Subjects and methods

Seven patients with cystic fibrosis (three boys and four girls) aged 4–11 years (mean, 8.0 years) from the cystic fibrosis clinic at Southampton University Hospitals NHS Trust were studied while on their normal habitual pancreatic enzyme replacement treatment. Patients with small bowel resection or other known factors that might limit absorption (for example Crohn’s disease) were excluded from the study. The cystic fibrosis patients were taking between 13 462 and 34 000 IU lipase/kg body weight/day (mean, 18 877); an enzyme dosage achieved by self-titration against gastrointestinal symptoms and bowel habit. No attempt was made to alter or intervene with the management of the pancreatic enzyme replacement treatment. Nine healthy children (six boys and three girls) aged 5–8 years (mean, 7.2 years) from local schools also participated in the study. Informed consent was obtained from all of the subjects and the study protocol was approved by the Ethical Committee of Southampton and South West Hampshire Health Commission.

After an overnight fast, the [1,1,1-¹³C]tripalmitin was administered orally to the subjects at a dose of 10 mg/kg body weight (99 atom % excess; Masstrace, Woburn, USA) as part of a controlled standard test meal consisting of 120 g white bread, 100 g orange juice, and 10 g butter of low ¹³C abundance (−25.5‰).8 The patients with cystic fibrosis took the same amount of enzymes with the test meal that they would usually take with a snack meal and this ranged from 1458 to 6849 IU lipase/kg body weight/day (mean, 3750). The ¹³C labelled tripalmitin was given as a glucose-sucrose-casein emulsion, using a modification of Emken et al,9 because emulsification before administration overcomes the problems associated with the physicochemical properties of the crystalline form of this label, which might otherwise result in poor digestion and absorption if given in a free, unemulsified form.10

Breath samples were collected using breath collection bags (Quintron) before consuming the test meal to provide a measure of baseline¹³C excretion on the breath, and were then collected at hourly intervals for a period of at least six hours and then after eight hours, 10 hours, and 24 hours. Specimen breath samples were transferred into evacuated gas sample containers (Exetainers; Isochem, Finchampstead, UK) for analysis in duplicate. All subjects were rested for the duration of the tests and were given constant supervision at the Clinical Nutrition and Metabolism Unit at Southampton General Hospital. No additional food or drink was permitted during the initial six hour period except for bottled mineral water. After six hours, the subjects ate a meal without any foods naturally enriched for¹³C. Whole body CO₂ excretion was measured by indirect calorimetry (GEM; Europa Scientific Ltd, Crewe, UK) for a period of 15 minutes at the same time points as breath sampling over the first six hours to calculate the amount of label excreted each hour. It was not possible to measure CO₂ excretion at eight,10, and 24 hours. The value for CO₂ excretion at six hours was used to calculate the amount of label excreted at eight and 10 hours. The proportion of label excreted on the breath at 24 hours was calculated using the baseline value for CO₂ excretion.

A stool sample was collected on the day before the labelled test meal to measure baseline ¹³C excretion. Thereafter, all stools passed were collected and processed individually for a period of up to five days. All the stool collections were carried out at home and monitored daily throughout the study period. All of the subjects and parents of children were cooperative, willing, and capable of following the details of the procedure. At no time was difficulty encountered nor was there any need for modification of the procedure to accommodate any of the subjects. All the stool collections were complete. The subjects were also instructed to avoid eating naturally ¹³C enriched foods (such as corn or maize based products and cane sugar) for at least two days before and during the stool collection period. None of the animal source protein foods eaten (such as eggs and meat) were naturally enriched with ¹³C.

BREATH AND STOOL ANALYSES

The methodology for processing stools has been described previously.8 Enrichment of ¹³C in the stool and as ¹³CO₂ on the breath was analysed by continuous flow isotope ratio mass spectrometry (ANCA-NT GSL; Europa Scientific Ltd). Total stool lipid was extracted from stools by a modification of the method of Folch and colleagues.11 The¹³C enrichment of samples of breath¹³CO₂ and stool was expressed as the “per ml relative difference from the reference standard Pee Dee Belemnite (PDB)” as defined by Craig (¹³C_PDB, ‰).12 The proportion of administered ¹³C label in the stools was calculated according to the formula presented by Schoeller and colleagues.13 The apparent absorption of the ¹³C label was determined from the difference between the amount of label administered and that excreted in the stool.

