Starch Digestion in Normal Subjects and Patients With Pancreatic Disease, Using a

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1 GASTROENTEROLOGY 1989;96:53-9 Starch Digestion in Normal Sbjects and Patients With Pancreatic Disease, Using a 13C2 Breath Test M. HIELE, Y. GHOOS, P. RUTGEERTS, and G. VANTRAPPEN Department of Internal Medicine, Laboratory of Gastroenterology, University Hospital Gasthisberg, University of Leven, Leven, Belgim A 13C2 starch breath test as developed to stdy starch digestion in 13 healthy volnteers and 16 patients ith pancreatic disease. As starch is hydrolyzed to glcose that is sbseqently oidized to CO2, the ecretion of 13C2 after starch intake, compared ith the ecretion of 13C2 after glcose intake, shold be a measre of starch hydrolysis. In normal sbjects both the horly 13C2 ecretion rate and the cmlative percentage of 13C2 ecretion after starch ingestion ere significantly different from those after glcose ingestion. Comparable differences ere fond ith the patient grop. In addition, the 13C2 ecretion after starch intake in normal sbjects as markedly different from that of patients ith pancreatic disease. Even after correction for endocrine pancreatic insfficiency by calclating the ratio of 13C2 ecretion after starch intake over 13C2 ecretion after glcose intake, the difference beteen the control grop and the patient grop as statistically significant. This stdy indicates that starch hydrolysis is the rate-limiting step in starch digestion. It also confirms recent findings sggesting that starch digestion is impaired in some patients ith pancreatic disease. The 13C2 starch breath test may prove to be sefl in evalating the digestibility of varios starch preparations in physiologic and pathological conditions. Recent stdies thro dobt on the classical vie (1) that starch digestion is complete in normal sbjects and only rarely affected in patients ith pancreatic disease. Measrements of breath Hz after intake of different kinds of starch led to the conclsion that starch absorption may be incomplete in gastrectomy patients (2), patients ith chronic pancreatitis or small boel disease (3), and even in normal sbjects (4,5). Stephen et al. (6) came to the same conclsion on the basis of ileal intbation eperiments in hich the recovery of starch and glcose from the distal ilem as stdied after ingestion of a meal containing starch. This ileal intbation techniqe is invasive and not easily applicable to large grops of volnteers or patients. The aim of this stdy as to evalate starch absorption by means of a 13C-starch breath test in normal sbjects and patients ith pancreatic disease. Corn starch and sgars derived from corn starch are natrally enriched in 13C, and can be sed as easily available sbstrates for 13COz breath tests (7-9). As starch is hydrolyzed to glcose, hich is oidized to COz, the cmlative 13C2 recovery as ell as the time-corse of 13COZ appearance in breath after 13C-labeled starch intake gives information on starch hydrolysis hen compared ith 13COz ecretion after 13C-glcose ingestion. Materials and Methods Sbjects Thirteen normal volnteers participated in the stdy, 8 men and 5 omen, aged 19-6 yr (mean age, 29 yr). The grop of patients ith pancreatic disease consisted of 16 sbjects, 14 men and 2 omen, aged 35 to 62 yr (mean age, 47 yr). Fifteen patients had proven chronic pancreatitis; in one of them a right hemipancreatectomy had been performed. The other patient had been treated by total pancreatectomy for carcinoma. The diagnosis of chronic pancreatitis as based on endoscopic retrograde pancreatography and compted aial tomography-scan data (Table 1). The endoscopic retrograde pancreatography data ere classified according to the score system of Braganza et al. (1), ith normal = 1, minimal change pancreatitis = 2, moderate change pancreatitis = 3, advanced change pancreatitis ith dilated dct = 4, advanced change pancreatitis ith obstrcted dct = 5, and advanced change pancreatitis ith segmented dct = by the American GastroenteroIogical Association /89/$3.5

