Abstract
Introduction
Diagnosing lactose malabsorption is usually based on hydrogen excretion in breath after a lactose challenge. However, a proportion of subjects with lactose malabsorption will not present a rise in hydrogen. Measuring excretion of methane or stable isotope labelled 13CO2 after ingestion of 13C-lactose have been proposed to mitigate this problem.
Objective
Assess the performance of measuring methane and 13CO2 in individuals with normal hydrogen excretion compared to a genetic lactase non-persistence test.
Methods
Individuals referred for lactose breath testing and healthy controls were included. Participants received 13C enriched lactose, performed breath testing and underwent genotyping for a marker of lactase non-persistence (13910C*T). Using genotype as gold standard the performance of measuring methane and 13CO2 excretion was assessed.
Results
151 subjects participated in the study, 50 of which presented a lactase non-persistent genotype. Of these, 72% were correctly diagnosed through hydrogen excretion of ≥20ppm above baseline. In subjects with normal hydrogen excretion, cumulative 13C excretion had an area under the curve (AUC) of the receiver operating characteristics (ROC) curve of 0.852. Sensitivity was 93% and specificity 51% for the current cutoff of 14.5%. The optimal cutoff was 12.65% (sensitivity 93%, specificity 70%). The ROC curve of peak methane had an AUC of 0.542 (sensitivity 14%, specificity of 91% for cutoff ≥10ppm).
Conclusions
In individuals with genetically demonstrated lactase non-persistence and negative hydrogen breath test, the use of 13C-lactose with measurement of 13CO2-excretion and hydrogen is a well-performing test to detect lactose malabsorption and performed better than methane in our cohort.
Keywords: Lactose intolerance, lactase, breath tests, hydrogen
Introduction
Lactose is a disaccharide that requires hydrolysis into its constituent monosaccharides galactose and glucose to enable intestinal absorption1. Hydrolysis of the disaccharide is performed by the lactase enzyme located on the small bowel brush border, expressed at high levels at birth in humans as it is integral in enabling the digestion of mother milk 2. After weaning, lactase expression is downregulated – the exception being populations with a long history of domesticated cows such as Caucasians; in these populations rates of lactase persistence beyond the weaning phase are high2. In the European population lactase persistence is mediated through the single nucleotide variant 13910C*T whereas individuals with a 13910C constellation (i.e. wildtype) present lactase non-persistence 3,4.
Lactase deficiency leads to an increased osmotic load in the small bowel and bacterial fermentation of the non-digested lactose which in turn may lead to symptoms in some but not all individuals after lactose ingestion2,5. Semantically the distinction is made between lactase persistence (i.e. presence of the aforementioned genetic variant in European populations) vs non-persistence (i.e. wildtype) while lactose malabsorption is the term for incomplete absorption of lactose in the small intestine which may occur due to various reasons, both primary (i.e. non-persistence of lactase) and secondary (e.g. villous atrophy in celiac disease) 2,5. The occurrence of gastrointestinal symptoms following lactose ingestion is termed lactose intolerance2,5. Breath testing is widely used to identify lactose malabsorption in patients presenting clinically with presumed lactose intolerance5,6. As secondary causes of lactose malabsorption (especially celiac disease and inflammatory bowel disease) are routinely ruled out in symptomatic individuals undergoing the test, breath testing aims to detect lactase non-persistence further indicating lifelong dietary adaptation aiming to reduce lactose exposure or supplementation of lactase before lactose ingestion 5–7. Although recent guidelines acknowledge that malabsorption documented using breath tests might not be a major determinant for the outcome of the diet, breath testing with symptom assessment is still considered to have a reasonable sensitivity and specificity by European experts 5.
The lactose breath test is performed by ingesting a lactose solution followed by collection of end expiratory breath samples to measure expiratory H2 5,6. H2 is not produced by human enzymes and thus reflects bacterial metabolization of lactose. An increase of >20 parts per million (ppm) of expiratory H2 after lactose ingestion is regarded indicative of lactose malabsorption 5,6. However, H2 production may not be increased despite the presence of lactose malabsorption in individuals with a microbial composition favoring methanogenic metabolism of lactose 8. These individuals might therefore not be diagnosed with lactose malabsorption when measuring H2 alone5. Additional measurements have been suggested for the detection of lactose malabsorption in subjects with low H2 breath tests including measuring CH4 and measuring 13CO2 after administration of 13C-labelled lactose 9,10. Small intestinal digestion of 13C-lactose results in 13C-glucose and 13C-galactose which are oxidized in the liver to 13CO2. Hence, the amount of 13CO2 that appears in breath reflects the digestion of lactose in the small intestine and a low 13CO2-excretion may indicate lactose malabsorption, even in the absence of hydrogen.
