Materials and Methods

Calves—Dehydrated calves with diarrhea admitted consecutively for treatment at the Veterinary Clinic of the University of Selcuk were enrolled in this study. Most calves were Swiss-Holstein breed, and all were < 45 days of age. Mean body weight of the calves was 38 kg (83.6 lb) on admission (range, 35 to 42 kg [77 to 92.4 lb]). Routine physical examination findings, including extent of eye recession into the orbit, were recorded, and jugular venous blood gas analysis was performed. A jugular venous catheter was placed aseptically and secured to the neck to permit collection of blood samples.

Calves (n = 20) were admitted to the study if they were mildly or moderately dehydrated (as assessed by eye recession into the orbit of 3 to 5 mm¹³) but did not have severe acidemia (ie, jugular venous pH was approx > 7.20). Calves with clinical evidence of shock or severe dehydration (eye recession > 5 mm) were excluded from the study. Enrofloxacin^a (5 mg/kg [2.3 mg/lb], IM) was administered daily for 3 days to all calves.

Experimental protocol—Calves were randomly allocated to receive 2 L of 1 of 4 treatments (n = 5 calves/treatment) administered at room temperature (20° to 22°C [68.0° to 71.6°F]) via oroesophageal intubation. A standardized method of fluid administration was used because the results of preliminary studies indicated that some calves would not suckle 2 L within 10 minutes, and a delay in suckling the allotted volume would make it difficult to accurately assess abomasal emptying rate via acetaminophen absorption. The 4 treatments were 150 mmol/L or 300 mmol/L sodium bicarbonate^b or sodium acetate^c solution (Table 1). All 4 test solutions contained acetaminophen^d (50 mg/kg [22.7 mg/lb]) and 50 g of glucose monohydrate^e (equivalent to 1.3 g/kg [0.6 g/lb]). The final osmolarity of the 150 mmol/L and 300 mmol/L ORT solutions was calculated as 426 mOsm/L and 726 mOsm/L, respectively.

Blood samples were collected anaerobically from the jugular vein into heparin-containing plastic syringes for blood gas analysis, which was performed within 10 minutes of collection. Venous blood samples for blood gas analysis, PCV determination, and serum biochemical analyses were obtained at 0 minutes (immediately before ORT solution administration) and at 30, 60, 90, 120, 150, 180, 240, 360, 480, and 720 minutes after ORT solution administration. Venous blood samples for determination of serum acetaminophen concentration were obtained at 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 240, 360, 480, and 720 minutes after oroesophageal intubation of 300 mmol/L sodium bicarbonate or sodium acetate ORT solution or at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150, 180, 240, 360, 480, and 720 minutes after oroesophageal intubation of 150 mmol/L sodium bicarbonate or sodium acetate ORT solution. These sampling times were selected in an attempt to have 6 data points before and after the anticipated time of maximal acetaminophen concentration to facilitate nonlinear regression analysis. Blood samples for serum biochemical analyses were collected into plain tubes, allowed to clot at room temperature, and centrifuged, and the serum was harvested and stored at −20°C until analyzed.

Calves were monitored closely for the first 12 hours after oroesophageal intubation. Calves did not have access to water for the first 4 hours after treatment but had access to water after 4 hours until 12 hours after treatment. At 12 hours, calves were offered 2 L of cow's milk in a bottle, and those that did not suckle 2 L within 10 minutes had the remaining volume of milk administered via oroesophageal intubation. Additional supportive treatment was administered as needed until the calf was discharged from the veterinary hospital.

Laboratory analysis—Blood gas analysis (pH and PCO₂) was performed with a blood gas analyzer,^f the values were corrected for rectal temperature, and the plasma bicarbonate concentration and extracellular base excess value were calculated. Serum sodium, potassium, and chloride concentrations were measured via potentiometry.^g Serum albumin concentration (bromcresol green assay) and total protein concentration (Biuret colorimetric assay) were measured in serum samples by use of a commercially available kit^g with a spectrophotometer.^h Packed cell volume was determined automatically.ⁱ Strong ion gap and AG were calculated by use of standard equations.

