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Improved clinicopathologic assessments of acute liver damage due to trauma in Indian ring-necked parakeets (Psittacula krameri manillensis).

Abstract: Increased activities of certain biochemical enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], lactate dehydrogenase [LDH], alkaline phosphatase [ALP]) have been associated with blunt liver injury in many species. To evaluate changes in plasma hepatic biochemical parameters in acute avian liver disease caused by trauma and to compare biochemical changes with histologic lesions in hepatic parenchyma, 30 healthy fasted Indian ring-necked parakeets (Psittacula krameri manillensis) were divided into 2 groups, and traumatic liver injury was caused by endoscopic liver biopsy (group 1) or by liver biopsy and crushing injury to the hepatic parenchyma with endoscopic forceps (group 2) in anesthetized birds. Blood samples were collected at baseline and at 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120 hours in alternate groups to compare analyte values after injury with those at baseline. Results showed consistently decreased plasma ALP activity (excluding 1 time point) throughout the study, which was thought to be associated with isoflurane administration. Plasma [micro]Lutamate dehydrogenase activity initially increased but rapidly declined thereafter and was attributed to acute focal hepatocellular injury. In both groups, increases in plasma AST, ALT, and LDH activities was most likely caused by muscle injury because creatine kinase activity was concurrently increased. Compared with baseline values, bile acid concentration and [gamma]-glutamyl transferase activity were not affected by liver biopsy or crush injury. Plasma sorbitol dehydrogenase activity was the most specific indicator of liver injury in both groups. Histologic changes correlated poorly with biochemical results, possibly because the small area of hepatic parenchyma that was damaged did not affect enzyme values substantially.

Key words: trauma, biopsy, liver, plasma biochemical values, liver enzymes, avian, Indian ringnecked parakeets, Psittacula krameri manillensis

Introduction

Traumatic liver injuries are often a result of blunt or penetrating abdominal trauma involving several organs; therefore, isolated liver injuries are uncommon. (1) In dogs and cats, the most common causes of trauma to the liver are falls, motor vehicle accidents, and blunt abdominal injuries. (1,2) However, an underlying hepatic disorder such as hepatic lipidosis, common in birds and cats, can increase the sensitivity of the liver to trauma and, subsequently, result in rupture of the livery

Traumatic injury to the liver can vary in severity and has an associated range of morbidity and mortality. (4) Significant blunt abdominal trauma can result in short- or long-term complications. In dogs and cats, blunt abdominal trauma can be life threatening from severe hemorrhage. Commonly, these animals present with depressed mentation, pale mucous membranes, delayed capillary refill time, tachycardia, and poor peripheral pulse quality. For dogs, cats, and birds, in some cases, survival is possible if the hemorrhage is confined to 1 hepatic peritoneal cavity (birds only) or to a subcapsular hematoma. (1,3)

Injuries to the liver can be challenging to diagnose because results of the initial abdominal examination can be unreliable. (5,6) However, increased activities of certain biochemical enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], lactate dehydrogenase [LDH], and alkaline phosphatase [ALP]) have been demonstrated to be associated with blunt liver injury in dogs, cats, and humans. (1,6-10) Increased activities of ALT, AST, and LDH are seen with acute hepatocellular damage and are more dramatic with blunt trauma to the liver, reflecting greater hepatocellular damage in people. (8) Furthermore, ALT and AST activities have been shown to correlate with the degree of hepatic injury. (5,7,9,10) The serum activities of ALT, AST, and LDH peak within the first 24 hours after hepatic injury and decrease rapidly 4 days after injury. (6,8) Hennes et al (6) reported a series of 87 pediatric patients with blunt abdominal trauma and found that significantly increased serum hepatic enzyme activities (AST > 450 U/L and ALT > 250 U/L) accurately identified children with hepatic injury and reliably correlated with the computed tomography results; therefore, it was concluded that activities of AST > 450 U/L and ALT > 250 U/L will accurately diagnose hepatic injury. To calculate the sensitivities and specificities of AST and ALT activities as predictors for blunt liver trauma in people, Ritchie et aP performed statistical analyses on Henries et al (6) data and data from 4 other retrospective studies; the mean sensitivity was 94.2% (range, 78%-100%), and the mean specificity was 88.6% (range, 77%-100%). In dogs, cats, and humans, serum ALP activity has been shown to gradually increase over time after trauma to the liverl. (1,10); however, in a study of experimental liver trauma in rabbits, (5) ALP activity was not increased except in cases of extensive liver damage.