In this proof of concept study we aimed to assess the performance characteristics of cumulative 13CO2 excretion and peak CH4 in subjects with a negative H2 breath test. Genetic testing was used as a gold standard although this test does not detect causes of secondary lactose malabsorption such as celiac disease, inflammatory bowel disease and infections. Since patients with suspected lactose intolerance routinely undergo investigations to exclude those disorders and none of our participants had such a disorder, the genetic test was considered a valid gold standard.
Methods
Patients
Between April 2017 and November 2020 patients were recruited using two approaches. 1/ Patients with a clinically indicated lactose breath test were prospectively invited to participate in the study. 2/Patients with an abnormal result on a clinically indicated lactose breath test were contacted to participate in the study. The patient population included patients from our tertiary care center and patients that had been referred by other hospitals for lactose breath testing.
This strategy was chosen as the proportion of pathological breath tests was initially low and the additional selection of participants based on the presence of pathological tests allowed inclusion of sufficient numbers of pathological results to ensure representative results.
Patients above the age of 3 undergoing a lactose breath test were invited to participate in the study. Exclusion criteria for participation were previous abdominal surgery (appendectomy allowed), medication influencing the gastrointestinal tract in the previous 2 weeks, antibiotic treatment within 1 month of participation and regular use of probiotics in the previous months.
The study was approved by the Ethics Committee of UZ/KU Leuven (S59823) and all subjects signed written informed consent.
All participants underwent both genetic testing and lactose breath testing as further described below.
Healthy volunteers
Healthy volunteers were recruited by advertisement and through the hospital’s intranet.
Breath test
Patients conducted the test at home after at least 8 hours of fasting. Before ingestion of the substrate, four baseline breath samples were obtained. After ingestion of 25g of 13C labelled Lactose (atom percent: 1.102%, (obtained by mixing 13C-lactose AP 99% (Campro Scientific GmbH, Germany) with 12C-lactose) or naturally enriched 13C lactose (atom percent: 1.097%; Hanze Nutrition, Groningen, The Netherlands) dissolved in 250 mL tap water, patients exhaled through a straw into 2 glass containers (Exetainers® (Labco Ltd., Ceredigion, UK) every 30 minutes for four hours. They were asked to refrain from strenuous physical activity and smoking during the test.
The analytical procedures used by our group to measure expiratory H2, 13CO2, CH4 and CO2 concentrations have been explained in detail elsewhere 11. In short, breath concentrations of H2, CH4 and CO2 were quantified in a single run using a gas chromatograph (GC, Trace GC Ultra, Thermo Scientific, Pittsburgh, PA, USA) coupled to a thermal conductivity detector for quantification of H2 (TCD, Thermoscientific, Pittsburgh, PA, USA) and a flame ionization detector (FID) for quantification of CH4 and CO2 (FID, Thermo Scientific, Pittsburgh, PA, USA). CO2 is measured as CH4 by the coupling of a methanizer in front of the FID. The 13CO2-content in breath samples was analyzed using a continuous flow isotope ratio mass spectrometer (IRMS, ABCA, Sercon, Crewe, UK) and expressed as δ13PDB value.
Cutoffs used for lactose malabsorption were a rise of H2 >20ppm above baseline or a cumulative 13C excretion of ≤14.5% 4 hours after test begin5,11. A rise of CH4 ≥10ppm above baseline was used as cutoff for CH4 as this is the cutoff previously reported in literature 6,10.
Genetic testing
Buccal mucosa was swabbed by the participants and the swabs sent to the laboratory for further analyses. DNA from swabs was extracted using the Magcore® Genomic DNA Tissue kit on a Magcore® HF16 Plus (RBC Bioscience) system and the genotype was assessed using a commercially available kit for the detection of the variant c.1917+326C>T (13910C*T) (LightMix® in-vitro diagnostics kit, TIB MOLBIOL, Cat no: 40-0307-64)).
