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Mixed triglyceride breath test: A noninvasive test of pancreatic lipase activity in the duodenum
GASTROENTEROLOGY
1989;96:1126-34
Mixed Triglyceride Breath Test: A Noninvasive Test of Pancreatic Lipase Activity in the Duodenum G. R. VANTRAPPEN,
P. J. RUTGEERTS,
Y. F. GHOOS,
and
M. I. HIELE Departments of Medicine and of Medical Research, Gastrointestinal Research Center. University Hospital Gasthuisberg, Leuven, Belgium
A synthetic “mixed” triglyceride (l,%distearyl, 2[13C]octanoyl glycerol), having a medium-chain fatty acid in the 2 position, was evaluated as a substrate for an exocrine pancreatic function test by comparing the 13C0, breath excretion with the lipase output in the duodenum in 25 normal subjects, 29 patients with pancreatic disease, and 22 patients with steatorrhea of nonpancreatic origin. Excellent correlation was found in normal subjects and patients with pancreatic disease (r = 0.89) between lipase output in the duodenum and the 6-h cumulative 13C0, excretion in breath, indicating that the mixed triglyceride breath test is an excellent noninvasive test of pancreatic lipase activity in the duodenum. As a test of exocrine pancreatic insufficiency, it has a sensitivity of 0.89 and a specificity of 0.81.
M
easurement of maximal pancreatic enzyme output by means of a marker-corrected perfusion technique is the gold standard of pancreatic positioning of a multifunction tests (1).It requires ple-lumen tube in the duodenum under fluoroscopic control, continuous infusion of the marker solution with aspiration of duodenal contents, and stimulation with cholecystokinin (CCK). This test is unpleasant for the patient and time-consuming for the investigator. Therefore, several indirect tests are used for the evaluation of pancreatic function. The bentiromide test and the pancreolauryl test (2) are based on urinary recovery of a test molecule that can only be absorbed after hydrolysis by pancreatic enzymes. The results depend on collaboration of the patient in collecting the urine. Fecal fat measurement detects fat malabsorption, but is insensitive and not specific as a test of exocrine pancreatic function (3). The same can be said
of several breath tests developed as screening tests for steatorrhea, in which triolein (4-6),trioctanoine (7,8), or tripalmitin (9,lO) are used as marker substances. We have developed a breath test in which a 14C-labeled “mixed triglyceride” is used (11). In this study we evaluated a new ‘“C-labeled test molecule (1 ,&distearyl, 2 [carboxyl-l”C]octanoyl glycerol), which contains a ‘“C-labeled medium chain fatty acid in the 2 position and long-chain fatty acids in the 1 and 3 positions. The rate-limiting step in the digestion of the mixed triglyceride is hydrolysis of the two stearyl groups by pancreatic lipase. The aim of the present study was to evaluate the mixed-triglyceride breath test as an exocrine pancreatic function test by comparing the excretion of labeled CO, in breath after oral administration of a labeled test meal with the lipase output in the duodenum upon “maximal” stimulation of the pancreas. Materials Patients studied.
and Methods and
Twenty-nine Twenty-one
Controls patients with pancreatic disease were patients (1 woman and 20 men, aged
28-59 yr) had chronic pancreatitis; in 19 it was due to alcoholism and in 2 patients no cause was identified. The diagnosis of chronic pancreatitis was based in all cases on the presence of pancreatic calcifications or pathologic findings at surgery, or both. Three patients [men, aged 5469 yr) had pancreatic cancer. Five patients had undergone pancreatic surgery for complicated chronic alcoholic pancreatitis. Small intestinal and hepatobiliary diseases were
Abbreviation used in this paper: CCK, cholecystokinin. 0 1989 by the American Gastroenterological Association 0016-5085/89/$3.50
“C-8KEATH TEST FOK PANCKEATIC: FIINCTION
April 1Y8Y
excluded on the basis of clinical, radiologic, and laboratory data. The control groups comprised 25 normal subjects (11 women and 14 men, aged 28-57 yr) and 22 patients with steatorrhea of nonpancreatic origin (15 women and 7 men, aged 27-73 yr). Both the lipase output in the duodenum upon maximal hormonal stimulation and the “‘CO, breath excretion were measured in these two control groups. The effect of metabolic disorders on the results of the mixedtriglyceride breath test was studied in other control groups, i.e.. 10 patients with insulin-dependent diabetes mellitus (3 women and 7 men, aged 23-79 yr), 10 obese subjects ,20”/, overweight (5 women and 5 men, aged 1854 yr). and 12 patients with chronic liver disease (4 women and 8 men aged 36-68 yr). In addition, the effect of therapy with pancreatic enzymes on ‘?ZO, excretion in breath and fat excretion in stools was studied in 11 patients with pancreatic insufficiency (10 men and 1 woman, aged 32-59 yr). Four patients presented with chronic pancreatitis. 1 with pancreatic carcinoma, 1 with pancreatic insufficiency after total gastrectomy, and 5 had undergone a partial or total pancreatic resection. In these patients the mixed-triglyceride breath test, fecal volume, and fecal fat were studied before and during treatment with pancreatic enzymes, i.e.. 4 tablets of 300 mg of pancreatin BP 1980 [protease 450 U. lipase 6000 U, amylase 6000 U (Enzypan forte: Norgine, Brussels. Belgium)], t.i.d. with meals.
Test
Molecule
and Test Meal
The mixed-triglyceride (1,3-distearpl. 2[carboxyl‘%]octanoyl glycerol] (S*OS) was purchased from the Belgian Institute of Isotopes (IRE, Fleurus, Belgium). It was 91% “‘C-enriched, and the chemical purity exceeded 980/O. The test meal consisted of 100 g of toast with 0.25 g of butter per kilogram of body weight, to which 16 mg of S*OS per gram of butter was added.
Experimental
Procedures
All subjects were hospitalized and put on a standard fat diet containing 90 g fat per day. After an equilibration period of 2 days, a 3-day. marker-corrected ([“Hlpolyethyleneglycol 4000) stool collection was performed for quantitative determination of fecal fat. The day after the stool collection, the mixed-triglyceride breath test was done and the next day the duodenal perfusion study was performed. Breath tests were carried out after an overnight fast of at least 12 h. End-tidal breath was sampled with a modified Haldane-Priestly tube (12). The samples were taken twice before the meal and at 30-min intervals for a period of 6 h after the meal. The “‘C enrichment was determined with an isotope ratio mass spectrometer (model 250; Finnigan MAT, Bremen, F.R.G.). All &values were expressed versus the PDB-international standard. after correcting for the oxygen isotope effect (13). The &value was calculated using the following formula:
1127
in the CO, of the sample and R,, = in which R,, = “C/“C ‘%I”% in the CO, of the standard. The results were expressed as the percentage of ‘%-recovery per hour and as cumulative values over 6 h. For this calculation the formula of Schoeller et al. (14) was used, and the CO, production was assumed to be 300 mmolim’ body surface area per hour. Body surface area was calculated by the weight-height formula of Haycock et al. (15). In normal subjects and patients with pancreatic disease, the estimated CO, production was compared with the CO, production measured by the CO,:argon method. For this measurement, the subject exhaled into a Haldane-Priestly tube connected to a spirometer until a constant tidal volume was reached (usually after about 5 min). A sample of expired air was then withdrawn from the tube and the CO, concentration was determined in the mass spectrometer using argon concentration as internal standard (16). The CO, production (CO, concentration x expired volume) was measured twice, at the onset and at the end of the test. Mean values were used to compute the 13C enrichment. Results obtained in this way will be called the by assuming a ‘*quantitative test,” and results obtained CO, production rate of 300 mmol per unit of body surface area per hour will be called the “simplified test.” Measurement of lipase, amylase. and trypsin output upon maximal stimulation of the pancreas was also carried out after an overnight fast of 12 h. A double-lumen duodenal tube and a separate gastric. tube were positioned under fluoroscopic control. The perfusion port of the duodenal tube was located near the ampulla of Vater and the aspiration site near the duodenojejunal flexure. Gastric contents were continuously aspirated. A warmed (37°C) mannitol-saline solution containing [ “Hlpolyethyleneglyco1 4000 as radioactive marker was infused at a constant rate of 10 mlimin at the proximal duodenal port. After a basal steady-state perfusion of 1 h. CCI;-pancreoxymin (Boots. 1 U/kg), was injected intravenously. The duodenal contents were collected on ice in four 15.min intervals. Lipase activity was measured titrimetrically at constant pH, using tributyrine as substrate (17). The [.‘H]polyethyleneglycol content in gastric and duodenal aspirates was measured by the oxidation method [Packard sample oxidizer, model 306: Packard, Downers Grove, Ill.), with subsequent liquid-scintillation counting [model 2450; Packard). Lipase output was calculated on the basis of the [“H]polyethyleneglycol recovery data and expressed as kilounits per hour. Trypsin activity was measured spectrophotometrically at 253 nm using p-toluene sulfonyl-2arginine methyl ester (18). cu-Amylast? activity was measured calorimetrically at 620 nm on a water-insoluble cross-linked starch polymer carrying a blue dye (Phadebas, Pharmacia. Sweden). The outputs of these enzymes were calculated as described for lipase.