Traumatic liver injury and its effects on biochemical parameters have been poorly studied in birds. Lierz et al (11) examined the effects of liver biopsy on blood biochemical results in 19 wild raptors. After liver damage, the activities of glutamine dehydrogenase (GLDH) and AST increased dramatically, peaked on day 3, and then returned toward baseline. The effects on ALT and ALP activities were less significant over the 11-day study; values of ALT changed minimally and those of ALP gradually increased from baseline. Jaensch et al (12) examined the effects of partial hepatectomy on plasma enzyme concentrations and galactose clearance tests in galahs (Eolophus roseicapillus). Early results revealed that ALP, AST, ALT, and creatine kinase (CK) activities were increased and consistent with muscle damage due to the surgery and not actual liver damage. By day 7, results were lower but not within reference intervals except for ALT. Bile acid levels were not affected and galactose clearance was affected only in the group that had an 18% hepatectomy performed.

The goals of this study were to evaluate changes in plasma hepatic biochemical parameters in acute avian liver disease caused by trauma and to compare biochemical changes with histologic lesions of the hepatic parenchyma. The induced lesions were anticipated to produce minimal hepatic trauma to determine the threshold for detecting liver injury by plasma biochemical testing.

Materials and Methods

The study protocol was approved by the University of Georgia's Institutional Animal Care and Use Committee (IACUC No. A2003-10199-0). Thirty flighted, juvenile to young adult Indian ring-necked parakeets (Psittacula krameri manillensis) of unknown sex (later determined) were purchased from licensed breeders in Georgia and Florida. The 30 birds were housed in 6 cages (4 cages of 5 birds, 1 cage of 4 birds, and 1 cage of 6 birds) and maintained in a room with an ambient temperature of approximately 23[degrees]C (73[degrees]F) at the University of Georgia, College of Veterinary Medicine's Animal Resources (Athens, GA). Birds were exposed to 12-hour light/dark cycles and the diet consisted of commercial parrot seeds with occasional fruit and millet seeds; water was available ad libitum. On arrival, all 30 birds were physically examined and were found to be clinically healthy. Although weights were not recorded, all birds were of similar size. The birds acclimatized to the research facility for approximately 2 months before the start of the study.

The 30 parakeets were randomly assigned to 2 groups. Group 1 (biopsy) consisted of 20 birds that underwent liver biopsy only. Group 2 (crush) consisted of 10 parakeets that received additional liver injury after biopsy by tissue crushing with forceps. Postsurgical phlebotomy was performed in all 30 birds at 24-hour time points starting at 12 hours after surgery. On the day of surgery, food was withheld from all parakeets overnight (for approximately 12 hours) before induction of anesthesia. The birds received ketoprofen (2 mg/ kg IM) 15-20 minutes before induction of anesthesia. Based on the American Society of Anesthesiologists score, each bird was considered class 1. (13) Anesthesia was induced with 3%-5% isoflurane in oxygen by use of an over-the-head mask that was connected to a nonrebreathing system. After induction, each bird was maintained at a surgical plane of anesthesia via the mask during the time necessary for phlebotomy and surgical preparation. Between 200 and 400 [micro]L of blood was drawn from the right jugular vein (occasionally the left jugular, left brachial, and/or right brachial veins) of all 30 parakeets (not exceeding 1 mL/100 g body weight) with a 3-mL syringe and 25-gauge needle, and placed into a lithium heparin Microtainer (Microtainer, Beeton Dickinson, Franklin Lakes, N J, USA) tube. (14) Blood samples were stored on ice during the surgical procedures; plasma was harvested and biochemical analyses were performed on an automated analyzer (Hitachi 912 Chemistry Analyzer, Roche Diagnostics, Indianapolis, IN, USA) at the end of the procedure.