2 54 HIELE ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part 1 Table 1. Patient Data 6-h 6-h Fecal cmla- cmla- 6-h Ratio Patient Age Glcose fat tive C tive cmla- starch mai Amylase No. (yr) Se ERp o CAT scan tolerance b (g/day) glcose starch tive ratio glcose ma (Ulh) 1 4 M 4 Psedocyst M 4 Psedocyst F M Atrophic pancreas M F Atrophic pancreas 7 41 M Psedocyst M M M Total pancreatectomy M 4 Atrophy of pancreas M M M Psedocyst M M (right hemipancreatectomy) CAT, compted aial tomography; ERP, endoscopic retrograde pancreatography. ERP findings: 3 = moderate pancreatitis, 4 = advanced change pancreatitis (ACP), ith dilated dct, 5 = ACP ith obstrcted dct (reference 1). b Glcose tolerance: 1 = normal. 2 = glcose intolerance by oral glcose tolerance test, 3 = overt diabetes. C 6h cmlative = 6-h cmlative percentage of 13C2 ecretion. All patients ere admitted to the h s p for i t the a l stdies and gave informed consent. Decreased pancreatic fnction as not an inclsion criterion. Data on glcose tolerance and fecal fat analysis are smmarized in Table 1. 13C_ Starch and 13C-glcose breath tests ere performed in all sbjects and patients. In 1 patients and 4 normal sbjects ho consented to this part of the stdy intbation of the dodenm for qantitative determination of pancreatic enzyme otpt after stimlation ith cholecystokinin as performed. The stdy protocol as approved by the ethical committee of the Leven University Hospital, Belgim (WH/MV/86/14S). Starch and Glcose Breath Tests Ra crystalline corn starch, a gift from CPC-Erope, Vilvoorde, Belgim, and 99% pre glcose, manfactred by hydrolysis of corn starch, a gift from Amylm, Aalst, Belgim, ere sed as sbstrates. Corn carbohydrates ere natrally enriched in Dc. The 13C enrichment of the glcose and starch sbstrates as fond to be and ,,131'8' respectively (here,,13 pbd is the l3c content in pro mille, epressed vs. 13C content in Pee Dee Belemnite limestone). After an overnight fast, all sbjects received an oral load of 5 g of glcose on 1 day and 5 g of starch on another day. The carbohydrates ere administered as a sspension in 25 ml of ater. There as an interval of at least 2 days beteen administration of both carbohydrates. The seqence of the tests as at random. The starch breath test as performed before the glcose test in 18 sbjects and vice versa in the remaining 11 sbjects. At the beginning of the second test, the fasting 13C2 vale had alays reached its basal level. The order of the tests did not inflence the reslts. Breath as sampled in a 3-L balloon, 1 and 5 min before intake of the test carbohydrate, and then every 3 min for 6 h. The sbjects rested qietly dring the test. A 2-ml sample of each balloon as injected in a H 2 monitor (GMt Renfre, U.K.) to measre the H2 concentration (epressed in parts per million). Before every test the H2 monitor as calibrated ith a gas containing 39 ppm of Hz (Matheson, Oevel, Belgim). The mean of the to fasting Hz readings as taken as the basal level. The area nder the crve of H2 ecretion as calclated, and epressed in parts per million hor above the basal level. As the basal H2 vales can be higher than the sbseqent Hz ecretion vales, this parameter can be negative (11). Pre COz as obtained by cryogenic trapping over liqid N z after removal of ater by a methanol-c2-ice trap. The ratio of B C to 12C in the sample as measred by isotope ratio mass spectrometry (model 25; Finnigan MAT, San Jose, Calif.) and epressed as the,,'3pdb vale (12). Assming a constant CO 2 prodction of 3 mm/m 2 of body srface area' h, the amont of BC recovered in breath as calclated as a percentage of the administered dose. The body srface area as estimated folloing the eqation of Haycock et al. (13).

