A number of tests are used to evaluate the liver; however, none are sensitive and specific for assessment of hepatic function. Abnormalities detected during abdominal palpation and radiography may indicate changes in liver size, but do not provide any indication of hepatic function.1 Although ultrasonography of the liver may reveal morphologic changes in hepatic structure, it does not permit definitive assessment of hepatic function.2
Other modalities commonly used to evaluate the liver include the measurement of serum hepatic enzyme activities and serum bile acid concentrations. Alterations in serum hepatic enzyme activities may indicate hepatocellular damage, which may be associated with a decrease in hepatic function. However, some of these enzymes are released from other tissues during nonhepatic disorders and make interpretation of hepatic function difficult.3 Presently, measurement of pre- and postprandial serum bile acid concentrations is the most clinically useful hepatic function test available; however, this test is neither sensitive for compromised hepatic function nor specific for identifying a particular hepatic disease.4 Patients with mild liver disease may have serum bile acids concentrations within the reference range. Additionally, serum bile acids concentrations may be increased in patients with cholestasis in which hepatic function is initially normal.4 In addition, serum bile acids concentrations do not correlate with the severity of hepatic disease.4
The indocyanine green, sulfobromophthalein, and ammonia tolerance tests have been developed to evaluate hepatic function. The indocyanine green and sulfobromophthalein tests evaluate hepatic perfusion and hepatobiliary function, but they use compounds with several disadvantages and are therefore rarely used.5 The ammonia tolerance test measures the ability of the liver to extract and detoxify ammonia.6 This test requires rapid processing of samples, and its clinical use is limited by its insensitivity. Therefore, a new test that is both sensitive and specific for assessment of hepatic function in dogs and cats is clearly needed.
In humans, the aminopyrine breath test has been developed to quantify hepatic microsomal enzyme function.7–11 Results of numerous studies9–12 indicate that this test is clinically useful and that test results correlate with disease severity as assessed by histopathologic evaluation of biopsy specimens obtained from patients with hepatic cirrhosis and chronic hepatitis.
In contrast to humans, breath tests are difficult to perform in dogs and cats, and thus a blood-based test would be preferable for use in these animals. The principles of the carbon 13 (13C)-labeled aminopyrine demethylation blood test have previously been reported13 and are similar to those of the breath test used in humans. Aminopyrine is administered orally or IV and is demethylated in the liver by microsomal enzymes. As a result, the methyl groups produced are then oxidized to CO2. The CO2 diffuses into the vascular space, is eventually carried to the pulmonary alveoli, and is released into the expiratory air.8,14,15 By use of aminopyrine labeled with various carbon isotopes, either 13C or carbon 14 (14C), the amount of 13C or 14C derived from the labeled aminopyrine can be measured in the expired air (breath test) as either 13CO2 or 14CO2, respectively.16 For the breath test, the amount of 13CO2 or 14CO2 is determined as a percentage of the oral or IV dose of 13C-aminopyrine or 14C-aminopyrine administered that is recovered in expiratory air.16 Similarly, the blood test involves measuring the amount of 13CO2 extracted from blood samples.13 Results of a preliminary study13 indicated that a 13C-aminopyrine demethylation blood test is technically feasible in dogs. In that study, 13C-aminopyrine administered orally at a dose of 2 mg/kg to healthy dogs resulted in a detectible increase in the PCD in all dogs. Another study17 was performed to evaluate the demethylation kinetics of 13C-aminopyrine administered IV in healthy dogs and determine an appropriate parameter for quantification of aminopyrine demethylation. Results of that study indicated that IV administration of 13C-aminopyrine, as previously detected for oral administration, does not result in any gross clinical adverse effects. In addition, PCD was found to be an appropriate parameter for the quantification of aminopyrine demethylation. Results of that study17 also indicated that a single blood sample collected 45 minutes after IV administration of 13C-aminopyrine is sufficient for assessment of hepatic demethylating capacity in dogs.
The purpose of the study reported here was to determine the optimal dose of 13C-aminopyrine for use in a 13C-aminopyrine demethylation blood test in healthy dogs. We used a dose of 2 mg/kg as an arbitrary starting dose because this dose is commonly used for the aminopyrine breath test in humans. The ideal dose would be one that gives the lowest variability of PCD values in healthy dogs and is cost-effective for use in a clinical setting.