The birds were then intubated by use of an uncuffed 2-mm endotracheal tube. During the surgical procedure, ventilation was assisted by the use of a pressure-limited mechanical ventilator (VT-5000, BAS Vetronics, Bioanalytical Systems Inc, West Lafayette, IN, USA); adjustments were made to maintain an end-tidal C[O.sub.2] reading between 35 and 45 mm Hg. A surgical depth of anesthesia was maintained in each bird by use of 1%-3% isoflurane in oxygen (flow rate: 1 L/min). The depth of anesthesia was monitored by evaluating reflexes, end-tidal capnography (ETCO2/Sp[O.sub.2] monitor, C[O.sub.2] SMO, Novametrix Medical Systems, Wallingford, CT, USA), cardiac Doppler ultrasonography (Ultrasonic Doppler, Parks Electric Laboratory, Aloha, OR, USA), and esophageal temperature measurement (Precision Thermometer, Tandy, Fort Worth, TX, USA). The risk of developing hypothermia was minimized by placing water-circulating heating pads under each bird and maintaining the surgical room at an ambient temperature of approximately 75[degrees]F (24[degrees]C). Each parakeet was positioned in right lateral recumbency with the wings secured dorsally and the left leg secured cranially; the surgical site was prepped by plucking the surrounding feathers and cleansed by alternating with povidone iodine and alcohol. A small skin incision was made on the left flank with a No. 15 scalpel blade and the underlying tissue was bluntly dissected with small straight mosquito hemostats until the left caudal air sac was penetrated. A rigid 30[degrees] viewing telescope (2.7 mm x 18 mm) housed within a 14.5-F (4.8 mm) operating sheath (Karl Storz Veterinary Endoscopy America Inc, Goleta, CA, USA) was introduced through the incision at the junction of the caudal edge of the eighth rib and the flexor cruris medialis muscle. (15) The telescope was connected to a xenon light source, camera, and monitor (Karl Storz Veterinary Endoscopy America). Endoscopically guided 1.7-mm scissors were used to gently dissect through the coalescent membranes of the left caudal thoracic air sac and the hepatic peritoneal cavity. The endoscope was positioned at different angles and depths, as described by Divers (16) to evaluate the health of various coelomic structures and to determine the sex of each bird (group 1 [biopsy]: 13 males and 7 females; group 2 [crush]: 7 males and 3 females). Two liver biopsy samples were collected from the lateral border of the liver in each bird by using 1.7-mm biopsy forceps through the instrument channel of the operating sheath. Each tissue specimen was gently transferred from the forceps to a biopsy cassette by a moistened cotton-tip applicator and placed in containers with neutral-buffered 10% formalin. Samples were routinely processed and embedded in paraffin blocks, and thin sections were placed on slides and stained for microscopic examination.

After the biopsies were completed, birds in group 2 received additional liver injury by positioning 1.7-mm retrieval forceps at 3 random sites and clamping down to crush the tissue. After the surgical procedures, the endoscopic instruments were removed, and the skin and muscle incisions were closed by use of a single, simple interrupted 4-0 absorbable suture (Monocryl, Ethicon, Somerville, NJ, USA). Subcutaneous fluids (8 mL of 2.5% dextrose) were administered in the right lateral flank before anesthetic recovery on 100% oxygen for approximately 5 minutes. All surgical instruments were sterilized by immersion in 2.4% alkaline [micro]Lutaraldehyde solution (CIDEX, Advanced Sterilization Products, Irvine, CA, USA) for 15 minutes and rinsed with sterile water between each surgical procedure. After recovery from anesthesia, the birds were returned to their cages and monitored for any change in behavior, food intake, or wound dehiscence; no abnormalities were observed.