Acetaminophen absorption—Plasma acetaminophen concentration was determined by use of a spectrophotometric technique as described.¹⁴ Acetaminophen absorption provides an accurate method of determining the abomasal emptying rate of liquid-phase meals in calves,¹⁴ with time to maximal acetaminophen concentration after oral administration primarily dependent on the rate of abomasal emptying.

Values for C_max and T_max were obtained from a plot of the plasma acetaminophen concentration–time data. The first derivative of the Siegel modified power exponential formula¹⁵ was used to describe the plasma acetaminophen concentration–time relationship, as follows:

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where C(t) is the acetaminophen concentration in plasma (Pg/mL) at time ≥ in minutes; m, k, and E are constants; and m is the total cumulative acetaminophen recovery when time is infinite. This model provided the best method for describing the acetaminophen absorption curve in suckling calves.¹⁴ Model T_max was obtained as follows:

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Model C_max was calculated by applying the values for m, k, E, and ≥ = model T_max to the cumulative dose curve.

Determination of percentage change in plasma volume—Percentage change in plasma volume at time i was calculated as follows:

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where SP₀ is the serum protein concentration at time = 0 minutes and SP_i is the serum protein concentration at time i.¹⁶

Statistical analysis—Data are expressed as least squares mean ± SEM, and values of P < 0.05 were considered significant. A software program was used for all statistical analyses.^j–l Models to describe the acetaminophen absorption curve were fit by use of nonlinear regression.¹⁴ The adequacy of model fit was assessed via visual examination of plots of observed versus predicted concentrations and by examination of residual plots. A general linear model procedure was used to compare the difference in acetaminophen absorption variables among groups. For this comparison, variables with unequal variances were log transformed or ranked before ANOVA was performed. When indicated by a significant F test for treatment, appropriate post tests were conducted between groups (150 mmol/L sodium acetate was compared with 150 mmol/L sodium bicarbonate, 300 mmol/L sodium acetate was compared with 300 mmol/L sodium bicarbonate, 150 mmol/L sodium acetate was compared with 300 mmol/L sodium acetate, and 150 mmol/L sodium bicarbonate was compared with 300 mmol/L sodium bicarbonate). The Bonferonni adjustment was used for multiple pairwise comparisons to maintain the experimentwise error rate for each family of comparisons; values of P < 0.0125 were considered significant for group comparisons.

A mixed model that used repeated measures was applied to compare the change in jugular venous blood acid-base variables; serum electrolyte, albumin, and total protein concentrations; PCV; and plasma volume, with the value at time = 0 as a covariate. Three covariance structures (compound symmetry, variance components, and first-order autoregressive) were compared by use of the Akaike information criterion; the covariance structure providing the lowest Akaike information criterion value was used in the final mixed models analysis. When indicated by a significant F test for a main effect or interaction effect, appropriate post tests were conducted within a group with the value at time = 0 or between groups (150 mmol/L sodium acetate was compared with 150 mmol/L sodium bicarbonate, 300 mmol/L sodium acetate was compared with 300 mmol/L sodium bicarbonate, 150 mmol/L sodium acetate was compared with 300 mmol/L sodium acetate, and 150 mmol/L sodium bicarbonate was compared with 300 mmol/L sodium bicarbonate). The Bonferonni adjustment was used for multiple pairwise comparisons to maintain the experimentwise error rate for each family of comparisons; values of P < 0.005 were considered significant for time comparisons at time = 0 within a group, and values of P < 0.0125 were considered significant for group comparisons at a specific time.

The primary variables of interest that examined the effect of treatment on acid-base status were jugular venous blood pH, PCO₂, base excess, plasma bicarbonate concentration, SIG, and AG. The primary variable of interest that examined the effect of treatment on abomasal emptying rate was acetaminophen model T_max. The primary variable of interest that examined the effect of treatment on extracellular fluid shifts was the calculated change in plasma volume.

Results

Calves—A substantial difference in clinical appearance was not observed among calves of the 4 groups during the 12-hour period after oroesophageal administration of the ORT solutions. The clinical appearance of most calves administered 150 or 300 mmol/L sodium bicarbonate ORT solutions or 150 mmol/L sodium acetate ORT solution was improved after approximately 3 hours, as judged on the basis of increased activity level and interest in the environment and partial improvement in their suckle reflex. However, a subsequent decrease in activity level was observed in calves fed 300 mmol/L sodium bicarbonate ORT solution from 6 to 12 hours. No obvious change in clinical appearance was observed in calves administered 300 mmol/L sodium acetate ORT solution during the 12-hour study period.

