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European Review for Medical and Pharmacological Sciences 2005; 9(Suppl 1): 3-8 13C-octanoic acid breath test for measuring gastric emptying of solids

F. PERRI, M.R. PASTORE*, V. ANNESE

Department of Internal Medicine, Gastroenterology Unit and *Department of Pediatrics, “Casa Sollievo della Sofferenza” Hospital, IRCCS – San Giovanni Rotondo, FG (Italy)

Abstract. – Non invasive evaluation of breath tests with stable isotopes have been gastric emptying is generally performed by developed to reliably assess gastric emptying which is, however, difficult to per- of both solids7 and liquids8,9. The main advan- form and not suitable to children and child- bearing women. A new method based on stable tage of breath test technology is represented isotope breath testing analysis has been intro- by the lack of radiation burden. Moreover, duced in clinical practice: the 13C-octanoic acid breath test are non invasive, non operator-de- breath test. In this paper, an overview of the pendent and can be performed several times current knowledge on this technique is given in the same subject without biological hazard. with special emphasis on the principle of the test, the mathematics used to analyse the re- Principle of 13C-octanoic Acid Breath Test sults, and the physiological, pathological, and 13 pharmacological aspects of gastric emptying The rationale of C-octanoic acid breath 13 studied with this new method. test ( C-OBT) to measure gastric emptying of solids is based on (1) the firm retention of Key Words: 13C-octanoic acid in the solid phase of a stan- Octanoic, Breath test, Gastric emptying, Scintigra- dard test meal during its passage through the phy. gastric environment, followed by (2) a rapid disintegration of the solid phase in the duo- denum with (3) subsequent absorption of 13C- octanoic acid and (4) hepatic oxidation to 13 7 CO2 (Figure 1). In vitro validation studies Introduction have shown that in gastric environment 13C- octanoic acid is firmly retained in the yolk of Gastric emptying is a highly coordinated scrambled egg used as test meal. Once the physiological response to the presence of meal reaches the duodenal lumen, 13C-oc- food which can be impaired in several patho- tanoic acid is rapidly absorbed through in- 13 logical conditions (Table I). Since its intro- testinal mucosa and oxidized to CO2 in . 13 duction in clinical practice, scintigraphy has The appearance of CO2 in breath after oral been considered the “gold standard” tech- administration of 13C-octanoic depends main- nique for measuring gastric emptying1. How- ly by the gastric emptying of the egg yolk into ever, expensive equipment is needed as well the (rate limiting step). The other as availability of qualified personnel of nu- metabolic steps (absorption and oxidation) 13 clear medicine department. Moreover, do not influence the rate of breath CO2 ex- scintigraphy induces a significant radiation cretion as shown by studies7 in which, after burden2 which limits its application in chil- duodenal instillation of 13C-octanoic acid, 13 dren, fertile women, and subjects undergoing CO2 appears in breath almost immediately repetitive measurements of gastric emptying with a very little inter-subject variability. in a short period of time. By all these reasons, 13 other non-invasive techniques such as ultra- Matematical Analysis of CO2 excretion sonography3, impedance epigastrography4, curves for gastric emptying applied potential tomography5, and magnetic A percentage of dose/h curve after inges- resonance6 have been proposed. Recently, tion of a 13C-octanoic acid labeled solid test

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F. Perri, M.R. Pastore, V. Annese

Table I. Delayed and accelerated gastric emptying states beled meal in the gastric area as a function of time. The scintigraphic retention curve is ex- Delayed gastric emptying states pressed by: PAcid-peptic related diseases -kt b Gastro-esophageal reflux disease y = 1- (1- e ) Gastric ulcer disease where “y” is the fractional dose of radioactiv- Atrophic gastritis associated or not with pernicious ity retained in the at time t, and “k” anaemia Viral gastro-enteritis (acute – chronic?) and “b’’ are estimated regression constants. Mechanical factors Two parameters are derived from this formu- Gastric carcinoma la: Idiopathic hypertrophic pyloric stenosis Metabolic and endocrine disorders • the gastric half emptying time: t1/2s = (- Diabetes (ketoacidosis, gastroparesis) (-1/b) Hypothyroidism 1/k) 5 ln (1-2 ) and Uraemia • the lag time: tlags = ln (b)/k Hypokaliemia, hypercalcemia