Statistical
Analysis
Relations between different parameters lated by regression analysis (linear regression
were calcuand satura-
1128
VANTRAPPEN
Table
1.
ET AL.
GASTROENTEROLOGY
Pancreatic Enzyme Output in Patients With Pancreatic Disease and Normal Controls Control group
Lipase (kU/h) Trypsin (kU/h) Amylase
Vol. 96, No. 4
Patient group
Mean I- SEM
Range
Mean + SEM
Range
182.5 + 10.3
107.9-335.3
35.6 + 7.2 11.3 + 2.6 6.7 ? 1.5
1.1-142.4 0.2-34.7 0.4-30.0
40.0 2 3.9 56.5 k 7.0
(kU/h)
tion kinetics). Differences between sample means were calculated using Student’s t-test. Sensitivity, specificity, and predictive values were calculated by ROC analysis (19). All values are given as mean ? SEM.
Results Enzyme
Output
The output of lipase, trypsin, and amylase activity after maximal pancreatic stimulation is given in Table 1 for both the normal control group and the patients with pancreatic disease. Figure 1 shows the lipase output in the duodenum of normal subjects as compared with that of patients with pancreatic disease. Baseline
10.2-63.9 9.2-111.6
13C Excretion
Rate
The “C excretion rate in normal subjects, expressed as a percentage of the dose excreted per hour, increased progressively, reached a plateau of 8% per hour between 4.5 and 5.5 h after administration of the label, and decreased thereafter. The 13C excretion rate was markedly different in patients with pancreatic disease; the 13C excretion rate rose more slowly and did not reach the high values obtained in normal subjects [Figure Za). The simplified test, in which the CO, production rate was assumed to be 300 mmol per unit body surface area
13C Excretion f
The baseline 13C enrichment in the control group was similar to that of the patient group, the +- 0.036 and -25.856 + &value being -26.163 0.030, respectively. The measured CO, production averaged 297.0 2 2.6 mmol per unit of body surface area per hour in normal subjects and 295.7 +- 3.7 in patients with pancreatic disease.
b-
x 8 Y
_ 4-
s2-
1
2
3
4
5
6
HOURS
I
1
Figure ;i
.iii: Figure
2
3
4
5
b HOURS
__--_
1. Lipase output (in kilounits per hour] for normals (open circles) and patients with pancreatic disease lfilled circles).
2. Excretion rate of ‘“CO, in breath after administration of ‘%-labeled mixed triglyceride. The upper part (a) represents the “quantitative test” in which CO, production is measured; the lower part (b) represents the “simplified test, ” in which CO, production is assumed to be 300 mmol per square meter of body surface area per hour. Open circles represent normal subjects: filled circles represent patients with pancreatic disease.
April
19811
’‘C-BREATH
TEST FOR PANCKEATIC
1129
FUNCTION
activity in the duodenum after maximal pancreatic stimulation was calculated in two different ways. Assuming a linear correlation between the two variables, the correlation coefficient (r) of the test was found to be 0.80 (p < 0.001). If it is assumed that the relation between the cumulative “‘CO, excretion at 6 h and the lipase activity is linear at low values and follows zero-order (saturation) kinetics at high lipase outputs, the mathematical expression of this relation can be written as follows: 1
2
3
L
6
5
‘“CO, excretion
(% cumulative
A [lipase) 6 h) =
(1) 1 + B (lipase)’
where A and B are constants (Figure 5a). From Equation (1) the following equation derived: Lipase -=_ ‘“CO,
1
2
3
L
5
6 HOURS
Figurrh
3. Cumulative I ‘CO, excretion in breath after administration of “C-labeled mixed triglyceride. The upper part ((I) represents the “quantitative test”; the lower part (b) represents the “simplified test.” Normal subjects are indicated bv open circles and patients with pancreatic disease bv filled circles.
per hour, resulted in a similar excretion the two study groups (Figure 2b). The between normal subjects and patients significant in both tests (p < 0.001). Cumulative
pattern in differences are highly
A
+ B (lipase) A
(21
Equation (2) is presented in Figure 5b. As the relation between lipase/‘“CO, excretion and lipase is very good (r = 0.89), and as Equation (2) is only a mathematical conversion of Equation (1). the assumption that the relation between “‘CO, excretion and lipase activity follows saturation kinetics seems to be valid. Sensitivity, Specificity, of the Mixed-Triglyceride Parameter of Pancreatic values
and Predictive Value Breath Test as a Lipase Activity
The sensitivity, specificity, and predictive of the mixed-triglyceride breath test for the
‘,‘C Excretion
The 6-h cumulative ‘“CO, excretion data are summarized in Figure 3. The excretion patterns of the two test procedures, quantitative and simplified, were similar. The 6-h cumulative ‘“CO, excretion, expressed as a percentage of the dose administered, amounted to 33.5% k 1.4% [quantitative) and 35.6% ? 2.8% (simplified] in normal subjects, and to 13.8% t 1.4% and 14.3”/, 2 1.8”/, in patients with pancreatic disease. The differences between normal subjects and patients are highly significant with both procedures (p < 0.001). The individual 6-h cumulative ‘“CO, excretion data for the two groups, together with the data for other control groups, are given in Figure 4. Relation Between “‘CO, Breath and Duodenal Lipase Output cretion
1
can be
Excretion
The relation between the 6-h cumulative exof “‘CO, in breath and the output of lipase
.. .. :. . : : :. .:: . ;. ; Figure
4. Cumulative 6-h ‘“CO, excretion in normal subjects (0). patients with pancreatic disease (0). patients with steatorrhea of nonpancreatic origin ( x 1,and metabolic controls; A indicates patients with liver disease; a. patients with diabetes: V. patients with obesity.
1130
g
U
X y
0"
VANTRAPPEN
ET AL
Vol. 96, No. 4
GASTROENTEROLOGY
40
!#_:I] -.
but 3 normal subjects. In Figure 6 the daily fecal fat loss is plotted against the lipase output. All but one of the subjects with a lipase output of >40 kU/h had a normal fat excretion in the stool, whereas all but three of those who had a lipase output of <40 kU/h had steatorrhea.
30
6h cum. “CO 2 -excr
Ie
I
=
1+ 0 027 [Lipase]
I
I
200
300 LIPASE (KU/h)
LIPASE (KU/h) (a]Cumulative ‘“CO2 excretion (in percentage of ingested dose) 6 h after intake of the [‘“Cl-labeled mixed triglyceride as a function of lipase activity (in kilounits per hour] in normal subjects (O] and patients with pancreatic disease (0). (b) Linearly modified saturation curve following the relation given by Equation (2). Open circles represent normal subjects; filled circles are patients with pancreatic disease. detection of impaired pancreatic lipase activity in the duodenum were determined by ROC analysis against the gold standard (lipase output in duodenum). The results, presented in Table 2, are based on data obtained in the patients with pancreatic disease and the various control groups. The lipase output was considered to be normal in all metabolic control patients, in whom duodenal intubation was not carried out. Relation Between Steatorrhea Three-day all but 1 patient
Lipase
stool collections with pancreatic
Output
and
were performed in disease, and in all
Effect of Pancreatic Enzymes on the Mixed-Triglyceride Breath Test in Patients With Pancreatic Insufficiency Eleven patients with pancreatic exocrine insufficiency were studied. The 6-h cumulative 13C0, excretion values before and during treatment with pancreatic enzymes (Enzypan Forte) are presented in Figure 7. Before treatment with pancreatic enzymes, the mean fat excretion in the feces amounted to 52.0 * 8.4 g/day. Enzyme treatment resulted in a decrease of stool fat to 19.8 2 4.0 g/day. All patients improved under therapy, but the fat excretion became normal in only 3 patients. The stool volume decreased from 727 g t 253 g before to 373 ? 68 g during enzyme treatment. The ‘“CO, excretion after administration of the ‘“C-labeled-mixed-triglyceride amounted to 6.3% +- 1.4% before treatment and increased to 17.6% * 3.3% during treatment with enzymes. Four patients showed little or no improvement when treated with 4 tablets t.i.d. When treated with a dose of 8 tablets t.i.d., 2 patients improved further. One of the patients who did not improve had a high basal gastric acid output (24.6 mEq H+/h). He was studied a third time while he was being treated with ranitidine (150 mg b.i.d.) and Enzypan Forte (8 tablets t.i.d.). This led to an improvement of the 13C0, excretion values.