Materials and Methods
Dogs—Nine young healthy adult dogs (4 males and 5 females) were enrolled in the study. Breeds of dogs included Labrador Retriever (n = 3), Brittany (2), Pointer (1), German Shepherd Dog (1), and Siberian Husky cross (2). Dogs were owned by and housed at an animal research facilitya and remained in the care of this facility for the duration of the study. All dogs were determined to be healthy on the basis of results of physical examination, CBC, and serum biochemistry analyses. None of the dogs had a history of receiving drugs known to alter hepatic enzyme function. Dogs were closely monitored during and for several hours after each experimental period. The animal care staff monitored dogs for development of gross evidence of clinically adverse effects for several days after each experimental period. The study protocol was approved by the Animal Care and Use Committee at The IAMS Company (protocol No. 990022).
Procedures—The study was divided into 4 experimental periods in which 13C-aminopyrine was administered at doses of 1, 2, 5, or 10 mg/kg. During the first experimental period, 13C-aminopyrine administered at a dose of 2 mg/kg was evaluated, followed by evaluation of 13C-aminopyrine administered at doses of 5, 1, and 10 mg/kg. Each dog was given the same dose of 13C-aminopyrine during each experimental period. There was a 2-week resting period between each experimental period.
Food was withheld from each dog for 12 hours prior to the initiation of each experimental period. A 2-mL baseline blood sample was collected from each dog and placed into an evacuated glass tubeb containing sodium heparin. The 13C-aminopyrinec was dissolved in deionized water, and the solution was sterilized by passage through a 0.1-μm pore-size syringe filterd and stored in an amber glass bottle at 4°C until administered. The 13C-aminopyrine was administered IV at the dose that had been predetermined for each experimental period. Additional blood samples (2 mL each) were collected from each dog 30, 45, 60, and 75 minutes after 13C-aminopyrine administration and stored in evacuated tubes containing sodium heparin. Samples were stored at 22°C and shipped overnight to the Gastrointestinal Laboratory at Texas A&M University for analysis.
The CO2 was extracted from each blood sample by addition of 1 mL of 6N hydrochloric acid.e Immediately after addition of the acid, samples were vortexed to prevent acid coagulation and to maximize CO2 release. Gas samples were then analyzed by use of fractional mass spectrometry using an automated breath-carbon analyzerf to measure the fraction of 13CO2 in the CO2 extracted from blood samples.
Data analysis—The PCD and CUMPCD values were calculated as previously described.13,18 The PCD values for each sample time in each experimental period were compared with the baseline sample for that experimental period by use of repeated-measures ANOVA. This was performed to determine whether there was a significant difference between PCD values for each sample time and the baseline value. The Dunnett multiple comparison test was used as a posttest to compare values for each sample time with the baseline value.
Mean ± SD and CV values were calculated for PCD and CUMPCD for each dose. Mean CV values for PCD and CUMPCD for all doses and sample times were compared by use of a t test. The PCD values for the various doses were compared by use of repeated-measures ANOVA and Bonferroni's multiple comparison tests. A statistical analysis packageg was used for data analysis; values of P < 0.05 were considered significant.
Results
Gross evidence of clinically adverse effects was not observed in dogs during, or for a period of several days after, each experimental period. The PCD values increased initially and then began to decrease with time for all dogs and all doses. Consequently, the mean PCD values for each dose also increased with time and peaked 45 minutes after administration of 13C-aminopyrine at doses of 1, 2, and 10 mg/kg and 30 minutes after administration of 13C-aminopyrine at a dose of 5 mg/kg (Figure 1). The CUMPCD values for all dogs and doses increased with time, and the mean CUMPCD values for each dose also increased with time (Figure 2).
Mean PCD values after IV administration of 13C-labeled aminopyrine at doses of 1 (diamond), 2 (square), 5 (triangle), and 10 (circle) mg/kg in 9 dogs. Time 0 = Baseline sample before administration of 13C-aminopyrine.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1110
Mean PCD values after IV administration of 13C-labeled aminopyrine at doses of 1 (diamond), 2 (square), 5 (triangle), and 10 (circle) mg/kg in 9 dogs. Time 0 = Baseline sample before administration of 13C-aminopyrine.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1110
Mean PCD values after IV administration of 13C-labeled aminopyrine at doses of 1 (diamond), 2 (square), 5 (triangle), and 10 (circle) mg/kg in 9 dogs. Time 0 = Baseline sample before administration of 13C-aminopyrine.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1110
Mean CUMPCD values after IV administration of 13C-aminopyrine at doses of 1 (diamond), 2 (square), 5 (triangle), and 10 (circle) mg/kg in 9 dogs. Time 0 = Baseline sample before administration of 13C-aminopyirne.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1110
Mean CUMPCD values after IV administration of 13C-aminopyrine at doses of 1 (diamond), 2 (square), 5 (triangle), and 10 (circle) mg/kg in 9 dogs. Time 0 = Baseline sample before administration of 13C-aminopyirne.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1110
Mean CUMPCD values after IV administration of 13C-aminopyrine at doses of 1 (diamond), 2 (square), 5 (triangle), and 10 (circle) mg/kg in 9 dogs. Time 0 = Baseline sample before administration of 13C-aminopyirne.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1110
As determined by repeated-measures ANOVA, the mean PCD values for all sample times differed significantly (P < 0.001 for all doses) with time for all 4 doses. When compared separately by use of the Dunnett multiple comparison test, mean PCD values after 13C-aminopyrine administration differed significantly (P < 0.01 for all 4 doses) from the baseline sample.