The proportion of ¹³C label excreted on the breath as¹³CO₂ was expressed as a percentage of absorbed¹³C label each hour and as the cumulative percentage dose excreted over 24 hours, according to the formula presented by Watkinset al.4

STATISTICS

The results are reported as mean (SD). Statistical comparisons between the data were performed using the unpaired Student’st test. Differences between means were considered significant when p < 0.05. Associations between variables were tested by the Pearson product moment correlation coefficient (R).

Results

In healthy children, 6.0% (3.7%) of the administered¹³C label was excreted in the stool (range, 1–12.7%) (table 1). In contrast, patients with cystic fibrosis excreted more¹³C label in the stool: 24.6% (22.3%) of the administered¹³C label was found in the stool, although there was a wide range (range, 0–64%; p > 0.05; table 1). Usually, the stools with the greatest ¹³C enrichment were found on the day after the test meal and enrichment returned to baseline values by day 3 or 4. In patients with cystic fibrosis, excretion of total lipid in the stool was four times greater than that seen in healthy children (13.2 g/day (3.1) v 2.95 g/day (1.0); p < 0.001). Analysing both groups together, there was only a weak association between the amount of ¹³C label and the total lipid in the stool (R = 0.53; p > 0.09).

In both groups, excretion of the label on the breath as¹³CO₂ peaked between one and six hours after the test meal, returning to baseline values in almost all subjects by 24 hours. In one of the healthy children and one cystic fibrosis patient, ¹³C enrichment on the breath was only 2 SD units greater by 24 hours than that observed at baseline. In healthy children, the proportion of administered ¹³C label excreted on the breath was 31.3% (10.6%) (range, 14.2–42.9%), whereas patients with cystic fibrosis excreted significantly less¹³C label on the breath (17.9% (9.5%); range, 0–31.3%; p < 0.05). In healthy children, the proportion of ¹³C label excreted on the breath was not altered if expressed directly as¹³C label administered rather than as label made available through digestion and absorption (33.1% (10.9%); range 15.5–43.3%; table 1). However, correcting excretion of the label on the breath for differences in digestion and absorption in patients with cystic fibrosis increased the differences seen between individuals (23.2% (15.2%) of absorbed label; range, 0–49.1%; p > 0.05). There was no association between the amount of ¹³C label in the stool and proportion of absorbed ¹³C label excreted on the breath (R = −0.15; p > 0.75).

Discussion

Our study aimed to examine both the gastrointestinal handling and metabolism of emulsified [1-¹³C]palmitic acid esterified into a triglyceride in healthy children and patients with cystic fibrosis by measuring excretion of the label in the stool and on the breath as ¹³CO₂. This is the first time that ¹³C labelled tripalmitin, orally administered as an emulsion, has been used to measure the digestion and absorption of dietary triglycerides in healthy children and patients with cystic fibrosis on habitual pancreatic enzyme replacement treatment by measuring directly the excretion of ¹³C label in the stool. In healthy children, only a small proportion of the ¹³C label was excreted in the stool, which is consistent with total stool lipid losses within the normal range of < 5 g/day.1Almost a quarter of the ¹³C label appeared in stools from patients with cystic fibrosis, with as much as 64% in one child, and there were substantially greater amounts of lipid in the stool when compared with healthy children. The differences seen in excretion of the ¹³C label in the stool between the patients with cystic fibrosis could be a result of poor digestion of the labelled triglyceride, which limits subsequent absorption. Alternatively, or in addition, the labelled products of digestion could have been malabsorbed because of a failure in the absorptive capacity of the gastrointestinal tract. These observations are in contrast to those shown previously by our group using [1-¹³C]palmitic acid presented as the free acid, where more of the label was absorbed in patients with cystic fibrosis compared with healthy children. It might be that these differences reflect the physicochemical properties of the crystalline form of saturated fatty acids, which appear to favour enhanced absorption in patients with cystic fibrosis compared with healthy subjects when the fatty acid is given as the free acid.