Mixed-Triglyceride Breath Test in Other Control Groups (Patients With Steatorrhea Nonpancreatic Origin and Patients With Metabolic Disorders Figure 4 shows the individual values mixed-triglyceride breath test in the various
Table
2.
of
of the study
Sensitivity, Specificity, and Predictive Values of the Mixed-Triglyceride Breath Test in Comparison With Intubation Test to Detect Disease-Diminished Lioase Outnut
Cut-off
(6 h cumulative percentage of ‘3C0,) Cut-off (lipase)
Sensitivity breath test Specificity breath test Positive predictive value Negative predictive value
22% 90 kU 0.89 0.81 0.63 0.95
24%
loo
kU
0.93 0.76 0.60 0.97
22%
loo kU 0.83 0.81 0.62 0.93
April
’'C-BKEATH TEST FOR PANCREATIC
1989
p
F‘UNCTION
1131
50 h
n F
ai
Figure
“, 2 k
6. Relation between daily fecal fat loss and duodenal lipase output. Open circles represent norma1 subjects: filled circles are patients with pancreatic disease.
.
. . 25
.
. -:
??
.
:*
I. . ,c-._______-------_________
. .
1
yr. 40
??
-------------___.
?? .
,
??
0
80
“q 120
O@$ 0 oooo 160
0
OOeFp“,;‘ ,“:;;; , 200
LIPASE
groups. The 6-h cumulative CO, excretion amounted to 25.5% ‘- 2.4% in diabetics, 25.5% + 4.1% in patients with hepatic insufficiency, and 30.5% t 1.9% in obese subjects. Eight patients had excretion values below the lowest value of “normal” control subjects. The 6-h cumulative CO, excretion in patients with steatorrhea of nonpancreatic origin was 26.3% ? 2.6%. Seven patients had 13C0, excretion values below the lowest “normal” value. Table 3 summarizes pertinent data on patients with steatorrhea of nonpancreatic origin. Five of 9 patients with celiac disease had subnormal 13C0, excretion values, had low duodenal lipase whereas only 1 patient activity upon maximal pancreatic stimulation. Two other patients, one with total gastrectomy and one
before
ENZYPAN FOiliF
Figure
3x4
tablets
3x8
tablets
3x8
tablets ??
ranhdme
7
Six-hour cumulative ‘“CO, excretion before and during treatment with pancreatic enzymes (Enzypan Forte). One patient was also treated with ranitidine.
/(KU/h)
with Crohn’s disease, had a very low ‘“CO, excretion and a normal lipase output.
Discussion Several indirect noninvasive tests have been proposed for the assessment of exocrine pancreatic function. Tests based on urinary recovery of a test molecule that can only be absorbed after hydrolysis by chympotrypsin (bentiromide test) or pancreatic esterases (pancreolauryl test) have a sensitivity and specificity of -8O%-90% in most studies (2). Other indirect pancreatic function tests that use14C- or 13C-labeled triglycerides, either alone or in combination, are proposed as tests of fat malabsorption and as possible substitutes for fecal fat determination (410,20-25). Steatorrhea is an insensitive test of exocrine pancreatic function, as it only occurs if pancreatic lipase output is reduced to 510% (26). Moreover, steatorrhea may be due to causes other than pancreatic insufficiency. Therefore, breath tests with good performance in the detection of steatorrhea are of much lesser value as pancreatic function tests. The mixed-triglyceride breath test was designed to measure intraluminal pancreatic lipase activity and not as a test of steatorrhea. The test molecule, 1,3distearyl, 2[carboxyl-‘“Cloctanoyl glycerol, consists of a medium-chain fatty acid in the 2-position and long-chain fatty acids in the 1 and 3 positions. The rationale of the test is that the two stearyl groups have to be split off the glycerol by lipase before [‘“Cloctanoyl monoglyceride or [l”C]octanoate, a medium-chain fatty acid, is absorbed and rapidly metabolized to l”CO, (27). The rate-limiting step in the oxidation to ‘“CO, is hydrolysis of the fatty acids in the I- and 3-positions of the mixed triglyceride. That the absorption and oxidation of free octanoate or octanoyl monoglyceride occur faster than the absorption and oxidation of a long-chain fatty acid or its monoglyceride is indi-
1132
VANTRAPPEN
Table
ET AL.
3. Data on Patients
GASTROENTEROLOGY
With Steatorrhea
of Nonpancreatic
Age Patient C.E. M.G. G.B. V.J. D.S. M.M. P.M. D.M. W.A. W.T. D.A. M.R. S.J. T.J. M.M. M.A. G.S. K.B. E.M. A.M.
Sex
lYd
F
27
M
68
F
31
F
49
F
32
M
49
M
36
M
26
F
73
M
36
F
58
F
59
F
48
F
52
M
35
F
31
F
33
F
26
F
54
F
42
E.A.
M
62
H.G.
