Several diagnostic tests to assess liver health in various animal species have been developed. However, none of the conventional indices of general hepatobiliary dysfunction, such as serum activities of alanine aminotransferase and alkaline phosphatase or concentrations of bile acids and total bilirubin, are both sensitive and specific indicators of the liver's functional capacity. Serum liver enzyme measurements reflect hepatocyte membrane integrity, hepatocyte or biliary epithelial necrosis, cholestasis, or enzyme induction, but do not reflect hepatic function in dogs and cats.1 Furthermore, results of the most clinically useful hepatic function test available, the pre- and postprandial measurements of serum bile acids concentrations, do not correlate with severity of disease; serum bile acids concentrations may increase substantially with the development of cholestasis, regardless of the liver's functional capacity.2,3 More sensitive and specific hepatic function tests, such as the indocyanine green, sulfobromophthalein, and ammonia tolerance tests, are available but they are generally impractical for routine use in dogs.4,5 There is a clear need for a test that not only is both sensitive and specific, but also practical for determining the functional capacity of the liver. Although it is still in the process of being evaluated in dogs, the 13C-ADBT may ultimately fill this void.
Aminopyrine demethylation tests quantify the excretion of isotope-labeled CO2 following administration of aminopyrine in which the N-methyl groups have been labeled with either a radioactive (14C) or nonradioactive (13C) carbon isotope. Aminopyrine is almost exclusively demethylated via the hepatic microsomal mixed-function oxidase system, and the resulting methyl groups are further oxidized to produce CO2 that diffuses into the vascular space. Isotope-labeled CO2 can be recovered ultimately from blood or expired air, quantified, and used as an indirect measure of functional hepatic mass. A breath aminopyrine demethylation test that involves oral administration of radiolabeled aminopyrine has been validated for use in humans.6 As such, the breath test is considered noninvasive and easy to perform with either stable or radioactive isotopes; its usefulness for the evaluation and staging of hepatic disorders, including alcoholic cirrhosis, chronic hepatitis, and liver injury resulting from acetaminophen overdose, has been demonstrated.6–13 Results of the breath aminopyrine demethylation test have also been used to evaluate hepatic induction in patients receiving anticonvulsant drugs and to determine surgical risk in patients with liver disease.14,15
Despite the apparent clinical usefulness of the breath aminopyrine demethylation test, its application in people has been somewhat limited because of concerns regarding potential but rare aminopyrine-induced agranulocytosis.16 In dogs, such adverse reactions have not been reported to date, and the potential usefulness of this test in this species is apparent. In dogs, a 13C-ADBT has been developed. This test involves IV administration of 13C-aminopyrine and assessment of the PCD; it has been analytically validated and shown to be technically feasible.17–19 In preliminary experiments in dogs,20 the results of the 13C-ADBT were similar to those obtained by use of the breath aminopyrine demethylation test in humans; there appeared to be an inverse correlation of PCD with increasing severity of hepatic disease (as determined histologically). However, the true usefulness of the 13C-ADBT in dogs has yet to be defined.
As part of the evaluation of the 13C-ADBT for routine use in dogs, the study reported here was undertaken to determine the optimal sample handling and processing conditions for the test and determine the reference range for PCD results in healthy dogs. By investigating the effects of various methods of blood sample preparation (such as anticoagulant use and interval from blood collection to gas extraction) on PCD values, it was anticipated that optimal procedures would be determined; these data would provide an indication of the practicability of the test for routine use and enable standardization among future clinical studies. Establishment of a reference range for canine PCD values derived by use of the 13C-ADBT would provide a basis for assessment of changes associated with hepatic dysfunction in dogs.