Hepatic coma in which t1/2s is the time at which half of the Collagen vascular diseases γ-emitting labeled meal is retained in the Pseudo-obstruction Chronic idiopathic intestinal pseudo-obstruction stomach and tlags is the time needed for the Secundary (amyloidosis, dermatomyositis, muscular antral contractions to grind solids into parti- dystrophies) cles small enough to pass through the py- Gastric surgery lorus. Both parameters are important since Postoperative ileus they properly describe the biphasic nature of Gastric resection gastric emptying of solids. For liquid test Neurological disorders meals, tlags is usually absent and only t1/2s is Central nervous system diseases, brainstem lesions, therefore calculated. spinal cord injuries In the formula by Ghoos7, the cumulative Parkinson’s disease dose of 13CO excreted as a function of time is Drugs 2 Anticholinergics, opioid drugs, tricyclic antide- given by: pressants, somatostatin, etc. Idiopathic z = m (1- e-kt)b Gastric dysrhythmias-tachygastria Gastroduodenal dissynchrony Severe exercise

Accelerated gastric emptying states Gastric emptying 13C-octanoic acid in egg yolk Zollinger-Ellison syndrome Vagotomy + pyloroplasty/antrectomy Passage through pylorus Drugs Motilin-receptor agonists, cisapride, domperidone, Intestinal absorption 13C-octanoic acid in metoclopramide, etc. duodenal lumen Mild exercise Passage through portal vein system

13 Liver oxidation CO2 production meal is characterized by an ascending slope, a in mitochondria peak of excretion, and a descending exponen- tial slope (Figure 2). In their validation paper, Plasmatic bicarbonate Ghoos et al9 developed two different formu- pool 13 las for analyzing the CO excretion curves 13 2 Breath excretion CO2 but, for simplicity, we describe only one of the two. This formula is derived by the 10 Figure 1. Sequential metabolic steps after ingestion of Siegel’s function used to describe the scinti- the 13C-labeled baked egg yolk. The rate limiting step of 13 graphic retention curve (Figure 2), i.e. the the breath CO2 excretion is represented by the gastric percentage of retention of a g-emitting la- emptying of the yolk.

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13C-octanoic acid breath test for measuring gastric emptying of solids

13 Figure 2. CO2 breath excretion curve (in % dose/h) and scintigraphic curve (in % gastric radioactivity retention) after administration of an egg in which the yolk has been labeled with 100 mg of 13C-octanoic acid and the white has been doped with 2 µCi of 99mTc-albumin colloid. Both curves are fitted by means of non linear regression analysis.

13 where z is the cumulative dose of CO2 ex- technique to evaluate gastric emptying of creted in breath per hour; t is the time (in solids. Both volunteers and dyspeptic subjects hours); m, k, and b are estimated regression were given standard test meal consisting of a 13 constants, with m the total amount of CO2 scrambled egg of which the yolk was doped recovered when time is infinite. As the curve with 100 mg 13C-octanoic acid and the egg z (% cum-dose curve) is obtained after inte- white was labeled with 110 MBq of 99mTc al- gration of a curve z’ (% dose/h curve), the bumin colloid. The egg was baked and ingest- curve z’ can be expressed as: ed within 10 minutes with two slices of white bread and 5 g of margarine, followed immedi- z’ = mkbe-kt (1- e-kt)b-1 ately by 150 ml of water. Immediately after the ingestion of the standard meal, each sub- Non linear regression analysis is performed ject underwent simultaneously scintigraphic on the data of the curve z’ to estimate m, k scans and breath collection at regular inter- and b, using the least square method. This vals of time for two hours. Breath sampling analysis can be easily performed by using the lasted another two hours with 15-minute in- “Solver” procedure in Excel (Office, Mi- tervals. Two curves were obtained for each crosoft). Two parameters can be derived from subject: the scintigraphic and the %dose/h this formula: curves. Using the formulas by Siegel10 and 7 Ghoos , four parameters were derived: t1/2s -1/b t1/2exc = (-1/k) ln (1-2 ) and t1/2exr, and tlags and tlagexc. By means of the regression analysis, two models were ob- i.e. the area under the fitted curve until half tained: of the dose of CO2 is excreted of the cumu- 13 lative CO2 excretion when time is infinite, t1/2exr = 66 + 1.12 5 t1/2s (with r-value = 0.89 and and p-value < 0.0001) and tlagexc = 60 + 0.94 5 tlags (with r-value = 0.92 β tlagexc = (ln )/k and p-value < 0.0001)

