Avian Biochemical Analytes for Hepatic Function and Injury Interpretation: A Review
Abstract:
This study aimed to synthesize and find a consensus in the most recent interpretations of hepatic function and injury analytes values in birds, within publications between 2010 and 2020. The review was conducted through article searching on Google Scholar and PubMed. From the literature found, seven articles were selected for having results considered relevant for discussion. The publications described the utilization of bile acids, albumin, alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transferase, sorbitol dehydrogenase, cholesterol, triglycerides, bilirubin, and creatine kinase. Interpretation and use of all these analytes were reviewed and discussed. Despite a few negative repetitive patterns and some remaining questions, results and discussion in this review observed a greater assertiveness and understanding of values and changes in different cases of liver disease when compared to theoretical framework, and also resulted in a requestable hepatic profile for specific clinical suspicions.
Keywords: Clinical Biochemistry; Veterinary Clinical Pathology; Avian medicine; Clinical enzymology; Metabolite evaluation.
1.1. INTRODUCTION
Laboratory diagnosis and other complementary exams in birds gradually become a widespread practice worldwide to wild animals and unconventional pets medicine (Harr, 2002). Among bird’s clinical conditions, hepatic diseases can be evidenced by measuring enzymes and metabolites related to the liver. However, there are still some obstacles such as: the need of laboratory techniques adaptation and a general cloudy knowledge about bird’s liver metabolism.
While consulting bibliographic references related to this subject, we found authors and information that appear to work as a basis for bird clinical biochemistry studies and laboratory routine, however on the downside some of these records date from more than 30 years ago. Analytical and interpretive methods for bird liver function and injury were reviewed through exploratory selection of articles from the last 10 years. Through this review, we target to condense and find a consensus in most recent interpretations of analytes used to liver function and injury evaluation, trying to determine which ones are more effective and under what circumstances. To achieve our objective, this work aims to answer the following questions:
I. According to primary studies, what is the contribution level of different hepatic profile analytes when monitoring liver health?
II. Which concepts on liver health analysis presented in the theoretical framework have been updated or corroborated?
III. According to literature, which analytes appear to be the most eligible to monitor bird liver function and injury, and under what clinical circumstances?
- THEORETICAL FRAMEWORK
Among the traditional bibliographic references, Lumeij’s work at the 6th Edition of Clinical Biochemistry of Domestic Animals (2008), as well as Harr’s (2002) and Jaensch’s (2000) bibliographic reviews synthesized fundamental information developed in anterior decades – which will be important to this review. In addition to these authors, Thrall’s work at Veterinary Hematology and Clinical Chemistry (2012, 2nd Ed), even being more a recent search way, worked as consultation to certain discussions, involving both theoretical frameworks and primary articles selected for this review.
Bird clinical pathology, as well as from other unconventional animals, is a relatively modern veterinary medicine field, since its theoretical frameworks began in the second half of the 20th century. Articles chosen for review had often selected the same theoretical frameworks as their own projects guide, corroborating our theoretical references and indicating a synthesis need over the ideas developed in the last decades – a growing moment for avian medicine.
Jaensch (2000) describes liver diseases in birds, as well as other clinical conditions in these animals, with considerable inconsistent clinical manifestations. Laboratory diagnosis methods must consider these animals’ physiological peculiarities, which makes use of previously stated parameters for mammals in clinico- pathological tests less specific or inapplicable in birds. There are numerous physiological processes where the liver has a metabolic importance, which makes its functionality determination useful when monitoring general clinical conditions, disease progress, or prognosis. Even injured, the liver’s high capacity for regeneration and its functional resistance can hinder early hepatic diseases diagnosis, as clinical manifestations are usually apparent only after having 80% of liver function compromised. However, the determination of an etiological diagnosis is possible by means of clinical biochemistry targeted at liver function and injury parameters measurement, bringing about which liver diseases can be indicated in both clinically affected and apparently healthy individuals.