Lactase non-persistence
We used the presence of the 13910C constellation as standard reference for lactase non-persistence and assessed the performance of the breath tests versus genetic testing to accurately identify individuals with lactase-non persistence 3,4.
Statistics
All statistical analyses were carried out using RStudio (RStudio 2022.07.1+554 "Spotted Wakerobin" Release (7872775ebddc40635780ca1ed238934c3345c5de, 2022-07-22)). Descriptive statistics are presented as median and interquartile range as values were not normally distributed. Performance of the respective tests were assessed with the pROC package (Version 1.18.0) using receiver operating characteristics (ROC) curves with the presence or absence of 13910C constellation as the reference test for lactose malabsorption (i.e. lactase non-persistence). Optimal cutoffs for maximal sensitivity and specificity were determined calculating the Youden index12. Performance of ROC curves were quantified using the area under the curve (AUC). AUC values take values from 0 to 1 with a value of 0 indicating a perfectly inaccurate test and 1 a perfectly accurate test13. A value of 0.5 suggests the test does not have any discriminatory ability. Values from 0.7-0.8 can be considered acceptable, 0.8-0.9 excellent and values above 0.9 can be considered outstanding 13. 95% confidence intervals (95% CI) were calculated by performing 2000 stratified bootstrap replicates.
Results
A total of 151 subjects completed the lactose breath test and underwent genetic testing. The baseline characteristics are provided in table 1. Median results of peak H2, peak CH4 and cumulative 13CO2 excretion are shown in table 2.
Table 1. Baseline characteristics of study participants.
| Overall | Adult | Pediatrics (<18 ys) |
|||
|---|---|---|---|---|---|
| N | 151 | 88 | 63 | ||
| Female (n) | 91 | 57 | 34 | ||
| Median age in years (IQR) | 26 (34) | 44 (25) | 12 (7) | ||
| Median BMI in kg/m2 (IQR) | 21.6 (6.8) | 24.5 (6.1) | 18.7 (4.5) | ||
| Lactase non-persistence (n) | 50 | 21 | 29 |
Table 2.
Values of breathtest parameters according to genotype. Presented are median and (interquartile ranges). Wildtype= homozygous 13910C, Heterzygous= heterozygous 13910T and 13910C, Homozygous=13910T homozgous
| Genotype | Peak H2 | Cumulative 13CO2 | Peak CH4 |
|---|---|---|---|
| Lactase non-persistant (13910C homozygous) | 45 ppm (18- 68 ppm) | 12,72% (7,62-18,47%) | 0.20 ppm (0.04-1.13 ppm) |
| Lactase persistant (13910T / 13910C heterozygous) | 1 ppm (0- 6 ppm) | 13,90% (11,33-19,90%) | 0.22 ppm (0.08-2.65 ppm) |
| Lactase persistant (13910T homozygous) | 2 ppm (0- 5 ppm) | 14.81% (11,24-20,72%) | 0.26 ppm (0.08-2.66 ppm) |
Genetic testing
Based on genetic testing, 33% (50/151) of the participants presented 13910C, i.e. lactase non-persistence (24% (21/88) in adults, 46% (29/63) in the pediatric population (<18ys)).
Peak H2 excretion
Overall performance of H2 measurement was excellent with an AUC of 0.885 on the ROC (95% CI 0.811-0.946). H2 measurement using a cutoff of peak H2 >20ppm above baseline had a true positive rate of 72% (71% in the adult, 72% in the pediatric population). Using this cutoff resulted in a specificity of 96% (95% CI 92-100%), sensitivity 72% (95% CI 58-84%), positive predictive value (PPV) of 90% and negative predictive value (NPV) of 87%.
Performance of 13 CO2 and CH4 measurement in H2 negative individuals
For further analyses, performance of 13CO2 and CH4 versus genotype for lactose non-persistence was assessed in all subjects with a negative H2 test- i.e. a peak H2 excretion of ≤20ppm above baseline.