i.e. the time corresponding to the maximum According to these model, t1/2b (i.e.: breath of the curve z’. test determined gastric half emptying time) and tlagb (i.e.: breath test determined lag Validation Versus Scintigraphy phase) were calculated: In the original paper7, Ghoos et al validat- 13 ed the C-OBT against scintigraphy which is t1/2b = t1/2exr - 66/1.12 and traditionally considered the gold standard tlagb = tlagexc - 60/0.94

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By definition, the correlation coefficients and adults; however, increasing the caloric between t1/2b and t1/2s is 0.89 and between tlagb content of the test meal (up to 250 kcal) gas- and tlags was 0.92. Based on data obtained tric emptying significantly decreased in a sim- from 42 normal healthy volunteers (22 ilar fashion in children and adults. Interest- women, 20 men; mean age 22 years), Ghoos7 ingly, when full cream milk (134 kcal) was calculated a mean value of 72 ± 22 min for used, a slower gastric emptying of the milk t1/2b and of 32 ± 20 min for tlagb. Interestingly, compared with the low caloric solid meal was both within-subject variability (interindivid- observed in children but not in adults. Intesti- ual variability) and between-subject variabili- nal neuro-endocrine mechanisms following ty (day to day variability) were small and not milk assumption could be different between statistically significant. children and adults. Gastric emptying of a mixed liquid-oil meal in healthy volunteers Clinical Applications was investigated in another study15: the emp- The 13C-OBT is a useful tool to evaluate tying of the oil phase of the meal occurred in- gastric emptying either in physiological and dependently from the fluid phase. The in- in pathological states. The test has been crease of the amount of fat led to a decreased proved to be sensitive enough to detect rate of emptying of the oil phase while the changes in gastric emptying induced by drugs emptying of the fluid phase was unchanged. such as motilin-receptor agonists, cisapride, In non dyspeptic pregnant women, the OBT anticholinergics, and octreotide. In one showed a gastric emptying of solids which study11, in which nine healthy volunteers was not significantly different from controls16. were tested, erythromycin lactobionate (200 In pre-term infants, results of OBT were mg given i.v., 30 minutes before the OBT) found to be similar to those reported using 17 significantly increased both t1/2 and tlag; on the other techniques . The OBT has been also contrary, propantheline, an anticholinergic employed to study the effects of gastric emp- drug (30 mg given p.o. 60 minutes before the tying on the rate of intraluminal lipolysis 13 OBT) significantly reduced t1/2 but not tlag. In (tested with C-“mixed trigliceride”-breath another study12, cisapride (10 mg p.o. the day test) in patients with pancreatic insufficien- before and 30 minutes before the OBT) was cy18 and the rate of ureolysis (tested with 13C- effective in decreasing significantly both t1/2 breath test) in infect- 19 and tlag of solids in six healthy and six dyspep- ed and uninfected subjects . In the last study, tic subjects with delayed gastric emptying. No gastric emptying was not significantly influ- influence of cisapride was seen on t1/2 and tlag, enced by ; on the contrary, the rate when a liquid test meal was used. Healthy of gastric emptying had some influence on subjects were also tested in two different days the urea breath test results modifying the without prokinetic drugs and intra-individual time of contact of labeled urea with the bac- variability was shown to be quite good (CV teria. of t1/2 for solids, 20%, and for liquids, 5%). In The test has been successfully used in dif- another study13, subcutaneous injection of a ferent pathological conditions. In patients single physiological dose (50 mg) of oc- with distal , an early dumping of treotide induced a marked delay in the gas- the oil phase of the meal was observed com- tric emptying of solids and liquids in healthy pared to healthy controls20. During recovery volunteers. This effect was not due to varia- from hyperemesis gravidarum, women had an tion of absorption or of octanoic accelerated gastric emptying compared to acid after administration of octreotide. All non dyspeptic pregnant women21 which these studies11-13 show that the OBT is able to strongly correlated with thyroid hyperfunc- assess the effect of drugs on gastric emptying tion22. This finding suggests that vomiting in parameters and represents a promising tool pregnancy is not correlated with delayed gas- for clinico-pharmacological studies. tric emptying, but rather with causes of cen- The OBT has been also used to evaluate tral origin. Gastric emptying has been also the physiology of gastric emptying of solids evaluated by OBT in patients with non-ulcer and liquids in both adults and children. In dyspepsia23. About 30% of patients with non one study14, gastric emptying of a low caloric ulcer dyspepsia had a delayed gastric empty- (150 kcal) test meal was similar in children ing as compared to controls. This figure is

