Dynamic carbon 13 breath tests for the study of liver function and gastric emptying

Abstract

In gastroenterological practice, breath tests (BTs) are diagnostic tools used for indirect, non-invasive assessment of several pathophysiological metabolic processes, by monitoring the appearance in breath of a metabolite of a specific substrate. Labelled substrates originally employed radioactive carbon 14 (¹⁴C) and, more recently, the stable carbon 13 isotope (¹³C) has been introduced to label specific substrates. The ingested ¹³C-substrate is metabolized, and exhaled ¹³CO₂ is measured by mass spectrometry or infrared spectroscopy. Some ¹³C-BTs evaluate specific (microsomal, cytosolic, and mitochondrial) hepatic metabolic pathways and can be employed in liver diseases (i.e. simple liver steatosis, non-alcoholic steato-hepatitis, liver fibrosis, cirrhosis, hepatocellular carcinoma, drug and alcohol effects).

Another field of clinical application for ¹³C-BTs is the assessment of gastric emptying kinetics in response to liquids (¹³C-acetate) or solids (¹³C-octanoic acid in egg yolk or in a pre-packed muffin or the ¹³C-Spirulina platensis given with a meal or a biscuit). Studies have shown that ¹³C-BTs, used for gastric emptying studies, yield results that are comparable to scintigraphy and can be useful in detecting either delayed- (gastroparesis) or accelerated gastric emptying or changes of gastric kinetics due to pharmacological effects. Thus, ¹³C-BTs represent an indirect, cost-effective and easy method of evaluating dynamic liver function and gastric kinetics in health and disease, and several other potential applications are being studied.

Introduction

Breath tests (BTs) are diagnostic tools based on the ingestion of various substrates that are processed at different levels in the gastrointestinal tract [1]. The principle of BTs relies on the concept that the metabolized substrate leads to the production of gases (e.g. CO₂, H₂) that pass into the blood, are excreted and quantified in expired air. The interest towards the use of BTs has increased since they are relatively simple, safe and non-invasive tools with potential applications in several clinical conditions including liver diseases, H. Pylori infection, gastrointestinal motility, small intestinal bacterial overgrowth, and sugar (fructose, lactose) malabsorption. BTs using specific substrates labelled with the stable (non-radioactive) isotope ¹³C have been also employed for the assessment of hepatic functional reserve and to measure gastric emptying. The potential applications of such ¹³C-BTs will be discussed in the present paper.

¹³C breath tests for the study of liver function

General features

A major challenge in clinical hepatology is the accurate and non-invasive assessment of liver function in patients with chronic liver disease. The evaluation of liver status is currently based on serum parameters of synthesis (prothrombin, cholesterol, albumin), hepatocellular integrity (transaminases), detoxification (ammonium), excretion and cholestasis (bilirubin, alkaline phosphatase, γGT), associated with imaging techniques (ultrasonography, computerized tomography, magnetic resonance and elastography) or scores such as the Child-Turcotte-Pugh classification, which combines clinical (ascites and degree of encephalopathy) and serum (bilirubin, albumin, and prothrombin time) parameters [2]. Such ‘static' tests of liver function may have limitations, especially when dealing with prediction of outcomes and in assessing liver dysfunction in critically ill patients [3]. In this respect, BTs represent novel indirect ‘dynamic' tools that provide additional insights in functional diagnosis and follow-up of patients with liver diseases [4].

The principles of BTs in hepatology are based on both biochemical and pharmacological considerations. Mechanisms of liver damage often include dysfunction of subcellular organelles, such as microsomal hypertrophy, mitochondrial abnormalities, and activation of peroxisomal metabolism (i.e. long chain fatty acids). Thus, assessing specific functions of such organelles through BTs may provide useful information to clinicians. Also, BTs allow the study of specific time-dependent metabolic processes by assessing the hepatic clearance of metabolically active substances [5]. In this context, for a given exogenous substrate:

$$\text{HEPATIC CLEARANCE}=\text{HEPATIC PERFUSION}\times \text{HEPATIC EXTRACTION}$$

(where HEPATIC EXTRACTION is the ratio of the difference between inflow and outflow concentration ÷ by inflow concentration of the probe) [6].