In individuals with a negative H2 test (111/151), the 13CO2 cumulative excretion performed excellently with an AUC of 0.852 (95% CI 0.739-0.938). Sensitivity for the usually used cutoff of cumulative excretion <14.5% was 93% (95% CI 79-100%), specificity 51% (95% CI 40-61%). The optimal cutoff overall was <12.62% with a sensitivity of 93% (95 % CI 79-100%) and specificity of 70% (95% CI 61-78%) in individuals with a normal H2 excretion. In adults, the optimal cutoff was 8.81% (sensitivity 100% 95% CI 100-100%, specificity 88% 95% CI 78-95%); in the pediatric population the optimal cutoff was 12.62% (sensitivity 88% 95% CI 63-100%; specificity 73%, 95% CI 58-88%).
In the same group (i.e. individuals with negative H2 breath tests), we also assessed the performance of measuring CH4.
The ROC curve of peak CH4 above baseline had an AUC of 0.542 (95% CI 0.384-0.711). Using a threshold of ≥10ppm above baseline resulted in a sensitivity of 14% (95% CI 0-36%), a specificity of 91% (95% CI 85-96%). The optimal cutoff was 0.185ppm with a sensitivity of 57% (95%CI 29-86%), specificity of 41% (95%CI 31-51%). Values for NPV and PPV are provided in table 2.
Thus, in our cohort, of the individuals with lactose malabsorption not diagnosed using the H2 excretion alone (9% of the total initial cohort, 28% of all lactase deficient individuals), 93% (13/14) were accurately diagnosed using a cumulative 13C excretion of less than 12.62% while 7% (1/14) remained undetected. Using this approach, 30% (29/97) of the H2 negative individuals were false positive. Using the proposed cutoff of peak CH4 >10ppm, 14% (2/14) of the individuals with lactose malabsorption were accurately diagnosed and 9% (9/97) were false positive- using a cutoff of 0.185ppm 57% (8/14) of the H2 negative individuals with lactose malabsorption were accurately diagnosed whereas 59% (57/97) were false-positive.
Overall performance
We finally calculated the overall performance of combining the measurement of H2 and the measurement of 13C in the population with normal H2 values. This approach, with a H2 cutoff of ≤20ppm and a cumulative 13C excretion of <14.5% (i.e. a breath test is considered positive if peak H2 ≥ 20ppm, if 13CO2 < 14.5% or both), resulted in a sensitivity of 98%, specificity of 49%, PPV 49% and NPV of 98%.
Applying the identified optimal cutoff of cumulative 13C excretion of <12.6% resulted in a sensitivity of 98%, specificity of 67%, PPV 60% and NPV of 99%.
Discussion
Lactose breath tests with H2 measurement are the most commonly used method to diagnose lactase deficiency in clinical practice in patients with suggestive symptoms after ingestion of dairy products. However, it is well-established that about 30% of individuals with lactase non-persistence do not exhale hydrogen after lactose ingestion resulting in a false negative test. In these individuals 13C or CH4 measurements can be used, but the performance of these tests and the optimal cut-offs are yet to be determined.
In our cohort, 28% of subjects with lactose malabsorption were not diagnosed with H2 measurements alone – this finding is within the range reported in literature 5,9,14. The subjects presenting a false negative H2 measurement is the population of interest in this study as the performance of additional analyses is of interest primarily in this subgroup. Measuring cumulative 13C diagnosed almost all of the individuals with lactose malabsorption missed with H2 alone at the cost of 49% false-positive individuals. The AUC of the ROC curve of 13C in H2 negative individuals was similar to that of H2 measurements for the entire cohort. In absolute measures, this led to the correct identification of 13 individuals in our cohort who might benefit from a dietary adaptation. The performance of measuring peak CH4 on the other hand was inferior with an AUC close to a random classifier. Both the proposed cutoff of 10ppm and the optimal cutoff of 0.185ppm were inferior to 13C measurements and hardly led to additional diagnoses or high rates of false positives, respectively. Of note, the identified optimal cutoff of 0.185 ppm is of merely hypothetical value, obtained by calculating the Youden index –measurement of CH4 below 1ppm is technically not feasible. We assessed the commonly reported cutoff of 10ppm but it has to be acknowledged that no uniform cutoff value has been agreed upon in international consensus5, however the identified optimal cutoff also performed poorly. Thus, in this observed cohort the measurement of CH4 was of no additional benefit. Recently, the added value of measuring CH4 has been reported only when adding the values of CH4 with the H2 values together 15. We did not assess the performance of combining values as to the best of our knowledge there are no recognized cutoffs for this approach but this is potentially a more promising approach than measuring CH4 peak values alone.