Materials and Methods
Dogs—Forty-four healthy dogs were included in the study. Nineteen (10 males and 9 females) were enrolled in the first phase of the study (evaluation of the effects of sample processing on PCD results). These 19 dogs and 25 additional dogs were used in the second phase of the study (determination of the PCD reference range); of these 44 dogs, 24 were male and 20 were female. The breeds of dogs used in phase 2 included mixed breed (n = 22), Labrador Retriever (9), Boxer (3), Doberman Pinscher (2), Rat Terrier (2), American Pit Bull Terrier (1), Australian Shepherd (1), Dachshund (1), German Shepherd Dog (1), Miniature Poodle (1), and Whippet (1). The ages of the 44 dogs ranged from 5 months to 9 years (median, 2.5 years). All dogs were client owned and were brought to the Texas A&M University Veterinary Medical Teaching Hospital for routine health examinations. Each dog was determined to be healthy on the basis of verbally obtained anamnesis, data from an owner-completed questionnaire, and results of a thorough physical examination of each dog. Dogs were excluded if they had any history of vomiting, diarrhea, anorexia, weight loss, polydipsia, polyuria, or other notable clinical signs in the preceding 3 months. Additionally, dogs were excluded if they had any known chronic or active disease. None of the dogs had a history of treatment with drugs known to alter hepatic enzyme function or were currently receiving dietary supplements, nutraceuticals, or any medications other than routine heartworm or flea preventative.
Dogs were closely monitored during and for 2 to 3 hours after aminopyrine administration for gross evidence of adverse reactions. After the dogs were released, their owners were instructed to report any signs of clinical abnormalities that developed within 7 days after study participation. The study protocol was approved by the Clinical Research Review Committee of the Texas A&M Veterinary Medical Center; all owners gave informed consent for their dog's participation in the study.
Procedures—A stock solution of 13C-aminopyrine (4 mg/mL) was prepared by dissolving 13C-aminopyrinea in deionized water. The solution was then sterilized via passage through a 0.2-μm-pore syringe filterb and stored in an amber glass bottle at 4°C until administered. Stock solutions were discarded after 2 weeks.
The study was comprised of 2 phases: evaluation of the optimal sample handling and processing conditions for the 13C-ADBT (phase 1) and determination of the reference range for PCD results in healthy dogs (phase 2).
For phase 1, a baseline 6-mL blood sample was obtained from each dog, from which 1 mL was immediately transferred into each of 6 collection tubesc (designated A, B, C, D, E, and F). 13C-aminopyrine (2 mg/kg) was then slowly administered into a peripheral vein. After 45 minutes, another 6-mL blood sample was obtained and transferred to 6 collection tubes in a similar fashion (day 0). Tubes A and B (5-mL evacuated glass tubes) contained vacuum-dried sodium heparin to which 2 mL of 6M hydrochloric acidd had been added prior to the start of the experiment; tubes C, D, and E (5-mL evacuated glass tubes) contained vacuum-dried sodium heparin alone; and tube F (4-mL evacuated plastic tube) contained sodium fluoride and potassium oxalate.
Two milliliters of 6M hydrochloric acid was added to tubes C, D, E, and F on days 7, 14, 21, and 21, respectively (acid was present in tubes A and B at the time of blood collection). To maximize acid-induced CO2 release, samples A and B were vortexed after collection and all other samples were vortexed after subsequent acid addition. To determine PCD, the baseline and 45-minute sample absolute 13C:12C ratios of the extracted CO2 were quantified via fractional mass spectrometry with an automated breath carbon analyzere on day 0 for tube A and on day 21 for all remaining tubes. All tubes were stored at room temperature (21° to 26°C) until analysis.
For determination of a reference range for PCD values in phase 2, PCD values were obtained from 2 groups of dogs. The first group consisted of the 19 dogs in phase 1. Only the PCD values obtained from analysis of sample F were used. For each of the remaining 25 dogs, a baseline 1-mL blood sample was obtained and immediately transferred into a tube containing sodium fluoride. Administration of 13C-aminopyrine was performed as in phase 1, and after 45 minutes, another 1-mL blood sample was obtained and transferred to another tube containing sodium fluoride. The addition of hydrochloric acid and quantification of absolute 13C:12C ratios of extracted CO2 via mass spectroscopy were performed within 1 week of collection of these samples according to the method described for phase 1.
