Ciinica Chimica Acta. 205 (1992) 51-64 © 1992 Elsevier .Science Publishers B.V. All riots reserved 0009-8981/92/$05.00

51

CCA05202

Limitations of the triolein breath test A n d r e w D u n c a n a, A l i s o n C a m e r o n a, Michael .I. Stewart b a n d R o b i n I. RusseU a aGastroenterology Unit and blnstitute of Biochemistry. Royal Infirmary. Glasgow, G31 2ER (UK) (Received 23 July 1991; revision received 31 October 1991; accepted 8 November 1991)

Key words: Malabsorption; Fat absorption test; Carbon dioxide output; Breath test; Dual isotope fat absorption test

Summary Patients being investigated for intestinal absorptive capacity were classified as norreals or malabsorbers on the basis of three fat absorption tests. Malabsorbers were further classified as mild, moderate, severe or gross according to severity of malabsorption. Using this classification system the triolein breath test was evaluated in 53 patients. Seventeen patients were excluded because their graph of percentage breath [14C]carbon dioxide versus time was exponential indicating that the peak [~4C]carbon dioxide may be occurring later than the six hour duration of the test. The sensitivity and specificity of the triolein breath test were found to be 100% and 96%, respectively and moderate correlations with the individual fat absorption tests were found. However, the breath test was limited in its capacity to predict the severity of malabsorption. Carbon dioxide output was also measured in order to determine the applicability of using an assumed value. The respiratory quotient and variability of results were high in nineteen patients indicating possible hyperventilation. In 32 patients with reproducible results and normal respiratory quotients the average carbon dioxide output was 8.66 mmol/kg per hour with a wide range of 5-12.4mmol/kg per hour. Consequently the use of an assumed carbon dioxide output can introduce considerable errors in the triolein breath test. This study highlights drawbacks of the triolein breath test, particularly problems in using an assumed carbon dioxide output for its calculation, its inability to predict the severity of malabsorption and the nature of the dietary load used.

Correspondence to: Andrew Duncan, Gastroenterology Unit, Royal Infirmary, Glasgow, G31 2ER, UK.

52 Introduction

In 1979 Newcomer et al., studied three triglycerides - - trioctanoate, tripaimitate and trioleate m as fat probes in J4C breath tests for malabsorption and found that trioleate gave the best results [1]. Since this report there has been considerable interest in the triolein breath test, which has now been assessed several times [2-9]. These evaluations, however, show a considerable divergence of opinion as to the clinical vall~e of this test (Table I). Possible reasons for these discrepancies are the varied designs and methods of analysing the various studies. Most of the reports use predictive values for evaluating the data with results varying from 42 to 100% for sensitivity and 86 to 100% for specificity. The evaluation of data on the basis of sensitivity and specificity alone can be misleading. For example, a small change in the reference range of the 'gold standard' (Table I shows that there is a wide variation in what level of faecal fat is considered normal) can have a sizeable effect on the rate of false positives and negatives. Some studies also calculate the degree of correlation with the reference method and wide variations have again been reported (Table I). For example, no correlation was found in a study which quotes good predictive values [5] and a good correlation in a study which shows much poorer predictive values [7]. The choice of control population also varies between studies. Healthy volunteers, non-gastroenterology patients and gastroenterology patients with normal faecal fat have variously been used. Another study recruited patients with irritable bowel disease as controls on the assumption that their absorptive capacity is normal, a premise which has since been shown to be false [10]. Finally, a variety of protocols has been employed with large quantitative and qualitative differences in both the fat load given and the duration of the test. Perhaps the greatest difficulty in evaluating the triolein breath test is the choice of an appropriate gold standard method. Unfortunately there is no test which can be considered definitive. In the absence of a more suitable test, the measurement of faecal fat has been mainly used t~r comparisons, despite its well known pitfalls [11]. In ~his study we have evaluated the triolein breath test using a reference system which classifies each patient as a normal or malabsorber on the basis of three fat absorption tests, quantitative faecal fat measurement and two dual isotope fat absorption (DIFA) tests [12,13]. The triolein breath test is usually ca|culated using an assumed carbon dioxide (CO2) value. In this study CO2 outputs were measured to determine the extent of any e~or introduced by making this assumption. Methods and Patients Methods For 2 days prior to the test and during the 3 days of the stool collection, the patient was given a high fat diet containing 70-100 g/day of fat. After an overnight fast the patient swallowed gelatin capsules containing 400 kBq of [tdC]triolein (18.5-30 GBq/mmol) and 2000 kBq of [3H]triether (970 GBq/mmol, both from

