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Rhabdomyolysis and artifactual increase in plasma bicarbonate concentration in an Amazon parrot (Amazona species).

Abstract: A 7-year-old male Amazon parrot housed outdoors presented with acute collapse, marked lethargy, and open-mouth breathing. The patient had stiffness of the pectoral muscles, and petechiation and ecchymosis noted around the eyes and beneath the mandible. Laboratory data revealed markedly increased aspartate aminotransferase, creatine kinase, and lactate dehydrogenase activity consistent with rhabdomyolysis, as well as markedly increased plasma bicarbonate concentration. Marked clinical improvement and resolution of laboratory abnormalities occurred with fluid therapy, administration of a nonsteroidal anti-inflammatory medication, and husbandry modifications, including indoor housing and dietary alteration. A spurious increase in bicarbonate measurement as documented in equine and bovine cases of rhabdomyolysis also occurred in this avian patient and must be considered for accurate interpretation of acid-base status in exotic species presenting with consistent clinical signs.

Key words: rhabdomyolysis, bicarbonate, lactate dehydrogenase, avian, Amazon parrot, Amazona species

Clinical Report

A 7-year-old 520 g male Amazon parrot (Amazona species; exact species not documented) was evaluated at the Louisiana State University Veterinary Teaching Hospital Zoological Medicine Service for an acute episode of collapse, marked lethargy, and open-mouth breathing. The bird had no previous history of health problems and was found by the owner, minimally responsive, on the floor of its outdoor enclosure. The animal's condition improved in transit, and although resting on its sternum, the bird was quiet, alert, and responsive on presentation. Abnormalities noted on physical examination were increased stiffness of the pectoral muscles, pale mucus membranes, dehydration (estimated at 5%-7%), areas of petechiation and ecchymosis around the margins of the eyelids and beneath the mandible, and obesity (body condition score 4/5). The parrot was housed in a metal wire enclosure of unspecified dimensions with a floor of unknown wood-chip substrate, which was cleaned monthly. The enclosure was located primarily on an outdoor porch and occasionally was moved inside during inclement weather. At presentation, ambient temperature was 26.7[degrees] to 32.2[degrees]C (80[degrees]-90[degrees]F) with a humidity of 60% to 100%, and the enclosure had remained outdoors. The bird was let out to play on the top of the enclosure under supervision for approximately 4 hours daily. It was fed a primary diet of seed that contained dried fruit and a vitamin supplement. Tap water and food were replenished daily. Although it was a single pet household, the bird's enclosure did allow contact with squirrels, which the owner observed recently eating from its food bowl.

Whole blood was collected via venipuncture of the right jugular vein and submitted in ethylene-diaminetetraacetic acid (EDTA) for a complete blood count (CBC) performed by a manual hemocytometer semidirect method (Leukopet system; Vetlab Supply, Palmetto Bay, FL, USA). (1) In the absence of in-house generated reference intervals, previously established reference intervals for Amazon parrots were used for result interpretation, when available. (2) The CBC results revealed a packed cell volume of 53% (reference interval, 40%-52% (2)) and a refractometric total solids value of 3.5 g/dL, and the plasma appeared moderately hemolyzed. The patient initially had a leukocytosis (33 100 cells/[micro]L; reference interval, 4300-12 500 cells/[micro][L.sup.2]) attributable to a marked mature heterophilia (31 400 cells/([micro]L) and mild monocytosis (700 cells/[micro]L). No morphologic abnormalities were observed among erythrocytes, leukocytes, or thrombocytes on visual blood smear evaluation, and thrombocyte numbers appeared adequate on manual slide estimate (1-5 cells/x100 objective). Moderately hemolyzed plasma collected in lithium heparin was submitted concurrently for a biochemical profile (Olympus AU400e; Olympus America Inc, Diagnostic Systems Division, Irving, TX, USA) and subsequently interpreted using previously established reference intervals for Amazon parrots (Table 1). (2) The most striking abnormalities on results of the plasma biochemical profile were markedly increased activities of aspartate aminotransferase (AST) and creatine kinase (CK) (>1000 and >2000 U/L, respectively), increased bicarbonate concentration (51.6 mmol/ L), and a negative anion gap value. The activity of AST and CK exceeded the upper limit of linearity for the analyzer. Automated dilutions were performed to obtain approximate values for these analytes (Table 1). Other clinically relevant abnormalities were a moderate, proportional hyponatremia and hypochloridemia, moderate to marked hypocalcemia and hypophosphatemia, mild hyperuricemia, and hyperglycemia. Based on clinical and laboratory findings, a working diagnosis of rhabdomyolysis and associated inflammation was made, the cause of which was unknown. The markedly increased bicarbonate concentration and negative anion gap calculated from this value were considered most likely artifactual changes due to an interfering substance released into plasma from rhabdomyolysis, such as lactate dehydrogenase (LDH) or pyruvate. To confirm the spurious increase in plasma bicarbonate from assay interference, the remainder of the heparinized plasma sample was submitted to the University of Miami Avian and Wildlife Laboratory for measurement of LDH. The measured LDH concentration was markedly increased (LDH >2150 U/L; reference interval, 155-425 U/[L.sup.2]) and exhibited reactivity greater than the dynamic range of the analyzer. Despite performance of multiple dilutions, a more accurate result was unable to be obtained.