Twelve hours after surgery, the birds in group I (biopsy) were divided into subgroups A and B with 10 birds in each group, and birds in group 2 (crush) were divided into subgroups A and B with 5 birds in each group. Phlebotomy was performed in subgroup A of both groups. Twenty-four hours after the surgery, phlebotomy was performed in subgroup B of both groups. Serial phlebotomy was then performed in group 2 (crush) subgroups A and B at alternating 12-hour intervals for a total of 120 hours. Between 200 and 400 [micro]L of blood was drawn from the right jugular vein (occasionally the left jugular, left brachial, and/or right brachial veins) of each bird. Plasma was harvested from the lithium heparin tubes after each time point and analyzed on an automated analyzer within 2 hours of phlebotomy. Biochemical testing was performed to evaluate ALP, ALT, AST, CK, GLDH, LDH, [gamma]-glutamyl transferase (GGT), and sorbitol dehydrogenase (SDH) activities, as well as bile acid (BA) and cholesterol (CHOL) concentrations. Throughout the study, the birds were housed in the same manner as described above. The health of the birds was observed and recorded; only minor abnormalities were observed, such as poor feather quality (including broken blood feathers) and hematomas (most consistent with venipuncture).

Data were analyzed with SAS 9.2 (SAS Institute, Cary, NC, USA) software. Because the distribution of data was highly skewed, an appropriate transformation (the natural logarithm [In]) on the response variables was needed to normalize the error terms and stabilize the variance. To determine which variables needed to be transformed, a comparison was performed for each variable's Q-Q plot of the original scaled variable to that of the transformed variable to identify which scale had a distribution closer to normal. Transformation appeared to be appropriate except for GGT, GLDH, and SDH. Therefore, changes were analyzed in In(ALP), ln(ALT), ln(AST), In(CK), ln(LDH), ln(BA), In(CHOL), GGT, GLDH, and SDH before and after liver damage in the crush study.

A Student's t test was used to compare effect of sex on measured variables for all 30 birds. To examine the effect of liver biopsy on the 10 variables, Student's t test was performed to compare mean results for the 10 birds in group 1 at baseline with results measured at 12 and at

24 hours after injury. A Student's t test was also used to compare mean results from group 1 with those from group 2 at 12 and at 24 hours. All 10 variables of group 2 were compared separately with the baseline data (t = 0) by repeated measure ANOVA. Results were considered significant at P < .05.

Results

Bird handling, anesthesia, surgical procedures, and phlebotomy were all completed and well tolerated with minor complications. One bird in group 2 developed bradycardia and was treated with 0.05 mL of atropine IV. Mild hemorrhage was observed during biopsy in some birds; however, no medical intervention was necessary.

At baseline (t = 0) of all 30 birds, the sample quantity was insufficient to determine concentration CHOL in 15 birds and BA in 7 birds and activities of GLDH in 3 birds and SDH in 1 bird. The 95% confidence interval (CI) was established for the mean of each of the 10 variables at baseline (Table 1). One bird had a very high SDH value of 67 U/L and was identified as an outlier. The distribution of SDH was close to normal if this outlier was excluded; therefore, the 95% CI for SDH was obtained without this outlier. Sex was determined not to be a significant factor for any of the 10 variables. Because of the ambiguity between immature and inactive reproductive organs, age was not statistically examined.

In group 1, the effect of liver biopsy on the 10 variables at 12 and 24 hours after biopsy were examined for significance (data not shown). At 12 hours, mean values of ALP, AST, CK, LDH, CHOL, and SDH were significantly higher than at baseline, and at 24 hours, mean values of ALT, AST, CK, LDH, and CHOL were significantly higher than baseline. The values of ALP and CHOL decreased significantly whereas those of ALT, AST, CK, LDH, and SDH increased significantly after 12 and 24 hours. In particular, ALP activity decreased significantly (P < .001) by approximately 21% after 12 hours compared with baseline.