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54 Amersham International, England). A 20 g fat load in the form of double cream and 1000 kBq of [StCr]chromium chloride (0.3-2.4 GBq/mol, Amersham) dissolved in 50 ml of water were taken at the same time. At 0, 1, 2, 3, 4, 5 and 6 h end-expiratory samples of breath were collected into methylbenzethonium hydroxide (Sigma Chemical Co, England) [14]. Factors known to affect CO2 production, such as smoking and exercise [15], were minimised. The percentage t4CO2 excretion in the hourly breath samples was calculated using an assumed CO2 output of 9 mmol/kg per hour [14]. The triolein breath test result was taken as the peak of 14CO2 excretion [1]. In patients who found difficulty in collecting end-expired breath, a mixture of endexpired and normally expired breath was usually collected. To determine whether these collections were valid, 24 samples each of end-expired breath and normal breath samples were collected from seven patients who could collect end-expired breath in the proper way. During the breath test, three or four 2-min samples of breath were collected into Douglas bags via a low dead-space Hans-Rudolph valve and the CO 2 and oxygen were measured. Hyperventilation was minimised by encouraging patients to watch television or read to reduce self-awareness of their breathing rate. The CO2 output and respiratory quotient were measured [16]. No patients had respiratory complaints s~,,ch as emphysema or kyphoscoliosis which could cause abnormal respiratory quotients. 'A 3 day stool collection was homogenised and 25 ml of 154 mmol/I saline and 37.5 ml of chloroform:methanol (2:1 by volume) was added to 3-6 g of homogenate. After refluxing for I h the refluxate was cooled and centrifuged (1500 x g for 5 min). Volumes of 0.5, 1.0 and 1.5 ml of the chloroform layer were transferred to Ocounting glass vials, evaporated off, and the dried extract exposed to light until decolourised, t4C and 3H were measured and quench corrections made using chemically quenched standards (Canberra Packard, Pangbourne, England). 5tCr was measured in 5 g portions of homogenate. The DIFA tests using [3H]triether or [5tCr]chromium chloride as non-absorbable marker (DIFA-3H, DIFA-stCr, respectively) were calculated as the percentage 14C absorbed [12,13]. Faecal fat excretion was measured by the van de Kamer method [17]. On the first twenty patient tests performed, "y-radioactivity was measured in the chloroform layer to determine if StCr was a potential interferent of liquid scintillation counting. Known amounts of [3Hltriether and [ t4C]triolein were added to eleven stools in order to measure their recovery. The extraction procedure was performed in duplicate on the first twenty-five patient tests to enable imprecision to be measured. Fifteen stool samples were analysed by this extraction procedure and by a biological oxidation method. Patients

The triolein breath test and the three reference tests of fat absorption were performed on 61 consecutive patients. Thirty-seven patients who had no evidence of fat malabsorption on the basis of DIFA-3H, DIFA-stCr and faecal fat results were

55

used as a control group. The reference ranges for these three fat absorption tests were greater than 95% for DIFA tests and less than 25 mmol/day (equivalent to about 7 g/day) for faecal fat. By selecting those patients with abnormal DIFA-3H, DIFA-S~Cr and faecal fat results, a corresponding group of sixteen patients with malabsorption was established. In the remaining eight patients DIFA results and faecal fat results did not agree. Malabsorbers were categorised into four groups according to the severity of malabsorption: (a) mild malabsorption-faecal fat of 25-35 retool/day and DIFA of 90-95%; Co) moderate malabsorption-faecai fat of 35-60 retool/day and DIFA of 75-90%; (c) severe malabsorption-faecal fat of 60-100 mmoFday and DIFA of 50-75%; and (d) gross malabsorption-faecal fat over 100 retool/day and DIFA less than 50%. Nine malabsorbers could be classified using this system and in four others the/'aecai fat and DIFA results were in adjacent groups. The latter results were not excluded but were placed in intermediate groups. Results Method assessment

No StCr was measurable in chloroform extracts and so it was concluded that 3H and t4C could be confidently measured in the chloroform layer without contamination from StCr which was found predominantly in the solid interphase. The recoveries of [3Hltriether and [~4Cltriolein were 104.7% (S.E.M. = 3.7) and 98.4% (S.E.M. = 3.1), respectively. The imprecision of the DIFA-3H test varied with different degrees of malabsorption (Table 11) because of the nature of the equation used for its calculation. Thus when absorption approached 100% large differences in the 31-I/14Cratio produ:,ed relatively small changes in the percentage absorption. When the percentage absorption is low the converse is the case. In general the imprecision of the test was acceptable. Fortunately the poor imprecision found at very low levels of absorption has no clinical implications. There was a good correlation (r = 0.99) between DIFA-3H results obtained by using the extractioa procedure described and biological oxidation (Spearman rank order correlation test). No statistical difference (P = 0.99) in ~4C was found between end-expired and normal-expired breath collections OVilcoxon matched-pairs signed-rank test).