Most calves had a weak suckle reflex at 12 hours and could not suckle 2 L of milk, necessitating oroesophageal intubation of the remaining milk volume. All 20 calves were discharged from the hospital in a healthy state.

Significant treatment effects existed for jugular venous blood PCO₂ and plasma bicarbonate concentration (P = 0.045; Figure 1), serum total protein and albumin concentrations (Table 2), and a number of acetaminophen absorption factors (C_max, T_max, model T_max, k, E, and m; Figure 2; Table 3). Significant time effects existed for jugular venous blood pH (P < 0.001; Figure 3), plasma bicarbonate concentration (P < 0.001), and base excess as well as for serum potassium, chloride, total protein, and albumin concentrations; PCV; and change in plasma volume from baseline (P = 0.003; Figure 4). Significant treatment × time interaction effects existed for jugular venous blood pH (P = 0.027) and plasma bicarbonate concentration (P < 0.001), base excess, serum potassium concentration, SIG, and AG.

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Change in jugular venous plasma bicarbonate concentration over time for calves (n = 5/group) administered 1 of 4 ORT solutions via oroesophageal intubation at time = 0 hours. All calves were treated with 2 L of an ORT solution containing 50 g of glucose monohydrate and 50 mg of acetaminophen/kg (22.7 mg/lb); various groups also received 300 mmol/L sodium bicarbonate (black diamonds), 300 mmol/L sodium acetate (black squares), 150 mmol/L sodium bicarbonate (black circles), or 150 mmol/L sodium acetate (black triangles). White symbols indicate that the mean value is significantly (P < 0.05) different from the value at time = 0 hours. Data are expressed as least squares mean. The bar in the upper left corner is the SEM.

Citation: Journal of the American Veterinary Medical Association 234, 7; 10.2460/javma.234.7.926

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Acetaminophen absorption curves for the same calves as in Figure 1. See Figure 1 for key.

Citation: Journal of the American Veterinary Medical Association 234, 7; 10.2460/javma.234.7.926

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Change in jugular venous blood pH over time for the same calves as in Figure 1. See Figure 1 for key.

Citation: Journal of the American Veterinary Medical Association 234, 7; 10.2460/javma.234.7.926

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Change in plasma volume from baseline over time for the same calves as in Figure 1. See Figure 1 for key.

Citation: Journal of the American Veterinary Medical Association 234, 7; 10.2460/javma.234.7.926

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Acid-base analysis—Almost all calves had a mild to moderate strong ion (metabolic) acidosis on admission, with jugular venous blood pH ranging from 7.16 to 7.34, PCO₂ ranging from 21 to 54 mm Hg, plasma bicarbonate concentration ranging from 8 to 26 mmol/L, base excess ranging from −21 to +2 mEq/L, SIG ranging from −32 to −1 mEq/L, and AG ranging from 16 to 42 mEq/L (Figures 1 and 2; Table 2). There were no significant differences in blood gas values at baseline among groups.

The rate of systemic alkalinization differed among the 4 treatments on the basis of the significant treatment × time interaction effects for jugular venous blood pH, plasma bicarbonate concentration, base excess, SIG, and AG (Figure 3; Table 2). Jugular venous PCO₂ for 150 mmol/L sodium bicarbonate was higher than that for 150 mmol/L sodium acetate throughout the study. Jugular venous PCO₂ for 300 mmol/L sodium bicarbonate was numerically greater than that for 150 mmol/L sodium acetate, but the difference was not significant (P = 0.091). Jugular venous bicarbonate concentration for 150 mmol/L sodium bicarbonate was numerically greater than that for 150 mmol/L sodium acetate throughout the study (Figure 1), and jugular venous bicarbonate concentration for 300 mmol/L sodium bicarbonate was numerically greater than that for 150 mmol/L sodium acetate, but the differences were not significant (P = 0.083 and 0.061, respectively). Mean plasma bicarbonate concentrations for calves administered sodium acetate–containing solutions were less than the reference range for calves at 12 hours. A large negative SIG, indicating the presence of unmeasured strong anions, and a high AG, indicating the presence of unmeasured anions, remained in a number of calves at 12 hours. Strong ion gap was significantly less negative in calves administered 300 mmol/L sodium bicarbonate than in calves administered sodium acetate at 12 hours.

