Materials and Methods

Animals—Twelve female Baluchi lambs were used for the study. Lambs were offspring of university-owned Baluchi ewes, and they were maintained in the flock to allow them to suckle from their dams. To train lambs to suckle from a nipple, animals were offered milk replacer from a nipple bottle once daily in the morning beginning approximately 1 week after birth. Lambs also had access to alfalfa hay at all times. All methods were in compliance with the guide for the care and use of agricultural animals in agricultural research and teaching.

Procedures—Two consecutive APATs were performed at a 1-week interval in the lambs at each of 3 developmental stages. Stage 1 (before weaning) was before lambs were weaned, stage 2 (at weaning) was at the time lambs were weaned, and stage 3 (after weaning) was after lambs were weaned. Mean ± SD age of lambs at stages 1, 2, and 3 was 10 ± 2 weeks, 16 ± 2 weeks, and 22 ± 2 weeks, respectively.

For the first APAT of each stage, lambs were allowed to suckle a test solution consisting of 20 mL of tap water, acetaminophen^a (50 mg/kg), and 30% barium sulfate solution (10 mL/kg) from a nipple bottle. The second APAT was performed 1 week later, but with the same test solution administered via an orogastric tube inserted into the rumen of each lamb.

On the morning of each APAT, lambs were weighed, and a 16-gauge catheter^b was aseptically inserted in a jugular vein. Blood samples were obtained immediately before administration of the test solution (time 0) and 30, 60, 90, 120, 150, and 180 minutes after administration. Collected blood samples were immediately transferred into 10-mL tubes that contained potassium oxalate; tubes were then centrifuged (1,800 X g for 15 minutes). Plasma was harvested and stored at −20°C until analyzed for plasma acetaminophen concentration by use of a spectrophotometric technique described elsewhere.⁷

Immediately after administration of the test solution, abdominal radiographs^c were obtained. The unsedated lambs were manually restrained in left lateral recumbency. Radiographs were examined to determine location of the barium sulfate in the forestomachs, thus allowing investigators to assess activation of the reticular groove reflex. All radiographs were interpreted by veterinary radiologists who were unaware of the treatment administered to each lamb. Only lambs that had proper closure of the reticular groove when suckling the test solution (as determined radiographically) were included for the analysis of stages 1 and 2. For stage 3, proper function of the reticular groove reflex was not used as an inclusion criterion because none of the lambs had complete closure of the reticular groove. Osmolalities of the test solutions with the lowest and highest acetaminophen concentration were determined by use of freezing-point depression.^d

Data analysis—The Cmax and Tmax were obtained from a plot of the plasma acetaminophen concentration-time data. The value for AUC_Acet was calculated by use of the trapezoidal rule to provide an index of the amount of acetaminophen absorbed for each treatment. An ROC analysis was performed to assess accuracy of the AUC_Acet and the plasma acetaminophen concentration at specific time points to determine function of the reticular groove reflex.

Statistical analysis—Data were expressed as mean ± SD. Normal distribution of data was tested by determining the Shapiro-Wilk W and associated P value and by screening the normal probability plots. When necessary, values were logarithmically transformed to achieve a normal distribution. A repeated-measures ANOVA was used to detect differences in measured or calculated variables among stages and over time. A paired 2-tailed t test was used to determine differences between treatments. Bonferroni adjusted P values were used to assess differences within and among stages whenever the F test was significant. Values of P < 0.05 were considered significant. The ROC curves were plotted for AUC_Acet and the Acet-T₆₀. The AUC for the ROC curve was calculated for both of these curves by use of the trapezoidal rule. A statistical software program^e was used for all analyses.

Results

All lambs remained healthy throughout the study. Volume of test solution administered to each lamb ranged from 87 mL for the smallest lamb (body weight, 6.7 kg) in stage 1 to 370 mL for the heaviest lamb (body weight, 35 kg) in stage 3. Osmolalities of the test solutions ranged from 199 to 218 mOsm/kg.

Suckling of the test solution resulted in complete closure of the reticular groove, as determined by use of radiography, in all 12 lambs at stage 1 (Figure 1). In contrast, examination of radiographs revealed no or incomplete closure of the reticular groove in 4 lambs at stage 2 (Figure 2). Therefore, data obtained from those 4 lambs from both APATs of stage 2 were not included in the data analysis. At stage 3, none of the lambs that suckled the test solution had radiographic evidence of radiopaque material in the forestomachs, which indicated no or incomplete closure of the reticular groove in all lambs. Because all lambs at stage 3 had failure of the reticular groove mechanism, data obtained for all lambs at stage 3 were included in the analysis.