The mean ± SD CV (21.5 ± 4.4%) for the PCD value from all doses was significantly (P < 0.01) lower than the mean ± SD CV (23.8 ± 4.6%) for the CUMPCD value for all the doses and sample times (Table 1). Therefore, the PCD value was used as an estimate of hepatic demethylation of 13C-aminopyrine for the remainder of the study.
Mean ± SD (CV) values for PCD and CUMPCD before (time 0; baseline) and after IV administration of 13C-aminopyrine at doses of 1, 2, 5, and 10 mg/kg in 9 dogs.
| Time (min) | PCD | CUMPCD | ||||||
|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 5 | 10 | 1 | 2 | 5 | 10 | |
| 0 | 0 (NA) | 0 (NA) | 0 (NA) | 0 (NA) | 0 (NA) | 0 (NA) | 0 (NA) | 0 (NA) |
| 30 | 0.1321 ± 0.0310 | 0.1251 ± 0.0223 | 0.1528 ± 0.0419 | 0.1334 ± 0.0437 | 1.9678 ± 0.4639 | 2.4878 ± 0.5391 | 2.2933 ± 0.6281 | 2.0011 ± 0.6557 |
| (23.6) | (17.8) | (27.4) | (32.7) | (23.6) | (21.7) | (27.4) | (32.8) | |
| 45 | 0.1383 ± 0.0311 | 0.1334 ± 0.0239 | 0.1500 ± 0.0339 | 0.1404 ± 0.0363 | 3.9889 ± 0.8888 | 4.4267 ± 0.8629 | 4.5644 ± 1.1921 | 4.0544 ± 1.2512 |
| (22.5) | (17.9) | (22.6) | (25.8) | (22.3) | (19.5) | (26.1) | (30.9) | |
| 60 | 0.1336 ± 0.0237 | 0.1242 ± 0.0227 | 0.1416 ± 0.0268 | 0.1347 ± 0.0317 | 6.0267 ± 1.2269 | 6.3600 ± 1.1551 | 6.7522 ± 1.6291 | 6.1178 ± 1.7469 |
| (17.8) | (18.2) | (18.9) | (23.6) | (20.4) | (18.2) | (24.1) | (28.6) | |
| 75 | 0.1322 ± 0.0239 | 0.1201 ± 0.0230 | 0.1319 ± 0.0211 | 0.1272 ± 0.0280 | 8.0222 ± 1.4936 | 8.1922 ± 1.4237 | 8.8022 ± 1.9658 | 8.0811 ± 2.1647 |
| (18.1) | (19.1) | (16.0) | (22.0) | (18.6) | (17.4) | (22.3) | (26.8) | |
NA = Not applicable.
No significant (P = 0.41, 0.73, 0.58, and 0.70) differences were detected in mean PCD values among the 4 doses (1, 2, 5, and 10 mg/kg, respectively) when compared by use of repeated-measures ANOVA. No dose was significantly (P > 0.05 for all comparisons) different from any other dose.
To compare interindividual variabilities, CV values were calculated for each dose. No significant (P = 0.07) differences in the mean CV values for PCD between doses were detected. There were also no significant (all values of P > 0.05) differences when the mean CVs for PCD for individual doses were compared.
Discussion
In the study reported here, gross evidence of clinically adverse effects was not observed in any dog during any experimental period. Although gross evidence of clinically adverse effects was not seen, subclinical adverse effects, such as subclinical organ damage, could not be definitively excluded. The safety of 13C-aminopyrine needs to be further evaluated in dogs with altered hepatic function.
A 2-week resting period was provided to dogs between experimental periods to eliminate any possible induction of hepatic demethylating enzymes caused by 13C-aminopyrine administration. We assumed that any microsomal enzyme induction that may have occurred after administration of a single dose of 13C-aminopyrine would have returned to baseline values during this 2-week resting period. To prove or disprove this contention, measurement of microsomal enzyme activities in hepatic biopsy specimens would have been required. However, obtaining hepatic biopsy specimens in dogs used in our study was not possible because of animal welfare guidelines at the facility in which dogs were housed. Additionally, assays for hepatic microsomal enzyme function in dogs were not available.