Serum biochemical analyses—There were no significant differences in serum biochemical concentrations at baseline (time = 0) among groups, except that total protein concentration was significantly greater in calves administered 150 mmol/L sodium acetate than those administered 150 mmol/L sodium bicarbonate. Serum albumin concentration for 150 mmol/L sodium bicarbonate was lower than that of 150 mmol/L sodium acetate and 300 mmol/L sodium bicarbonate throughout the study. Serum total protein concentration for 150 mmol/L sodium bicarbonate was lower than that of 300 mmol/L sodium bicarbonate throughout the study. Serum total protein concentration for 300 mmol/L sodium acetate was lower than that of 300 mmol/L sodium bicarbonate and 150 mmol/L sodium acetate throughout the study.

Serum sodium concentrations were similar for all 4 groups and did not change over time (Table 2). Small changes in serum chloride concentrations were detected over time. Small differences in serum potassium, serum albumin, total protein, and total albumin concentrations were occasionally detected among groups at different time points.

PCV—Calves administered 300 mmol/L sodium bicarbonate had decreased PCV from 2.5 hours onward (Table 2). Calves administered 150 mmol/L sodium bicarbonate had a significantly lower PCV at 4 hours, compared with the baseline value.

Abomasal emptying rate—Pharmacokinetically determined values for k, E, and m were determined (Table 3). The mean value for C_max for serum acetaminophen concentration in calves suckling 150 mmol/L sodium acetate was significantly lower than that for calves suckling 150 mmol/L sodium bicarbonate (Figure 2). Mean values for model C_max were similar for all 4 treatments.

Mean values for T_max and model T_max were similar for 150 mmol/L sodium acetate and sodium bicarbonate; however, both solutions had significantly lower mean values than those of 300 mmol/L sodium acetate and 300 mmol/L sodium bicarbonate (Table 3). This result indicated that 150 mmol/L solutions were emptied significantly faster than 300 mmol/L solutions, with no significant difference between equimolar solutions of sodium acetate and bicarbonate in emptying rate.

Plasma volume—Plasma volume was significantly increased in calves administered 300 mmol/L sodium acetate from 6 to 12 hours after treatment. No change was detected in the other 3 groups (Figure 4).

Discussion

The major finding of the study reported here in calves with naturally acquired diarrhea and mild to moderate strong ion acidosis and dehydration was that sodium bicarbonate ORT solutions increased plasma bicarbonate concentrations at a slightly faster rate than did sodium acetate ORT solutions of similar osmolality, although the differences were not significant. Despite the differences in plasma bicarbonate concentration–time profiles (Figure 1), there were no consistent differences in the rate of increase of venous blood pH between sodium bicarbonate and sodium acetate ORT solutions (Figure 3). We also confirmed our findings in euhydrated healthy calves that the rate of abomasal emptying was slowed when the ORT solution osmolality was increased.^11,12 However, we found that a slower rate of abomasal emptying did not have a clinically important effect on the rate of systemic alkalinization, presumably because the rate that sodium bicarbonate or sodium acetate arrived at the site of absorption in the small intestine was similar for 150 mmol/L and 300 mmol/L solutions of each agent. Of additional interest was the finding that 300 mmol/L sodium acetate expanded plasma volume, whereas 300 mmol/L sodium bicarbonate did not alter plasma volume.

