Performance of the [13C]-acetate gastric emptying breath test during physical exercise

BACKGROUND: The gastric emptying rate of liquids can be determined non-invasively using the [13C]-acetate breath test at rest. The aims of our study were to validate this test during physical exercise against the double-sampling method and to evaluate the time needed for intestinal absorption and the delay between absorption and appearance of 13CO2 in breath, both at rest and during exercise. DESIGN: Fifteen well-trained male subjects were investigated. Gastric emptying was determined simultaneously measuring the 13CO2 breath enrichment after intragastric administration of 0.5 L of carbohydrate solution with 150 mg of [13C]-acetate added and by the double-sampling technique (n = 9). In separate tests, 150 mg of [13C]-acetate was also applied intraduodenally and intravenously (n = 6), both at rest and during exercise. Time-to-peak (TTP) 13CO2 enrichment was determined using a curve fit and was considered as the parameter for gastric emptying. RESULTS: TTP enrichment derived from the breath test significantly correlated with the gastric emptying half-time obtained from the gastric aspirates. During exercise, median TTP enrichment values after intragastric, intraduodenal (i.d.) and intravenous (i.v.) administration of [13C]-acetate were 22.3, 10.3 and 5.4 min respectively. During exercise, i.d. and i.v. values were reached significantly earlier than at rest. CONCLUSION: The [13C]-acetate breath test can be used as a non-invasive method to determine relative gastric emptying rates of liquids during exercise, but the results are influenced by the rate of absorption and the time needed for subsequent oxidation of [13C]-acetate and exhalation of 13CO2.     

Limitations of the triolein breath test.

Patients being investigated for intestinal absorptive capacity were classified as normals 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 [14C]-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 5-12.4 mmol/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.     

Comparison of the [13C]glucose breath test to the hyperinsulinemic-euglycemic clamp when determining insulin resistance

OBJECTIVE: With increasing emphasis on the recognition of the metabolic syndrome and early type 2 diabetes, a clinically useful measure of insulin resistance is desirable. The purpose of this study was to evaluate whether an index of glucose metabolism, as measured by (13)CO(2) generation from ingested [(13)C]glucose, would correlate with indexes from the hyperinsulinemic-euglycemic clamp. RESEARCH DESIGN AND METHODS: A total of 26 subjects with varying degrees of insulin sensitivity underwent both the [(13)C]glucose breath test and the hyperinsulinemic-euglycemic clamp. Results from the [(13)C]glucose breath test were compared with measures of insulin sensitivity from the glucose clamp as well as with other commonly used indexes of insulin sensitivity. RESULTS: There was a strong correlation between the [(13)C]glucose breath test result and the glucose disposal rate (r = 0.69, P < 0.0001) and insulin sensitivity index (r = 0.69, P < 0.0001) from the insulin clamp. The magnitude of these correlations compared favorably with QUICKI and were superior to the homeostasis model assessment. CONCLUSIONS: The [(13)C]glucose breath test may provide a useful noninvasive assessment of insulin sensitivity.     

Erythromycin breath test as an assay of glucocorticoid-inducible liver cytochromes P-450. Studies in rats and patients.

The major P-450IIIA gene family member present in human liver is HLp which, like its rat liver orthologue P-450p, is inducible by glucocorticoids and catalyzes erythromycin N-demethylation. To develop a practical method to estimate the amounts of HLp in patients [14C]N-methyl erythromycin was injected into rats that had been pretreated with dexamethasone or with inducers of other forms of cytochrome P-450. The rate of demethylation of this substrate, measured simply as 14CO2 in the breath, correlated well with the concentrations of immunoreactive P-450p protein (r = 0.70), holocytochrome P-450p (r = 0.70), or with erythromycin N-demethylase activity (r = 0.90) determined in the liver microsomes prepared from each rat. Next, [14C]N-methyl erythromycin was administered to 30 patients and there was a sixfold interindividual variation in breath 14CO2 production seemingly unrelated to medications, smoking status or age. However, the average breath test values were twofold greater in female as compared to male patients (P less than 0.01). Breath 14CO2 production rose in patients retested after treatment with the P-450IIIA inducers dexamethasone (P less than 0.05) or rifampicin (P less than 0.05) and was decreased after treatment with the HLp inhibitor triacetyloleandomycin (P less than 0.05). We conclude that the erythromycin breath test provides a convenient assay of P-450IIIA cytochromes in rats and in some patients.     