Hepatic clearance is defined as flow-limited (range 0.7–1.0) or enzyme-limited (<0.3) [7]. The general characteristics of the ‘ideal' substrate for the study of liver function are depicted in Table 1 [8].

Table 1.

General characteristics required of an ideal substrate for studying dynamic liver function

Pharmacokinetic and metabolic aspects

Rapidly and consistently absorbed when administered orally

Primarily metabolized in the liver

Low (20–30%) hepatic extraction ratio (i.e. metabolism  independent from liver blood flow)

Clear metabolic pathway; simple pharmacokinetic; short  elimination half-life

13CO2 generated should be distributed in the body, not  compartmentalized

Methodological aspects

Safe

Simple to prepare and administer

No- or minimal interaction with extra-hepatic tissues  (i.e. adipose tissue or muscle)

Reproducible over time and repeatable (useful for follow-up)

Costs

Low-priced

The intrinsic complexity of liver metabolic pathways does not allow a single functional test to explore the whole liver function. Different substrates are therefore used to assess cytosolic, microsomal or mitochondrial function (Figure 1). Such substrates are marked with the natural stable isotope of carbon, carbon 13 (¹³C; currently the most widely used isotope). After intestinal absorption, the given substrate undergoes liver metabolism at different levels, which ultimately results in the production and appearance of ¹³CO₂ in exhaled air, as a marker of specific liver metabolic functions [4, 9] (Figure 2).

Figure 1.

Sites where metabolic processes may be explored by breath test in hepatocytes. In particular, ¹³C-α-ketoisocaproic acid, ¹³C-methionine, and ¹³C-octanoate are the three substrates more widely employed for the dynamic assessment of mitochondrial function (see text for details).

Figure 2.

General methodology of breath test analysis using ¹³C-labelled substrates for the dynamic study of liver function and of gastric emptying, both depending on time-dependent concentration of exhaled ¹³CO2. The estimation of liver function is accurate if gastric emptying, duodenal absorption, portal transfer of the substrate to the liver, ¹³CO₂ distribution in the body compartments, and lung function are preserved. The estimation of gastric emptying is accurate if liver function is also preserved.

Breath tests for the study of liver cytosolic function

Phenylalanine is an aromatic amino acid, converted to tyrosine in the cytosol of the hepatocytes by the enzyme phenylalanine hydroxylase. Phenylalanine oxidation is affected when liver function is impaired and by using a ¹³C-phenylalanine BT, a reduced ¹³CO₂ release in the exhaled breath is recorded [10, 11]. The ¹³C-phenylalanine BT has been used to predict post-operative complications and to monitor liver regeneration after partial hepatectomy, and to predict the severity of liver cirrhosis, staged according to the Child-Turcotte-Pugh score [12]. However, in a study by our group that compared the hepatic functional mass in chronic liver disease classified according to Child-Turcotte-Pugh score and serum bile acid levels, the diagnostic power of ¹³C-phenylalanine BT was less than that of ¹³C-methacetin BT [13].

Galactose is a carbohydrate primarily metabolized by the liver. The metabolism of galactose investigates the activity of galactose kinase, which catalyses the ATP-dependent phosphorylation of galactose to galactose 1-phosphate. Originating from the radioactive carbon 14 (¹⁴C)-galactose BT, the non-radioactive ¹³C-galactose BT has been also developed [14]. The performance of the ¹³C-galactose BT relates inversely with the severity of liver disease—and in particular with cirrhosis—reaching 71% sensitivity, 85% specificity, and 84% accuracy [15, 16]. Combination with another BT investigating the microsomal function (aminopyrine) further increases the diagnostic sensitivity and specificity [15]. The ¹³C-Galactose BT has also been used to assess progressive decline in liver mass function in patients with chronic liver disease due to Hepatitis C virus (HCV) infection [17].