3 Febrary 1989 A 13C-LABELED STARCH BREATH TEST 55 Measrement of Amylase Otpt After Maimal Stimlation of the Pancreas The amylase otpt as measred by means of a marker-corrected dodenal perfsion techniqe in 1 of the 16 patients ith pancreatic disease and 4 of the 13 normal sbjects. After an overnight fast, a doble-lmen dodenal tbe as positioned nder floroscopic control. Throgh the proimal orifice located near the amplla of Vater, a armed (37 C) mannitol-saline soltion, containing [3HJpolyethylene glycol 4 as marker, as infsed at a constant rate of 1 mllmin. The dodenal contents ere aspirated throgh the distal orifice of the tbe located near the dodenojejnal flere. After a basal perfsion period of 1 h, 1 U/kg of cholecystokinin-pancreozymin (Boots Pharmaceticals, Shreveport, La.) as injected intravenosly. The dodenal aspirate as collected for 1 h in for intervals of 15 min. Amylase activity as measred colorimetrically at 62 nm on a ater-insolble cross-linked starch polymer carrying a ble dye (Phadebas; Pharmacia Laboratories, Piscataay, N.J.). The [3HJpolyethylene glycol content in the aspirate as measred by the oidation method (Packard Sample Oidizer, model 36; Packard Instrment, Doners Grove, 111.), ith sbseqent liqidscintillation conting (Packard, model 245). Amylase otpt as calclated according to the [3H]polyethylene glycol recovery and as epressed in nits per hor. Statistical Analysis Vales are given as mean ± SD, nless otherise mentioned. Mann-Whitney-Wilcoon statistics ere sed for comparing grops of data. Reslts 13C2 Breath Tests Rate of 13C2 ecretion. The horly rate of 13C2 appearance in breath after starch and glcose intake by normal sbjects and patients ith pancreatic disease is illstrated in Figre 1. This figre shos that (a) in both grops the 13COZ ecretion rate as significantly sloer after starch intake as compared ith glcose intake, (b) the metabolization rate of 13C-glcose as sloer in the patient grop as compared ith the control grop, and (c) the difference beteen normal sbjects and patients in the metabolization rate of 13C-starch is more significant than the difference in the metabolization rate of glcose. Cmlative percentage of 1.1C 2 ecretion. Figre 2 shos the cmlative 13COZ ecretion in breath after starch and after glcose intake in each of the stdy grops. The 6-h cmlative 13COZ ecretion as highest after glcose intake in normal sbjects and loest after starch intake in patients. Figre 3 shos the 6-h cmlative percentage of 13C2 ecretion vales in individal patients. To z 6 a:: ON r;jw 4 c -. VI a CJ ";l. 8r , glcose normals o glcose patients starch normals o starch patients o 2 Figre 1. Rate of l3c2 ecretion (in percentage of dose per hor) after ingestion of 13C-Iabeled starch or glcose by normal sbjects and patients ith pancreatic disease (mean ± SEM). eliminate the effect of impaired glcose metabolism, the ratio of the 6-h cmlative percentage of 13C2 ecretion after starch intake to the 6-h cmlative percentage of 13C2 ecretion after glcose intake as calclated. By sing this ratio, the starch digestion test becomes independent of glcose metabolism, as is shon by the absence of correlation beteen the ratio and the 6-h cmlative percentage of 13C2 ecretion after glcose intake in the patient grop (r =.24). The vales of this ratio for the individal patients are shon in Figre 4. In patients ith pancreatic disease the ratio as.51 ±.24, hereas in normal sbjects it amonted to.89 ±.24 (p =.1). 3 t a:: ON 2 ";l. > i= <: :3 1 :L ::::> glcose normals starch normals, J TIME (h) o glcose patients o starch patients 4 6 Figre 2. Cmlative l3c2 ecretion (in percentage of dose) after ingestion of l3c-labeled starch or glcose by normal sbjects and patients ith pancreatic disease (mean ± SEM).