Because of suspected heat stroke and rhabdomyolysis, the patient initially received a balanced crystalloid solution (Plasma-Lyte 148; Baxter International Inc, Deerfield, IL, USA) subcutaneously at a rate of 160 mL/kg/day

to replace the dehydration deficit and provide maintenance requirements. After the diagnostic test results were evaluated, fluid therapy was changed to treatment with 0.9% NaCl to address the hyponatremia and hypochloremia. Upon correction of fluid deficits, meloxicam was prescribed at 1 mg/kg, initially intramuscularly followed by PO every 24 hours. A seed/pelleted diet mix and tap water were offered ad libitum. The parrot's attitude, appetite, respiratory pattern, and activity level continued to improve while hospitalized and it began to exhibit normal perching activity, with eventual resolution of the petechiations and ecchymoses. The CBC and plasma biochemical profile were repeated on day 3 of treatment. Clinically relevant differences as compared to the initial CBC included a decrease in cell concentrations for total leukocytes (13 600 white blood cells [WBCs]/[micro]L), mature heterophils (11 400 cells/[micro]L), and monocytes (300 cells/[micro]L), consistent with resolving inflammation. The PCV (35%) and refractometric total solids (2.6 g/dL), as well as the total protein concentration on the biochemical panel also had decreased since previous measurement. All other analyte derangements observed on the initial plasma biochemical panel had resolved except for increases in CK and AST activities, increased bicarbonate concentration, and decrease in anion gap. Values for these analytes did, however, appear markedly improved and were trending towards the reference interval, consistent with resolving rather than sustained muscle injury. Based on improvement in clinical and laboratory findings, the patient was discharged with instructions to continue oral meloxicam therapy until recheck physical examination, and repeat plasma biochemical panel in 7 days. The owner also was instructed to move the enclosure indoors to avoid contact with wildlife and to negate heat stress, which may have precipitated the patient's episode. A recommendation also was made to provide the patient with a primarily pelleted diet for more balanced nutrient intake. The patient did not return for a 1-week recheck appointment and subsequently was lost to follow-up as the owner could not be reached for rescheduling.