Breath tests for the study of liver microsomal function

Aminopyrine is known as an antipyretic analgesic drug. The ¹³C-labelled methyl groups of aminopyrin are demethylated with transformation into formate, formaldehyde, and the produced bicarbonate releases ¹³CO₂ which is exhaled in breath. After oral administration, aminopyrine is completely absorbed and has a low hepatic extraction (E = 0.2), seen to be independent of liver blood flow and not disturbed by hepatic vascular shunts. Aminopyrine is metabolized by the hepatic microsomal cytochromes and provides an index of functional hepatic mass. Aminopyrine BTs have been used to provide prognostic information, to study, graft rejection, severity of paracetamol intoxication and to detect alcoholic liver injury. When used to discriminate the degree of liver fibrosis, aminopyrine BT displayed good specificity and sensitivity for advanced fibrosis or cirrhosis but performed poorly for intermediate fibrosis stages [7, 18, 19]. Some limitations need to be considered in the interpretation of aminopyrine BT: (i) the age of the subject can affect the P450-dependent N-demethylation, (ii) the simultaneous use of drugs with enzyme inductive effect or inhibiting the microsomal enzymatic activity might alter the findings of the BT, and (iii) female sex hormones can produce a negative effect on aminopyrine metabolism [6, 20].

Methacetin is a derivative of phenacetin which is metabolized rapidly by the hepatic microsomal enzyme systems CYP1A2 into acetaminophen and ¹³CO₂ by a single O-dealkylation step. Since methacetin has a high extraction (E > 0.8) [21] and undergoes extensive first-pass clearance, its metabolism can be altered by hepatic blood flow alterations and by hepatic 'first-pass' effect. Capacity for metabolizing methacetin is lower in elderly people than in other adults [22]. Methacetin BT was shown to accurately assess the degree of liver damage in patients with histologically proven chronic liver diseases and to distinguish chronic aggressive hepatitis from liver cirrhosis; it also distinguished early cirrhosis (Child A) from non-cirrhotic patients. Methacetin BT was a useful predictive marker of clinical outcomes in chronic HCV patients [23–25]. Moreover, methacetin BT can better estimate the degree of fibrosis in patients with chronic HCV infection than biochemical parameters (i.e. aspartate aminotransferase-to-platelet ratio and aspartate aminotransferase-to-alanine aminotransferase ratio) or Fibroindex [26, 27].

Breath tests for the study of liver mitochondrial function

Ketoisocaproate (KICA) is an intermediate in the metabolism of leucine. The decarboxylation of KICA and the generation of CO₂ reflects the mitochondrial branched-chain amino acid decarboxylation function [28]. This step is observed when transamination to leucine (the major competing pathway for KICA elimination) is suppressed by the simultaneous administration of fixed doses of leucine (Figure 3a). This metabolic pathway of KICA has been tested in experimental models, in isolated mitochondria, in healthy subjects treated with acetyl salicylic acid or with low ethanol intake, and in patients with liver diseases [8, 9, 28, 29]. ¹³C-KICA decarboxylation is lower in alcoholic patients than in patients with non-alcoholic fatty liver disease (NAFLD) or controls [29, 30]. We found that the mitochondrial decarboxylation capacity of KICA was lower in patients with advanced non-alcoholic steatohepatitis (NASH) than in healthy subjects and patients with simple liver steatosis. Notably, the ¹³CO₂ cumulative recovery values following ¹³C-KICA were inversely related to the extent of fibrosis, to serum hyaluronate, and to body size in NASH patients [31]. We extended the studies with ¹³C-KICA BTs and found that KICA decarboxylation was significantly lower in cirrhotic patients with hepatocellular carcinoma (HCC) than in cirrhotic patients without HCC and identical Child-Pugh score. Moreover, KICA decarboxylation was deranged following radiofrequency ablation, but not after transarterial chemoembolization. Finally, the recurrence of HCC was associated with an early decrease of KICA decarboxylation [32]. In a different context, we recently found that a ¹³C-KICA BT was abnormal (and therefore suggesting mitochondrial malfunction) in a female patient suffering from massive liver echinococcosis. Notably, mitochondrial liver function improved following pericystectomy and limited hepatectomy [33]. KICA BT is useful for the assessment of drug effects on liver mitochondrial function. Liver injury might occur following the use of such drugs, which accumulate in the mitochondria and interfere with respiratory complexes or electron transfer [34, 35]. KICA BT may be helpful in ascertaining the integrity of these organelles before the administration of potentially toxic drugs and in detecting drug-induced mitochondrial damage before the appearance of symptoms, in order to manage patients in a timely manner and prevent adverse effects. Examples are tacrolimus, aspirin, and ergot alkaloids. There are also potential applications with amiodarone, valproate, and retroviral drugs [36, 37].