A previous study has shown that doubling the dose of enzymes could increase ¹³C recovery on the breath after oral administration of a mixed triglyceride, which is assumed to reflect a reduction in excretion of the label in the stool.14 In our study, the habitual dose of enzymes was taken with the test meal, a dose that might not have been optimal for this particular meal, and this could have contributed to poor digestion of the labelled tripalmitin. Owing to concern over the adverse effects of high doses of pancreatic enzyme on the large intestine,15 any increase in the intake of pancreatic enzymes would be supported by further studies examining the nature of species bearing the ¹³C label to discriminate between maldigestion and malabsorption. The appearance of the label in the stool predominantly as a triglyceride would indicate a failure of digestion (impaired lipase activity arising from pancreatic insufficiency, or solubilisation, or poor pancreatic enzyme replacement treatment management), while appearance of the label as free fatty acid, 2-monoacylglycerol, or as soaps would indicate a failure in absorption of hydrolysed trigylceride.

Our study revealed that ¹³C labelled tripalmitin was not oxidised to the same extent in both groups, whether it was expressed as a proportion of the ¹³C label administered or the proportion of that absorbed. This could be examined in two ways. First, the value of characterising the extent of digestion and absorption in cystic fibrosis using emulsified ¹³C labelled tripalmitin in breath tests needs to be considered. While patients with cystic fibrosis excreted more ¹³C label in their stools, recovery of the label on their breath was lower than in healthy children, although there was some overlap between the two groups. For example, in patient 1 (table 1), although excretion of the ¹³C label in the stool was raised, no label was detected in the breath; other patients not only had raised stool losses but also a greater recovery of the label on the breath, comparable to healthy children (patients 3 and 5; table 1). This suggests that in some individuals emulsified¹³C labelled tripalmitin does not sufficiently reflect the processes involved in the digestion and absorption of dietary long chain saturated triglycerides. However, it should be noted that measurements of ¹³C label in the stool have not been reported for other ¹³C labelled mixed triglycerides and medium chain triglycerides used in breath tests. The other consideration is the extent of recovery of the absorbed label. In both groups, there was a large variability between subjects in the proportion of absorbed ¹³C label recovered on the breath. Having corrected for differences in digestion and absorption, it is not clear what other factors might account for this observation. The metabolic competence of an individual influenced by either genetic, nutritional, or lifestyle factors would be important determinants of the partitioning and subsequent oxidation of ¹³C labelled fatty acids. Other factors such as physical activity might also influence the extent of breath ¹³CO₂ recovery, particularly when performed throughout the postprandial period,16 although it is unlikely that they contributed much towards the differences between the subjects in our study. All subjects were measured under similar resting conditions and avoided eating and drinking for at least 12 hours before label administration and for six hours during the period of the breath test. The subjects also avoided any foods naturally enriched for ¹³C over the period of the study.

Previous studies have shown that not all of the CO₂generated from the label would have been excreted as breath¹³CO₂. A proportion of the¹³CO₂ formed from the oxidation of labelled substrate is not excreted directly on the breath but might be retained in the body pools or excreted via the urine, skin,17 or stool.18 Previous studies have either presented uncorrected data4-6 14 or have used correction factors to predict more accurately the extent of oxidation of labelled substrates.8 19 Precisely how much of the¹³CO₂ is retained in the body pools remains unresolved, with estimates ranging from 10% to 50% in normal individuals.20 Because there is no specific information on this issue in patients with cystic fibrosis, we have not attempted to correct the oxidation data. It is possible that the ¹³C labelled products of maldigestion and malabsorption provide a substrate for colonic bacterial metabolism. However, little is known about the extent to which fermentation of fatty acids by colonic bacteria influences measurements of ¹³C recovery on the breath. Fermentation of these labelled products might result in the excretion of the label as flatus gases (such as methane or carbon dioxide), which would result in an underestimation of oxidation. Alternatively, any such ¹³C labelled carbon dioxide from fermentation might be absorbed across the colonic mucosa and could contribute to the amount of ¹³CO₂ excreted on the breath. The potential contribution made by colonic bacterial fermentation of ¹³C labelled lipids to differences in the recovery of ¹³C on the breath requires further study.

In conclusion, after oral administration of emulsified ¹³C tripalmitin, patients with cystic fibrosis excreted more of the¹³C label in the stool when compared with healthy children, although a wide difference in excretion was observed between these patients. This suggests that in cystic fibrosis there are differences in the functional capacity of the gastrointestinal tract to handle saturated fatty acids when esterified into triglycerides, despite pancreatic enzyme replacement treatment. It should not be assumed that excretion of the label on the breath reflects the gastrointestinal handling of emulsified ¹³C labelled tripalmitin. Further studies are needed to examine whether breath tests alone can predict with confidence the extent of digestion and absorption in cystic fibrosis of other ¹³C labelled triglycerides and fatty acids.