We evaluated the 2 most common alkalinizing components in commercial ORT solutions, bicarbonate and acetate; most of the commercially available ORT solutions in the United States contain bicarbonate. Systemic alkalinization is required to treat strong ion (metabolic) acidosis,^17–19 which is frequently present in calves with diarrhea.^2,8 Sodium bicarbonate is an effective alkalinizing agent that does not require metabolism to exert its effect. However, sodium bicarbonate has the disadvantage that alkalinization of the gastrointestinal tract can facilitate growth of pathogenic enteric bacteria.^1,10,11 Acetate is metabolized in 10- to 80-day-old calves similarly to other domestic animals,²⁰ with the disappearance rate being slightly faster in 11- to 18-day-old calves than in 2- to 5-day-old calves.²¹ Acetate has a net alkalinizing ability similar to bicarbonate on an equimolar basis, whereby acetate (CH₃COO⁻) is oxidized to CO₂ and H₂O within mitochondria, via the reaction: CH₃COO⁻ + H⁺ + O₂ o 2CO₂ + H₂O.²¹ Studies in dehydrated calves²² and euhydrated dogs²³ reveal that IV administration of bicarbonate increases plasma bicarbonate concentration and pH at an initially faster rate than does IV administration of an equimolar solution of acetate; this result is attributed to the time needed for acetate oxidation within mitochondria. However, after 30 to 60 minutes of IV administration, bicarbonate and acetate have similar blood pH–time and plasma bicarbonate–time profiles.^22,23 In contrast, a study²¹ in euhydrated healthy calves revealed that IV administration of bicarbonate increased plasma bicarbonate concentration and pH at a rate similar to that of IV administration of an equimolar solution of acetate. Acetate does have an advantage over bicarbonate in that it provides energy but does not excessively alkalinize the abomasal fluid or interfere with milk clotting.^10,11,24 Acetate also stimulates sodium and water absorption in the small intestine²⁵ and is metabolized readily by peripheral tissues.

To the authors' knowledge, this is the first study in calves with naturally acquired diarrhea to compare the initial alkalinizing effects of an acetate-based ORT solution with that of a bicarbonate-based ORT of similar osmolality. The only other study¹⁹ to compare acetate- and bicarbonate-based ORT solutions in calves used an experimental diarrhea model and measured the change in blood pH 48 hours after initiation of treatment. On the basis of an approximate mean euhydrated body weight of 40 kg (88 lb), a distribution space for bicarbonate of approximately 60% of body weight,^9,26 a calculated mean plasma bicarbonate concentration for the 4 groups of 14 to 18 mmol/L, and a plasma bicarbonate concentration goal of 28 mmol/L, 240 to 336 mmol of bicarbonate was required to correct the metabolic acid-base abnormality of the calves in the present study. The calculated mean amount of bicarbonate assumed immediate correction of the acid-base abnormality and maintenance of acid-base balance. Because calves were administered 300 to 600 mmol of bicarbonate (2 L of 150 or 300 mmol/L sodium bicarbonate) or bicarbonate precursor (2 L of 150 or 300 mmol/L sodium acetate), the 4 treatments should have provided sufficient alkalinizing ability to normalize acid-base status in most calves during the 12-hour study period. Inspection of the change in blood pH (Figure 3) indicated that this was true.

As expected, bicarbonate-containing ORT solutions provided a faster method for increasing plasma bicarbonate concentration than acetate-containing ORT solutions (although not significantly) because of the time required for absorbed acetate to be metabolized to bicarbonate. It is interesting that the more rapid increase in plasma bicarbonate concentration following administration of isotonic bicarbonate-containing ORT solution (Figure 1) was not accompanied by a more rapid increase in blood pH (Figure 3). This somewhat unexpected result was attributable to a higher PCO₂ in calves administered the isotonic bicarbonate-containing ORT solution because of proton buffering as indicated by the Henderson-Hasselbalch equation (HCO₃⁻ + H⁺ l H₂CO₃ l H₂O + CO₂). Although it would be preferable for an ORT solution to increase both blood pH and plasma bicarbonate concentration, any increase in plasma bicarbonate will be clinically beneficial because bicarbonate is an important extracellular buffer.

