Breath tests for the assessment of gastroparesis

Introduction

Many providers are often faced with the decision of selecting the best test available from their armamentarium to diagnose gastroparesis. This can be challenging with recent evidence showing poor correlation between symptom relief and gastric emptying . There are currently many options on the market, as discussed in other chapters. The Breath Test for gastroparesis is one of the newer tests for gastroparesis available to practitioners, and therefore, one of the least well known. Given its potential utility and efficacy, the Breath Test may supersede Scintigraphy for diagnosing gastroparesis. This chapter will serve as a review of the history of the Breath Test, how it is applied clinically, and its disadvantages and advantages as it relates to its competitors. It will also provide a basic understanding and describe the current utilization of the Breath Test for diagnosing abnormalities in gastric emptying.

History of breath testing in medicine

Analysis of a patients’ breath for detection of various diseases goes back to the time of Hippocrates, the pioneer of medicine. He asked his students to identify diseases by the smell of a patients breath – for example, the odor of rotten apples suggested diabetic ketoacidosis and a urine like odor to the breath suggested renal failure. In the 1770s, Antoine Lavoisier a French chemist discovered the presence of CO ₂ in breath. The adaptation of these findings into medicine specifically, gastroenterology, began during the latter part of the 19th century. Shortly thereafter, Linus Pauling’s milestone discovered the presence of 250 distinct substances within a single exhaled breath, hence presenting hopeful insight into breath testing , thus considered the starting point in the development of exhaled breath analysis.

The physiology behind breath testing

The exhaled human breath consists of well over 3000 different volatile organic compounds (VOCs), and an entire breath cycle consists of around 500 various VOCs, which are reported in part per million (ppm) or part per billion (ppb) . The exhaled breath has long been considered a “breath-print” which can be as unique a fingerprint, which is commonly used as a personalized key. The basic principle of breath testing is that this uniform and standard ratio of substances excreted in a single breath can only change in certain diseased states. The conventional methods for detecting these changes are based on spectrometry techniques, although recently there has been the introduction of gas sensors . While the pathophysiology and therefore methodology of each available test is different, the basic principle remains the same. This principle is that there are abnormal ratios of a certain compound measured in the exhaled breath when there is altered human physiology. This compound is produced by the metabolization of a substrate either in the intestines or in the liver. The product of this reaction contains the gas which is labeled with the stable isotope (Urea and Gastric Emptying Breath test). In the case of the hydrogen breath test there is no tracer, but the test relies on the principle that hydrogen is only made from bacterial fermentation in the gut. The same applies to methane which is a gas excreted by some but not all patients. The gas produced is then transported to the lungs via the blood stream and from the blood it is exchanged by diffusion though the pulmonary alveolar membrane. This exhaled air can easily and painlessly be obtained and analyzed as seen in Fig. 13.3 .

Nuances to remember regarding this process, would be that the first portion of the patient’s exhalation is dead space whereas the latter portion contains alveolar air which contains most of the valuable information . In addition, the anatomy of all breath tests rely on the appropriate function of multiple organ systems (at least the lung, liver, intestine and stomach) and therefore excludes certain patient populations from its usage. Regardless, the field of breath testing continues to grow enormously with evolving technologies in sampling, sensor design, standardization, and analytical methods breath analysis. This is due in part to its simplicity in application.

Current breath tests in clinical practice for gastroenterology

Breath tests have been an important tool for decades for gastroenterologists to explore a number of prevalent diseases related to the gastrointestinal tract. The most common breath tests used can be subdivided into two categories: The Urea Breath Test and The Carbohydrate Breath tests.

The Urea Breath Test (UBT) is the most accurate and best validated of the currently available breath tests . It is a very well-known tool used to diagnose H pylori since the early 2000s. Many diagnostic methods have been developed over the past 2 decades to detect Helicobacter pylori (H pylori) infection—some invasive (rapid urease test, histology, culture, and polymerase chain reaction) because they cannot be performed without endoscopy, and others non-invasive (serology, UBT and more recently, fecal H pylori antigen). The UBT relies upon the oral administration of urea labeled with ¹³ C to identify urease, an enzyme produced in large amounts by H pylori. Urease cleaves the labeled urea to ammonia and labeled bicarbonate, which is subsequently converted to labeled carbon dioxide that is then measured in exhaled breath. The ratio of carbon dioxide would therefore be increased given to its increased production by the bacteria.