4 56 HIELE ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part 1 4 z Q a:: 3 DO ' I 2 DO L ::J 8.J::: 1 8 "-D DO go fo- P:O.56 P:O.OOO3 GLUCOSE I STARCH normals patients normals patients Figre 3. Individal vales for 6-h cmlative percentage of l3c2 ecretion after ingestion of 13C-labeled glcose or starch by normal sbjects and patients ith pan creatic disease Vl => l;;l < ::r: "- :I: a::.4 < Vl <.2 y:o.515 (amylase} / 1 O. ::r: OOO582(amylase} AMYLASE ( 1 3 ) Units/h Figre 5. Relation beteen pancreatic amylase otpt after stimlation ith cholecystokinin-pancreozymin and starch hydrolysis, measred by the ratio of maimal l3c2 ecretion after starch vs. glcose intake. 13C2 breath test verss amylase otpt. The relation beteen starch hydrolysis and pancreatic otpt of amylase activity as stdied by comparing the maimal 13C2 ecretion rate after starch intake ith the amylase otpt measred by means of a marker-corrected dodenai perfsion techniqe. To correct for the possible decrease in glcose oidation : D. c 1.2.S! (lj. 1. (lj N l:!.8 E :::l.6 V1. ::J.4 -J \,;:J :I:.2 :: < P:O.OO1 V1 normals patients Figre 4. Individal va:les for ratio of 6-h cmlative percentage of 13C 2 ecretion after DC-labeled starch intake and 6-h cmlative percentage of l3c2 ecretion after 13C-Iabeled glcose intake. in some patients, the ratio of the maimal 13C2 ecretion after starch intake and the maimal 13C2 ecretion after glcose intake as calclated. The reslts are shon in Figre 5. The relation beteen amylase activity and the ratio of maimal 13COZ ecretion after starch verss glcose intake has the shape of a satration crve ith a steep first part, folloing the eqation Ratio = a amylase/(l + b amylase), here a and b are constants. By dividing amylase by each term of this eqation, one obtains Amylase/ratio = l/a + (b/a) amylase. In both eqations the "ratio" = the ratio of the maimal rate of 13C2 ecretion after starch intake and the maimal rate of 13COZ ecretion after glcose intake. Figre 6 shos the crve of this second eqation. The fit of the crve is ecellent (r =.96). H2 Ecretion After Starch Intake Table 2 shos the reslts of the Hz measrements in the patient grop and in the control grop. One patient (No. 1) had an elevation of breath Hz of 134 ppm above the basal level after 6 h. Three other patients (No.2, 4, and 12) shoed a slight increase in Hz ecretion (13, 1, and 1 ppm, respectively) above the basal level, 2, 2.5, and 3.5 h, respectively,

5 Febrary 1989 A 13C-LABELED STARCH BREATH TEST 57 a I.LJ VI a ::;) -' l:j «L 1 8 "- 6 L - <:> J : 4 VI 2 o 2 y=1943 (!712S) (!O.9) I amylasel r=o AMYLASE ( Units/h Figre 6. Linear modification of Figre 5. The good fit of the crve proves that the relation beteen dodenal amylase activity and starch hydrolysis can be considered as a satration crve. natre of starch, and interactions ith other sbstances in ntrition, ere recently revieed (14,15). Normal sbjects and patients ith pancreatic disease sho significant differences in the 6-h cmlative percentage of 13COz recovery after starch ingestion. Hoever, the 13COz ecretion after glcose intake is also different beteen the to grops. Part of the difference in 13COZ ecretion after starch intake, therefore, cold be de to endocrine pancreatic insfficiency ith sbseqent diminished glcose oidation. For this reason the 13COZ ecretion after starch intake cannot be considered an independent parameter of starch hydrolysis. By sing the starch to glcose ratio, Le., by dividing the 13COZ ecretion after starch intake by the 13COZ ecretion after glcose intake, a ne parameter is obtained that is no longer dependent on glcose metabolism. The ratio of the patient grop is significantly different from that of the normal grop. Moreover, 7 of the 16 patients ith pancreatic disease had a vale less than the loest vale fond in the normal grop. Therefore, if the loest vale of the normal grop is after starch intake. In 1 of these patients (No.4), the peak as only observed at one measrement, and the area nder the Hz ecretion crve as lo. To other patients (No. 14 and 16) had a high fasting Hz level, hich makes the interpretation of a sbseqent rise nreliable. If the area nder the Hz ecretion crve is taken as a