Data analysis—The PCD values were generated by use of a series of calculations21 (Appendix). All data sets were evaluated for normality by use of the D'Agostino-Pearson omnibus normality test. Mean PCD values for the various sample groups were compared by use of a repeated-measures ANOVA and Dunnett post hoc test, with sample A as the control. Correlation of PCD and covariables was determined via calculation of the Pearson product-moment correlation coefficient. Statistical analysis softwaref was used for data analysis. For all analyses, the level of significance was set at a value of P < 0.05. From the PCD data, the reference range was established by use of the central 95th percentile.
Results
No clinically obvious adverse effects were evident during or 2 to 3 hours after aminopyrine administration. None of the owners reported any signs of adverse effects in their dogs in the 7 days following participation in the study. Extravasation of aminopyrine occurred in 1 dog that participated in phase 2 of the study, but no adverse effects developed locally or systemically within the 2- to 3-hour poststudy observation period. Two weeks later, the dog returned for a follow-up appointment. At that time, a full physical examination was repeated and no abnormalities were detected. The owner had not detected development of any adverse effects associated with extravasation of aminopyrine during the 2-week period. Although a 13C-ADBT was performed at the recheck visit, the derived PCD was later excluded from data analysis to avoid any influence the extravasation might have had.
As determined via repeated-measures ANOVA, the mean PCD values differed significantly (P < 0.001) across all processing conditions (samples A through F; Figure 1). When compared separately by use of the Dunnett post hoc test, the mean PCD was significantly lower in samples C, D, and E, compared with that in the control sample (P < 0.001 for sample C; P < 0.01 for samples D and E). There was no significant difference in mean PCD between the control sample and samples B or F. No statistical correlation could be made between PCD value and age or weight of the dogs on the basis of calculated Pearson product-moment correlation coefficients.
Effect of handling conditions and processing time on PCD values in blood samples collected from 19 healthy dogs for analysis via the 13C-ADBT. A blood sample from each dog was collected at time 0 and 45 minutes after IV administration of 13C-aminopyrine (2 mg/kg; day 0); aliquots were immediately transferred into tubes containing sodium heparin and hydrochloric acid (samples A and B), sodium heparin alone (samples C, D, and E), or sodium fluoride (sample F). Hydrochloric acid was added to samples C through F at days 7, 14, 21, and 21, respectively. The PCD was calculated by use of baseline and post–aminopyrine administration absolute 13C:12C ratios of sample extracted CO2 via fractional mass spectrometry on day 0 (control sample A) or 21 (samples B through F). The solid line represents the mean value for each data set. *Value was significantly (P < 0.05) different from the control sample (A) value.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
Effect of handling conditions and processing time on PCD values in blood samples collected from 19 healthy dogs for analysis via the 13C-ADBT. A blood sample from each dog was collected at time 0 and 45 minutes after IV administration of 13C-aminopyrine (2 mg/kg; day 0); aliquots were immediately transferred into tubes containing sodium heparin and hydrochloric acid (samples A and B), sodium heparin alone (samples C, D, and E), or sodium fluoride (sample F). Hydrochloric acid was added to samples C through F at days 7, 14, 21, and 21, respectively. The PCD was calculated by use of baseline and post–aminopyrine administration absolute 13C:12C ratios of sample extracted CO2 via fractional mass spectrometry on day 0 (control sample A) or 21 (samples B through F). The solid line represents the mean value for each data set. *Value was significantly (P < 0.05) different from the control sample (A) value.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
Effect of handling conditions and processing time on PCD values in blood samples collected from 19 healthy dogs for analysis via the 13C-ADBT. A blood sample from each dog was collected at time 0 and 45 minutes after IV administration of 13C-aminopyrine (2 mg/kg; day 0); aliquots were immediately transferred into tubes containing sodium heparin and hydrochloric acid (samples A and B), sodium heparin alone (samples C, D, and E), or sodium fluoride (sample F). Hydrochloric acid was added to samples C through F at days 7, 14, 21, and 21, respectively. The PCD was calculated by use of baseline and post–aminopyrine administration absolute 13C:12C ratios of sample extracted CO2 via fractional mass spectrometry on day 0 (control sample A) or 21 (samples B through F). The solid line represents the mean value for each data set. *Value was significantly (P < 0.05) different from the control sample (A) value.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
Because of the lack of significant difference in PCD values in samples collected in tubes containing sodium fluoride alone versus those collected in tubes containing sodium heparin with hydrochloric acid, tubes containing sodium fluoride were used exclusively for phase 2 of the study, in which a reference range for PCD was derived by use of the 13C-ADBT. On the basis of the central 95th percentile, the reference range for PCD from the 13C-ADBT in healthy dogs was 0.08% to 0.20% (mean PCD, 0.13%; Figure 2).