The Carbohydrate Breath Test takes advantage of different naturally occurring substrates to assess orocecal transit, carbohydrate malabsorption, or to diagnose patients with small intestinal bacterial overgrowth (SIBO) . To effectively evaluate these disorders the testing depends on the ability of intestinal bacteria to metabolize various carbohydrate substrates (glucose, lactulose and fructose) by releasing hydrogen and/or methane and resulting in the release of quantifiable levels of these gases in exhaled air from the lungs .

The Lactulose Hydrogen Breath test can be used to diagnose Oro-cecal Transit Time, SIBO and lactose intolerance . The Glucose breath test has become more utilized for the diagnosis for SIBO, due to its increased sensitivity. Under normal physiological conditions, glucose is readily absorbed in the small intestine however if there is presence of bacterial overgrowth the flora ferments the glucose prior to it being absorbed. Hence, a breath test showing an inappropriate rise in the hydrogen after consumption of a meal is consistent with the diagnosis of SIBO. The Lactose Breath test uses the principle that the disaccharide is metabolized by the brush border enzyme lactase to glucose and galactose . Reduction or absence of this enzyme leads to excessive exposure of the colon to lactose, where fermentation can result in excessive gastrointestinal symptoms. Fructose is a naturally occurring monosaccharide found in fruits as well as in the disaccharide sucrose. The Fructose breath test is performed to evaluate fructose intolerance. Malabsorption of fructose can lead to its fermentation in the large bowel by the natural flora.

The development of the gastric emptying breath test

While scintigraphy has historically remained the gold standard in confirming abnormal gastric emptying there remained a need for a radioactive free and cheaper alternative. Choi et al, compared simultaneous scintigraphy and ¹³ C-octanoic acid breath test among 15 healthy volunteers and demonstrated that although widely different values were observed in some subjects, repeated breath tests showed a high degree of reproducibility within individuals .

Szarka et al, demonstrated that ¹³ C-Spirulina platensis GEBT is as reproducible as scintigraphy; and that imprecision with both tests reflects physiologic variation . Gastric emptying can be divided into normal, accelerated as seen in dumping syndrome or decelerated such as in gastroparesis. These rates can be affected in healthy subjects with usage of atropine and erythromycin. A recent meta-analysis found that promotility agents can significantly accelerate gastric emptying and to produce significant improvements in UGI symptoms .

The American and European Neurogastroenterology and Motility Society concluded that ¹³ C-GEBT is a simple, safe, radiation-free and validated test for assessing gastric emptying .

The FDA approved the GEBT as a diagnostic tool for gastroparesis in 2015. This was after they performed their rigorous validation trial in which the overall diagnostic agreement between the GEBT and gastric scintigraphy was established . A direct comparison can be seen in Table 13.1 . This trial also allowed for establishing the time points used in the current GEBT. Effectiveness of the GEBT was based on the comparison of the test vs scintigraphy at six different time points. This study also showed a relationship of higher concordance at earlier time points ranging from 77% to 87% at 45–150 minutes post GEBT meal ingestion. Furthermore, the GEBT demonstrated specificity values of 89–98% between 45 and 240 minutes and Positive Predictive Values (PPV) values of 73–97% between 45 and 240 minutes across all time points post meal ingestion (The values can be seen depicted in Table 13.2 ; demographics Table 13.3 ). This reiterates the importance of these time points when interpreting each patients results since these were the points of highest concordance and PPV. The GEBT is currently available nationwide (Cairn Diagnostics, Brentwood, TN). To date, it has been in use in community and university based practices nationally, and currently gathering recognition among practitioners

Table 13.1

A direct comparison of the GEBT to its competitor nuclear scintigraphy for evaluating gastroparesis.