parameter, 6 patients had an Hz ecretion above the highest vale fond in the control grop. Among them are the 2 patients ith a high Hz backgrond. None of the sbjects in the control grop shoed a marked Hz increase in breath after the starch meal. Discssion Starch derived from some plants is natrally enriched in 13C and may be sed as a sbstrate for a 13COZ breath test. The reslts of this stdy sho that this breath test allos semiqantitative measrement of starch hydrolysis in normal sbjects and patients ith pancreatic disease. The statistically significant difference in 13COz ecretion after starch intake as compared ith glcose intake indicates that starch hydrolysis and not monosaccharide absorption is the rate-limiting step. This finding is in contrast ith the classical vie that monosaccharide absorption is rate-limiting (1). Stdies of the glycemic response after intake of different starchy foods shoed that many factors other than monosaccharide absorption determine the rate of starch absorption. These factors, inclding particle size, chemical Table 2. H2 Ecretion After 5 g Starch Intake Peak above Fasting fasting H2 Time of Area nder H2 level level peak H2 the H2 crve (ppm) (ppm) (h) (ppm h) Normals A B C D E F 4-12 G H I J K L M Patients

6 58 HIELE ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part 1 considered as the loer limit of normal, only 7 of the 16 patients had a diminished starch hydrolysis. This lo sensitivity for pancreatic disease is not srprising. Pancreatic lipase otpt also has to be decreased by 9% before steatorrhea develops (16). Clearly, the 13COz starch breath test is not a diagnostic test of pancreatic disease bt evalates starch absorption in a ne ay. Only 6 of the 16 patients had a detectable and persistent increase in breath Hz dring the stdy period. In 1 of these patients (No. 1) the increase as very prononced (134 ppm). In 2 other patients (No.2 and 12), there as only a slight increase of 13 and 1 ppm, respectively, above fasting level. In 1 patient (No.3) the peak rise above basal level as even loer (3 ppm), bt the area nder the Hz crve as above the highest level observed in normals. In 2 patients (No. 14 and 16) the Hz backgrond as high, hich makes the interpretation of a sbseqent rise nreliable. Conseqently, only 4 of the patients had a reliably positive Hz breath test. Three of these 4 patients had an abnormal 13COz test; 1 patient had an abnormal Hz test ith a normal 13COZ test. In contrast, the 13COZ test as abnormal in 4 patients ith a normal Hz test. These reslts indicate that the 13COz breath test may be more sensitive than the Hz breath test in detecting diminished starch digestion. It cold be arged that elevated Hz levels in breath old have been fond in more patients if the test period had lasted longer. The time-corse of the breath Hz ecretion in the patients in hom an elevation of breath Hz as fond (the increase in breath Hz alays occrred ithin 4 h after intake of starch), and the figres pblished in the literatre (3,5) strongly sggest that increases in breath Hz ecretion ere not missed by ending the stdy after 6 h. In the stdy by Levitt et al. (5), one of the sbstrates as corn starch, hich as also sed in this stdy, and the elevation of breath H2 as fond to be maimal after 5-6 h, hich falls ithin the limits of or stdy period. It is possible that in some of the patients ith a lo 13C2 ecretion, the H2 ecretion as not increased becase starch absorption as delayed and not becase the starch as malabsorbed. This problem cannot be solved ithot intbation stdies. Stephen et al. (6), sing intbation stdies, fond that maimal starch recovery in the terminal ilem of normal sbjects occrred beteen the second and the fifth hor after ingestion of the test meal. It is interesting to note that one of their sbjects did not sho an increase in breath Hz ecretion althogh 2% of the ingested starch as not absorbed. This observation indicates that breath Hz determination is probably not a very sensitive test for starch malabsorption. As e did not perform intbation stdies in or patients, e can- not discriminate beteen delayed absorption in the small intestine and malabsorption, i.e., colonic metabolism, in or