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13C-octanoic acid breath test for measuring gastric emptying of solids

13 very similar to that found in a group of dys- perimental and the fitted CO2 excretion peptic patients studied by the OBT24: almost curve for each subject was given. Fourth, an- one third of patients displayed a significant other study27 performed by our group has ob- delay in gastric emptying which was signifi- tained the same results of the Leuven group, cantly correlated with a group of symptoms confirming that the OBT is a reliable test to such as postprandial fullness, nausea, and assess gastric emptying. Finally, in a paper vomiting. In the same study, patients were al- published in 2000 by the same Mayo Clinic so evaluated by means of the urea breath test group, the Authors28 used a new test meal in- but no correlation between delayed gastric corporating a 13C-labelled alga (Spirulina emptying and presence of Helicobacter pylori platensis). Breath test measurements and infection was found. Children with celiac dis- scintigraphic scans were performed simulta- ease have been studied by the OBT before neously. US researchers found that this and after instituting a gluten-free diet25. A “new” [13C] S platensis breath test was able to significantly delayed gastric emptying was ob- measure gastric emptying t1/2 for solids with served in celiac patients at diagnosis com- results comparable with those obtained from pared with healthy age-matched controls. Af- scintigraphy. ter 6 months of gluten-free diet, gastric emp- In conclusion, the OBT has been proposed tying parameters returned to normal range in in 1993 as a non invasive, reliable, and safe celiac patients. This finding suggests that test for measuring the gastric emptying of gluten-related mucosal damage causes, solids. In view of the use of 13C-labeled meals, through a neuro-hormonal mechanism, a sig- the test has the potential for widespread clini- nificant alteration in gastric emptying of cal application in centers where gamma-cam- solids which normalizes when small intestinal era facilities are not available. Moreover, mucosa restores. pregnant women and children may be tested without biological hazard. Results from the Potential Drawbacks OBT correspond to those obtained from The OBT has been validated against scintigraphic method which is currently re- scintigraphy and a good correlation between garded as the “gold standard” for evaluating the results obtained from the two techniques gastric emptying. The close correlation be- has been achieved. However, a report from tween breath test and scintigraphy has been Mayo Clinic in US found that this correlation definitely proven even by those who ques- was not so close as previously suggested. tioned on the validity of the test in the past. Choi et al26 have repeated the same experi- However, new studies are needed to validate ence made in Leuven by performing simulta- a simple, “office-based” OBT with standard- neous OBT and scintigraphy in 15 healthy ized 13C-enriched test meals. subjects. They found that gastric emptying parameters from the two methods were not significantly correlated. Moreover, increasing breath test duration (up to 5 or 6 hours) References yielded decreasing estimates of both t1/2 and lag phase. A good reproducibility, however, 1) GRIFFITH GH, OWEN GM, KIRKMAN S, SHIELDS R. Mea- was obtained with a mean coefficient of vari- surement of rate of gastric emptying using chromium-51. Lancet 1966; i: 1244-1245. ation of t1/2 of 20% between individuals and 12% within the same individual. They con- 2) SIEGEL J, WU R, KNIGHT L, ZELAC RE, STERN HS, MAL- cluded that the OBT required further valida- MUD LS. Radiation dose estimates for oral agents used in upper gastrointestinal disease. J Nucl tion but it seemed useful for intra-individual Med 1983; 24: 835-837. comparisons. Different critiques have been made to this study27. First, data obtained 3) BOLONDI L, BORTOLOTTI M, SANTI V, C OLLETTI T, G AIANI S, LABO G. Measurement of gastric emptying by from only 15 subjects should be regarded real-time ultrasonography. Gastroenterology with caution especially when regression 1985; 89: 752-759. analysis is used. Second, US researchers used 4) MCCLELLAND GR, SUTTON JA. Epigastric impedance: a labeled tracer which was different from that a non-invasive method for the assessment of originally used by Ghoos et al7. Third, no da- gastric emptying and motility. Gut 1985; 26: 607- ta on r2 coefficient obtained between the ex- 614.