Comparison of the proportion of unconjugated to total serum cholic acid and the [14C]-xylose breath test in patients with suspected small intestinal bacterial overgrowth.

The proportion of unconjugated to total cholic acid in fasting serum and the 1-gram [14C]-xylose breath test were determined in 36 patients with suspected bacterial overgrowth of the small intestine. Twenty-two patients had an abnormal [14C]-xylose breath test, indicating bacterial overgrowth. The proportion of unconjugated to total cholic acid was significantly higher in the patients with an abnormal breath test compared with those displaying a normal breath test (47 +/- 5% vs 16 +/- 3%). A good correlation was obtained between the proportion of unconjugated to total cholic acid and the breath test (r = 0.63, n = 36). Provided the [14C]-xylose breath test is reliable as a test of bacterial overgrowth, determination of the proportion of unconjugated to total cholic acid in fasting serum had a sensitivity of 73% and a specificity of 94%. It is suggested that determination of the proportion of unconjugated to total cholic acid in peripheral venous blood may be useful as a simple screening test for detection of bacterial contamination of the upper small intestine provided the patients do not have bile acid malabsorption.     

The glyceryl [14C]tripalmitate breath test: a reassessment

Several reports have been published commending the use of 14C-labelled triglyceride breath tests in the assessment of fat malabsorption. We report further studies using gyceryl [14C]tripalmitate. Corrections for age, weight or metabolic rate failed to improve the test's ability to discriminate between malabsorbers and control subjects. A correction for respiratory quotient improved the linear correlation observed between the breath test results and daily faecal fat excretion. The significance of these findings is discussed and a number of problems identified which, at present, are preventing the introduction of breath tests for fat malabsorption into routine clinical practice.     

Bile acid synthesis in man. In vivo activity of the 25-hydroxylation pathway.

During biosynthesis of bile acid, carbons 25-26-27 are removed from the cholesterol side-chain. Side-chain oxidation begins either with hydroxylation at the 26-position, in which case the three-carbon fragment is released as propionic acid, or with hydroxylation at the 25-position, in which case the three-carbon fragment is released as acetone. We have previously shown in the rat that the contribution of the 25-hydroxylation pathway can be quantitated in vivo by measuring production of [14C]acetone from [14C]26-cholesterol. In the present study, we adapted this method to human subjects. 4 d after oral administration of 100 microCi of [14C]26-cholesterol and 1 d after beginning a constant infusion of 16.6 mumol/min unlabeled acetone, three men and two women underwent breath collections. Expired acetone was trapped and purified as the 2,4 dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied by the acetone infusion rate to calculate production of [14C]acetone. [14C]Acetone production averaged 4.9% of total release of 14C from [14C]26-cholesterol, estimated by 14CO2 output. The method was validated by showing that [14C]acetone production from [14C]isopropanol averaged 86.9% of the [14C]-isopropanol infusion rate. We conclude that in man, as in the rat, the 25-hydroxylation pathway accounts for less than 5% of bile acid synthesis.     