Figure 3.

Mitochondrial metabolism of a) methionine; b) α-ketoisocaproic acid; c) octanoic acid. CO₂ is invariably produced at the end of the process. The use of ¹³C-labelled substrates ultimately leads to production of ¹³CO₂ following mitochondrial metabolism.

Methionine is an essential amino acid, principally metabolized in the liver with production of CO₂ [38]. ¹³C-methionine, either in the form of L-(1-¹³C) methionine or (methyl-¹³C)-methionine, investigates the oxidative capacity of the liver [39] (Figure 3b). In human studies, ¹³C-methionine BT has been used to assess mitochondrial function during acute intoxication and in patients with chronic liver diseases. For example, acute ethanol consumption impairs ¹³C-methionine decarboxylation in healthy volunteers [39], whereas metabolism of methionine is decreased in patients with liver cirrhosis, and especially in those with ethanol aetiology, in patients with NAFLD, in those taking high-dose valproate or nucleoside analogues for the treatment of HIV, and in patients with Friedreich ataxia [37, 40–44]. A defective methionine metabolism has also been reported in hepatitis C-infected cells [45].

Octanoate is a medium chain fatty acid that enters mitochondria independently of the carnitine transport system. Within the mitochondria, octanoate undergoes β-oxidation, which generates acetyl co-enzyme A (AcCoA). AcCoA enters the Krebs cycle and is oxidized to CO₂ unless utilized for the synthesis of other energy-rich compounds (Figure 3c). The ¹³C-octanoate BT has been employed for the study of gastric emptying (see below) but the test should also reflect hepatic mitochondrial function when gastric emptying and duodenal absorption are not severely deranged. Octanoate is therefore a potential substrate for non-invasive BT of hepatic mitochondrial β-oxidation. In fact, in animal models, the decarboxylation of octanoate was decreased in rats developing thioacetamide-induced acute hepatitis and liver cirrhosis [46]. In patients with NASH, the oxidation of octanoate was unchanged or increased [47, 48], and unchanged in those with early stage and advanced cirrhosis with and without porto-systemic shunt [49].