Discussion

Rhabdomyolysis refers to cellular breakdown of skeletal muscle and the subsequent release of intracellular contents systemically. In our case, the cause of rhabdomyolysis in this parrot was unknown. Rhabdomyolysis can be precipitated by many events and disease processes that act through conserved mechanisms to incite cell damage, most notably adenosine triphosphate (ATP) depletion. The ATP depletion may occur secondary to electrolyte derangements (eg, hypokalemia, hyponatremia, hypernatremia, hypophosphatemia), excessive muscular activity, and ischemic injury (eg, shock, embolism, compression/prolonged immobilization), among other causes. (3) Muscle damage also may occur directly with some inciting factors as in the case of temperature extremes (eg, hyperthermia, hypothermia), trauma, burns, and high voltage electrical injury. (3) Exertional rhabdomyolysis secondary to aggravation by a predator or other wildlife and exacerbated by heat stress was considered plausible, as the owner recently had observed squirrels eating from the bird's enclosure, and at the time of presentation environmental temperature and humidity were high. Given that the event precipitating this patient's condition was unobserved, other causes, such as prior seizure activity or trauma, could not be excluded. Additionally, while no feed analysis was performed, the bird was consuming an undesirable diet for this species, and some of the noted electrolyte derangements on the initial biochemical panel (eg, hypophosphatemia) may have been preexisting and predisposed the animal to development of rhabdomyolysis. While the bird in this report recovered uneventfully with supportive care, rhabdomyolysis can have life-threatening sequelae. Release of large quantities of intracellular substances, such as potassium, sulfur-containing proteins, lactate, and myoglobin, may lead to cardiac arrhythmias, acidosis, and renal injury and failure, respectively. (3) Additionally the degree of tissue injury may precipitate disseminated intravascular coagulation (DIC), as well as ongoing local inflammation and tissue damage, resulting in compartment syndrome. (3)

In people, DIC is a documented potential sequela of rhabdomyolysis and is speculated to develop secondary to widespread release of tissue factor and other constituents from damaged muscles, favoring activation of the coagulation cascade. (3,4) Additionally, certain causes of rhabdomyolysis (eg, sepsis, systemic inflammatory response syndrome [SIRS]) are inherently inflammatory, and ongoing inflammation can result in increased expression of tissue factor by cells within the vasculature and downregulation of coagulation inhibitors, further contributing to perturbations in the hemostatic system. (5) Indeed, many of the recognized causes of rhabdomyolysis also are independently recognized inciting factors for DIC. Of note, heat stroke, a clinical differential in this case, may cause coagulation disturbances and, in some instances, overt DIC; information from human clinical cases as well as research using animal models have implicated increased expression of tissue factor as a likely contributor. (4,6)

It is unknown if our patient may have had disturbances of the hemostatic system secondary to rhabdomyolysis. While thrombocytes appeared adequate on both CBCs, no additional coagulation testing was performed. The decrease in plasma protein and hematocrit values with treatment may have been in part from fluid therapy, although clinically unobserved hemorrhage secondary to coagulopathy or leakage from intramuscular capillary destruction could not be excluded. (4) As hypoproteinemia was present before fluid therapy, inflammation, dietary factors, underlying intestinal or other disease also may have affected this value. However, lack of additional protein parameters and follow-up information in this case precluded further speculation as to cause. Petechiation and ecchymosis observed around the eyes and mandible at presentation remain unexplained, and while appearing focal, these lesions may have been more widely distributed but only appreciated in poorly feathered areas. Some considerations for these findings were possible trauma, endothelial injury secondary to heat stroke, as well as coagulopathy including DIC.

Artifactually increased serum bicarbonate values have been reported in cases of rhabdomyolysis in horses and cattle. (7,8) Like our case, horses and cattle have markedly increased enzyme activities associated with skeletal muscle damage, markedly increased bicarbonate concentration, negative anion gap, and clinical signs consistent with rhabdomyolysis. The presence of a markedly increased bicarbonate and negative anion gap in a serum or plasma sample should merit investigation for presence of a machine error or assay interference. In cases of rhabdomyolysis, endogenous substances released from damaged muscle have an increased presence within the plasma. These substances include LDH and pyruvate, which have been shown to cause positive interference with bicarbonate measurement by certain methodologies, including that used within our laboratory. The Olympus bicarbonate assay, like methodologies used by some other large automated chemistry analyzers, uses a 2-step enzymatic method for measurement of bicarbonate

The first step of this enzymatic reaction is catalyzed by phosphoenolpyruvate carboxylase and proceeds as follows:

Phosphoenolpyruvate + [HC03.sup.-] [right arrow] oxaloacetate + [H2P04.sup.-]

The second step of the reaction is catalyzed by malate dehydrogenase that converts oxaloacetate to malate and oxidized NADH:

Oxaloacetate + NADH + [H.sup.+] [right arrow] malate + [NAD.sup.+]