When comparing results from group 1 (biopsy) and group 2 (crush) birds, the mean value of LDH in group 2 birds increased significantly by approximately 215% (P < .001) at 12 hours, and mean ALT activity in group 2 birds increased significantly at 24 hours (P < .04). No other significant differences were found. Only 1 bird in group 2 had a baseline CHOL measurement; therefore, a comparative analysis was not performed on this variable.

In group 2 subgroups A and B, the postoperative data were compared with the corresponding baseline data. A separate analysis was performed for the 2 subgroups over time (0-120 hours) because results in the 2 groups were not similar to each other with respect to most of the 10 variables. Consequently, the means for hours 12, 36, 60, 84, and 108 were not compared with the means for hours 24, 48, 72, 96, and 120. The results are listed in Tables 2 and 3.

Biopsy procedures were successfully performed in all cases, with minimal hemorrhage. The liver biopsy specimens from each parakeet were routinely stained with hematoxylin and eosin (H&E) stain (60 sections total). Sections were stained with acid-fast (AF) stain (30 sections total) and Perls Prussian blue stain (26 sections total) when deemed necessary. all sections were examined by light microscopy, and severity of histologic changes was scored as ND (not determined), 0 (normal, 0%-5%), 1+ (mild, 5%-25%), 2+ (low moderate, 26%-50%), 3+ (high moderate, 51%-74%), or 4+ (severe, >75%). In the H&E sections for all 30 birds, crush artifact affected 0%-5% of liver sections from 6 birds, 6%-25% of liver sections in 13 birds, 26%-50% of liver sections in 5 birds, 51%-74% of liver sections in 5 birds, and >75% of the liver section in 1 bird. In most cases, the crush artifact was confined to the periphery of the section, leaving a sufficient amount of undamaged tissue for evaluation. The microscopic changes observed in all 30 birds were hepatocellular anisocytosis (ND in 26 birds, 0 in 1 bird, 1+ in 3 birds) and anisokaryosis (ND in 1 bird, 0 in 4 birds, 1+ in 18 birds, 2+ in 6 birds, 3+ in 1 bird), hepatic lipidosis (0 in 24 birds, 1+ in 4 birds, 2+ in 1 bird, 4+ in 1 bird), hepatocellular necrosis (0 in 26 birds, 1+ scattered necrosis in 1 bird, and focal necrosis in 3 birds), heterophilic hepatitis with variable degrees of lymphocytic or histiocytic infiltrates (0 in 6 birds, 1+ in 20 birds, 2+ in 4 birds), glycogen-laden hepatocytes (0 in 3 birds, 1+ in 14 birds, 2+ in 4 birds, 3+ in 7 birds, 4+ in 2 birds), and iron-laden hepatocytes (0 in 13 birds, 1+ in 7 birds, 2+ in 8 birds, 3+ in 2 birds). In the HE sections for group 2 birds, crush artifact affected 0%-5% of liver sections from 3 birds, 6%-25% of liver sections in 6 birds, and 26%-50% of liver sections in 1 bird. The histologic changes in group 2 birds were hepatocellular anisocytosis (ND in 9 birds, 0 in 1 bird) and anisokaryosis (0 in 2 birds, 1+ in 7 birds, 2+ in 1 bird), hepatic lipidosis (0 in 7 birds, 1+ in 1 bird, 2+ in 1 bird, 4+ in 1 bird), hepatocellular necrosis (0 in 9 birds, 1+ focal necrosis in 1 bird), heterophilic hepatitis with variable degrees of lymphocytic infiltrates (0 in 1 bird, 1+ in 8 birds, 2+ in 1 bird), glycogen-laden hepatocytes (0 in 1 bird, 1+ in 1 bird, 2+ in 2 birds, 3+ in 5 birds, 4+ in 1 bird), and iron-laden hepatocytes (0 in 4 birds, 1+ in 4 birds, 2+ in 1 bird, 3+ in 1 bird). Neither hepatic fibrosis nor bile duct hyperplasia was observed in any of the liver specimens. The AF-stained sections were negative for mycobacteria.