The acetaminophen absorption test provides an accurate and practical method for assessing the fluid emptying rate in suckling calves.¹⁴ Acetaminophen absorption has been used in a number of studies evaluating the effect of erythromycin, tylosin, tilmicosin, and gentamicin administration^27,28 and ORT solution osmolality, formulation, and route of administration^10–12 on the rate of abomasal emptying in healthy calves. We believe this is the first time that the acetaminophen absorption test has been used to evaluate the rate of abomasal emptying in diarrheic and dehydrated calves. Acetaminophen is a lipophilic compound with low molecular weight (151 g/mol) that is poorly bound to plasma proteins and evenly distributed throughout the body water compartments. After oral administration, the serum concentration–time relationship of acetaminophen is dependent on its absorption, metabolism, distribution, and clearance.²⁹ Absorption of a drug from the gastrointestinal tract is related to the gastric emptying rate, luminal pH, presence of bacteria or binding matter in the intestinal lumen, surface area available for absorption, mucosal blood flow, and metabolism of drug by gastrointestinal epithelial cells.^29,30 Elimination of a drug from the central compartment is dependent on the glomerular filtration rate and the rate of metabolism. Obviously, the presence of disease can affect a number of these factors and thereby alter the serum acetaminophen concentration–time relationship. Potentially important determinants of the serum acetaminophen concentration–time relationship in calves with naturally acquired diarrhea and moderate dehydration are the rate of abomasal emptying (major influence on T_max), the small intestinal surface area available for absorption (major influence on C_max and area under the curve), the volume of distribution (major influence on C_max), and the rate of elimination (major influence on T_max and C_max). Because calves were randomly assigned to treatment groups, it is reasonable to assume that the volume of distribution and glomerular filtration rate were similar for all 4 groups. Support for this assumption is provided by similar values for PCV and serum albumin concentration before treatment.

Our finding that sodium acetate did not slow the rate of abomasal emptying relative to that of sodium bicarbonate was expected in that 80 mmol/L acetate in an ingested solution does not alter the gastric emptying rate in humans³¹ and because 150 mmol/L acetate in an ORT solution does not alter emptying rate in euhydrated healthy calves, compared with 150 mmol/L sodium bicarbonate.¹⁰ As expected, 300 mmol/L ORT solutions were emptied at a slower rate than 150 mmol/L solutions; this finding is similar to that recently reported in euhydrated calves.¹² The clinical importance of a slower emptying rate after administering a hypertonic ORT solution remains uncertain because a slower rate of emptying can be beneficial if the net effect is sustained delivery of fluid, electrolytes, and nutrients to the small intestine.³² It should be noted that the rate of systemic alkalinization was similar for calves treated with 300 mmol/L or 150 mmol/L solutions (Figure 3), despite significant differences in abomasal emptying rates (Figure 2; Table 3).

On the basis of a calculated dehydration percentage of 5% to 8% of body weight and a euhydrated body weight of approximately 40 kg, 2.0 to 3.2 L of free water was required to correct the dehydration of the calves in this study, assuming immediate correction of the free water abnormality. For comparison, calves were administered 2 L of free water via oroesophageal intubation, suggesting that an additional administration of an ORT solution at 6 hours may have been clinically beneficial. Our results also suggested that sodium acetate ORT solutions are more effective in expanding the plasma volume than are sodium bicarbonate ORT solutions, although there was considerable calf-to-calf variability (Figure 4). Because the extracellular fluid of diarrheic calves is hypotonic, administration of slightly hypertonic ORT solutions should be more efficacious than isotonic ORT solutions because hypertonic solutions facilitate movement of free water from the intracellular space to the extracellular space.²⁶ The apparent difference in plasma volume expansion between sodium acetate and sodium bicarbonate may have been caused by differences in the number of effective osmoles; bicarbonate is an ineffective osmole because of its rapid conversion to CO₂, whereas acetate remains an effective osmole until metabolized.

Hypertonic ORT solutions have the theoretical disadvantage that they may create a transient dehydration after feeding because the hypertonicity of abomasal fluid contents can cause movement of water from the extracellular space into the abomasum. Administration of a hypertonic ORT solution (698 mOsm/kg) at 37 mL/kg (16.8 mL/lb) induced a transient dehydration for < 30 minutes in calves with dehydration and diarrhea³³ but not in euvolemic calves.¹⁴ For comparison, we administered a hypertonic ORT solution (calculated osmolality, 726 mOsm/kg) to dehydrated calves at approximately 53 mL/kg (24.1 mL/lb) and did not detect transient dehydration. Taken together, these findings indicated that transient dehydration caused by suckling a hypertonic ORT solution, if present, is not likely to be clinically important. Results indicated that clinically important differences in the resuscitative response to 300 mmol/L or 150 mmol/L ORT solutions of sodium acetate or sodium bicarbonate were not identified.