¹³C breath tests for the study of gastric emptying

General features

A subgroup of patients may develop disturbed gastric motility disorders, namely accelerated ('dumping syndrome') or delayed gastric emptying, which is the most frequently investigated condition. Gastroparesis implies a delayed gastric emptying without mechanical obstruction and is a condition frequently associated with symptoms such as nausea, vomiting, bloating, early satiety, and or upper abdominal pain [50]. After excluding mechanical obstruction by means of upper endoscopy, computed tomography, barium follow-through examination or magnetic resonance enterography, the diagnosis of gastroparesis is based on functional studies to assess gastric motility in response to liquids and/or solid test meals. Investigations include the traditional scintigraphic studies [50], functional ultrasonography [51, 52], magnetic resonance imaging and, recently, wireless motility capsule [53] and BTs (see below). Major limitations of such techniques consist of radiation hazards (scintigraphy), duration of the exam and experience of the operator (ultrasonography), and costs (magnetic resonance imaging and wireless capsule). To establish the aetiology of gastroparesis a further step should take into account the idiopatic form (affecting about half of the patients), diabetes mellitus, and post-surgical, viral, neurological, autoimmune, viral causes, scleroderma, as well as effect of medications (e.g. calcium channel blockers, dopamine antagonists, octreotide, etc.). To establish the aetiology, laboratory tests, gastroduodenal manometry, autonomic testing and additional studies—including single photon emission computed tomography and full thickness biopsies of the stomach and small intestine—might be necessary from case to case.

Breath tests for the study of gastric emptying

Gastric emptying has also been investigated by BTs using the radioactive isotope ¹⁴C and more recently, the non-radioactive stable isotope ¹³C, both labelling substrates dissolved in liquids (acetate in water or fruit juice) or solid test meals (octanoic acid in egg yolk). Octanoic acid, a medium-chain fatty acid fully retained in egg yolk during mixing and grinding in the stomach, is rapidly liberated in the duodenum and quickly transported to the liver via the vena portae. The ¹³C-octanoic acid rapidly enters the mitochondria without requiring carnitine (see also above: liver BTs) and is rapidly oxidized and metabolized to ¹³CO₂. The assumption of the ¹³C-BT is that small intestinal absorption of the substrate, liver metabolism, and pulmonary excretion of ¹³CO₂ must be preserved and rapid. Thus, a delay in the appearance of ¹³CO₂ in breath samples is exclusively due to a reduced solid transit pace between the stomach and the duodenum, i.e. gastric emptying becomes the rate-limiting step when the substrate is rapidly absorbed. An important diagnostic parameter is the half-emptying time, i.e. the time at which half of the solid meal has moved from the stomach to the duodenum [54–59]. Breath samples are taken at baseline and every 30 min over a period of 240 min, using a non-linear regression formula [55] or, less frequently (e.g. at 45, 150 and 180 mins) during a period of 180 min using the generalized linear regression model [60] of the ¹³CO₂ excretion-time curve. Measurement of exhaled ¹³CO₂ is performed by mass spectrometry or infrared spectroscopy. Smoking, physical exercise, drinking and eating are prohibited during the test, and gastric motility-altering drugs are not allowed. The retention of ¹³C-octanoic acid in the solid meal should be confirmed by in vitro incubation studies [57, 61].

Several researchers have employed the *C-BTs (where * denotes the variable isotope number) for the study of gastric emptying, as summarized in Table 2. Results vary according to the number of healthy subjects and type of patients enrolled, methodology and type of *C isotope, test meal characteristics (liquid, solid, calorie content or preparation), and interpretation of results. Two mathematical models—the non-linear regression model [55] and the general linear regression model [62]—have been proposed for the calculation of the BT half-emptying time, starting from the time-dependent ¹³CO₂ excretion curve. Several studies have compared BT with scintigraphy and a few have performed studies investigating the absorption kinetics of *C-octanoic after duodenal instillation or the retention of octanoic in specific meals.

Table 2.

Principal studies investigating gastric emptying of liquids and solids by breath tests using carbon ¹⁴C and ¹³C