The consumption of NADH in this second reaction causes a decrease in the absorbance of ultraviolet light measured spectrophotometrically, and the rate of change of absorbance is directly proportional to the concentration of HCO3" in the absence of interfering substances. As consumption of NADH ultimately is what is measured to calculate [HC0.sub.3.sup.-] with this methodology, any reaction that consumes NADH occurring within the sample will artifactually increase the reported HC[O.sub.3.sup.-]. Such is the case when pyruvate and/or LDH is present in markedly increased amounts, fueling a side reaction within the sample that is catalyzed by LDH and consumes NADH:

Pyruvate + NADH + [H.sup.+] [right arrow] L - lactate + [NAD.sup.+]

Presumably these substances are not a major source of interference outside of the context of severe rhabdomyolysis because their plasma concentrations are low in healthy animals. While measurement of plasma pyruvate also may have been of interest in our case, it was not performed because of the labile nature of this analyte and lack of an available reference laboratory for sample testing. If a spurious increase in bicarbonate concentration due to rhabdomyolysis is suspected, the astute clinician and laboratory personnel should regard any other enzymatic reactions that rely on NADH consumption to determine analyte concentration as suspect.

Blood gas analysis provides an acceptable methodology to assess more accurately acid-base status in patients suspected of having analytic interference using enzymatic HC[O.sub.3.sup.-] determination. Most blood gas analyzers calculate HC[O.sub.3.sup.-] concentration based on values for pH and pC[O.sub.2] obtained via ion-selective electrodes that measure [H.sup.+]; thus, this methodology is not significantly affected by increased concentrations of pyruvate and LDH or consumption of NADH. (9) While it is unlikely that laboratory personnel or clinicians would attribute a markedly increased serum or plasma bicarbonate concentration and negative anion gap to physiologic or pathophysiologic changes, in cases where the degree of assay interference is less pronounced, the possibility exists for incorrect interpretation of acid-base status. In such cases, HC[O.sub.3.sup.-] determination by blood gas analysis provides a quick and easy confirmatory testing option. This was not used in the current case because of limited plasma sample for analysis, although comparison with a calculated HC[O.sub.3.sup.-] concentration would have been ideal to confirm the spurious nature of the value obtained by enzymatic method, and lack of these supportive data is a limitation of this case report.

Moderate sample hemolysis was not considered a major contributor to any of the observed plasma biochemical abnormalities in this parrot. Results of a previous study demonstrated a positive percent change for some biochemical analytes in markedly hemolyzed plasma samples in Amazon parrots. (10)

The extremely high values for relevant analytes in our case could not be attributed to hemolysis. (10) While the source of hemolysis of the sample was not determined, preanalytical hemolysis was suspected and is not uncommon in avian blood samples, especially those collected through small needles. Any plasma discoloration from free myoglobin was considered unlikely, as myoglobin is rapidly cleared from the plasma and generally not appreciated grossly in plasma or serum samples from patients with rhabdomyolysis. (3)

Intracellular shifting into damaged or necrotic myocytes was considered a likely cause of the initial hyponatremia, hypochloridemia, and hypocalcemia, although the possibility exists that one or more of the conditions may have been preexisting and contributed to development of rhabdomyolysis (Table 1). With depletion of ATP, dysfunction of the myocyte calcium and sodium/potassium ATPase pumps ensue, allowing for intracellular influx of sodium, chloride, calcium, and water. (3) Hypocalcemia is a documented sequela of rhabdomyolysis in people, attributed to deposition of calcium within necrotic myocytes, and along with hyperkalemia may contribute to arrhythmias in patients with rhabdomyolysis. (3) Certain electrolyte derangements also may be a cause rather than a sequela of rhabdomyolysis and include hypophosphatemia as well as hyponatremia, a documented cause of rhabdomyolysis in some human patients with psychogenic polydipsia and congestive heart failure. (3,11)

Hyperkalemia and hyperphosphatemia from extracellular shifting of potassium and phosphorus also may be expected with rhabdomyolysis but were not seen in this case. A review of reported cases in the veterinary literature suggests such changes in electrolytes and minerals are not conserved findings, as some animals in previous reports also presented with serum potassium and phosphorus concentrations within reference intervals. (7,8,12) This may be a result of variability in disease severity and presentation, time of sampling as related to disease onset, or other patient-related factors, including comorbidities.