patients. Kerlin et al. (3) fond a rise in breath Hz in 12 of 17 patients ith pancreatic disease. Hoever, the carbohydrate intake in their stdy as 1 g, hereas or patients ingested only 5 g of starch. The difference in starch load may eplain the loer figres of Hz ecretion in or stdy. The initial part of the crve relating pancreatic amylase activity ith starch digestion is very steep. This indicates that starch digestion reaches a maimal level ith an amylase otpt of even less than 5 U/h. Belo this level the crve is so steep that the amylase otpt has only a limited vale in predicting the rate of starch digestion. From this stdy it can be conclded that starch hydrolysis and not monosaccharide absorption is the rate-limiting step in starch digestion and absorption. The differences fond beteen normal sbjects and patients ith pancreatic disease confirm recent findings that starch digestion may be impaired in patients ith pancreatic disease. Hoever, this occrs only if amylase otpt is etremely lo. The 13COZ starch breath test appears sitable to stdy, in a noninvasive manner, the effect of diseases sch as chronic pancreatitis and small boel injry on starch absorption. References 1. Gray GM. Intralminal and srface membrane digestion of dietary carbohydrate and protein. In: Go VLW, Gardner JO. Brooks FP, Lebenthal E, OiMagno EP, Scheele GA, eds. The eocrine pancreas: biology, pathobiology, and diseases. Ne York: Raven, 1986: Bond JH, Levitt MD. Use of plmonary hydrogen (H2) measrements to qantitate carbohydrate absorption. Stdy of partially gastrectomized patients. J Clin Invest 1972;51: Kerlin P, Wong L, Harris B, Capra S. Rice flor, breath hydrogen, and malabsorption. Gastroenterology 1984;87: Anderson IH, Levine AS, Levitt MD. Incomplete absorption of the carbohydrate in all-prpose heat flor. N Engl J Med 1981;34: Levitt MD, Hirsh P, Fetzer CA, Sheahan M, Levine AS. H2 ecretion after ingestion of comple carbohydrates. Gastroenterology 1987;92: Stephen AM, Haddad AC, Phillips SF. Passage of carbohydrate into the colon: direct measrements in hmans. Gastroenterology 1983;85: Lacroi M, Mosora F, Ponts M, Lefebvre P, Lyck A, Lopez-Habib G. Glcose natrally labeled ith carbon-13: se for metabolic stdies in man. Science 1973;181: Shlman RJ, Wong WW, Irving CS, Nichols BL, Klein PD. Utilization of dietary cereal by yong infants. J Pediatr 1983;13: Hiele M, Ghoos Y, Rtgeerts P, Vantrappen G. Measrement of the rate of assimilation of oligo- and polysaccharides by

7 Febrary 1989 A J3C-LABELED STARCH BREATH TEST 59 locoz breath tests and isotope ratio mass spectrometry. Biomed Environ Mass Spectrom (in press). 1. Braganza JM, Hnt LP, Warick F. Relationship beteen pancreatic eocrine fnction and dctal morphology in chronic pancreatitis. Gastroenterology 1982;82: Hiele M, Ghoos Y, Rtgeerts P, Vantrappen G, Carchon H, Eggermont E. A l3c2 breath test sing natrally 13C-enriched lactose for the detection of lactase deficiency in patients ith gastrointestinal symptoms. J Lab Clin Med 1988;112: Schoeller DA, Klein PD, Watkins JB, Heim T, MacLean We. l3c abndances of ntrients and the effect of variations in l3c isotopic abndances of test meals formlated f1\ l3c2 breath tests. Am J Clin Ntr 198;33: Haycock GB, Schatz GJ, Wisotsky DH. Geometric method for measring body srface area: a height-eight formla validated in infants, children, and adlts. J Pediatr 1978;93: Jenkins DJA, Jenkins AL, Wolever TMS, Thompson LH, Rao AV. Simple and comple carbohydrates. Ntr Rev 1986;44: Cmmings JH, Englyst HN. Fermentation in the hman large intestine and the available sbstrates. Am J Clin Ntr 1987;45: DiMagno EP, Go VLW, Smmers kill WHJ. Relations beteen pancreatic enzyme otpts and malabsorption in severe pancreatic insfficiency. N Engl J Med 1973;288: Received December 3, Accepted Agst 31, Address reqests for reprints to: Professor G. Vantrappen, Internal Medicine, University Hospital Gasthisberg, B-3, Leven, Belgim. This stdy as spported by grant No of the Fnd for Medical Scientific Research, Belgim. M. Hiele is a research assistant of the National Fnd for Scientific Research, Belgim. The athors thank P. Bois, D. Clas, B. Geypens, S. Rtten, and L. Sinnen for their laboratory ork.