F
35
Diagnosis Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Short bowel Short bowel Short bowel Short bowel Crohn’s disease Crohn’s disease Crohn’s disease Bacterial overgrowth Bacterial overgrowth Bacterial overgrowth Total gastrectomy Extrahepatic cholestasis Hyperthyroidism
Mean
lJeca1 volume (g/24 hl 322
compare favorably with other indirect pancrefunction tests, such as the pancreolauryl test. mixed-triglyceride breath test, like the intuba-
Fecal fat lgi2-1 hl 21.5
Breath test (% ’'C. cumulative
Lipase activity 6 h)
(kU/h)
32
353
23
162
449
11
7.8
132
2813
51
14.1
210
19
252
28
240
380 285
8
17.5
623
47
320
7
25
347
48
385
8.3
18
149
1133
11.7
27
144
1109
33
34
126
491
55
29
233
938
20
46
124
45
254
1165 772
25
390
8
39
163
573
13
29
238
734
20
23
95
644
27
44
98
830
79
3.3
6.3
186
125
140
28
244
580
40
26%
t119
cated by the observation that 20% of our normal subjects had their peak ‘“CO, excretion in the first 3 h after intake of the substrate, whereas in the study by Newcomer et al. (4) and in a recent study by Turner et al. (28), none of the normal subjects had their peak 14C0, excretion during the first 3 h after intake of 14C-triolein. Recently, Hofmann’s group (29,30) presented cholesteryl octanoate as a new substrate for a ‘“CO, pancreatic function test. In their healthy controls the peak 14C0, excretion occurred at 60-90 min in all subjects. As they used the same medium-chain fatty acid as we did, the difference in peak excretion must be due to the slower hydrolysis of the stearyl groups in the 1 and 3 position of the medium-chain triglyceride in comparison with the hydrolysis of the cholesteryl octanoate. The present study shows that the mixed-triglyceride breath test is a sensitive noninvasive test of pancreatic lipase activity in the duodenum. The 13C0, production correlates very well with the duodenal intubation test in which the lipase output upon maximal pancreatic stimulation is measured by means of a marker-corrected perfusion technique. It allows detection of pancreatic dysfunction with an acceptable sensitivity (0.89) and specificity (0.81)
(n = 22)
228
696
SEM
that atic The
Origin
Vol. 96, No. 4
22
26.3
t4.2
22.6
190 217
tion test and other indirect tests, will not detect pancreatic disease when pancreatic function is normal, despite the presence of anatomic changes. However, the test is much more sensitive in detecting impaired pancreatic function than fecal fat determionly occurs if nation. Figure 6 shows that steatorrhea lipase output falls below 40 kU/h, whereas the mixed-triglyceride breath test detects a lipase output of <90 kU/h with a sensitivity of 89%. It was only below a ‘“CO, excretion of 7.5% that steatorrhea was always present, whereas the limit value of the ‘“CO2 test for discrimination between normal function and abnormal exocrine pancreatic function was 22%. The mixed-triglyceride breath test, like other indirect pancreatic function tests, may be used to replace the invasive intubation test for the detection of exocrine pancreatic disease, to follow the evolution of pancreatic disease, and to monitor the effect of therapy. That the test is suitable to follow the effect of enzyme supplementation is also suggested by the results presented in Figure 7. Therapy with pancreatin (Enzypan Forte) in patients with pancreatic insufficiency resulted in marked improvement of the breath test, concurrently with improved fecal volume and fecal fat. The specificity of the test for the detection of pancreatic disease is not as good as that of the intubation test. The mixed-triglyceride breath test yielded false-positive results in 1 gastrectomy patient, possibly due to insufficient mixing of nutri-
April
‘%BREATH
1989
ents with pancreatic enzymes, and in 4 patients with celiac disease. Although the pancreatic enzyme output after stimulation with CCK can be normal in patients with celiac disease, the pancreas may be insufficiently stimulated by a meal, which yields “false-positive” mixed-triglyceride breath tests. It has been shown that stimulation of the pancreas with CCK results in normalization of the labeled CO, excretion in patients with celiac disease (11).In this respect, the present breath test seems to be more physiologic than the CCK intubation test. Abnormal breath tests are not unexpected in patients with hepatic and metabolic diseases. In diabetes, two factors may be responsible for a lowered [13C]octanoic acid oxidation. Slowed gastric emptying due to neuropathy may delay digestion of the mixed-triglyceride even when pancreatic function is normal. On the other hand, the [13C]octanoic acid is probably diluted in a larger pool of short-chain fatty acids, resulting in slower oxidation. Obesity does not interfere with the mixed-triglyceride test. In contrast, severe liver disease may result in abnormal ‘“CO, exhalation after administration of ‘“C-labeled mixed triglyceride, probably because of impaired octanoic acid oxidation in the decreased liver cell mass. This is true not only for the mixed-triglyceride breath test. A high incidence of abnormal triolein breath tests was also described in alcoholics with test and the liver damage (31).Also, the bentiromide pancreolauryl test were found to give false-positive results in patients with liver disease (32,~~). In conclusion, the excellent correlation between the ‘“CO, excretion in breath and the duodenal lipase output in normal subjects and in patients with pancreatic disease (r = 0.89). and the good sensitivity and specificity (0.89 and 0.81,respectively) in comparison with the CCK test, indicate that the mixed-triglyceride breath test is a promising noninvasive test of pancreatic lipase activity in the duodenum. Unlike tests involving urine collection, the cooperation of the patient can be controlled.
EP. Assessment of exocrine intubation. Clin Gastroenterol
pancreatic 1984:13:
Scharpe S. Illiano L. Two indirect tests of exocrine pancreatic function evaluated. Clin Chem 1987:33:5-12. DiMagno EP. Clain JE. Chronic pancreatitis. In: Go VLW. Gardner JD. Brooks FP, Lebenthal E. DiMagno EP. Scheele GA, eds. The exocrine pancreas: biology, pathology, and diseases. New York: Raven. 1986:541-75. Newcomer AD, Hofmann AF, DiMagno EP, Thomas PJ, Carlson GL. Triolein breath test-a sensitive and specific test for fat malabsorption. Gastroenterology 1979;76:6-13. Einarsson K. Bjorkhem I. Eklof R, Blomstrand R. “‘C-triolein
test as a rapid
and
convenient
FUNCTION
screening
1133
test for fat
malabsorption. Stand J Gastroenterol 1983;18:9-12. breath 6. Benini L, Senro LA, Menini E. et al. Is the “C-triolein test useful in the assessment of malabsorption in clinical practice. Digestion 1984;29:91-7. SM. Estima7 Schwabe AD, Cozetto FJ, Bennett LR. Mellinkoff
8
9
10
11
12
13
14
tion of fat absorption by monitoring of expired radioactive carbon dioxide after feeding a radioactive fat. Gastroenterology 1962;42:285-91. Watkins JB, Schoeller DA, Klein PD. Ott DG. Newcomer AD, Hofmann AF. r3C-trioctanoin: a nonradioactive breath test to detect fat malabsorption. J Lab Clin Med 1977:90:422-30. Kaihara S. Wagner H. Measurement of intestinal fat absorption with carbon-14 labelled tracers. J Lab Clin Med 1968;71: 400-11. Burrows P, Fleming JS. Garnett ES, Ackery- DM, Colin-Jones DC. Bamforth J. Clinical evaluation of the ‘“C fat absorption test. Gut 1974;15:147-50. Ghoos YF. Vantrappen GR. Rutgeerts PJ. Schurmans PC. A mixed triglyceride breath test for intraluminal fat digestive activity. Digestion 1981:22:239-47. Douwes A. Respiratory hydrogen excretion as a parameter for sugar absorption in children. Thesis. Rijksuniversiteit Groningen, 1979. Mook WG, Grootes PM. The measuring procedure and corrections for the high-precision mass-spectrometric analysis of isotopic abundance ratios, especially referring to carbon, oxygen and nitrogen. Int 1Mass Spectr Ion Phvs 1973;12:27398. Schoeller DA, Klein PD. Maclean WC, Watkins JB, Van
Santen E. Fecal “C analysis for the detection and quantitation of intestinal malabsorption. [ Lab Clin Med 1981:97:439-X8. 15. Haycock G. Schwartz G. Wisotsky D. Geometric method for measuring body surface area: a height-weight formula validated in infants, children and adults. 1Pediatr 1978:93:62-6. 16
17
18
19 20
21.
References Go VLW. DiMagno function by duodenal 701-15.
breath
TEST FOR PANCREATIC
22.
23.
24.
25.
Ghoos Y, Rutgeerts P, Vantrappen G. Hiele M. Measurement of “C glucose oxidation rate using mass spectrometric determination of the CO,:Ar ratio and spirometry. Biomed Environ Mass Spectrom 1988:15:447-51. Erlanson C, Borgstrom B. Tributyrine as a substrate for determination of lipase activity of pancreatic juice and small intestinal content. Stand J Gastroenterol 1970:5:293-5. Pelot D. Grossman MI. Distribution and fate of pancreatic enzymes in small intestine of the rat. Am 1Physiol 1965:202: 285-8. Metz CE. Basic principles of ROC analysis. Semin Nucl Med 1978:8:283-98. Chen WI. Azmudeh K. Connell AM. Saenger EL. “C-tripalmitin breath test as a diagnostic aid for fat malabsorption due to pancreatic insufficiency. J Nucl Med 1974:15:1125-9. Goff JS. Two-stage triolein breath test differentiates pancreatic insufficiency from other causes of malabsorption. Gastroenterology 1982:83:44-6. Adlung J. Grazikowske H. Diagnosis of fat absorption with l’C-tripalmitate/‘H-palmitic acid. Stand 1Gastroenterol 1979; 14:587-92. Pedersen NT, Andersen BN. Ivlarqversen J. Estimation of “C-triolein assimilation as a test of lipid assimilation, Breath test or measurement of serum radioactivity. Stand J Gastroenter01 1982;17:309-16. Pedersen NT. Halgreen H. Simultaneous assessment of fat maldigestion and fat malabsorption bv double-isotope method using fecal radioactivity. Gastroenterology 1983;88: 47-54. Watkins JB, Klein PD, Schoeller DA, Kirschner BS, Park R. Perman JA. Diagnosis and differentiation of fat malabsorption
1134
VANTRAPPEN
in children palmitic
using
tests.
enzyme
outputs
27. Schwabe
AD, Bennett
tion and oxidation GKT.
diagnosis
LR, Bowman S. Fellows
[‘4C]-triolein
of malabsorption. test. Preliminary pancreatic
WHJ. Relations
and
between
in severe
LP. Octanoic
J Appl
Physiol
pan-
acid absorp1964;19:335-7.