Fate of oleate in the colon of the rat

To study the fate of oleate in the colon, oleate labeled with carbon 14 was instilled into the cecum of four rats through chronically implanted cecal cannulas. Fecal recovery of 14C and 14CO2 excretion were measured over a 3-day period. A mean of only 57% +/- 6% of the dose of [14C(U)]oleate was recovered as 14C in fecal lipid. About 8% was recovered in the aqueous phase of feces and 4% was recovered as 14CO2. Occlusion of the terminal ileum did not diminish 14CO2 excretion, excluding ileal reflux with small bowel absorption. Studies in two germ-free rats showed no conversion of [14C(U)]oleate to fecal water-soluble compounds, indicating that [14C]oleate is converted into water-soluble compounds by bacterial metabolism. The metabolism of [14C]oleate to 14CO2 was also observed in germ-free rats, indicating oxidation in the colon or other host tissues. We conclude that the colonic absorption of lipid or lipid metabolites plus conversion to water-soluble fecal compounds or volatile metabolites results in a fecal fat measurement that appreciably overestimates small bowel absorption of lipid.     

Hepatic phenylalanine metabolism measured by the [13C]phenylalanine breath test

BACKGROUND: The amino acid clearance test including phenylalanine is known to reflect liver functional reserve, which correlates with surgical outcome; however, the procedure is not clinically useful because of its laborious and time-consuming nature. This study evaluates whether phenylalanine oxidation capacity measured by a breath test could reflect liver functional reserve. DESIGN: We determined phenylalanine oxidation capacity in 42 subjects using the L-[1-13C]phenylalanine breath test (PBT). The 13CO2 breath enrichment was measured at 10-min intervals for 120 min after oral administration of 100 mg of L-[1-13C]phenylalanine. Subjects were divided into the following three groups according to their plasma retention rate of indocyanine green at 15 min (ICG R15): Group I (ICG R15 < 10%), Group II (ICG R15 10--20%), and Group III (ICG R15 > 20%). First, we determined the parameters of the phenylalanine oxidation capacity that differentiated these groups and then, using these parameters, we compared the PBT with the ICG clearance test, Child-Pugh classification score and standard liver blood tests. RESULTS: The %13C dose h(-1) at 30 min and cumulative excretion at 80 min were significantly different among the three groups (P < 0.05). These two parameters significantly correlated with the ICG R15, Child-Pugh classification score (P < 0.0001) and results of standard liver blood tests (P < 0.05). CONCLUSIONS: Phenylalanine oxidation capacity measured by the PBT was reduced according to the severity of liver injury assessed by the ICG clearance test, Child-Pugh classification, and standard liver blood tests. These results indicate that the PBT can be used as a noninvasive method to determine liver functional reserve.     

The caffeine CO2 breath test: dose response and route of N-demethylation in smokers and nonsmokers

The optimal conditions for performing the caffeine CO2 breath test (CBT) were investigated in smokers and nonsmokers. Caffeine labeled with 13C or 14C in all three (1, 3, and 7) methyl groups or specifically in the 1-, 3-, or 7-methyl groups were orally administered to healthy adults and the expiration of labeled CO2 was measured for 8 or 24 hr. The absolute rate of labeled CO2 excretion from trilabeled caffeine was proportional to the dose up to 3 mg/kg in all subjects. In smokers, the rate of labeled CO2 excretion averaged twice that in nonsmokers at all doses. A correlation was observed between the 2-hr cumulative CO2 excretion from trilabeled caffeine and the apparent oral metabolic clearance rate (MCR) of caffeine (R = 0.90). Monolabeled CBTs in smokers and nonsmokers demonstrated that 80% +/- 4% of labeled CO2 expired in the breath during the first 2 hr of a trilabeled CBT was derived from the 3 position; at 6 to 8 hr equal amounts were derived from the 3 and 7 positions. Little N-demethylation was observed from the 1 position at any time during the 8-hr test. The results indicate that the 2-hr cumulative excretion of labeled CO2 could be used to accurately predict the metabolic clearance rate of caffeine is the best CBT parameter for detecting the effect of smoking on caffeine N-demethylation. The data suggest that the primary routes of caffeine metabolism are 3-N-demethylation and ring hydroxylation and confirm that caffeine metabolites are N-demethylated primarily in the 3 and 7 positions.     

