Pathology in Practice

Danielle L. Gordon Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL

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Alessandro C. Migliorisi Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL

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Michael F. Rosser Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL

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History

A 3-year-old American Quarter Horse gelding was presented to the University of Illinois Equine Medicine and Surgery service with a 3-day history of lethargy, facial edema, and soft tissue swelling of the distal limbs. The horse was presented in the fall, and all other horses in the herd were clinically normal.

Clinical and Clinicopathologic Findings

Upon presentation, the gelding was quiet but responsive with a rectal temperature of 38.6 °C (reference interval, 37.5 to 38.5 °C). Hyperemic mucous membranes, tachycardia (80 beats/min; reference interval, 28 to 40 beats/min), and tachypnea (40 breaths/min; reference interval, 8 to 15 breaths/min) with a shallow breathing pattern were noted. All distal aspects of the limbs were mildly cool on palpation with soft tissue swelling, and digital pulses were slightly increased in the hind limbs. Marked and symmetric gluteal muscle atrophy was observed, and the patient had moderate swelling of the masseter muscles bilaterally.

A CBC revealed mild leukocytosis (14.00 X 103 cells/μL; reference interval, 5.50 X 103 to 12.00 X 103 cells/μL) characterized by mild mature neutrophilia (13.44 X 103 neutrophils/μL; reference interval, 3.00 X 103 to 7.00 X 103 neutrophils/μL) and moderate to marked lymphopenia (0.42 X 103 lymphocytes/μL; reference interval, 1.50 X 103 to 5.00 X 103 lymphocytes/μL). The erythron and thrombon were unremarkable. Upper respiratory tract endoscopy and abdominal and noncardiac thoracic ultrasonography were unremarkable. The horse was hospitalized, and results of serial plasma biochemical analyses (AU680 Clinical Chemistry Analyzer; Beckman Coulter Inc) from the time of presentation and throughout hospitalization were compiled (Table 1). Dipstick urinalyses on days 1, 2, and 4 showed a 4+, 4+, and 3+ heme reaction and negative, 2+, and 3+ bilirubin, respectively. All other urine dipstick parameters were within reference limits.

Table 1

Results of plasma biochemical analysis performed on days 1, 3, 5, 7, and 10 of hospitalization for a 3-year-old American Quarter Horse gelding evaluated because of a 3-day history of lethargy, facial edema, and soft tissue swelling of the distal aspects of all 4 limbs.

Analyte Day 1 Day 3 Day 5 Day 7 Day 10 Reference Interval
Creatinine (mg/dL) 1.1 0.8 1.0 0.9 1.0 0.8–1.8
BUN (mg/dL) 19 16 22 19 19 11–23
Total protein (g/dL) 6.9 6.7 7.2 6.1 6.1 5.5–7.3
Albumin (g/dL) 3.4 3.3 3.7 3.3 3.4 2.7–3.7
Globulin (g/dL) 3.5 3.4 3.5 2.8 2.7 2.0–4.6
Calcium (mg/dL) 10.9 11.5 11.9 11.8 11.6 10.5–13
Phosphorus (mg/dL) 4.2* 2.5 4.3* 3.7 4.4* 1.5–3.9
Sodium (mmol/L) 134 135 132* 136 133 133–142
Potassium (mmol/L) 3.8 3.6 3.7 2.6 4.4 2.5–5.0
Chloride (mmol/L) 96* 98 96* 96* 97* 98–105
Glucose (mg/dL) 187* 122* 115* 116* 107 71–111
Alkaline phosphatase 262* 233* 232* 185* 180* 41–137
Aspartate aminotransferase (U/L) 15,040* 11,190* 6,550* 3,292* 1,392* 150–294
γ-Glutamyltransferase (U/L) 32* 32* 39* 34* 34* 4–20
Total bilirubin (mg/dL) 1.9 1.4 2.2 1.3 1.3 0.5–2.3
Creatine kinase (U/L) 40,416* 7,324* 3,219* 1,669* 404* 71–300
Cholesterol (mg/dL) 137 116 110 88 83 60–172
Glutamate dehydrogenase (U/L) 14.9* 22.3* 38.1* 22.7* 6.6* 1.0–5.0
Bicarbonate (mmol/L) 40* 44* 39* 35* 32 24–33
Magnesium (mg/dL) 1.9 1.9 1.5 1.8 2.0 1.5–2.1
Triglycerides (mg/dL) 381* 92* 87* 67* 18 11–56
Anion gap 2.0* –3.4* 1.0* 8.0 8.0 6.4–14.6
Lipemic indicator 0 0 0 0 0
Icteric indicator 1 0 1 0 0
Hemolytic indicator 0 0 0 0 0

Important abnormalities.