IW, Johnson
absorption:
I. Hill
a useful
test
PG.
in the
Gut 1987;28:694-700.
29. Cole SG, Rossi S. Stern A, Hofmann human
triolein,
1982:82:911-7.
and malabsorption
in humans.
JM, Lawrence
breath
trioctanoin,
N Engl J Med 1973;288:813-5.
insufficiency.
28. Turner Holmes
lipids:
Gastroenterology
EP. Go VLW, Summerskill
pancreatic creatic
“C-labeled
acid breath
26. DiMagno
GASTROENTEROLOGY
ET AL.
studies
exocrine
AF. Cholesteryl Gastroenterology
test of 1987;
93:1372-80. 30. Mundlos noate
S, Rhodes
breath
insufficiency 31. Korsten
JB, Hofmann
test: a new procedure in the rat. Pediatr
MA, Klapholz
AF. The cholesteryl for detection
octa-
of pancreatic
Res 1987;22:257-61.
MB, Leaf MA, Lieber
triolein breath test in alcoholics with liver damage. J Lab Clin Med 1987;109:62-6. I. Fehr C, Vonderschmitt D, 32. Gyr K. Stalder GA. Shiffmann Fahrlaender H. Oral administration of a chymotrypsin-labile peptide: a new test of exocrine pancreatic function in man (PFT). Gut 1976:17:27-32. P. Biichler M, Muller A, Ditschuneit H. Influ33. Malfertheiner ence of extrapancreatic digestive disorders on the indirect pancreatic function test with fluorescein dilaurate. Digestion 1983;28:45-6.
octanoate
on a new non-invasive
function.
Vol. 96, No. 4
CS. Use of the
Received March 30, 1988. Accepted October 31. 1988. Address requests for reprints to: Prof. Dr. G.R. Vantrappen, University Hospital Gasthuisberg, B-3000 Leuven, Belgium. This study was supported by the Fund for Medical Scientific Research, Brussels, Belgium. The authors thank L. Swinnen, S. Rutten, and D. Claus for skillful technical assistance.
1989;96:1126-34
Mixed Triglyceride Breath Test: A Noninvasive Test of Pancreatic Lipase Activity in the Duodenum G. R. VANTRAPPEN,
P. J. RUTGEERTS,
Y. F. GHOOS,
and
M. I. HIELE Departments of Medicine and of Medical Research, Gastrointestinal Research Center. University Hospital Gasthuisberg, Leuven, Belgium
A synthetic “mixed” triglyceride (l,%distearyl, 2[13C]octanoyl glycerol), having a medium-chain fatty acid in the 2 position, was evaluated as a substrate for an exocrine pancreatic function test by comparing the 13C0, breath excretion with the lipase output in the duodenum in 25 normal subjects, 29 patients with pancreatic disease, and 22 patients with steatorrhea of nonpancreatic origin. Excellent correlation was found in normal subjects and patients with pancreatic disease (r = 0.89) between lipase output in the duodenum and the 6-h cumulative 13C0, excretion in breath, indicating that the mixed triglyceride breath test is an excellent noninvasive test of pancreatic lipase activity in the duodenum. As a test of exocrine pancreatic insufficiency, it has a sensitivity of 0.89 and a specificity of 0.81.
M
easurement of maximal pancreatic enzyme output by means of a marker-corrected perfusion technique is the gold standard of pancreatic positioning of a multifunction tests (1).It requires ple-lumen tube in the duodenum under fluoroscopic control, continuous infusion of the marker solution with aspiration of duodenal contents, and stimulation with cholecystokinin (CCK). This test is unpleasant for the patient and time-consuming for the investigator. Therefore, several indirect tests are used for the evaluation of pancreatic function. The bentiromide test and the pancreolauryl test (2) are based on urinary recovery of a test molecule that can only be absorbed after hydrolysis by pancreatic enzymes. The results depend on collaboration of the patient in collecting the urine. Fecal fat measurement detects fat malabsorption, but is insensitive and not specific as a test of exocrine pancreatic function (3). The same can be said
of several breath tests developed as screening tests for steatorrhea, in which triolein (4-6),trioctanoine (7,8), or tripalmitin (9,lO) are used as marker substances. We have developed a breath test in which a 14C-labeled “mixed triglyceride” is used (11). In this study we evaluated a new ‘“C-labeled test molecule (1 ,&distearyl, 2 [carboxyl-l”C]octanoyl glycerol), which contains a ‘“C-labeled medium chain fatty acid in the 2 position and long-chain fatty acids in the 1 and 3 positions. The rate-limiting step in the digestion of the mixed triglyceride is hydrolysis of the two stearyl groups by pancreatic lipase. The aim of the present study was to evaluate the mixed-triglyceride breath test as an exocrine pancreatic function test by comparing the excretion of labeled CO, in breath after oral administration of a labeled test meal with the lipase output in the duodenum upon “maximal” stimulation of the pancreas. Materials Patients studied.
and Methods and
Twenty-nine Twenty-one
Controls patients with pancreatic disease were patients (1 woman and 20 men, aged
28-59 yr) had chronic pancreatitis; in 19 it was due to alcoholism and in 2 patients no cause was identified. The diagnosis of chronic pancreatitis was based in all cases on the presence of pancreatic calcifications or pathologic findings at surgery, or both. Three patients [men, aged 5469 yr) had pancreatic cancer. Five patients had undergone pancreatic surgery for complicated chronic alcoholic pancreatitis. Small intestinal and hepatobiliary diseases were
Abbreviation used in this paper: CCK, cholecystokinin. 0 1989 by the American Gastroenterological Association 0016-5085/89/$3.50
“C-8KEATH TEST FOK PANCKEATIC: FIINCTION
April 1Y8Y
excluded on the basis of clinical, radiologic, and laboratory data. The control groups comprised 25 normal subjects (11 women and 14 men, aged 28-57 yr) and 22 patients with steatorrhea of nonpancreatic origin (15 women and 7 men, aged 27-73 yr). Both the lipase output in the duodenum upon maximal hormonal stimulation and the “‘CO, breath excretion were measured in these two control groups. The effect of metabolic disorders on the results of the mixedtriglyceride breath test was studied in other control groups, i.e.. 10 patients with insulin-dependent diabetes mellitus (3 women and 7 men, aged 23-79 yr), 10 obese subjects ,20”/, overweight (5 women and 5 men, aged 1854 yr). and 12 patients with chronic liver disease (4 women and 8 men aged 36-68 yr). In addition, the effect of therapy with pancreatic enzymes on ‘?ZO, excretion in breath and fat excretion in stools was studied in 11 patients with pancreatic insufficiency (10 men and 1 woman, aged 32-59 yr). Four patients presented with chronic pancreatitis. 1 with pancreatic carcinoma, 1 with pancreatic insufficiency after total gastrectomy, and 5 had undergone a partial or total pancreatic resection. In these patients the mixed-triglyceride breath test, fecal volume, and fecal fat were studied before and during treatment with pancreatic enzymes, i.e.. 4 tablets of 300 mg of pancreatin BP 1980 [protease 450 U. lipase 6000 U, amylase 6000 U (Enzypan forte: Norgine, Brussels. Belgium)], t.i.d. with meals.
Test
Molecule
and Test Meal
The mixed-triglyceride (1,3-distearpl. 2[carboxyl‘%]octanoyl glycerol] (S*OS) was purchased from the Belgian Institute of Isotopes (IRE, Fleurus, Belgium). It was 91% “‘C-enriched, and the chemical purity exceeded 980/O. The test meal consisted of 100 g of toast with 0.25 g of butter per kilogram of body weight, to which 16 mg of S*OS per gram of butter was added.