The moderate-to-marked hypophosphatemia in our patient at presentation, however, was not readily explainable, particularly given evidence of severe muscle damage, which would be expected to increase phosphorus. Nutritional deficiency was considered plausible, given improvement in this value with supportive care including dietary management. It also is possible that the severity of hypophosphatemia may have been more profound than appreciated, as release of phosphorus from intracellular stores may have occurred with rhabdomyolysis. While the mild hyperuricemia at presentation may have been reflective of dehydration, it also could have occurred from degradation of purines released from necrotic cells, as has been documented in mammalian species with rhabdomyolysis. (3) The initial mild hyperglycemia was considered consistent with a glucocorticoid or epinephrine-mediated stress response, which also is common in avian patients.

While leukocytosis is common in avian patients with systemic inflammation, it is not a conserved feature of reported mammalian cases of rhabdomyolysis. In our case, the transient nature of the patient's inflammatory leukogram and marked improvement in diagnostic and clinical findings without use of antimicrobials supported sterile inflammation, with damage or necrosis of skeletal muscle considered a likely contributor. The cause of petechiation and ecchymosis in this case also is unclear, although trauma and platelet and/or endothelial dysfunction secondary to heat stress were considered.

While the cause of this patient's episode of rhabdomyolysis was never fully delineated, the biochemical findings in this case illustrated that artifactually increased plasma bicarbonate concentration secondary to rhabdomyolysis occurs within a wider subset of species than has been reported previously and can occur in species that are not heavily muscled. Additionally, this case highlights the importance of familiarity with sources of analytical interference and the working methodologies of commonly used laboratory assays, as this understanding may affect accurate interpretation of laboratory data and case management.

References

(1.) Campbell TW, Ellis CK. Hematology of birds. In: Avian and Exotic Animal Hematology and Cytology, 3rd ed. Ames, IA: Blackwell Publishing; 2007:24-33.

(2.) Avian Reference Ranges: Chemistry. Available at: http://cpl.med.miami.edu/documents/avian_ reference_ranges_121815.pdf. Accessed May 25. 2016.

(3.) Khan FY. Rhabdomyolysis: a review of the literature. Neth J Med. 2009:67(9):272-283.

(4.) Warren JD, Blumbergs PC, Thompson PD. Rhabdomyolysis: a review. Muscle Nerve. 2002;25(3): 332-347.

(5.) Stokol T. Laboratory diagnosis of disseminated intravascular coagulation in dogs and cats: the past, the present, and the future. Vet Clin North Am Small Anim Pract. 2012:42(1): 189-202.

(6.) Jilma B. Derhaschnig U. Disseminated intravascular coagulation in heat stroke: a hot topic. Crit Care Med. 2012:40(4): 1370-1372.

(7.) Overmann JA, Finno C, Sharkey LC. What is your diagnosis? Increased total C02 concentration and negative anion gap in a foal. Vet Clin Pathol. 2010: 39(4):515-516.

(8.) Collins ND, LeRoy BE, Vap L. Artifactually increased serum bicarbonate values in two horses and a calf with severe rhabdomyolysis. Vet Clin Pathol. 1998;27(3):85-90.

(9.) Stockham SL. Scott MA. Blood gases, blood pH, and strong ion difference. In: Fundamentals of Veterinary Clinical Pathology. 2nd ed. Ames, IA: Blackweli Publishing; 2008:565-568.

(10.) Hawkins MG, Kass PH. Zinkl JG, Tell LA. Comparison of biochemical values in serum and plasma, fresh and frozen plasma, and hemolyzed samples from orange-winged Amazon parrots (Amazona amazonica). Vet Clin Pathol. 2006;35(2):219-225.

(11.) Sasaki M. Yuzawa M, Saito T, et al. Clinical and laboratory features of hyponatremia-induced myopathy. Clin Exp Nephrol. 2007; 11(4):283-286.

(12.) Perkins G, Valberg SJ, Madigan JM, et al. Electrolyte disturbances in foals with severe rhabdomyolysis. J Vet Intern Med. 1998; 12(3): 173-177.

