;15:503-507. Dutta SK, Anand K, Gadacz TR. Bile salt malabsorption in pancreatic insufficiency secondary to alcoholic pancreatitis. Gastroenterology 1986;91:1243-1249. Weber AM, Roy CC, Chartrand L, Lepage G, Dufour OL, Morin CL, Lasalle R. Relationship between bile acid malabsorption and pancreatic insufficiency in cystic fibrosis. Gut 1976;17:295-299. Siperstein MD, Chaikoff IL, Reinhardt WO. C?*-cholesterolV. obligatory function of bile in intestinal absorption of cholesterol. J Biol Chem 1952;198:111-114. Tilvis RS, Miettinen TA. Serum plant sterols and their relation to cholesterol absorption. Am J Clin Nutr 1986;43:92-97. Vuoristo M, Tilvis R, Miettinen TA. Serum plant sterols and lathosterol related to cholesterol absorption in coeliac disease. Clin Chim Acta 1988;174:213-223. Kesaniemi YA, Miettinen TA. Cholesterol absorption efficiency regulates plasma cholesterol level in Finnish population. Eur J Clin Invest 1987;17:391-395. Miettinen TA. Detection of changes in human cholesterol metabolism. Ann Clin Res 1970;2:300-320. Miettinen TA. Effects of bile acid feeding and depletion on plasma and biliary squalene, methyl sterols and lathosterol. In: Paumgartner G, Stiehl A, Gerok W, eds. Bile acids and lipids. Lancaster, England: MTP Press, 1981:225-262. Bjiirkhem I, Miettinen TA, Reihner E, Ewerth S, Angelin B, Einarsson K. Correlation between serum levels of some cholesterol precursors and activity of HMG-CoA reductase in human liver. J Lipid Res 1987;28:1137-1143. Strandberg TE, Tilvis RS, Miettinen TA. Variations of hepatic cholesterol precursors during altered flows of endogenous and exogenous squalene in the rat. Biochim Biophys Acta 1989;1001:150-156. Crouse JR, Grundy SM. Evaluation of a continuous isotope feeding method for measurement of cholesterol absorption in man. J Lipid Res 1978;19:967-971. Roschlau P, Berut W, Gruber W. Enzymatische Bestimmung des gesamt Cholesterius im Serum. Z Klin Chem Klin Biothem 1974;12:403-410. Wahlefeld AW. Triglyceride determination after enzymatic hydrolysis. In: Bergmeyer HU, ed. Methods of enzymatic analysis. 2nd ed. San Diego, CA: Academic, 1971:1831-1835. Department of Health. Lipid research clinics program. Manual of laboratory operations. Education and Welfare Publication No. NIH 75-628, 1974. Ehnholm C, Lukka M, Kuusi T, Nikkila E, Utermann G. Apolipoprotein E polymorphism in the Finnish population: gene frequencies and relation to lipoprotein concentrations. J Lipid Res 1986;27:227-235. Miettinen TA, Koivisto P. Non-cholesterol sterols and bile acid production in hypercholesterolaemic patients with ileal bypass. In: Paumgartner G, Stiehl A, Gerok W, eds. Bile acids

23.

24.

25.

26. 27.

28. 29. 30.

31.

32.

33. 34.

35.

36. 37.

38.

39.

40.

41.

and cholesterol in health and disease. Lancaster: MTP Press, 1983:183-187. Van de Kamer JH, ten Bokkel Huinink H, Weyrs HA. Rapid method for the determination of fat in feces. J Biol Chem 1949;177:347-355, Bolin DW, King RP, Klosterman EW. A simplified method for the determination of chromic oxide (Cr,O,) when used as an index substance. Science 1952;116:634-635. Miettinen TA, Ahrens Jr EH, Grundy SM. Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J Lipid Res 1965;6:411-424. Grundy SM, Ahrens Jr EH, Miettinen TA. Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J Lipid Res 1965;6:397-410. Miettinen TA. Gas-liquid chromatographic determination of fecal neutral sterols using a capillary column. Clin Chim Acta 1982;124:245-247. Miettinen TA. Hyperlipidemia, bile acid metabolism and gallstones. Ital J Gastroenterol 1978;10:53-55. Samuel P, McNamara DJ. Differential absorption of exogenous and endogenous cholesterol in man. J Lipid Res 1983;24:265-278. Vuoristo M, Miettinen TA. Increased biliary lipid secretion in celiac disease. Gastroenterology 1985;88:134-142. University of California. BMDP. Statistical software. Los Angeles: University of California Press, 1981. Miettinen TA, Keslniemi YA. Cholesterol absorption: regulation of cholesterol synthesis and elimination and withinpopulation variations of serum cholesterol levels. Am J Clin Nutr 1989;49:629-635. Miettinen TA, Tilvis RS, Kesaniemi YA. Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population. Am J Epidemiol 1990;131:20-31. Dutta SK, Rubin J, Harvey J. Comparative evaluation of the therapeutic efficacy of a pH-sensitive enteric coated pancreatic enzyme preparation with conventional pancreatic enzyme therapy in the treatment of exocrine pancreatic insufficiency. Gastroenterology 1983;84:476-482. Borgstrom B. Studies on intestinal cholesterol absorption in the human, J Clin Invest 1960;39:809-815. Hofmann AF, Borgstrom B. Physico-chemical state of lipids in intestinal content during their digestion and absorption. Gastroenterology 1962;2:43-50. Zentler-Munro PL, Fitzpatrick WJF, Batten JC, Northfield TC. Effect of intrajejunal acidity on aqueous phase bile acid and lipid concentrations in pancreatic steatorrhoea due to cystic fibrosis. Gut 1984;25:500-507. Crouse JR, Grundy SM. Effects of sucrose polyester on cholesterol metabolism in man. Metabolism 1979;28:994-1000. Dunaif G, Schneeman BO. The effect of dietary fiber on human pancreatic enzyme activity in vitro. Am J Clin Nutr 1981;34:1034-1035. Isaksson G, Lundquist I, Ihse I. Effect of dietary fiber on pancreatic enzyme activity in vitro: the importance of viscosity, pH, ionic strength, adsorption, and time of incubation. Gastroenterology 1982;82:918-924. Isaksson G, Lundquist I, Akesson B, Ihse I. Effects of pectin and wheat bran on intraluminal pancreatic enzyme activities and on fat absorption as examined with the triolein breath test in patients with pancreatic insufficiency. Stand J Gastroenterol 1984;19:462-467. Borgstrljm B, Dahlqvist A, Lundh G, Sjovall J. Studies on intestinal digestion and absorption in the human. J Clin Invest 1957;36:1521-1536. Borgstriim B. Studies on intestinal cholesterol absorption in the human. J Clin Invest 1960;39:809-815.