Discussion

In this study, liver injury was produced by an endoscopic biopsy or crush of hepatic parenchyma. Microscopic examination of the liver biopsy samples indicated normal variance within clinically healthy birds. To our knowledge, this was the first study conducted in birds that examined the effects of minimal traumatic liver injury via endoscopy on certain biochemical parameters.

Plasma biochemical analysis of all 30 birds was performed before any fiver damage; reference intervals were established for 10 hepatic analytes. In this study, sex did not have an effect on any of the variables. In birds that underwent biopsy only (group I), ALP activity decreased significantly at 12 hours after biopsy; however, ALP activity was not significantly different from baseline at 24 hours after biopsy. The mean CHOL concentration was decreased at 12 and 24 hours, with no change in BA concentration. Because BA concentration is considered sensitive and specific for liver disease, (17,18) the decrease in CHOL concentration is likely secondary to a nonhepatic cause such as decreased dietary intake. (19) Other significant biochemical abnormalities were increased mean activities of ALT (t = 24), AST (t = 12, 24), LDH (t = 12, 24), SDH (t = 12), and CK (t = 12, 24). The increase in SDH activity 12 hours after liver damage with a rapid return toward baseline levels supports its liver specificity and short elimination half-life. (20) The increased activities of AST, LDH, and CK at 12 and 24 hours with SHD activity returning to baseline levels after biopsy are most consistent with muscle damage associated with surgical manipulation and handling. Furthermore, LDH activity is present in many cell types and is a relatively nonspecific indicator of cellular injury.

When comparing the results of plasma biochemical analysis of the 2 groups, a significant increase in the mean activities of LDH at 12 hours and ALT at 24 hours was observed only in the birds that received crush injury (group 2). Neither of these biomarkers is fiver specific, and muscle injury is the primary consideration for the increased activities of these 2 enzymes. (17,19-21) In both groups, mean CK activity was significantly increased, which further supports muscle damage as the cause of increased activities of LDH and ALT.

In group 2 (crush) birds, all variables (except BA) were significantly different from baseline values at least at 1 time point during the study. With 1 exception, ALP activity was consistently decreased after surgery in group 2 birds. Although the specific mechanism is uncertain, a significant decrease in ALP activity has been reported with isoflurane administration in humans. (22) However, in other reports in humans, (23,24) as well as in dogs, (25) cats, (26) goats, (27) rabbits, (28) and American kestrels (Falco sparverius), (29) ALP activity did not change in association with isoflurane anesthesia. In humans, the decrease in ALP activity with anesthesia is speculated to result from decreased enzymatic induction from nonhepatic organs. (22) Because the liver was deliberately injured during surgery, ALP activity might also have decreased with the loss of hepatocytes, but this effect was unlikely given the minimal number of hepatocytes affected.

The activities of AST, ALT, and LDH were significantly increased in group 2; however, CK values always increased concurrently. In birds, these enzymes (AST, ALT, and LDH) have a broad tissue distribution, with the highest levels of AST and ALT in skeletal muscle, liver, and kidneys and of LDH in skeletal muscle, liver, kidneys, and brain; therefore, these enzymes can be increased with nonhepatic disease. (18,30,31) Creatine kinase is specific and sensitive for muscle damage in birds and thus should be measured simultaneously with the other enzymes. (32) Because CK values were concurrently increased without significant change in the other variables measured, the increased activities of AST, ALT, and LDH in birds receiving crush injuries were most likely the result of muscle damage from the surgical approach or from venipucture-associated tissue trauma rather than hepatic disease.

At 24 hours after surgery, a significant decrease in mean CHOL concentration was observed in group 2 birds; however, comparisons could not be analyzed statistically because of low numbers of samples with enough sample volume at the other time points. Even with low sample numbers, CHOL concentration did not appear to change substantially throughout the study and was likely unaffected by liver injury.