(13.) Tell LA, Citino SB. Hematologic and serum chemistry reference intervals for Cuban Amazon parrots (Amazona leucocephala leucocephala). J Zoo Wildl Med. 1992;23(1):62-64.

Mary K. Leissinger, DVM, MS, Dipl ACVP, James G. Johnson III, DVM, Thomas N. Tully Jr, DVM, MS, Dipl AVBP (Avian), Dipl ECZM, and Stephen D. Gaunt, DVM, PhD, Dipl ACVP

From the Departments of Pathobiological Sciences (Leis-singer. Gaunt), and Veterinary Clinical Sciences, (Johnson, Tully). School of Veterinary Medicine. Louisiana State University. Skip Bertman Drive, Baton Rouge. LA 70803, USA.

Present address: Department of Physiological Sciences, University of Florida College of Veterinary Medicine, Gainesville, FL 32610. USA (Leissinger); Columbus Zoo and Aquarium. 9990 Riverside Drive. Powell. OH 43065. USA (Johnson).
Table 1. Results of serial heparinized plasma biochemical panels in
an Amazon parrot with acute rhabdomyolysis.

Analyte                                     Day 1

Alkaline phosphatase, U/L                    13
Anion gap, mmol/L                           -15.0
Aspartate aminotransferase, U/L    >1000 (b) (34 100 (c))
Bicarbonate, CO2, mmol/L                    51.6
Calcium, mg/dL                               5.0
Chloride, mmol/L (d)                         80
Creatine kinase, U/L               >2000 (b) (837 000 9c))
Creatinine, mg/dL                           0.21
Gamma glutamyl transferase, U/L               5
Glucose, mg/dL                               391
Phosphorus, mg/dL                            1.4
Potassium, mmol/L                            3.6
Sodium, mmol/L                               113
Total protein, g/dL                          2.4
Uric acid, mg/dL                             9.6

Analyte                                    Day 3

Alkaline phosphatase, U/L                    59
Anion gap, mmol/L                           0.3
Aspartate aminotransferase, U/L    >1000 (b) (29 600 (c))
Bicarbonate, C02, mmol/L                    41.9
Calcium, mg/dL                              8.5
Chloride, mmol/L (d)                        109
Creatine kinase, U/L               >2000 (b) (45 900 (c))
Creatinine, mg/dL                           0.12
Gamma glutamyl transferase, U/L              5
Glucose, mg/dL                              236
Phosphorus, mg/dL                           4.8
Potassium, mmol/L                           3.2
Sodium, mmol/L                              148
Total protein, g/dL                         1.9
Uric acid, mg/dL                            2.5

Analyte                              Rcference
                                   interval1 (a)

Alkaline phosphatase, U/L             15-150
Anion gap, mmol/L                       --
Aspartate aminotransferase, U/L       141-347
Bicarbonate, CO2, mmol/L               13-26
Calcium, mg/dL                       8.2-10.9
Chloride, mmol/L (d)                  101-123
Creatine kinase, U/L                  125-345
Creatinine, mg/dL                     0.1-0.4
Gamma glutamyl transferase, U/L        1-12
Glucose, mg/dL                        221-302
Phosphorus, mg/dL                     3.1-5.5
Potassium, mmol/L                     3.0-4.5
Sodium, mmol/L                        125-155
Total protein, g/dL                   3.0-5.2
Uric acid, mg/dL                      2.1-8.7

(a) Reference interval values were obtained from the University of
Miami Avian and Wildlife Laboratory. Data is from 24-hour-old
heparinized plasma samples from Amazon parrots run on an Ortho 250XR
analyzer. (2)

(b) Values obtained before sample dilution, reported as greater than
the upper limit of detection.

(c) Values subsequently obtained by automated dilution.

(d) Reference interval values determined from serum samples of 37
Cuban Amazon parrots analyzed using the Kodak Ektachem 700XR. (13)
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Title Annotation:Clinical Report
Author:Leissinger, Mary K.; Johnson, James G., III; Tully, Thomas N., Jr.; Gaunt, Stephen D.
Publication:Journal of Avian Medicine and Surgery
Date:Sep 1, 2017
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