February

CHOLESTEROL MALABSORPTION

1992

42. Siperstein

MD, Chaikoff IL, Reinhardt WO. Obligatory function of bile in intestinal absorption of cholesterol. J Biol Chem 1952;198:111-114. 43. Morgan RGH, Borstrom B. The mechanism of fat absorption in the bile fistula rat. Q J Exp Physiol 1969;54:228-243. 44. Farkkila MA, Miettinen TA. Plasma lathosterol and campesterol in detection of ileal dysfunction. Stand J Gastroenterol 1988;23:19-25. 45. Toskes PP, Hansel1 J, Cerda J, Deren JJ. Vitamin B,, malabsorption in chronic pancreatic insufficiency: studies suggesting the presence of a pancreatic “intrinsic factor.” N Engl J Med 1971;284:627-634.

52.

53.

54.

46. Meihoff WE, Kern F. Bile salt malabsorption

in regional ileitis, diarrhea. J Clin Invest

47.

48.

49.

50. 51.

ileal resection, and mannitol-induced 1968;47:261-267. Mattson FH, Jandacek RJ, Webb MR. The effect of a nonabsorbable lipid, sucrose polyester, on the absorption of dietary cholesterol by the rat. J Nutr 1976;106:747-752. Vuoristo M, Tarpila S, Miettinen TA. Serum lipids and fecal steroids in patients with celiac disease: effects of gluten-free diet and cholestyramine. Gastroenterology 1980;78:15181525. Miettinen TA, Siurala M. Micellar solubilization of intestinal lipids and sterols in gluten enteropathy and liver cirrhosis. Stand J Gastroenterol 1971;6:527-535. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34-47. Vuoristo M, Kesaniemi YA, Miettinen TA. Effects of glutenfree diet (GFD) on LDL turnover, cholesterol metabolism and

55. 56.

IN PANCREATIC

INSUFFICIENCY

655

plant sterols in patients with coeliac disease (CD). In: Abstract book. XIII1 International Congress of Gastroenterology. Rome: International University Press, 1988;I(suppl I):922. Vuoristo M, Miettinen TA. Effects of cholesterol feeding on cholesterol metabolism and plasma lipoproteins in vegetarians. In: Abstracts. Holland Digestive Disease Week, 21-24 June, 1989. 75th Anniversary of the Netherlands Society of Gastroenterology. No. 258:299. Katan MB, Beynen AC. Characteristics of human hypo- and hyperresponders to dietary cholesterol. Am J Epidemiol 1987;125:387-399. Miettinen TA. Effects of neomycin alone and in combination with cholestryamine on serum methyl sterols and conversion of acetate and mevalonate to cholesterol. Stand J Clin Lab Invest 1982;42:189-196. Salen G, Ahrens Jr EH, Grundy SM. Metabolism of /3-sitosterol in man. J Clin Invest 1970;49:952-967. Den Besten L, Connor WE, Kent TH, Lin D. Effect of cellulose on the recovery of dietary plant sterols from feces. J Lipid Res 1970;11:341-345.

Received October 19, 1990. Accepted July 8, 1991. Address requests for reprints to Tatu A. Miettinen, M.D., Second Department of Medicine, University of Helsinki, SF-00290 Helsinki, Finland. Supported by a grant from the Paulo Foundation. The authors thank Leena Kaipiainen, Eeva Gustafsson, Liisa Kuhta, Pia Hoffstrom, Anja Salolainen, Marja Aarnio, and Jaana Rabina for their assistance.