Assessment of liver function by the aminopyrine breath test

The aminopyrine breath-test (ABT) has been proposed as a non-invasive quantitative test of liver function and reserve. To evaluate its usefulness, we compared the ABT with standard liver function tests, Child's classification of liver disease and ICG clearance, as means of assessing liver function in 30 patients with cirrhosis. The cumulative output of 14CO2 in breath during the 6 h following [14C]aminopyrine administration was significantly decreased in the cirrhotic group as compared with control subjects. The severity of liver dysfunction, as assessed by Child's classification, was associated with a progressive and statistically significant impairment of the ABT. There was a good correlation between the ABT and ICG systemic clearance (r = 0.770, P less than 0.001) and also between the ABT and ICG intrinsic clearance (r = 0.885, P less than 0.001), a measure which is independent of hepatic blood flow variations. These results further strengthen the concept that the ABT is a simple non-invasive method to assess quantitatively liver function and reserve, and could be useful in following the evolution of patients with liver disease.     

Erythromycin breath test predicts oral clearance of cyclosporine in kidney transplant recipients.

It has been shown recently that cyclosporine is largely metabolized by P450IIIA (CYP3A), an enzyme whose catalytic activity varies significantly among patients. To determine whether heterogeneity in P450IIIA activity contributes to interpatient differences in cyclosporine dosing requirements, the oral pharmacokinetics of the drug were determined in 20 stable kidney transplant recipients. P450IIIA activity was then measured in each patient by use of the erythromycin breath test. In the 16 patients who were at steady state, the logarithm of the apparent oral clearance of cyclosporine correlated significantly with the rate of 14CO2 exhaled in breath after intravenous administration of [14C N-methyl]erythromycin (r = 0.55, p = 0.03). No significant correlations existed between apparent oral clearance and age, high-density lipoprotein cholesterol or low-density lipoprotein cholesterol, or hematocrit in these patients. We conclude that heterogeneity in P450IIIA activity significantly contributes to interpatient differences in dosing requirements of cyclosporine in kidney transplant patients.     

The erythromycin breath test as a predictor of cyclosporine blood levels

The daily dose of cyclosporine required to attain a desired blood level can vary greatly among patients. Because elimination of cyclosporine depends on its metabolism in the liver by an enzyme (cytochrome P-450IIIA) that also demethylates erythromycin, we reasoned that the ability of patients to demethylate a test dose of erythromycin might be useful in estimating their appropriate daily doses of cyclosporine. Accordingly, the [14C-N-methyl] erythromycin breath test was administered to 32 patients before they received 3.0, 5.0, or 7.5 mg/kg/day cyclosporine to treat psoriasis. We found that a simple mathematical equation incorporating just the 14CO2 production, the age of the patient, and the daily dose of cyclosporine accounted for almost 80% (R2 = 0.78) of the interpatient variability in cyclosporine blood levels we observed. Our data indicate that P-450IIIA activity largely accounts for the relationship between dose of cyclosporine and blood levels for an individual patient. We conclude that the erythromycin breath test may be a convenient guide for cyclosporine dosing.     

Phenylacetic oil: a possible simple test for fat absorption.

The preparation of a phenyl[1-14C]acetic acid oil is described. The absorption pattern of the phenylacetate in this oil is similar to that of oleic acid in triolein when examined in rats under conditions that may interfere with digestion and/or absorption. The safety of this compound, which is a normal metabolite, was established by the absence of the 14C label in various tissues five days after its administration to rats. When tested in 18 normal human volunteers 66.6 +/- 16.0 (S.D.) percent of the phenylacetate was recovered in a five hour urine collection. In two patients with malabsorption, less than 30% of the label was recovered in the urine over the same period. The half-life of the phenylacetate was determined to be 1-1.5 h. Our preliminary results indicate that phenylacetic oil may serve as a useful simple clinical test for fat absorption.     