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Lateral radiographic view of the abdomen of a lamb at the first stage (mean ± SD age, 10 ± 2 weeks) immediately after suckling a test solution containing acetaminophen and barium sulfate. Notice that the barium sulfate in the test solution is located in the abomasum and not in the reticulum, which indicates complete closure of the reticular groove.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.820

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Lateral radiographic view of the abdomen of a lamb at the second stage (mean ± SD age, 16 ± 2 weeks) immediately after suckling a test solution. Notice that the barium sulfate in the test solution is located in the forestomachs, which indicates no or incomplete closure of the reticular groove.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.820

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Mean plasma acetaminophen concentrations at the various stages and for the 2 treatments over time were plotted (Figure 3). The Cmax values determined for lambs when they suckled the test solution and when they were tube fed the test solution at each developmental stage were summarized (Table 1). The Cmax and AUC_Acet were significantly higher for lambs when they suckled the test solution than when they were tube fed the test solution at stages 1 and 2, but were in a similar range for both treatments at stage 3 (Table 2). The mean ± SD AUC_Acet of lambs when suckling the test solution decreased (but not significantly) in stages 1 and 3 (61.4 ± 26.7 mg·h/mL, 63.9 ± 17.1 mg·h/mL, and 48.8 ± 24.3 mg·h/mL for stages 1, 2, and 3, respectively), whereas mean AUC_Acet increased (but not significantly) when lambs were tube fed in stages 1 and 3 (36.4 ± 15.7 mg·h/mL, 35.4 ± 28.0 mg·h/mL, and 52.9 ± 24.0 mg·h/mL for stages 1, 2, and 3, respectively).

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Plasma acetaminophen concentration over time for lambs when suckling a test solution (solid line) and when tube fed the test solution (dashed line) at stage 1 (A), stage 2 (B), and stage 3 (C) of the study. Notice that the scale on the y-axis differs among panels. The onset of administration of the test solution is indicated (arrow) and is designated as time 0. In all 3 panels, a proposed cutoff value of 25 μg/mL is indicated (horizontal dotted line). *Value for this time point differs significantly (P < 0.05) from the value for time 0. †Within a time point, values differ significantly (P < 0.05) between treatments.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.820

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Results of ROC analysis that compared the suitability of Acet-T₆₀ and AUC_Acet to the criterion-referenced standard (ie, radiography) were determined (Figure 4). The AUC for the ROC curve for Acet-T₆₀ was 0.70, and the AUC for the ROC curve for AUC_Acet was 0.59. From these data, a cutoff value for Acet-T₆₀ with optimal sensitivity and specificity was determined; the cutoff value was 24.9 μg/mL, which yielded a sensitivity of 0.80 and a specificity of 0.89.

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The ROC plots for Acet-T₆₀ (solid line) and AUC_Acet (dotted line) determined for 12 lambs at stage 1, 8 lambs at stage 2, and 12 lambs at stage 3. Sensitivity (horizontal dashed lines) and specificity (vertical dashed lines) were determined for suggested cutoff values of 24.9 μg/mL (point A) and 26.3 μg/mL (point B) for Acet-T₆₀.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.820

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Discussion

The primary objective of the study reported here was to compare results of APATs in young ruminants when acetaminophen was administered with and without stimulation of the reticular groove reflex. Analysis of the results indicated that Cmax and AUC_Acet after stimulation of the reticular groove mechanism were nearly double the values when lambs were fed with an orogastric tube feeder to circumvent closure of the reticular groove. The ROC analysis indicated that Acet-T₆₀ is a variable with reasonable accuracy for use in determining function of the reticular groove reflex and that a single blood sample obtained to determine Acet-T₆₀ (with a cutoff value of 25 μg/mL) can be used to characterize function of the reticular groove mechanism in lambs between 8 and 24 weeks of age.

The APAT has been validated as an alternative to other more invasive and costly methods for investigating emptying rates of the stomach in humans, horses, and other species and of the abomasum in calves.^6–8 Because acetaminophen is readily absorbed in the duodenum but not in the stomach, the transit of this substance through the stomach is considered to be the rate-limiting step for its absorption and thus its appearance in plasma.^5,12

In ruminants, pharmacokinetics of acetaminophen depend on abomasal motility as well as the reticular groove mechanism that considerably impacts the transit time of ingesta from the esophagus to the duodenum. Therefore, ODT was proposed as a more accurate term than abomasal emptying rate when describing results obtained by use of the APAT in ruminants.⁹ The importance of the effect of the reticular groove mechanism on ODT (as determined by results of the APAT) has been reported in a study⁹ in which tube-fed calves had significantly lower peak plasma acetaminophen concentrations, AUCs, and delayed Tmax for acetaminophen in plasma, compared with results for suckling calves. In monogastric species, there is little evidence that relevant amounts of acetaminophen are absorbed from the stomach,⁵ but to our knowledge, the absorption of acetaminophen from the ruminant forestomachs has not been investigated. For the study reported here, we assumed that acetaminophen would not be absorbed from the rumenoreticulum or abomasum; however, it would be possible that a larger fraction of acetaminophen would be absorbed from the forestomachs in tube-fed lambs than in lambs suckling the test solution because of the prolonged transit time of liquid ingesta through the forestomachs.