Author (year)	Subjects/patients	Isotope	Test meal (kcal)	Main diagnostic outcomes
Ghoos et al. (1993) [55]	16 healthy subjects 20 functional dyspepsia	14C-OA in egg yolk	Solid (320 kcal)	Validation studies of OA incorporation into egg yolk Validation with scintigraphy Non-linear regression model Subtraction of 66 min from ‘real' BT T1/2 calculated by mathematical modela Tlag and GEC (Normal GEC = 3.1)
	5 healthy individuals	14C-OA and 13C-OA in egg yolk	Solid (320 kcal)	Repeated studies (3 times) Inter-individual variability NS Day-to-day variability NS Coefficient variation mean 27%
	41 healthy individuals	aC-OA in egg yolk	Solid (320 kcal)	Studies for normal range 72 ± 22SD min 83 min (75th percentile)
Maes et al. (1994) [73, 74]	9 healthy subjects: accelerated emptying (i.v. 200 mg erythromycin); delayed emptying (i.v. 30 mg propantheline) 36 healthy subjects 20 dyspeptic patients	14C-OA and 13C-OA in egg yolk	Solid (250 kcal)	Validation with scintigraphy Non-linear regression model T1/2 Normal 71 ± 27SD min Accelerated 37 ± 21 min Delayed 141 ± 88 min
Braden et al. (1995) [64]	20 healthy subjects 16 functional dyspepsia	13C-acetate in 100 mL water	Liquid (0 kcal)	Validation with scintigraphy Normal T1/2: 78 ± 14 min Dyspepsia T1/2: 100 ± 21 min
Duan et al. (1995) [63]	6 healthy subjects 6 dyspeptic patients With or without cisapride (10 mg x 2) 12 healthy subjects (day-to-day variability)	13C-acetate in water 13C-OA in egg yolk	Liquid (0 kcal) Solid (250 kcal)	Liquid T1/2 83 ± 6SD min (control); 96 ± 21 min (dyspepsia) Solid T1/2 148 ± 35 min (control); 203 ± 41 min (dyspeptic) Cisapride T1/2 117 ± 27 min (control); 166 ± 58 min (dyspeptic) In healthy controls, T1/2 for liquids and solids were reproducible on the two different days
Choi et al. (1997) [62]	15 healthy subjects	13C-OA in egg yolk	Solid (240 kcal)	Validation with scintigraphy Non-linear linear regression model High intra-individual variability, dependent also on collection periods (4, 5, 6 hours, delta 43-63 min) Low intra-individual variability (on 3 different days) T1/2 median (range) 186–191 min (167–210)
Choi et al. (1998) [76]	30 healthy subjects	13C-OA in egg yolk	Solid (240 kcal)	Validation with scintigraphy T1/2: different between subjects but highly reproducible within subjects Normal T1/2 median 191 min (range 120–386)
Lee et al. (2000) [60]	30 healthy subjects	13C-S. Platensis	Solid (220 kcal) or Pre-packed biscuit	Validation with scintigraphy General linear regression model Normal T1/2 100 ± 20SD min (solid meal) T1/2 91 ± 15 min (biscuit)
Lee et al. (2000) [67]	6 healthy subjects 50 healthy subjects (additional) 22 symptomatic diabetic patients	13C-OA in egg yolk	Solid (420 kcal)	Validation with scintigraphy Non-linear regression model: high variability and T1/2 BT longer than T1/2 scintigraphy General linear regression models: more accurate results Normal T1/2 median 118 min (range 72–188) Diabetic gastroparesis in 3 patients (1 misclassified according to scintigraphy)
Viramontes et al. (2001) [68]	50 healthy subjects Accelerated emptying (i.v. erythromycin) Delayed emptying (i.v. atropine)	13C-S. Platensis	Solid (220 kcal)	Validation with scintigraphy General linear regression model T1/2 ‘Normal' range 70–150 min
Hellmig et al. (2006) [65]	90 healthy subjects	13C-acetate in water 13C-OA in egg yolk	Liquid (250 mL apple juice) Solid (310 kcal)	Liquid T1/2 81 ± 22SD min (range 43–51) Solid T1/2 144 ± 55 (median 127 min; 25–75% percentiles: 112.0–168 min) No influence of age, sex or BMI
Szarka et al. (2008) [69]	38 healthy subjects 5 healthy subjects (i.v. atropine) 124 patients (suspicion of delayed gastric emptying)	13C-S. Platensis	Solid (238 kcal)	Validation with scintigraphy Normal T1/2 68 ± 15SD Accelerated if <10th percentile (52 min) Delayed if >90th percentile (86 min)
Perri et al. (2010) [57]	131 healthy subjects 8 diabetic gastroparesis 11 untreated celiac patients	13C-OA	Solid (378 kcal) (EXPIROGer®) pre-packed muffin	T1/2 Normal 88 ± 29SD min Normal upper cut-off value 146 min Gastroparesis 179 ± 50 min Celiac disease 151 ± 20 min