Harr (2002) and Lumeij (2008) reviewed many known enzymes used in birds clinical biochemistry. Aspartate aminotransferase (AST) is considered a non-specific indicator of hepatocellular damage, but it proves to be highly sensitive and is therefore combined with other muscle-specific enzymes, such as Creatine kinase (CK), to differentiate between muscle and liver damage. Alanine aminotransferase (ALT) is an enzyme with long plasma half-life, but besides being found in hepatocyte cytosol, it is also present in other body cells – therefore, its specificity is even lower than AST, impairing clinical relevance and making it often omitted for birds. Glutamate dehydrogenase (GLDH), an enzyme found in hepatocytes mitochondria, is considered the most specific test for hepatocellular damage. Despite this, GLDH plasma half-life is short (especially when compared to enzymes with low specificity such as AST and ALT) and its sensitivity is low, as it is only released into plasma after hepatocyte irreversible lesions, such as necrosis. Lactate dehydrogenase (LDH) has many tissue isoenzymes, which makes it less specific for monitoring liver health, which causes the clinical use of LDH uncommon. Gamma-glutamyl transferase (GGT), as well as in domestic animals, appeared to be highly specific for liver and kidneys in birds. Despite being considered non-sensitive for the diagnosis of liver disease alone, high concentrations of GGT were reported in birds under cholestatic conditions and in plasma of pigeons experimentally induced to liver disease. GGT then appears to be a quality analyte for liver damage assessment in birds if measured together with other enzymes specific to extrahepatic tissues, such as CK. Although present in the liver, Alkaline phosphatase (ALP) is specifically related to osteoblastic activity (growth, trauma, reparative osteomyelitis, neoplasia, nutritional hyperparathyroidism, and preparation for egg laying).
Thrall (2012) describes Sorbitol Dehydrogenase (SDH), also called Iditol dehydrogenase, as cytosolic extravasation enzyme mainly used for large animals given its hepatic specificity. There are not many studies about SDH diagnosis capability related to hepatocellular injury in birds, a possible reason for similarity with mammal reference values; SDH’s short plasma half-life in relation to AST and other enzymes appears to be its main disadvantage. Currently, SDH is not available in most veterinary clinical laboratories.
Lumeij and Harr also cite metabolites related to liver activity. Plasma bile acids (BA) can be measured in clinical laboratories through automatic spectrophotometry, and is considered the best-isolated liver function marker. BA has the advantage of being more hepato-specific, with higher sensitivity and specificity (when sampled from a correctly fasting animal) – compared to AST. Hypoalbuminemia appears to indicate liver failure, nephropathies, nutritional and digestive disorders. Albumin (ALB) is considered a liver function indicator protein despite not having high specificity because of being fully converted from transiterin (pre- albumin) into the liver. The presenting of known physiological ALB increases in females preparing for laying is an important stand out to conventional laboratory methods – which can present doubtful variations for the measurement of ALB in birds. Biuret method is presented by Lumeij (2008) as the most reliable ALB measurement method. Harr (2002) stated that cholesterol metabolism (CHOL) in birds seems to be similar to mammalian, however, the presence of abnormal values and physiological increase in the concentration of this analyte in the plasma of females in vitellogenesis and egg formation (even before it visibility on radiographic image) are important to distinguish.
Lumeij (1988a; 1988b) determined different plasma analytes specificity and sensitivity rates for diseases in liver and muscle tissue based on pigeons’ experimental studies (Table 1).
- MATERIALS AND METHOD
Bibliographic review arises from the need for information synthesis in birds clinical pathology, since most well-known references are relatively out of date, apart from the rising of new methods and general veterinary and avian medicine development. New interpretations of analytes that indicate liver injury and function in birds can be found in more recent studies. Through this review, we aim to synthesize this theme knowledge, indicate still existing gaps, and seek a consensus between these research questions and results found in literature.
Articles from the last 10 years related to this review subject were selected to compose this work. Materials selected for review come from different data- bases and scientific journals since the search was carried out directly by Google Scholar and PubMed through automatic search.
Selection criteria involved the following key themes, which also served as search strings: bird’s serum analytes, bird’s plasma biochemistry, bird’s liver assessment, bird’s liver analytes, bird’s liver profile, liver function and injury in birds, monitoring of liver health in birds, bird’s plasma enzymes, bird’s plasmatic proteins.
In total, 12 articles were selected for this review, which five eliminated for any of the following reasons: I. The article hada different chosen theme from proposed for this review; II. Although having a correlated theme, it has no interesting results or raised relevant discussions for this review. All 7 articles approved for this review are identified in Table 2.
- RESULTS
4.1. ARTICLE 1
VasHist’s (2011) aimed to analyze the responsive liver profile of Gallus gallus, Linnaeus, 1758 experimentally infected with Plasmodium juxtanucleare (Versiani & Gomes, 1941), the causing agent of avian malaria. Chemical analytes related to liver function were AST and ALT.
The results obtained showed differences related to liver damage caused by infection between the two groups, indicated by ALT increase activity, and inflammatory infiltrates found in liver tissue. Such data indicated parameters reliability for ALT to predict the peak of infection, in addition to a possible function as stress markers for ALT and also AST. AST variation was not decreased between the two groups, possibly due to the non-specificity of the enzyme in relation to liver injuries.