From a clinical standpoint, high sensitivity and specificity in the lactose breath test are of importance as both ruling in and ruling out of lactose malabsorption have clinical implications. Accurately ruling in the diagnosis allows a dietary modification or intake of lactase-containing capsules thus improving symptoms in many patients. Accurately ruling out the diagnosis avoids the subject from undergoing a restrictive and likely ineffective and more expensive lactose-free diet and allows further diagnostic steps in deciphering the origin of food-related symptoms such as functional disorders or sensitivity to FODMAPs. Adding the measurement of 13CO2 effectively diminished false negative results of the lactose breath test by detecting H2 negative individuals with lactose malabsorption – albeit at the price of a high rate of false positive results. Interpreting these results and weighing the advantages of virtually eliminating false negative results versus increasing false positive results remains a clinical challenge and will certainly depend on the clinical setting. While the proposed values presented here suggest that measuring 13CO2 increases the diagnostic yield of the lactose breath test, real life performance characteristics and optimal cutoffs require validation in an all-comer cohort that has not been selectively recruited.
Furthermore, it is important to emphasize that lactose malabsorption will not result in lactose intolerance in all subjects and that dairy products might lead to symptoms in absence of lactose malabsorption – several factors may contribute including visceral hypersensitivity and possibly local allergic reactions in some patients5,16,17. Of note, these conditions would likely not benefit from a diet merely excluding lactose but would require a broader reduction of non-absorbed carbohydrates or dairy proteins respectively. Clinically approaching this complex interplay of various factors thus remains a challenge in the daily routine and research unravelling the mechanisms of food-related symptoms is ongoing. Most recent European guidelines pragmatically suggest that a restrictive diet should only be recommended when the relationship between ingestion of lactose and symptoms has been established5.
This study has some limitations. First and foremost, the recruitment strategy of retrospectively contacting patients with pathological results hampers the generalizability of the results, especially with respect to negative- and positive predictive values. Thus, the result merely serves as a proof of concept that using 13C labelled lactose may substantially improve detection rates of lactase deficiency in breath tests in individuals without rise in H2.
The strength of this study lies in the use of genetic testing as a gold standard in this large sample size comprising of adult and pediatric subjects. Furthermore, using a stratified bootstrapping approach with 2000 replicates allowed us to calculate confidence intervals which are typically not available from ROC statistics alone, further reinforcing the validity of our results.
In conclusion, in this group of subjects referred for a lactose breath test, additional measurement of 13CO2 aided in the diagnosis of lactose malabsorption in H2 negative individuals. In this group, it outperformed CH4 measurements. Larger studies done in all-comers are required to confirm this finding and determine optimal cutoff values.
Figure 1. ROC curves for 13C and CH4 in H2 negative individuals.
Table 2.
Performance parameters of 13CO2 and CH4 using various cutoff values in the subgroup with a normal H2 breath test. 95% confidence interval in brackets, NPV= negative predictive value, PPV= positive predictive value
| Peak H2 excrection ≤20ppm | ||||
|---|---|---|---|---|
| Cutoff for Lactose malabsorption | Sensitivity | Specificity | NPV | PPV |
| Cumulative 13C ≤14.5% | 93% (79%-100%) | 51% (40%-60%) | 98% | 21% |
| Cumulative 13C ≤12.62% | 93% (79%-100%) | 70% (61%-78%) | 99% | 31% |
| Peak CH4 ≥10ppm | 14% (0%-36%) | 91% (85%-96%) | 88% | 18% |
| Peak CH4 ≥0.185ppm | 57% (29%-86%) | 41% (31%-51%) | 87% | 12% |
Acknowledgements
The authors wish to thank Geert Verbeke and Steffen Fieuws for statistical assistance and Anniek Corveleyn Bruno Vankeirsbilck, Nicole Gorris, Christine Dewit, Helga Ceulemans en Nicole Pieters for technical assistance.
Funding
LMB received funding from a Postdoc.Mobility grant from the Swiss National Science foundation. TV is supported by a senior clinical research mandate of the Flanders Research Foundation (FWO Vlaanderen). JT received funding through a Methusalem grant from the KU Leuven.
Footnotes
Conflicts of interest: None
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