= Reference intervals are not maintained in our laboratory; however, interference was not expected when the indicator was < 4.

Additional Clinicopathologic Findings

Plasma samples from days 1, 3, 5, 7, and 10 were obtained and stored at –80 °C until shipment (approx 2 weeks) to a veterinary diagnostic lab (Animal Health Diagnostic Center, Cornell University) for measurement of lactate dehydrogenase (LDH) activity, and all results were high (14,486 U/L, 8,837 U/L, 5,892 U/L, 2,470 U/L, and 1,436 U/L, respectively; reference interval, 218 to 555 U/L). Venous blood gas analyses (Stat Profile pHOx Ultra; Nova Biomedical) were performed on days 2, 4, and 6 of hospitalization and suggested mild hypochloremic metabolic alkalosis and respiratory acidosis on day 4 (Table 2).

Table 2

Key results for serial venous blood gas analyses performed for the horse described in Table 1 on days 2, 4, and 6 of hospitalization.

Analyte Day 2 Day 4 Day 6 Reference interval
pH 7.44 7.41 7.44 7.40–7.45
Pco2 (mm Hg) 38.1* 53.1* 42.7 38.2–48.7
Bicarbonate (mmol/L) 25.9* 35.2* 30.8 27.1–32.9
Lactate (mmol/L) 2.8* 0.8 0.9 0–1.2
Chloride (mmol/L) 102.6 101.3* 103.0 101.9–105.5
Sodium (mmol/L) 135.6 136.2 135.7 134.2–138.4
Potassium (mmol/L) 4.14 4.20 4.76* 3.79–4.53
Ionized calcium (mmol/L) 1.50 1.51 1.51 1.48–1.65

Important abnormalities.

Electrocardiography revealed a ventricular arrhythmia and echocardiographic examination was unremarkable. A serum sample was submitted for Streptococcus equi M protein ELISA testing (Equine Diagnostic Solutions LLC), and the result was weakly positive at 1:400, indicating an equivocal result.

Diagnosis and Case Summary

Artifactual increase in plasma bicarbonate (HCO3) concentration resulting in a low or negative anion gap, associated with myopathy of unknown origin (possible boxelder toxicosis) in a horse.

Comments

The most important findings on serial plasma biochemical profiles included markedly high creatine kinase (CK) and aspartate aminotransferase (AST) activities and high HCO3 concentration in conjunction with low or negative anion gap. The high CK and AST activities were consistent with severe muscle damage or necrosis.1 The positive heme reaction on the urine dipstick raised suspicion for myoglobinuria1; however, confirmatory testing was not pursued. High HCO3 concentration is usually compatible with a metabolic alkalosis, often secondary to selective chloride loss, or, less commonly, due to hypoalbuminemia or a compensatory response to a primary respiratory acidosis.2 The patient had evidence of mild hypochloremic metabolic alkalosis and respiratory acidosis on day 4. Selective chloride loss or a compensatory response to a respiratory acidosis may have minimally contributed to the high HCO3 concentration on day 4, although these changes were not consistent with the magnitude of HCO3 increase across all time points. A spurious increase in HCO3 concentration was therefore suspected.

The low anion gap was an unusual finding because most patients with metabolic alkalosis have an anion gap within the reference interval.2 The anion gap is a calculated parameter ([Na+ + K+] – [Cl + HCO3]) that reflects the difference in serum or plasma concentrations of the “measured” cations and “measured” anions. In this case, the artifactual increase in HCO3 concentration resulted in a low or negative anion gap. Other causes of a decreased anion gap, such as hypoalbuminemia and administration of fluids rich with HCO3, were ruled out in this case.2

Artifactually high plasma HCO3 concentrations have been reported in cases of rhabdomyolysis in horses, cattle, and in an Amazon parrot.35 In such cases, release of LDH and pyruvate from damaged muscle results in positive interference with HCO3 measurement in some biochemical analyzers.3 Several automated biochemical analyzers, including the one used with this case, have a 2-step enzymatic method for HCO3 measurement.6 The second step in this reaction involves oxidation of nicotinamide adenine dinucleotide (NADH), and the resultant decrease in NADH causes a decrease in absorbance proportional to the HCO3 content of the sample.6 Therefore, any reaction consuming NADH can result in artifactually high HCO3 concentration. High concentrations of LDH and pyruvate interfere with this assay by supplying a side reaction that results in oxidation of NADH, causing a spurious increase in the HCO3 concentration.3 Spuriously high plasma HCO3 concentration was confirmed in this case by concurrent markedly high LDH activity.