In contrast to results of a study⁷ in which investigators evaluated abomasal emptying rate in calves and found that Tmax for acetaminophen was the best proxy for assessing abomasal emptying, our results suggested that the Cmax for acetaminophen at a specific time is best suited for determining function of the reticular groove mechanism. This conclusion is in agreement with the observation that the delay in acetaminophen absorption in calves with disturbed reticular groove mechanism is largely attributable to prolonged retention in the ruminoreticulum rather than to decreased abomasal motility.¹⁰ Accordingly, the effect of a disturbed reticular groove reflex is more important on Cmax than on Tmax.¹¹

Factors affecting the abomasal emptying rate, such as osmolality or pH of the solution containing the acetaminophen, are likely to affect results of the APAT.¹³ In the study reported here, osmolality of the test solutions was assumed to vary because differing acetaminophen concentrations were measured. The measured osmolality of each test solution was approximately 200 mOsm/kg; thus, they were considerably less than the typical osmolality of milk or commercial milk replacers.¹⁴ The range of variation for the osmolality of the test solution was 19 mOsm/kg. Therefore, we deem it unlikely that a change in the composition of the test solution may have biased results of the APAT by affecting the abomasal emptying rate.

The numeric increases of the AUC_Acet detected from stage 1 to stage 3 suggested that pharmacokinetics of acetaminophen are affected by age-dependent changes in the volume of distribution and possibly also by renal and hepatic function responsible for acetaminophen clearance. In calves, the volume of distribution in extracellular fluid, abomasal emptying rate, and capacity to metabolize acetaminophen change with age; these facts suggest that Tmax should be the most stable variable over time because Cmax and AUC will be influenced by other factors, including volume of distribution and rate of elimination.^15,16

Analysis of results of several experiments performed on preweaned calves administered acetaminophen (50 mg/kg) mixed in whole milk or casein-based milk replacer revealed a considerably lower Cmax than that in the study reported here and also revealed biphasic curves for plasma acetaminophen concentrations.⁷ Results of an in vitro study¹¹ in which investigators evaluated characteristics of acetaminophen binding in clotted milk suggest that when acetaminophen is mixed in milk or casein-based milk replacer, a large fraction of acetaminophen transits the abomasum rapidly with the whey fraction of milk, and the remainder of the acetaminophen is bound to curd. It is assumed that these characteristics of acetaminophen in clotted milk are the underlying cause for the biphasic passage of acetaminophen through the abomasum and thus a biphasic plasma acetaminophen concentration. Because the test solution did not contain milk or milk replacer, a monophasic plasma acetaminophen concentration curve and a higher Cmax would have been anticipated. This assumption is supported by results of a study¹³ in which investigators compared plasma acetaminophen concentrations in calves fed casein-based milk replacer and various electrolyte solutions; a considerably lower Cmax and delayed Tmax were detected in calves receiving acetaminophen in milk replacer.

To our knowledge, the safety of acetaminophen in ruminants after oral administration has not been directly investigated. Intravenous administration of acetaminophen to calves at dosages up to 50 mg/kg does not result in clinically appreciable adverse effects of hepatotoxicosis.^17,18 In sheep, acetaminophen toxicosis has been associated with injectable formulations. The main pathologic finding is hepatopathy, which has been identified in sheep administered a single dose of acetaminophen (400 mg/kg), IV. Other changes in the serum biochemical profile of sheep with acetaminophen toxicosis are hypoglycemia, which is associated with interference of normal carbohydrate metabolism; a decrease in the albumin-to-globulin ratio; and hypercholesterolemia.¹⁷ Therefore, we believe that an APAT conducted by use of a dosage of 50 mg/kg in young ruminants does not pose any risk of toxic effects.

Analysis of our results indicated that the APAT can be a valuable diagnostic tool for use in determining function of the reticular groove mechanism. Evaluation of the Acet-T₆₀ revealed the strongest differences between lambs with and without closure of the reticular groove. Analysis of our results suggested that this variable is reasonably accurate for use in determining function of the reticular groove with an AUC for the ROC curve of 0.70. We propose that a cutoff value for Acet-T₆₀ of 25 μg/mL (which yielded a sensitivity of 0.80 and a specificity of 0.89) be used for evaluation of lambs between 8 and 22 weeks of age.