Experimental
Procedures
All subjects were hospitalized and put on a standard fat diet containing 90 g fat per day. After an equilibration period of 2 days, a 3-day. marker-corrected ([“Hlpolyethyleneglycol 4000) stool collection was performed for quantitative determination of fecal fat. The day after the stool collection, the mixed-triglyceride breath test was done and the next day the duodenal perfusion study was performed. Breath tests were carried out after an overnight fast of at least 12 h. End-tidal breath was sampled with a modified Haldane-Priestly tube (12). The samples were taken twice before the meal and at 30-min intervals for a period of 6 h after the meal. The “‘C enrichment was determined with an isotope ratio mass spectrometer (model 250; Finnigan MAT, Bremen, F.R.G.). All &values were expressed versus the PDB-international standard. after correcting for the oxygen isotope effect (13). The &value was calculated using the following formula:
1127
in the CO, of the sample and R,, = in which R,, = “C/“C ‘%I”% in the CO, of the standard. The results were expressed as the percentage of ‘%-recovery per hour and as cumulative values over 6 h. For this calculation the formula of Schoeller et al. (14) was used, and the CO, production was assumed to be 300 mmolim’ body surface area per hour. Body surface area was calculated by the weight-height formula of Haycock et al. (15). In normal subjects and patients with pancreatic disease, the estimated CO, production was compared with the CO, production measured by the CO,:argon method. For this measurement, the subject exhaled into a Haldane-Priestly tube connected to a spirometer until a constant tidal volume was reached (usually after about 5 min). A sample of expired air was then withdrawn from the tube and the CO, concentration was determined in the mass spectrometer using argon concentration as internal standard (16). The CO, production (CO, concentration x expired volume) was measured twice, at the onset and at the end of the test. Mean values were used to compute the 13C enrichment. Results obtained in this way will be called the by assuming a ‘*quantitative test,” and results obtained CO, production rate of 300 mmol per unit of body surface area per hour will be called the “simplified test.” Measurement of lipase, amylase. and trypsin output upon maximal stimulation of the pancreas was also carried out after an overnight fast of 12 h. A double-lumen duodenal tube and a separate gastric. tube were positioned under fluoroscopic control. The perfusion port of the duodenal tube was located near the ampulla of Vater and the aspiration site near the duodenojejunal flexure. Gastric contents were continuously aspirated. A warmed (37°C) mannitol-saline solution containing [ “Hlpolyethyleneglyco1 4000 as radioactive marker was infused at a constant rate of 10 mlimin at the proximal duodenal port. After a basal steady-state perfusion of 1 h. CCI;-pancreoxymin (Boots. 1 U/kg), was injected intravenously. The duodenal contents were collected on ice in four 15.min intervals. Lipase activity was measured titrimetrically at constant pH, using tributyrine as substrate (17). The [.‘H]polyethyleneglycol content in gastric and duodenal aspirates was measured by the oxidation method [Packard sample oxidizer, model 306: Packard, Downers Grove, Ill.), with subsequent liquid-scintillation counting [model 2450; Packard). Lipase output was calculated on the basis of the [“H]polyethyleneglycol recovery data and expressed as kilounits per hour. Trypsin activity was measured spectrophotometrically at 253 nm using p-toluene sulfonyl-2arginine methyl ester (18). cu-Amylast? activity was measured calorimetrically at 620 nm on a water-insoluble cross-linked starch polymer carrying a blue dye (Phadebas, Pharmacia. Sweden). The outputs of these enzymes were calculated as described for lipase.
Statistical
Analysis
Relations between different parameters lated by regression analysis (linear regression
were calcuand satura-
1128
VANTRAPPEN
Table
1.
ET AL.
GASTROENTEROLOGY
Pancreatic Enzyme Output in Patients With Pancreatic Disease and Normal Controls Control group
Lipase (kU/h) Trypsin (kU/h) Amylase
Vol. 96, No. 4
Patient group
Mean I- SEM
Range
Mean + SEM
Range
182.5 + 10.3
107.9-335.3
35.6 + 7.2 11.3 + 2.6 6.7 ? 1.5
1.1-142.4 0.2-34.7 0.4-30.0
40.0 2 3.9 56.5 k 7.0
(kU/h)
tion kinetics). Differences between sample means were calculated using Student’s t-test. Sensitivity, specificity, and predictive values were calculated by ROC analysis (19). All values are given as mean ? SEM.
Results Enzyme
Output
The output of lipase, trypsin, and amylase activity after maximal pancreatic stimulation is given in Table 1 for both the normal control group and the patients with pancreatic disease. Figure 1 shows the lipase output in the duodenum of normal subjects as compared with that of patients with pancreatic disease. Baseline
10.2-63.9 9.2-111.6
13C Excretion
Rate
The “C excretion rate in normal subjects, expressed as a percentage of the dose excreted per hour, increased progressively, reached a plateau of 8% per hour between 4.5 and 5.5 h after administration of the label, and decreased thereafter. The 13C excretion rate was markedly different in patients with pancreatic disease; the 13C excretion rate rose more slowly and did not reach the high values obtained in normal subjects [Figure Za). The simplified test, in which the CO, production rate was assumed to be 300 mmol per unit body surface area
13C Excretion f
The baseline 13C enrichment in the control group was similar to that of the patient group, the +- 0.036 and -25.856 + &value being -26.163 0.030, respectively. The measured CO, production averaged 297.0 2 2.6 mmol per unit of body surface area per hour in normal subjects and 295.7 +- 3.7 in patients with pancreatic disease.
b-
x 8 Y
_ 4-
s2-
1
2
3
4
5
6
HOURS
I
1
Figure ;i
.iii: Figure
2
3
4
5
b HOURS
__--_
1. Lipase output (in kilounits per hour] for normals (open circles) and patients with pancreatic disease lfilled circles).
2. Excretion rate of ‘“CO, in breath after administration of ‘%-labeled mixed triglyceride. The upper part (a) represents the “quantitative test” in which CO, production is measured; the lower part (b) represents the “simplified test, ” in which CO, production is assumed to be 300 mmol per square meter of body surface area per hour. Open circles represent normal subjects: filled circles represent patients with pancreatic disease.
April
19811
’‘C-BREATH
TEST FOR PANCKEATIC
1129
FUNCTION
activity in the duodenum after maximal pancreatic stimulation was calculated in two different ways. Assuming a linear correlation between the two variables, the correlation coefficient (r) of the test was found to be 0.80 (p < 0.001). If it is assumed that the relation between the cumulative “‘CO, excretion at 6 h and the lipase activity is linear at low values and follows zero-order (saturation) kinetics at high lipase outputs, the mathematical expression of this relation can be written as follows: 1
2
3
L
6
5
‘“CO, excretion
(% cumulative
A [lipase) 6 h) =
(1) 1 + B (lipase)’
where A and B are constants (Figure 5a). From Equation (1) the following equation derived: Lipase -=_ ‘“CO,
1
2
3
L
5
6 HOURS
Figurrh
3. Cumulative I ‘CO, excretion in breath after administration of “C-labeled mixed triglyceride. The upper part ((I) represents the “quantitative test”; the lower part (b) represents the “simplified test.” Normal subjects are indicated bv open circles and patients with pancreatic disease bv filled circles.
per hour, resulted in a similar excretion the two study groups (Figure 2b). The between normal subjects and patients significant in both tests (p < 0.001). Cumulative
pattern in differences are highly
A
+ B (lipase) A
(21
Equation (2) is presented in Figure 5b. As the relation between lipase/‘“CO, excretion and lipase is very good (r = 0.89), and as Equation (2) is only a mathematical conversion of Equation (1). the assumption that the relation between “‘CO, excretion and lipase activity follows saturation kinetics seems to be valid. Sensitivity, Specificity, of the Mixed-Triglyceride Parameter of Pancreatic values
and Predictive Value Breath Test as a Lipase Activity
The sensitivity, specificity, and predictive of the mixed-triglyceride breath test for the
‘,‘C Excretion
The 6-h cumulative ‘“CO, excretion data are summarized in Figure 3. The excretion patterns of the two test procedures, quantitative and simplified, were similar. The 6-h cumulative ‘“CO, excretion, expressed as a percentage of the dose administered, amounted to 33.5% k 1.4% [quantitative) and 35.6% ? 2.8% (simplified] in normal subjects, and to 13.8% t 1.4% and 14.3”/, 2 1.8”/, in patients with pancreatic disease. The differences between normal subjects and patients are highly significant with both procedures (p < 0.001). The individual 6-h cumulative ‘“CO, excretion data for the two groups, together with the data for other control groups, are given in Figure 4. Relation Between “‘CO, Breath and Duodenal Lipase Output cretion
1
can be
Excretion
The relation between the 6-h cumulative exof “‘CO, in breath and the output of lipase
.. .. :. . : : :. .:: . ;. ; Figure
4. Cumulative 6-h ‘“CO, excretion in normal subjects (0). patients with pancreatic disease (0). patients with steatorrhea of nonpancreatic origin ( x 1,and metabolic controls; A indicates patients with liver disease; a. patients with diabetes: V. patients with obesity.