Parameter	The GEBT	Scintigraphy
Test meal and active ingredient	Powdered scrambled egg meal containing non-radioactive ¹³C-labeled Spirulina	Radio-labeled food (typically egg) Technetium (Tc)-99m sulfur colloid
Data acquisition	To provide breath samples, patients blow through a straw into screw-capping tubes before ingesting the test meal and at intervals after	Gamma cameras take images in anterior and posterior positions
Time intervals measured	0 (pre-meal), 45, 90, 120, 150, 180 and 240 min	Typically 0, 60, 120, 240 min
Setting of care	Physician’s office/clinic Clinical lab patient service center Motility clinic	Nuclear medicine department
Analysis and quantification of gastric emptying	The patient’s ¹³C ₂excretion rate is determined at each post-meal measurement time by gas isotope ratio mass spectrometry; the ¹³C ₂excretion rate is proportional to the amount of meal emptied from the stomach	The amount of radio-labeled food emptied from/retained in stomach is measured at given time intervals
Setting of analysis	Samples shipped to manufacturers lab results in 24–48 h	Analysis of radio-labeled emptying conducted at imaging facility
Reporting of results	Graphic display of kPCD results over the course of 4 h ¹³CO ₂excretion rates using kPCD metric: KPCD=1000 x [percent ¹³C dose excreted as ¹³CO ₂	Scintigaphic fraction of test meal emptied at each time point over 4 h Scintigraphic t _1/2

Table 13.2

The results of the FDA validation trial Comparing the GEBT to GES at distinct time points after meal consumption.

			Time	Points
Classification (N=115)	45	90	120	150	180	240
True positive	16	30	31	29	20	11
True negative	84	63	57	60	57	67
False positive	6	2	1	2	7	2
False negative	9	20	26	24	31	35
Performance statistics (%)
Specificity	93.3	96.9	98.3	96.8	89.1	97.1
Sensitivity	64	60	54.4	54.7	39.2	23.9
Concordance	87	80.9	76.5	77.4	67.0	67.8
Positive predictive value	72.7	93.8	96.9	93.5	74.1	84.6
Negative predictive value	90.3	75.9	68.7	71.4	64.8	65.7

Table 13.3

Demographics of the patients used in the FDA validation trial.

Subjects (134 screened, 132 enrolled, 129 completed test)	New Motility Clinic Patients = 99 Previously tested motility clinic patient = 24 Healthy subjects (atropine-induced) = 5 Asymptomatic diabetics (Type 1 or II) = 1
Age demographics of enrolled subjects	Range 21–77 Median age 50
Sex demographics of enrolled subjects	Male 37 Female 95
Race demographics	American Indian/Native Alaskan = 1 Asian = 1 Black = 2 White = 127 Other = 1

The chemistry of gastric emptying breath test

In recent years, breath tests using stable isotopes have been developed to reliably assess gastric emptying of both the solid phase ( ¹³ C-Spirulina or S. platensis) and liquid phase ( ¹³ C-acetate breath test). S. platensis (synonym Arthrospira platensis) is an edible, naturally abundant, nutritionally enriched filamentous, water cyanobacterium used as a protein source in many parts of the world and used as a food supplement in many health stores in the United States . S platensis, which contains 98.9% ¹² C and 1.1% ¹³ C, was acknowledged by the FDA as a legally marketed food in 1981 and is recognized as safe when contained in foods at levels from 0.5 to 3.0 g per serving. S platensis is well known and marketed for its claims in anti-oxidant and anti-cancerous properties as well as its ability to amend the carcinogen damaged DNA . It is made up of 50–60% protein, 30% starch and 10% lipid . The natural level of ¹³ C in S platensis and in all living things is approximately 1%. Despite its safety and nutritional value, only 0.1 g of S platensis per test meal is required for the procedure .

S. platensis can be labeled with two separate isotopes of carbon: ¹⁴ C and ¹³ C. The earliest versions of the UBT (for Helicobacter Pylori) used ¹⁴ C however ¹³ C has now superseded and become the more commonly used isotope for breath tests to date . The main reason being that ¹⁴ C is a radioactive and although the radiation dose delivered by the standard ingested activity is equal to or less than daily background radiation (and therefore, scintigraphy) radiation mistrust can be an issue with certain patients and providers. Due to its radio-activity, testing with ¹⁴ C requires an available nuclear department (with a scintillation counter), certain licensures for storage/disposal, shipping issues, and a large quantity of tracer required to perform larger studies. In contrast, ¹³ C is a non-radioactive/stable isotope that can be measured by mass spectrometry, easily delivered using the mail system and safely used for repeated testing in children and fertile females (allowing for large scale epidemiological studies). The advantages of ¹⁴ C (which would be lost with the use of ¹³ C) would include its significantly lower price and that it does not require a test meal, use of a gelatin capsule would suffice. Both ¹³ C and its measurement tool (mass spectrometry) are also more expensive. However, another future advantage of ¹³ C would be the ability to administer the test at home given its stability and ease of distribution through a courier service. Thus ¹³ C is used for the GEBT.