TABLE !i Precision of DIFA-3H at differing degrees of malabsorption DIFA-3H range

N

Mean

S.D.

C.V.

90-100% 75-900/0 < 20%

8 12 5

97.8% 83.3% 9.6%

0.78 3.3 2.2

0.800/0 3.9% 23%

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Hence, it is not imperative that the breath samples collected are end-expiratory. Nevertheless for the purposes of this study, end-expiratory breath samples were collected whenever possible although results from patients who had difficulty in providing such samples were not excluded. Triolein breath test

The initial results of the triolein breath test in controls and malabsorbers showed a considerable overlap between the control and malabsorption groups (Fig. I). Such a degree of overlap would make the triolein breath test of little value but before vilifying it we w¢re keen to ensure that such unimpressive results could not have been caused by any technical errors. In particular we were concerned that the 6-h duration of the test might have been insufficient to 'catch" all of the peaks. When the times of peak t4CO2 excretion were studied it was found that these were reached during the fourth hour in 2 (3%) and during the fifth hour in 12 (20%), but that in most patients (77%) the peak in t4CO2 excretion came at the sixth, and last, hour of collection. It was possible therefore that in some patients a peak might have been reached during the seventh or eighth hour had these samples been collected. The possibility of having missed late peaks prompted us to analyse the pattern of t4CO2 excretion in more detail and so the graph of t4CO2 with time was plotted for each test. In general, three graph types were produced showing either a peak in 14CO2 excretion (Fig. 2A), plateauing of the curve at about 6 h (Fig. 2B), or a late exponential rise in breath t4CO2 (Fig. 2C). It seemed likely that in those patients whose breath t4CO2 excretion was still apparently increasing at 6 h the peak might have been missed. In addition, with this exponential peak type the earlier t4CO2

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Fig. I. Peak 14CO2 triolein breath test results in control patients and malabsorbers: (+) t4C02 peaks excreted during the six hours of the test; (0) probable late t4CO2 peaks.

57

results at the second and third hours were significantly lower, P < 0.01 and P < 0.001, respectively (Mann-Whitney U-test), than with the other two peak forms also suggesting that there was a delay in 14CO2 excretion. With the resultant suspicion that some results were underestimates, all patient data were reviewed in order to select those t4CO2/time curves which corresponded

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Fig. 2. Typical graphs of breath t4CO~ excretion versus time found in the triolein breath test: (A) obvious peak of breath t4CO2 during the 6-h time course of the test: (B) plateau of breath t4co2 at 6 h; (C) late exponential rise of breath t4CO2.

58

to Fig. 2C. Of the 53 patients investigated 17 were found to have exponential graphs of this type. In Fig. 1 these patients are represented by open circles. In the control group, 14 patients gave an exponential type of t4CO2 excretion suggestive of a late t4CO2 peak. Of the 10 control results which overlapped with the malabsorption group eight could be explained on the basis of a probable late 14CO2 peak. In the malabsorption group, three '4CO2/time graphs were increasing at 6 h suggesting a late peak. In only one of these was the result high enough (2.2% t4CO2 dose/hour) that it might have been misclassified as a false negative had a later breath sample been collected. The control and malabsorption groups were sub-divided into two categories in which (a) the t4CO2 peak was late and had probably been missed and (b) those whose peaks were valid since they fell within the six hours of the test. The means of these data and statistical comparisons of groups are shown in Table III. In the case of the control group the late peaks are significantly lower (Mann-Whitney Utest). This provided further indirect evidence that patients with exponential graphs of "SCO2excretion versus time produced late t4co 2 peaks resulting in their triolein breath test results being underestimated. The results of triolein breath tests in controls and malabsorbers were replotted omitting all results in which the peak t4CO2 excretion had probably been missed. When this was done there was an improved resolution between controls and malabsorbers (Fig. 3). The lower limit of normal was set at 2.6 (i.e. mean minus two S.D.s). Figure 3 also shows the results of the triolein breath test in patients with differing severities of malabsorption. A poor degree of correlation (r = -0.34) was found between the various severities of malabsorption and peaks of t4CO2 (Spearman rank order correlation test). The triolein breath test was correlated with the DIFA-3H and DIFA-StCr tests and with the quantitative excretion of faecal fat. A moderate correlation was produced for each: r = 0.64 for triolein breath test versus DIFA-3H; r = 0.51 for triolein breath test versus DIFA-stCr; and r = -0.60 for triolein breath test versus faecal fat excretion (Spearman rank order correlation test).

TABLE I!I Comparisons of triolein breath test results characterised by late or valid peaks in controls and malabsorbers Group

Menn (% dose/hour)

S.D.

Controls (valid peaks) Controls (late peaks)

5.18% ~.03%

1.29 ) 0,9~

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