Bile acid concentrations were not significantly increased at any time after endoscopy in birds with crush injury. The lack of significant change in BA concentration was most likely a result of insufficient damage to functional hepatocytes. In other words, the crush injury applied was not extensive enough to cause a high enough reduction in functional hepatocytes necessary to observe a significant increase in BA concentration. (20,30-33) Instead, the mild changes in BA concentration from baseline (fasted sample) to 120 hours were most likely caused by postprandial effects. (20,34)

Significantly increased GGT activity was present at 4 time periods (t = 24, 60, 84, and 96 hours) in group 2 birds. The significance of the mean GGT activity in subgroup B at t = 96 was questionable because an extreme outlier was present. If the outlier was excluded, the trend of the mean GGT activity would have most likely followed a pattern of gradual decline. Usually GGT activity increases with biliary hyperplasia or cholestasis in domestic animals and with biliary disease in birds. (20) However, in this study, neither biliary hyperplasia nor cholestasis was observed microscopically. Therefore, the cause of the initial increase in GGT activity cannot be explained by these mechanisms.

At only 1 time point (t = 24) and in only 1 subgroup (B) was the mean GLDH activity significantly different from baseline in group 2. Focally extensive hepatocellular necrosis resulting from surgery could be the reason for the significant increase in GLDH activity. This enzyme has an extremely short biological half-fife (< 1 hour) in racing pigeons (Columba livia domestica). (35) However, in a study by Lierz et al, (11) GLDH activity peaked at 72 hours in wild raptors after liver injury. This analyte was not measured at 12 hours in subgroup B; therefore, values at 12 hours might have demonstrated a more dramatic increase.

A markedly significant increase in mean SDH activity was observed at 12 hours but returned to baseline at subsequent time points. This pattern of SDH activity is consistent with its biological half-life (< 12 hours) and suggests acute hepatocellular injury as the cause. (20) Interestingly, a significant decrease was noted at 48 hours; however, the reason for this decrease is unclear. Typically, decreases in hepatic enzyme activities do not have clinical relevance except in the cases of end-stage fiver disease. Obviously, end-stage liver disease as a cause of decreased SDH activity in this study is unlikely.

Overall, the histologic and biochemical changes did not correlate well after liver injury. This is most likely because of the small area of hepatic parenchyma that was damaged during biopsy or crush injury. Values of many of the analytes indicated minor muscle damage from the surgical approach for biopsy or difficult venipuncture and not from the liver damage induced by the forceps. Further studies with an increased level of liver damage might give a more accurate assessment of which biochemical tests are best to determine significant liver trauma compared with minor changes from performing endoscopic liver biopsy.

Acknowledgments: We thank Jien Chen at the Statistical Consulting Center at the University of Georgia for performing the statistical analysis.

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Susan M. Williams, DVM, PhD, Dipl ACPV, Lisa Holthaus, DVM, MS, Heather Wilson Barron, DVM, MS, Dipl ABVP, Stephen J. Divers, BVetMed, DZooMed, Dipl ACZM, Dipl ECZM, FRCVS, Michael McBride, DVM, Frederic Almy, DVM, MS, Dipl ACVP, Sharon Bush, BS, and Kenneth S. Latimer, DVM, PhD, Dipl ACVP

From the Department of Population Health (Williams), the Department of Pathology (Holthaus, Almy, Bush, Latimer), and the Department of Small Animal Medicine & Surgery (Zoological Medicine) (Barron, Divers, McBride), College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. Present address: Clinic for Rehabilitation of Wildlife, Sanibel Island, FL 33957, USA (Barron); Roger Williams Park Zoo, Providence, RI 02907, USA (McBride); Johnson and Johnson, Langhorne, PA 19047-0726, USA (Almy); Covance Laboratories Inc, Pathology Department, Madison, WI 53704, USA (Latimer).
Table 1. Results of plasma biochemical analysis for
fasted Indian ring-necked parakeets (n = 30) at time
0 (baseline).