Lactate dehydrogenase catalyzes the conversion of lactate to pyruvate and is found in several tissues including skeletal and cardiac muscle and the liver.1 High LDH activity in this case was likely due to release from damaged muscle. Results for serial plasma CK and AST activities revealed a dramatic decrease in CK activity from day 1 to 10. The decrease in AST activity was more gradual, consistent with its longer half-life compared to CK.7,8 Serial LDH results showed a similar decreasing trend, compatible with resolving muscle injury throughout hospitalization.

As the LDH activity decreased, the anion gap increased due to less interference with the HCO3 assay. However, the LDH activity was greatest on day 1, but the highest HCO3 concentration and lowest anion gap were noted on day 3. If LDH was the only contributing factor, we would have expected the highest HCO3 concentration and lowest anion gap to be seen on day 1. Hyperlactatemia was considered the most likely cause for this discrepancy. Although not specifically measured on days 1 and 3, hyperlactatemia was present on day 2 and normalized on subsequent measurements (Table 2). Lactate accumulation titrates HCO3, causing a decrease in the HCO3 concentration and an increase in the calculated anion gap.3 It was suspected that the patient also had marked hyperlactatemia on day 1.

Blood gas analysis is preferred to measure HCO3 concentration when analytic interference is suspected using enzymatic methodology. Most blood gas analyzers, including the one used in this case, determine HCO3 concentration using the Henderson-Hasselbalch equation in which pH and Pco2 are measured.1 This method is independent of NADH oxidation and is not impacted by LDH concentration. Blood gas analysis was only performed on the days a biochemical profile was not obtained; therefore, direct comparison of HCO3 concentrations by use of both methods was not available. The blood gas analyzer reported lower HCO3 concentrations on days 2 and 4, compared with the results obtained by use of the biochemistry analyzer on days 1, 3, and 5. These discrepancies further supported an artifactual high HCO3 concentration on the biochemical panel.3

High glutamate dehydrogenase, alkaline phosphatase, and γ-glutamyltransferase activities and bilirubinuria supported concurrent mild hepatobiliary disease and cholestasis.1 Muscle injury may have minimally contributed to the increase in glutamate dehydrogenase activity.1 The intermittent hyperphosphatemia may have been associated with skeletal muscle injury or less likely decreased glomerular filtration rate.1 The hypertriglyceridemia was likely associated with a negative energy balance or less likely equine metabolic syndrome.1 The patient’s mild mature neutrophilia and moderate to marked lymphopenia were most compatible with a corticosteroid-mediated stress response, and mild hyperglycemia provided additional support.1

A definitive cause of muscle injury or necrosis was not determined in this case; however, on the basis of clinical findings and ancillary testing, the patient had skeletal and likely concurrent cardiac muscle disease. A specific etiology for our patient’s ventricular arrhythmia was not obtained but presumed multifactorial and associated with the patient’s myopathy. The arrhythmia resolved following treatment with intravenous lidocaine, magnesium supplementation, and oral sotalol. It was indicated that the horse may have access to boxelder trees (Acer negundo) in the pasture. Hypoglycin A in the seeds of boxelder trees is suspected to cause seasonal pasture myopathy typically in the fall,9 corresponding with the onset of this horse’s clinical signs. Oftentimes, only few of many exposed horses develop the disease.9 Therefore, boxelder toxicosis was considered as a possible cause for the patient’s disease.

Streptococcus equi infection was initially considered as a possible inciting cause of the patient’s myopathy because infarctive purpura hemorrhagica, acute rhabdomyolysis, and immune-mediated polymyositis are possible complications of S equi infection.10,11 However, it was ultimately considered unlikely given the low S equi M protein titer, lack of retropharyngeal lymphadenopathy, guttural pouch empyema, and hyperfibrinogenemia as well as a clinical response to immunosuppressive corticosteroid treatment.

The patient received supportive care, and biochemical parameters began to normalize throughout hospitalization. Upon discharge, a recheck examination was recommended in 3 weeks, but the patient was lost to follow-up. The biochemical findings in this case supported that artifactually high HCO3 concentration should be considered in horses with severe myopathy, especially when the anion gap is low or negative.

References

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