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12) DUAN L, BRADEN B, CASPARY W, L EMBKE B. Influence 24) PERRI F, C LEMENTE R, FESTA V, A NNESE V, Q UITADAMO of cisapride on gastric emptying of solids and liq- M, RUTGEERTS P, A NDRIULLI A. Patterns of symptoms uids monitored by 13C breath tests. Dig Dis Sci in functional dyspepsia: role of Helicobacter pylori 1995; 40: 2200-2206. infection and delayed gastric emptying. Am J Gastroenterol 1998; 93: 2082-2088. 13) MAES B, GHOOS Y, G EYPENS B, HIELE M, RUTGEERTS P. Influence of octreotide on the gastric emptying of 25) PERRI F, P ASTORE M, ZICOLELLA A, ANNESE V, Q UITADAMO solids and liquids in normal healthy subjects. Ali- M, ANDRIULLI A. Gastric emptying of solids is de- ment Pharmacol Ther 1995; 9: 11-18. layed in celiac disease and normalizes after gluten withdrawal. Acta Paediatr 2000; 89: 921-925. 14) MAES B, GHOOS Y, G EYPENS B, HIELE M, RUTGEERTS P. Relation between gastric emptying rate and ener- 26) CHOI M, CAMILLERI M, BURTON D, ZINSMEISTER A, gy intake in children compared with adults. Gut FORSTROM L, SREEKUMARAN NAIR K. 13C-octanoic acid 1995; 36: 183-188. breath test for gastric emptying of solids: accura- cy, reproducibility, and comparison with scintigra- 15) GHOOS Y, M AES B, GEYPENS B, HIELE M, RUTGEERTS P. phy. Gastroenterology 1997; 112: 1155-1162. Gastric emptying of a mixed liquid-oil meal in healthy volunteers, as measured by breath tests. 27) PERRI F, C LEMENTE R, FESTA V, A NDRIULLI A. 13C-octanoic Gastroenterology 1994; 106: A503. acid breath test: valueless test for gastric empty- ing? Gastroenterology 1998; 114: 857-859. 16) MAES B, GHOOS Y, S PITZ B, RUTGEERTS P, V AN ASSCHE A, VANTRAPPEN G. Gastric emptying rate of solids in 28) LEE JS, CAMILLERI M, ZINSMEISTER AR, BURTON DD, non-dyspeptic pregnant women, using the 13C-oc- KOST LJ, KLEIN PD. A valid, accurate, office based tanoic acid breath test. Gastroenterology 1993; non-radioactive test for gastric emptying of solids. 104: A546. Gut 2000; 46: 768-773.

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