Absorption of 13C-labeled stearic, oleic, and linoleic acids in humans: application to breath tests

Intestinal absorption of ingested [1-13C]stearic, [1-13C]oleic, and [1-13C]linoleic acid was compared in six healthy men. A bolus of each [1-13C]-labeled fatty acid was ingested in random order at 72-hour intervals with the breakfast meal. Subjects consumed fixed diets during a 9-day fecal collection period. Pooled 9-day fecal samples were homogenized and total fat extracted. Fat extracts were saponified and methylated, and individual fatty acids were quantitated by gas-liquid chromatography. Preparative high-performance liquid chromatography was used to obtain fractions containing stearic, oleic, and linoleic acid for combustion to CO2 and assay of 13C enrichment over background. Prelabel period 24-hour samples were treated similarly to measure background 13C abundance. Total fatty acid and stearic, oleic, and linoleic acid excretion (+/- SEM) in the six volunteers over the 9-day period was 41.5 +/- 7.3, 10.0 +/- 1.3, 8.8 +/- 2.9, and 0.8 +/- 0.1 mg/day/kg body weight, respectively. The absorption efficiency for [1-13C]stearic, [1-13C]oleic, and [1-13C]linoleic acid was 78.0% +/- 4.5%, 97.2% +/- 1.7%, and 99.9% +/- 0.1%, respectively. The reduced absorption of [1-13C]stearic acid observed emphasizes the importance of correcting breath test oxidation data for fecal loss of 13C substrate. The potential application of our method to other areas of intermediary metabolism is discussed.     

The aminopyrine breath test, an inadequate early indicator of methotrexate-induced liver disease in patients with psoriasis

The aminopyrine breath test has been shown to be a sensitive noninvasive indicator of liver cell dysfunction. In a search for a noninvasive method of monitoring the effects of methotrexate therapy, we have investigated the use of the aminopyrine breath test in patients receiving methotrexate for the treatment of severe psoriasis. The [14C]-aminopyrine breath test was performed in 20 normal control subjects, 32 patients with psoriasis receiving methotrexate therapy, and 8 patients with histologically confirmed cirrhosis of differing etiology. Eighteen patients on methotrexate had liver biopsies classified as grade I changes, 6 patients as grade II, and 8 patients as grade III. The normal value for the breath test was 11.0 +/- 1.6% (mean +/- 1 SD). The mean [14C]-CO2 excretion (8.3 +/- 4.4%) of the 8 patients with grade III liver disease was significantly different from the control subjects (p less than 0.02), and those with grade I liver changes (p less than 0.04). The aminopyrine breath test was only able to detect the later severe stages of methotrexate hepatotoxicity, grade III, when fibrosis occurs, before established cirrhosis was present. Our data suggests that the aminopyrine breath test is not a sensitive indicator for the detection of early methotrexate-induced hepatotoxicity, (stages I and II), but will detect the precirrhotic stage III change. Consequently, we recommend that a liver biopsy should be performed annually in all psoriatic patients receiving methotrexate, to detect histological damage, especially when the aminopyrine breath test score falls below the 95% confidence limits of normal.     

Utilization for protein synthesis of 2-ketoisocaproate relative to utilization of leucine, as estimated from exhalation of labelled CO2

1. We have previously shown that the ratio (RWBP) of incorporation of label from 2-ketoisocaproate (KIC) into the leucine of whole-body protein to the simultaneous incorporation of label from leucine itself into protein is a measure of the nutritional efficiency of KIC as a substitute for leucine. 2. In order to determine whether RWBP can be estimated indirectly from measurement of labelled CO2 excretion, rats were injected orally or intravenously with [4,5-3H]leucine and either [1-14C]leucine or [1-14C]KIC. Expired CO2 was collected for 6 h. 3. The results show that 9-14% of KIC underwent first-pass oxidation after oral administration. When isotopes were given intravenously, the mean rate of excretion of 14CO2 from KIC, after 20 min, remained 1.8 times the mean rate of excretion of 14CO2 from leucine. 4. Mean RWBP, measured in whole-body protein in rats given isotopes orally or intravenously along with small or large doses of carriers, was the same as mean RWBP estimated from mean cumulative CO2 excretion. 5. We conclude (1) that nutritional efficiency of KIC relative to leucine can be estimated from measurement of labelled CO2 excretion, and (2) that the relative inefficiency of KIC as a substitute for leucine in the rat is attributable to first-pass oxidation of 9-14% (when given orally) and 80% greater susceptibility to systemic oxidation than leucine.     