Analyte                95% Confidence interval

ALP, U/L                        65-102
ALT, U/L                         8-14
AST, U/L                       182-282
BA, (a) [micro]mol/L            14-30
CHOL, (b) mg/dL                307-367
CK, U/L                        682-981
GGT, U/L                         3-5
GLDH, (c) U/L                    1-2
LDH, U/L                        73-103
RDH (d) U/L                      7-17

Abbreviations: ALP indicates alkaline phosphatase; ALT,
alkaine aminotransferase; AST, aspartate aminotransferase;
CK, creatine kinase; LDH, lactate dehydrogenase; CHOL,
cholesterol; BA, bile acids; GGT, [gamma]-glutamyl transferase;
GLDH, glutamate dehydrogenase; SDH, sorbitol dehydrogenase.

(a) n= 23.

(b) n = 15.

(c) n=27.

(d) n=28.

Table 2. Results of plasma biochemical analysis for Indian ring-
necked parakeets after liver biopsy and crush injury  in group 2
subgroup A (n = 5) taken over time (0-108 hours).

                                  Analyte mean
       ALP,    ALT,   AST,    BA,            CHOL,      CK,     GGT,
Time   U/L     U/L    U/L     [micro]mol/L   mg/dL      U/L     U/L

0      186     8      219     21             QNS        773     3
12     131 *   14     512     25             230 (a)   4541 *   4
36     123 *   14     457 *   27             263       2309 *   4
60     134 *   12     257     39             265       1396 *   5 *
84     143 *   24 *   196     29             285       1072 *   6 *
108    145 *   13     209     27             QNS        844     5

          Analyte mean
       GLDH,   LDH,    SDH,
Time   U/L     U/L     U/L

0      1       65      5
12     2       251 *   21 *
36     1       101     2
60     0.4     74      7
84     1       48      6
108    0.1     51      5

Abbreviations: ALP indicates alkaline phosphatase; ALT, alanine
aminotransferase; AST, aspartate aminotransferase; CK,  creatine
kinase; LDH, lactate dehydrogenase; CHOL, cholesterol; BA, bile acid;
GGT, [gamma]-glutamyl transferase; GLDH, glutamate  dehydrogenase; SDH,
sorbitol dehydrogenase; QNS, quantity not sufficient.

(a) This result might be unreliable because of several missing
baseline values.

* Significantly different from baseline (t = 0) value (P < .OS).

Table 3. Results of plasma biochemical analysis for Indian
ring-necked parakeets after liver biopsy and crush injury
in group 2 subgroup B (n = 5) taken over time (0-120 hours).

                               Analyte mean
       ALP,   ALT,   AST,        BA,        CHOL,        CK,     GGT,
Time   U/L    U/L     U/L    [micro]mol/L   mg/dL        U/L     U/L

0      58     14     307          25        370          532     5
24     47 *   20     663 *         9        272 (a) *   3549 *   7 *
48     51     12     497 *        42        300         1995 *   7
72     47 *   12     383          31        294         1328 *   6
96     51     10     370          28        318         1054 *   8 *
120    47 *   8      288          33        304          812 *   5

          Analyte mean
       GLDH,   LDH,    SDH,
Time    U/L     U/L    U/L

0      1       81      10
24     2 *     149 *   9
48     1       81      2 *
72     1       54      6
96     1       49 *    9
120    1       45 *    6

Abbreviations: ALP indicates alkaline phosphatase; ALT, alanine
aminotransferase; AST, aspartate aminotransferase; CK,  creatine
kinase; LDH, lactate dehydrogenase; CHOL, cholesterol; BA, bile acid;
GGT, [gamma]-glutamyl transferase; GLDH, glutamate  dehydrogenase;
SDH, sorbitol dehydrogenase.

(a) This result might be unreliable because of several missing
baseline values.

* Significantly different compared with baseline (time 0) (P < .05).
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Title Annotation:Original Studies
Author:Williams, Susan M.; Holthaus, Lisa; Barron, Heather Wilson; Divers, Stephen J.; McBride, Michael; Al
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Geographic Code:9INDI
Date:Jun 1, 2012
Words:6209
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