1130
g
U
X y
0"
VANTRAPPEN
ET AL
Vol. 96, No. 4
GASTROENTEROLOGY
40
!#_:I] -.
but 3 normal subjects. In Figure 6 the daily fecal fat loss is plotted against the lipase output. All but one of the subjects with a lipase output of >40 kU/h had a normal fat excretion in the stool, whereas all but three of those who had a lipase output of <40 kU/h had steatorrhea.
30
6h cum. “CO 2 -excr
Ie
I
=
1+ 0 027 [Lipase]
I
I
200
300 LIPASE (KU/h)
LIPASE (KU/h) (a]Cumulative ‘“CO2 excretion (in percentage of ingested dose) 6 h after intake of the [‘“Cl-labeled mixed triglyceride as a function of lipase activity (in kilounits per hour] in normal subjects (O] and patients with pancreatic disease (0). (b) Linearly modified saturation curve following the relation given by Equation (2). Open circles represent normal subjects; filled circles are patients with pancreatic disease. detection of impaired pancreatic lipase activity in the duodenum were determined by ROC analysis against the gold standard (lipase output in duodenum). The results, presented in Table 2, are based on data obtained in the patients with pancreatic disease and the various control groups. The lipase output was considered to be normal in all metabolic control patients, in whom duodenal intubation was not carried out. Relation Between Steatorrhea Three-day all but 1 patient
Lipase
stool collections with pancreatic
Output
and
were performed in disease, and in all
Effect of Pancreatic Enzymes on the Mixed-Triglyceride Breath Test in Patients With Pancreatic Insufficiency Eleven patients with pancreatic exocrine insufficiency were studied. The 6-h cumulative 13C0, excretion values before and during treatment with pancreatic enzymes (Enzypan Forte) are presented in Figure 7. Before treatment with pancreatic enzymes, the mean fat excretion in the feces amounted to 52.0 * 8.4 g/day. Enzyme treatment resulted in a decrease of stool fat to 19.8 2 4.0 g/day. All patients improved under therapy, but the fat excretion became normal in only 3 patients. The stool volume decreased from 727 g t 253 g before to 373 ? 68 g during enzyme treatment. The ‘“CO, excretion after administration of the ‘“C-labeled-mixed-triglyceride amounted to 6.3% +- 1.4% before treatment and increased to 17.6% * 3.3% during treatment with enzymes. Four patients showed little or no improvement when treated with 4 tablets t.i.d. When treated with a dose of 8 tablets t.i.d., 2 patients improved further. One of the patients who did not improve had a high basal gastric acid output (24.6 mEq H+/h). He was studied a third time while he was being treated with ranitidine (150 mg b.i.d.) and Enzypan Forte (8 tablets t.i.d.). This led to an improvement of the 13C0, excretion values.
Mixed-Triglyceride Breath Test in Other Control Groups (Patients With Steatorrhea Nonpancreatic Origin and Patients With Metabolic Disorders Figure 4 shows the individual values mixed-triglyceride breath test in the various
Table
2.
of
of the study
Sensitivity, Specificity, and Predictive Values of the Mixed-Triglyceride Breath Test in Comparison With Intubation Test to Detect Disease-Diminished Lioase Outnut
Cut-off
(6 h cumulative percentage of ‘3C0,) Cut-off (lipase)
Sensitivity breath test Specificity breath test Positive predictive value Negative predictive value
22% 90 kU 0.89 0.81 0.63 0.95
24%
loo
kU
0.93 0.76 0.60 0.97
22%
loo kU 0.83 0.81 0.62 0.93
April
’'C-BKEATH TEST FOR PANCREATIC
1989
p
F‘UNCTION
1131
50 h
n F
ai
Figure
“, 2 k
6. Relation between daily fecal fat loss and duodenal lipase output. Open circles represent norma1 subjects: filled circles are patients with pancreatic disease.
.
. . 25
.
. -:
??
.
:*
I. . ,c-._______-------_________
. .
1
yr. 40
??
-------------___.
?? .
,
??
0
80
“q 120
O@$ 0 oooo 160
0
OOeFp“,;‘ ,“:;;; , 200
LIPASE
groups. The 6-h cumulative CO, excretion amounted to 25.5% ‘- 2.4% in diabetics, 25.5% + 4.1% in patients with hepatic insufficiency, and 30.5% t 1.9% in obese subjects. Eight patients had excretion values below the lowest value of “normal” control subjects. The 6-h cumulative CO, excretion in patients with steatorrhea of nonpancreatic origin was 26.3% ? 2.6%. Seven patients had 13C0, excretion values below the lowest “normal” value. Table 3 summarizes pertinent data on patients with steatorrhea of nonpancreatic origin. Five of 9 patients with celiac disease had subnormal 13C0, excretion values, had low duodenal lipase whereas only 1 patient activity upon maximal pancreatic stimulation. Two other patients, one with total gastrectomy and one
before
ENZYPAN FOiliF
Figure
3x4
tablets
3x8
tablets
3x8
tablets ??
ranhdme
7
Six-hour cumulative ‘“CO, excretion before and during treatment with pancreatic enzymes (Enzypan Forte). One patient was also treated with ranitidine.
/(KU/h)
with Crohn’s disease, had a very low ‘“CO, excretion and a normal lipase output.
Discussion Several indirect noninvasive tests have been proposed for the assessment of exocrine pancreatic function. Tests based on urinary recovery of a test molecule that can only be absorbed after hydrolysis by chympotrypsin (bentiromide test) or pancreatic esterases (pancreolauryl test) have a sensitivity and specificity of -8O%-90% in most studies (2). Other indirect pancreatic function tests that use14C- or 13C-labeled triglycerides, either alone or in combination, are proposed as tests of fat malabsorption and as possible substitutes for fecal fat determination (410,20-25). Steatorrhea is an insensitive test of exocrine pancreatic function, as it only occurs if pancreatic lipase output is reduced to 510% (26). Moreover, steatorrhea may be due to causes other than pancreatic insufficiency. Therefore, breath tests with good performance in the detection of steatorrhea are of much lesser value as pancreatic function tests. The mixed-triglyceride breath test was designed to measure intraluminal pancreatic lipase activity and not as a test of steatorrhea. The test molecule, 1,3distearyl, 2[carboxyl-‘“Cloctanoyl glycerol, consists of a medium-chain fatty acid in the 2-position and long-chain fatty acids in the 1 and 3 positions. The rationale of the test is that the two stearyl groups have to be split off the glycerol by lipase before [‘“Cloctanoyl monoglyceride or [l”C]octanoate, a medium-chain fatty acid, is absorbed and rapidly metabolized to l”CO, (27). The rate-limiting step in the oxidation to ‘“CO, is hydrolysis of the fatty acids in the I- and 3-positions of the mixed triglyceride. That the absorption and oxidation of free octanoate or octanoyl monoglyceride occur faster than the absorption and oxidation of a long-chain fatty acid or its monoglyceride is indi-
1132
VANTRAPPEN
Table
ET AL.
3. Data on Patients
GASTROENTEROLOGY
With Steatorrhea
of Nonpancreatic
Age Patient C.E. M.G. G.B. V.J. D.S. M.M. P.M. D.M. W.A. W.T. D.A. M.R. S.J. T.J. M.M. M.A. G.S. K.B. E.M. A.M.
Sex
lYd
F
27
M
68
F
31
F
49
F
32
M
49
M
36
M
26
F
73
M
36
F
58
F
59
F
48
F
52
M
35
F
31
F
33
F
26
F
54
F
42
E.A.
M
62
H.G.