The S. platensis for the GEBT procedure is typically grown as a pure monoculture in a closed hydroponics chamber in a medium containing inorganic salts, and purged with pure ¹³ CO ₂ . The result of which are cells that are uniformly labeled with ¹³ C. The natural level of ¹³ C in the environment is approximately 1% this is raised to 99% in the substrate. When metabolized, the proteins, carbohydrates and lipids of the S. platensis then give rise to respiratory CO ₂ that is enriched in ¹³ C which can be detected by mass spectrometry such as in Fig. 13.4 .

The conduit used for the ¹³ C-labeled S. platensis consists of egg whites combined with 200 mg of ¹³ C–S. platensis. Since the contents of the algal cells are not freely diffusible, incorporation of labeled S. platensis into egg whites provides a means to assess the emptying of the solid phase of the meal. Thus, ¹³ C can only be released from the algal cells after the egg white is emptied, the cells are digested, and the ¹³ C-labeled substrates (protein, lipid, carbohydrate) absorbed. In this way, ¹³ C-S platensis gives rise to respiratory CO ₂ that is saturated with ¹³ C. The egg meal is cooked and placed on a slice of whole-wheat bread (substitutions include saltine crackers) , and is consumed with a glass of skimmed milk or water for a total caloric value of no more than 220–240 kcal with a nutrient composition of 35% protein, 40% carbohydrate and 25% fat . The quantity and quality of the meal is selected to guarantee the meal remains stable at room temperature, palatable, and has a calorie content that would be consumed entirely, even by patients with suspected gastroparesis and upper abdominal symptoms (nausea, vomiting, bloating, post-prandial fullness, pain).

To avoid false negative and false positive results, the instructions and preparations for the GEBT are similar to that of scintigraphy. The patient must be fasted overnight or at least 8 hours as to not “dilute” the substrate which can give rise to false negative results. Furthermore, these patients are at risk for emesis and so an empty stomach may reduce these risks. It is important to counsel patients on the avoidance of medications that alter emptying rates (anticholinergics, narcotics, erythromycin, metoclopramide and domperidone) for at least 48–72 hours prior to the test . The use of proton pump inhibitors less than 2 weeks before the urea breath test has been well documented to adversely affect the sensitivity of the UBT . The mechanism by which it causes a false negative result still remains ill-defined. Due to the theoretical change in metabolism and absorption of the test meal in the presence of a pH altering medication we recommend stopping the proton pump inhibitor 2 weeks prior to administering the test. The most common reasons overall for GEBT failure include subjects vomiting or inability to complete the test meal and failure to collect the samples at the time marks outlined, so careful attention is needed to avoid these mishaps .

Before the meal is prepared, the baseline breath samples are obtained in two of the glass capped test tubes provided. Two separate samples are taken in case one is damaged in travel; as it is imperative to have at least one baseline sample for comparison. After the test meal is administered, the breath samples are taken following the same sequence as scintigraphy, hence45, 90, 120, 150, 180 and 240 minutes from the end of test meal consumption. The samples are then returned by courier to the central laboratory for analysis by mass spectrometry to determine the change in the ratio of ¹³ CO ₂ / ¹² CO ₂ (reported as a delta value) from baseline in each test tube . After calculating the delta value over time and comparing it to the patient’s pre-meal baseline sample the rate of ¹³ CO ₂ can be computed and therefore the patients gastric emptying rate. If the institution has a mass spectrometer and the expertise to use and interpret the results, the calculations can be done in house. Tables 13.4–13.7 have summarized the instructions and administration of the GEBT.

Table 13.4

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