Altered liver function in diabetes: model experiments with aminopyrine in the rat

The aminopyrine breath test is a valuable quantitative liver function test. However, it may be influenced by factors not related primarily to liver disease. For instance, it has been published that diabetes affects microsomal demethylation of aminopyrine in vitro. The relevance of these findings for the in vivo situation, however, is ill defined. Aminopyrine disposition was evaluated, therefore, by performing an in vivo-in vitro comparison of its kinetics in control, diabetic and insulin-treated diabetic rats. Diabetes was induced by streptozotocin (75 mg/kg i.v.). A tracer dose of [14C]aminopyrine was injected i.p. (40 mu Ci/kg, 0.7 mg/kg) and the kinetics of 14CO2 in breath as well as disappearance of aminopyrine in blood were followed simultaneously for 2 hr. Diabetes increased the 14CO2 elimination rate constant in breath by 90%, whereas total recovery of 14CO2 in breath was decreased by 30% (P less than .001). Aminopyrine clearance in blood was doubled in diabetic rats compared to control (48.9 +/- 11.3 vs. 21.4 +/- 3.3 ml/min X kg, P less than .001). This was due to an increased volume of distribution (1.99 +/- 0.31 in diabetic rats vs. 0.96 +/- 0.11 liters/kg in control). In vitro aminopyrine kinetics in hepatic microsomal preparations showed a 52% higher Vmax of aminopyrine demethylase in D (P less than .001), whereas Km remained unchanged. The diabetes-induced changes were reversible by insulin. It is concluded that diabetes alters the aminopyrine breath test by interfering with demethylation rate and distribution of aminopyrine, and by changing the fate of the cleaved C1-fragments.     

The aminopyrine breath test as a measure of liver function. A quantitative description of its metabolic basis in normal subjects

The APBT is used widely as a measure of liver function. The development of the APBT into a liver function test of greater diagnostic value requires quantitative information on the processes involved in aminopyrine disposition and metabolism in man and on how APBT values reflect changes in these processes. A dual-isotope kinetic study of aminopyrine disposition and metabolism has been carried out on five normal adult subjects. Oral administration of 13C-aminopyrine (2 mg/kg) accompanied by simultaneous intravenous injection of 14C-aminopyrine was followed by serial measurements of aminopyrine and monomethylaminopyrine in plasma and urine over 6 hr. Timed collections of respiratory CO2 were analyzed for the content of excess 13CO2 and for 14CO2. On separate days, an intravenous bolus of 13C-labeled NaHCO3 was administered to obtain estimates of the kinetic parameters of CO2 elimination in each subject. These data were fitted simultaneously to a multicompartmental model that, in addition to providing hitherto unavailable quantitative information, has revealed that (1) demethylation is the major elimination pathway for aminopyrine; (2) a major alternative pathway not involving demethylation exists for monomethylaminopyrine; and (3) only 50% of the labeled carbon generated by demethylation eventually is oxidized to HCO3-. The sensitivity of seven types of APBT scores to 50% reductions in the rates of aminopyrine absorption, metabolism of monomethylaminoantipyrine, intermediate carbon metabolism, and bicarbonate kinetics was evaluated with breath test curves simulated using the APBT model. Every APBT score currently in use was affected by variations in both gastrointestinal output of aminopyrine and bicarbonate kinetics. There is a need for further development of selective scoring methods in the aminopyrine breath test.