F
35
Diagnosis Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Celiac disease Short bowel Short bowel Short bowel Short bowel Crohn’s disease Crohn’s disease Crohn’s disease Bacterial overgrowth Bacterial overgrowth Bacterial overgrowth Total gastrectomy Extrahepatic cholestasis Hyperthyroidism
Mean
lJeca1 volume (g/24 hl 322
compare favorably with other indirect pancrefunction tests, such as the pancreolauryl test. mixed-triglyceride breath test, like the intuba-
Fecal fat lgi2-1 hl 21.5
Breath test (% ’'C. cumulative
Lipase activity 6 h)
(kU/h)
32
353
23
162
449
11
7.8
132
2813
51
14.1
210
19
252
28
240
380 285
8
17.5
623
47
320
7
25
347
48
385
8.3
18
149
1133
11.7
27
144
1109
33
34
126
491
55
29
233
938
20
46
124
45
254
1165 772
25
390
8
39
163
573
13
29
238
734
20
23
95
644
27
44
98
830
79
3.3
6.3
186
125
140
28
244
580
40
26%
t119
cated by the observation that 20% of our normal subjects had their peak ‘“CO, excretion in the first 3 h after intake of the substrate, whereas in the study by Newcomer et al. (4) and in a recent study by Turner et al. (28), none of the normal subjects had their peak 14C0, excretion during the first 3 h after intake of 14C-triolein. Recently, Hofmann’s group (29,30) presented cholesteryl octanoate as a new substrate for a ‘“CO, pancreatic function test. In their healthy controls the peak 14C0, excretion occurred at 60-90 min in all subjects. As they used the same medium-chain fatty acid as we did, the difference in peak excretion must be due to the slower hydrolysis of the stearyl groups in the 1 and 3 position of the medium-chain triglyceride in comparison with the hydrolysis of the cholesteryl octanoate. The present study shows that the mixed-triglyceride breath test is a sensitive noninvasive test of pancreatic lipase activity in the duodenum. The 13C0, production correlates very well with the duodenal intubation test in which the lipase output upon maximal pancreatic stimulation is measured by means of a marker-corrected perfusion technique. It allows detection of pancreatic dysfunction with an acceptable sensitivity (0.89) and specificity (0.81)
(n = 22)
228
696
SEM
that atic The
Origin
Vol. 96, No. 4
22
26.3
t4.2
22.6
190 217
tion test and other indirect tests, will not detect pancreatic disease when pancreatic function is normal, despite the presence of anatomic changes. However, the test is much more sensitive in detecting impaired pancreatic function than fecal fat determionly occurs if nation. Figure 6 shows that steatorrhea lipase output falls below 40 kU/h, whereas the mixed-triglyceride breath test detects a lipase output of <90 kU/h with a sensitivity of 89%. It was only below a ‘“CO, excretion of 7.5% that steatorrhea was always present, whereas the limit value of the ‘“CO2 test for discrimination between normal function and abnormal exocrine pancreatic function was 22%. The mixed-triglyceride breath test, like other indirect pancreatic function tests, may be used to replace the invasive intubation test for the detection of exocrine pancreatic disease, to follow the evolution of pancreatic disease, and to monitor the effect of therapy. That the test is suitable to follow the effect of enzyme supplementation is also suggested by the results presented in Figure 7. Therapy with pancreatin (Enzypan Forte) in patients with pancreatic insufficiency resulted in marked improvement of the breath test, concurrently with improved fecal volume and fecal fat. The specificity of the test for the detection of pancreatic disease is not as good as that of the intubation test. The mixed-triglyceride breath test yielded false-positive results in 1 gastrectomy patient, possibly due to insufficient mixing of nutri-
April
‘%BREATH
1989
ents with pancreatic enzymes, and in 4 patients with celiac disease. Although the pancreatic enzyme output after stimulation with CCK can be normal in patients with celiac disease, the pancreas may be insufficiently stimulated by a meal, which yields “false-positive” mixed-triglyceride breath tests. It has been shown that stimulation of the pancreas with CCK results in normalization of the labeled CO, excretion in patients with celiac disease (11).In this respect, the present breath test seems to be more physiologic than the CCK intubation test. Abnormal breath tests are not unexpected in patients with hepatic and metabolic diseases. In diabetes, two factors may be responsible for a lowered [13C]octanoic acid oxidation. Slowed gastric emptying due to neuropathy may delay digestion of the mixed-triglyceride even when pancreatic function is normal. On the other hand, the [13C]octanoic acid is probably diluted in a larger pool of short-chain fatty acids, resulting in slower oxidation. Obesity does not interfere with the mixed-triglyceride test. In contrast, severe liver disease may result in abnormal ‘“CO, exhalation after administration of ‘“C-labeled mixed triglyceride, probably because of impaired octanoic acid oxidation in the decreased liver cell mass. This is true not only for the mixed-triglyceride breath test. A high incidence of abnormal triolein breath tests was also described in alcoholics with test and the liver damage (31).Also, the bentiromide pancreolauryl test were found to give false-positive results in patients with liver disease (32,~~). In conclusion, the excellent correlation between the ‘“CO, excretion in breath and the duodenal lipase output in normal subjects and in patients with pancreatic disease (r = 0.89). and the good sensitivity and specificity (0.89 and 0.81,respectively) in comparison with the CCK test, indicate that the mixed-triglyceride breath test is a promising noninvasive test of pancreatic lipase activity in the duodenum. Unlike tests involving urine collection, the cooperation of the patient can be controlled.
EP. Assessment of exocrine intubation. Clin Gastroenterol
pancreatic 1984:13:
Scharpe S. Illiano L. Two indirect tests of exocrine pancreatic function evaluated. Clin Chem 1987:33:5-12. DiMagno EP. Clain JE. Chronic pancreatitis. In: Go VLW. Gardner JD. Brooks FP, Lebenthal E. DiMagno EP. Scheele GA, eds. The exocrine pancreas: biology, pathology, and diseases. New York: Raven. 1986:541-75. Newcomer AD, Hofmann AF, DiMagno EP, Thomas PJ, Carlson GL. Triolein breath test-a sensitive and specific test for fat malabsorption. Gastroenterology 1979;76:6-13. Einarsson K. Bjorkhem I. Eklof R, Blomstrand R. “‘C-triolein
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malabsorption. Stand J Gastroenterol 1983;18:9-12. breath 6. Benini L, Senro LA, Menini E. et al. Is the “C-triolein test useful in the assessment of malabsorption in clinical practice. Digestion 1984;29:91-7. SM. Estima7 Schwabe AD, Cozetto FJ, Bennett LR. Mellinkoff
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tion of fat absorption by monitoring of expired radioactive carbon dioxide after feeding a radioactive fat. Gastroenterology 1962;42:285-91. Watkins JB, Schoeller DA, Klein PD. Ott DG. Newcomer AD, Hofmann AF. r3C-trioctanoin: a nonradioactive breath test to detect fat malabsorption. J Lab Clin Med 1977:90:422-30. Kaihara S. Wagner H. Measurement of intestinal fat absorption with carbon-14 labelled tracers. J Lab Clin Med 1968;71: 400-11. Burrows P, Fleming JS. Garnett ES, Ackery- DM, Colin-Jones DC. Bamforth J. Clinical evaluation of the ‘“C fat absorption test. Gut 1974;15:147-50. Ghoos YF. Vantrappen GR. Rutgeerts PJ. Schurmans PC. A mixed triglyceride breath test for intraluminal fat digestive activity. Digestion 1981:22:239-47. Douwes A. Respiratory hydrogen excretion as a parameter for sugar absorption in children. Thesis. Rijksuniversiteit Groningen, 1979. Mook WG, Grootes PM. The measuring procedure and corrections for the high-precision mass-spectrometric analysis of isotopic abundance ratios, especially referring to carbon, oxygen and nitrogen. Int 1Mass Spectr Ion Phvs 1973;12:27398. Schoeller DA, Klein PD. Maclean WC, Watkins JB, Van
Santen E. Fecal “C analysis for the detection and quantitation of intestinal malabsorption. [ Lab Clin Med 1981:97:439-X8. 15. Haycock G. Schwartz G. Wisotsky D. Geometric method for measuring body surface area: a height-weight formula validated in infants, children and adults. 1Pediatr 1978:93:62-6. 16
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Received March 30, 1988. Accepted October 31. 1988. Address requests for reprints to: Prof. Dr. G.R. Vantrappen, University Hospital Gasthuisberg, B-3000 Leuven, Belgium. This study was supported by the Fund for Medical Scientific Research, Brussels, Belgium. The authors thank L. Swinnen, S. Rutten, and D. Claus for skillful technical assistance.