Values of PCD determined by use of the 13C-ADBT in blood samples collected in tubes containing sodium fluoride from 44 healthy dogs. The solid line represents the mean value. The dotted lines represent the upper and lower limits of the central 95th percentile; these limits were used to establish a reference range for 13C-ADBT–derived PCD values in dogs.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
Values of PCD determined by use of the 13C-ADBT in blood samples collected in tubes containing sodium fluoride from 44 healthy dogs. The solid line represents the mean value. The dotted lines represent the upper and lower limits of the central 95th percentile; these limits were used to establish a reference range for 13C-ADBT–derived PCD values in dogs.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
Values of PCD determined by use of the 13C-ADBT in blood samples collected in tubes containing sodium fluoride from 44 healthy dogs. The solid line represents the mean value. The dotted lines represent the upper and lower limits of the central 95th percentile; these limits were used to establish a reference range for 13C-ADBT–derived PCD values in dogs.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
Discussion
Consistent with findings of previous studies,17–20 no clinically obvious adverse effects developed in any of the dogs during the study and the following 7 days. Despite the apparent safety of the 13C-ADBT in the 75 dogs used in all studies17–20 to date, no long-term hematologic or serum biochemical analyses have been performed as part of a more rigorous safety evaluation. Mild organ damage may be more important in dogs with compromised hepatic function. Because this test is ultimately intended for use in that patient group, evaluation of its safety for use in those dogs would appear prudent.
In the present study, extravasation of approximately half the calculated dose of 13C-aminopyrine occurred in 1 dog. The dog did not react adversely to that event and no extravasation-related clinical signs developed either immediately after or during the weeks that followed the event. The extravasation did preclude use of the samples collected on that day and ultimately those collected at the recheck visit because the pharmacokinetics of aminopyrine following SC administration in relation to the 13C-ADBT has not been reported to our knowledge. This issue may be important in routine use of the 13C-ADBT—careful attention to IV administration of aminopyrine may be crucial to ensure the accuracy of results. Extravasation may result in a falsely decreased PCD and erroneously indicate suspect hepatic function because of delayed absorption and metabolism of 13C-aminopyrine.
A potential limitation of our study is that the health status of the dogs was not assessed beyond what could be ascertained via history and findings of a thorough physical examination. Thus, the PCD reference range was derived from apparently healthy dogs rather than from truly clinically normal dogs in which hematologic and serum biochemical variables were known to be within reference limits. A more complete investigation including measurement of serum liver enzyme activities and pre- and postprandial bile acids concentrations, abdominal ultrasonography, and other assays for hepatic microsomal function may have identified individuals among the study dogs that had subclinical disease or enzyme induction, which could potentially alter the results of the 13C-ADBT. However, even if such dogs were included in the study, the results of phase 1 of the study would not be affected because the absolute PCD value is relatively inconsequential, compared with its variation among the different sample handling methods and processing times. Nevertheless, it is possible that PCD values from dogs with subclinically decreased hepatic function could have been included in the generation of the reference range. Although we suspect that this is highly unlikely, if this had occurred, the lower limit of the derived PCD reference range would most likely be falsely low, resulting in a concordant decrease in the sensitivity of the 13C-ADBT for detection of compromised hepatic function. It is also theoretically possible that some dogs had enzyme inductions, which would have resulted in falsely high PCD values. This was also considered to be highly unlikely because drug administrations are most likely to cause enzyme inductions and owners were carefully questioned to ensure that none of the dogs were given any medications (other than flea or heartworm preventative), nutraceuticals, or dietary supplements prior to study commencement. Endocrinopathies that could also result in enzyme induction would most likely have been detected on the basis of history and physical examination findings. Ultimately, if the 13C-ADBT is found to lack sensitivity for identification of impaired hepatic function on the basis of the data obtained in the present study, a more robust reference range for proven clinically normal rather than apparently healthy dogs may need to be established.