Although the mean PCD value for 13C-aminopyrine administered at a dose of 5 mg/kg peaked earlier than that for 13C-aminopyrine administered at a dose of 2 mg/kg (as well as at the other 2 doses), we do not believe that this was caused by induction of hepatic microsomal enzyme function. If this were the case, we would have expected the mean PCD value for 13C-aminpyrine administered at a dose of 1 mg/kg to peak earlier than that for 13C-aminopyrine administered at a dose of 2 mg/kg and perhaps at a dose of 5 mg/kg. Because mean PCD values for 13C-aminopyrine administered at doses of 1 and 10 mg/kg did not peak earlier than or at the same time as that for 13C-aminopyrine administered at a dose of 2 mg/kg, we are confident in our assumption that hepatic microsomal induction did not lead to the delayed peak time for the PCD value detected after administration of 13 C-aminopyrine at a dose of 5 mg/kg.
Data for 13C-aminopyrine administered at a dose of 2 mg/kg were collected during a kinetic study performed previously.17 The remaining 3 doses of 13C-aminopyrine were evaluated during various experimental periods so that doses were actually evaluated in the following order: 2, 5, 1, and 10 mg/kg. All dogs received the same dose during each experimental period to enhance the possibility of identifying potential adverse effects of repeated 13C-aminopyrine administration.
Intravenous administration of 13C-aminopyrine resulted in an increase in the PCD value of gas extracted from blood samples in all 9 dogs and for all experimental periods. For all 4 doses, the mean PCD value at each sample time after 13C-aminopyrine administration was significantly greater than the mean PCD value at baseline.
One of the parameters used to assess the potential clinical usefulness of a new diagnostic test is interindividual variability of that test in a group of healthy animals. This is based on the assumption that the lower the interindividual variability in healthy dogs, the easier it would be to differentiate between clinically healthy dogs and dogs with disease. Interindividual variability can be assessed by calculating CV. In the study reported here, the CV values for PCD and CUMPCD were calculated and compared. Results of our study indicated that the mean CV value for PCD was significantly (P < 0.01) lower than the mean CV value for CUMPCD. Taking into consideration the relative ease of collecting a single blood sample after administration of 13C-aminopyrine for determination of the PCD value, compared with collection of multiple samples as necessary for determination of CUMPCD, this finding indicates that determination of PCD is preferable to CUMPCD for assessment of hepatic 13Caminopyrine demethylation. These findings are consistent with that of another study.17 However, whether determination of CUMPCD would be clinically more useful than determination of PCD in dogs with altered hepatic function cannot be definitively determined on the basis of results of the study reported here.
Significant differences in CV values among the 4 doses were not detected. However, 13C-aminopyrine administered at a dose of 2 mg/kg had the lowest CV value, compared with the other doses. As previously mentioned, a low interindividual variability in healthy dogs is desirable. Use of 13C-aminopyrine at a dose of 2 mg/kg would also be more cost-effective than use of 13C-aminopyrine at doses of 5 and 10 mg/kg because less 13C-aminopyrine is needed for the test. Thus, we concluded that administration of 13C-aminopyrine at a dose of 2 mg/kg is appropriate for use in the 13C-aminopyrine demethylation blood test in healthy dogs. Unfortunately, in the study reported here, dogs with hepatic dysfunction were not evaluated. Demethylation kinetics may be severely altered in dogs with hepatic dysfunction; therefore, administration of 13C-aminopyrine at a dose of 2 mg/kg may not be optimal for use in dogs with hepatic dysfunction. Additional studies are required to fully evaluate the clinical usefulness of a 13C-amionpyrine demethylation blood test in dogs with hepatic disease. An initial clinical study19 has been reported since the completion of the study reported here, but further studies are required.
ABBREVIATIONS
| PCD | Percentage dose of carbon 13 administered as carbon 13-aminopyrine and recovered in gas extracted from blood samples |
| CUMPCD | Cumulative PCD values up to a given sampling time |
| CV | Coefficient of variation |
Nutritional Research Center, The IAMS Co, Lewisburg, Ohio.
BD vacutainer, Preanalytical Solutions, Franklin Lakes, NJ.
4-dimethyl-13C2-aminoantipyrine, Isotech Inc, Miamisburg, Ohio.
Gelman Sciences, Supor Acrodisc, 0.1 μm, sterile, VWR Scientific Products Corp, West Chester, Pa.
Hydrochloric acid, Sigma Chemical Co, St Louis, Mo.
Automated breath carbon analyzer, Europa House, Crewe, UK.
GraphPad Prism, version 3.0, GraphPad Software Inc, San Diego, Calif.
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