^aProbably dependent on the observed half-time for absorption and oxidation of OA after intraduodenal instillation (i.e. 62 min).

BMI = body mass index; BT = breath test; GEC = gastric emptying coefficient; NS = not significant; OA = octanoic acid; SD = standard deviation; T1/2 = half-emptying; Tlag = calculation of lag phase time (min).

A 150 mg dose of ¹³C-acetate has been used in the liquid test [63–65]. In solid test meals, different formulations have been employed:

1. One egg yolk labelled with 91–100 mg ¹³C-octanoic acid, served with 50 g of ham, 10 g of butter, two slices of white bread and a glass of water [equalling 324 kcal with 26 g carbohydrates (32%), 16 g fats (44%), and 19 g proteins (24%)] [54–56, 66] or slightly different calorie composition ranging from 220 kcal [60] to 420 kcal [67].

2. An edible blue-green alga protein-enriched food supplement (Spirulina platensis) labelled with 100–200 mg ¹³C-spirulina. The supplement is incorporated into egg white and the meal consists of either a cooked egg served with skimmed milk and wheat bread (220 kcal, protein 35%, carbohydrate 40%, fat 25% and 2.6 g of fibre) or as biscuit meal with a rye roll (160 kcal), cream cheese (90 kcal) and white grape juice (80 kcal) [60, 68, 69].

3. A new gluten-, glucose-, and lactose-free muffin (EXPIROGer®, Sofar, Milano, Italy) pre-labelled with 100 mg ¹³C-octanoic acid (equalling 378 kcal with 57 g carbohydrate (61%), 14 g fat (33%), and 6 g protein (6%)) [57].

4. Three small gluten-, glucose-, and lactose-free pre-packed muffins [equalling 390 kcal with 47 g carbohydrate (48%), 20 g fat (46%), and 5.5 g protein (6%)] ingested two before and one after a soluble capsule containing 75 mg ¹³C-octanoic acid (AB Analitica SrL, Padua, Italy) [70].

Table 2 also shows that BTs have been tested in patients with functional 'dysmotility-like' non-ulcer dyspepsia, idiopatic or diabetic gastroparesis, celiac disease, and accelerated or delayed emptying. Other studies have dealt with patients suffering from connective tissue disorders, gastro-oesophageal reflux disease, post-gastric surgery, dumping syndrome, HIV infection, gastric ulcer or obesity [55, 57, 64, 69]. The effects of drugs (cisapride, erythromycin atropine, propantheline and capsaicin) on gastric kinetics have been also investigated [63, 65, 68, 71–73].

The results depicted in Table 2 show a wide range of subjects/patients ranging in number from 6 to more than 130 and different outcomes in both health and disease. For example, for a liquid test meal, ¹³C-acetate yielded a mean half-emptying time of 78 ± 14 min in a study with 20 healthy subjects (coefficient of variation 10–36%) and showed a highly reproducible positive linear correlation with technetium-99 (^99mTc)-albumin colloid scintigraphy [64]. Data were confirmed in subsequent studies [63, 65].

In patients with functional dyspepsia, the mean half-emptying time was significantly delayed (Table 2) and showed a greater inter-individual variability [63, 64]. The authors concluded that the ¹³C-acetate BT is a reliable, widely applicable diagnostic tool to assess the gastric emptying of liquids and liquid phases in semi-solid test meals.