As the major goal of the present study, our intent was to establish an effective but practical method of sample handling and processing for the 13C-ADBT to increase the likelihood that its application would become clinically feasible. To date, protocols for the test have involved the immediate addition of hydrochloric acid to blood samples to extract CO2. Unfortunately, if the 13C-ADBT is to be used anywhere other than the location of the reference laboratory, this protocol requires shipping collection tubes containing hydrochloric acid. Because of government regulations, shipment of blood collection tubes that contain a class 8 hazardous substance such as hydrochloric acid is more costly and labor intensive than mailing of routine collection tubes. Additionally, breakage of the tubes (either accidentally or intentionally) could pose a serious health hazard for practitioners and their staff. Results of our study indicated that if the addition of hydrochloric acid to blood samples collected in tubes containing sodium heparin is delayed for a week or more, PCD values do significantly change. We postulated that continued ex vivo erythrocyte and leukocyte glycolysis in the blood samples collected in tubes containing sodium heparin produces enough CO2 to effectively dilute the 13C-labeled CO2 present, thereby altering the PCD. Thus, for purposes of the 13C-ADBT, this effect precludes the need to add acid to blood samples collected in standard collection tubes containing sodium heparin blood after delivery to the laboratory.
In an attempt to eliminate this effect and the necessity of shipping hydrochloric acid, we also examined the use of tubes containing sodium fluoride for collection of 13C-ADBT blood samples. Exposure of erythrocytes to fluoride induces a variety of metabolic alterations, most of which result from the secondary effects of enzyme inhibition that lead to a reduction of pyruvate synthesis and interference with the regeneration of diphosphopyridine nucleotide.22 The end result, as it relates to the 13C-ADBT, is that the ex vivo production of CO2 in the blood samples is halted. In the present study, there was no significant difference in PCD values between the control samples and samples collected in tubes containing sodium fluoride in which acid extraction of CO2 was delayed for as long as 3 weeks. Thus, use of blood collection tubes containing sodium fluoride would circumvent the need to add hydrochloric acid to the tubes prior to shipment. Additionally, a 3-week period in which to complete the assay provides ample time to batch samples for shipment without significantly affecting results. Furthermore, the results of our study indicated that storage of blood samples in tubes containing sodium fluoride at room temperature did not significantly alter PCD values. This fact also makes collection and shipment of 13C-ADBT blood samples easier. As sample handling and shipment procedures become simplified, further studies to rigorously evaluate the clinical usefulness of the 13C-ADBT are needed.
ABBREVIATIONS
| 13C-ADBT | Carbon 13-labeled aminopyrine demethylation blood test |
| PCD | Percentage dose of carbon 13 administered as carbon 13-labeledaminopyrine and recovered in CO2 extracted from blood samples |
4-dimethyl-13C2-aminoantipyrine, Isotech Inc, Miamisburg, Ohio.
Gelman Sciences Supor Acrodisc (0.2 μm, sterile), VWR Scientific Products Corp, West Chester, Pa.
BD vacutainer, Preanalytical Solutions, Franklin Lakes, NJ.
Hydrochloric acid, Sigma Chemical Co, St Louis, Mo.
Automated breath carbon analyzer, Europa House, Crewe, England.
GraphPad Prism, version 5.0, GraphPad Software Inc, San Diego, Calif.
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Appendix
Formulas21 adapted for calculation of PCD from data obtained by use of the 13C-ADBT.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
Formulas21 adapted for calculation of PCD from data obtained by use of the 13C-ADBT.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
Formulas21 adapted for calculation of PCD from data obtained by use of the 13C-ADBT.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1385