Starting from a seminal study in healthy subjects and dyspeptic patients, Ghoos et al. used ¹⁴C- and ¹³C-OA in egg yolk to validate the BTs according to the non-linear regression model, providing three kinetic parameters: half-emptying time, lag phase and gastric emptying coefficient [55]. Inter-individual and day-to-day variabilities were acceptable. The normal range of gastric emptying in healthy subjects was 72 ± 22SD min. Further studies have used BTs during pharmacologically-induced accelerated or delayed gastric emptying [72, 73].

When validating the ¹³C-Spirulina platensis solid test meal in 30 healthy volunteers, the Mayo Clinic group found optimal correlation vs. scintigraphy, even when restricting the measurements to a few time points (i.e. baseline, 75, 90 and 180 min, i.e. the Mayo 'reduced model') [60]. The half-emptying time was 100 ± 20SD min the solid meal and 91 ± 15 min with the S. platensis biscuit. In a subsequent study in 57 healthy subjects, simulated disturbances of gastric emptying were induced by either accelerating (using i.v. erythromycin) or delaying (using i.v. atropin) gastric emptying. As compared with scintigraphy and normal half-emptying values (95 ± 24 min) the sensitivity and specificity of ¹³C-Spirulina platensis BT using the solid meal were 86% and 80%, respectively, in detecting either accelerated (71 ± 14 min) or delayed emptying (207 ± 44 min), using a normal range for half-emptying time of 70–150 min [68].

In the multicentre Italian study employing the pre-packed solid meal EXPIROGer®, we recently found that the reference range of half-emptying time in 131 healthy subjects was 88 ± 29SD min with a normal upper cut-off value of 146 min. There was no significant difference between subjects sorted by sex or age. The within-subject variability of half-emptying time was 17%. By contrast, the half-emptying values were significantly delayed, showing 179 ± 50 min in diabetic patients with gastroparesis and 151 ± 20 min in untreated celiac disease patients [57]. In the initial series of healthy subjects using the novel three-muffins test, the ¹³C-octanoic BT yielded a half-emptying time of 108 ± 18SD (Portincasa et al.: unpublished observations) [70].

Using the ¹³C stable isotope represents a simple, non-invasive, office- or field-based technique [60, 74]. The accuracy of BTs for the study of gastric emptying appears to be comparable with scintigraphic studies [60, 64, 69, 73], but the technique, although easy to perform, fully non-invasive, and extendable also to children and pregnant women, represents an indirect method of assessing gastric emptying. In other words, variability in the rate of absorption, metabolism, and excretion of the marker between individuals must be considered in the interpretation of data. Imprecision with both stable isotope and scintigraphy in measuring gastric emptying, however, does reflect pathophysiological variations. The use of BTs for the study of gastric emptying appears promising for intra-individual comparisons. Further studies, however, might further improve standardization of the methodology in terms of statistical analysis, time-test, and sampling frequency [57, 60, 66, 75, 76].

Conclusions and future perspectives

BTs represent valuable diagnostic non-invasive tools for in vivo assessment of various enzyme activities, transport processes, or organ functions. By monitoring the metabolization of several ¹³C-labelled substrates and the consequent appearance of ¹³CO₂ in breath, it is possible to study the liver function dynamically (focussing on different intracellular pathways) as well as gastric emptying kinetics (similarly to scintigraphy, regarded as the ‘gold standard'). BTs currently use the non-radioactive stable isotope carbon ¹³C as tracer, which is safe in children, during pregnancy and while breast-feeding. BTs are simple, non-radioactive office- or field-based tools which, in epidemiological and pharmachodynamic studies, can better define pathophysiologically relevant abnormalities in the fields of hepatology and gastroenterology. Interpretation of BTs requires a knowledge of methodological limitations and potential pitfalls before routinely extending such studies in the clinical setting.

Conflict of interest statement: none declared.