4.2 ARTICLE 2
The purpose of Williams et al. (2012) study was to evaluate changes in liver biochemical parameters (GGT, GLDH, SDH, ALP, ALT, AST, CK, LDH, BA and CHOL) after an acute liver injury caused by trauma originated by endoscopy biopsy. The values were subsequently compared with the lesions observed in histological findings. The birds were divided into two groups: group 1 suffered injuries from direct trauma, while group 2 suffered injuries caused by crushing. At the first sample collection time, 12 hours after, all variables studied underwent a significant increase compared to reference values; at 24 hours, ALT, AST, CK, LDH, and CHOL values remained significantly higher than reference values – ALP and CHOL decreased significantly while ALT, AST, CK, LDH, and SDH increased at 12 and 24 hours intervals. No changes in BA values have been reported. Increased values of enzymes related to extrahepatic tissues (AST, LDH, and CK) between 12 and 24 hours correlated with the return of SDH to normality are more consistent with muscle damage associated with surgical manipulations and physical restraint of the animals – which we can highlight the presence of significantly higher values of LDH and ALT in group 2, the one who suffered compression. Despite being considered sensitive and specific for liver analysis, slightly altered BA values correlated with little variation in CHOL concentration indicate that lesion was probably not extensive enough to reach sufficiently functional hepatocytes. ALP increasing was related to reports of reduced plasma concentration after isoflurane administration in humans, but little evidence has made this hypothesis consistent. Significant increase in GGT activity occurred at four moments (5, 24, 60, 84, and 96 hours later) in group 2, with a gradual decline (excluding the 96 hours value, considered questionable). The cause of increase concentration couldn’t be explained by the mechanism of this study, since GGT increased activity is commonly related to hepatic hyperplasia or cholestasis in domestic animals – both conditions not re- ported or induced. Corroborating with reports of extremely short plasma half-life, there was only once significantly increase in GLDH in group 2 after 24 hours value, which was related to possible extensive hepatocellular necrosis caused by surgi- cal procedure. A significant increase in SDH activity was seen 12 hours later but returned to reference values subsequently – consistent with its plasma half-life of 12 hours, despite its high liver specificity reported by literature. These SDH values suggest acute hepatocellular lesions. Many analytes indicated that the surgically induced liver damage wasn’t sufficient to cause major changes.
4.3 ARTICLE 3
Matos (2016) sought to establish infection models with Fowl aviadenovirosis (FAdV) by experimental induction of FAdV strains from recent inclusion body hepatitis field outbreaks, selecting chemical analytes with clinical importance relations regarding liver, pancreatic and renal integrity and function, comparing the biochemical values found over study period, by days post-infection (dpi) associated with clinical signs, pathological lesions, and viral load in organs of infected birds and characterizing the pathogenicity of different strains of FAdV.
Animals were divided into six groups of 26 birds each, one control group and five infected, and in each group, 20 were inoculated with determined strains, and the remaining six animals were kept as sentinels. Structural hepatic analytes, as AST and GLDH, and functional hepatic analytes, like BA, total plasma proteins, and albumin were analyzed.
Most biochemical changes occurred between the 7th and 10th dpi. A significant increase in cytoplasmic enzyme AST and mitochondrial GLDH were obtained at 7dpi compared to control group, being associated respectively with recent cell degeneration and tissue necrosis. Increased BA concentration, and decreased plasma albumin coincided with changes in functional level, and later with histological and macroscopic findings during necropsy.
Clinical biochemistry appeared to be a valuable complementary tool for assessment and monitoring of infected birds, promoting a greater understanding on pathophysiological mechanisms of FAdV infections.
4.4 ARTICLE 4
Ou (2017) main objective was to investigate the course and immune response of adult ducks infected by hepatitis Avirus, as well as related liver injury, fibrosis, and regeneration processes. Throughout course of infection successive increases and decreases in AST, ALT, and total bilirubin were noted, which were related to liver damage at early stages of infection. Increased levels of triacylglycerols were associated with severe steatosis, confirmed later by hematoxylin-eosin staining. Below normal albumin levels also suggested modification in liver protein synthesis.
4.5 ARTICLE 5
Plaza (2018) sought to determine whether health status of Coragyps atratus (Bechstein, 1793) (black-headed vultures) residing in dumps was affected in any way compared to health level of vultures living in wild areas (steppes). The hypothesis built was that a diet in rubbish dumps would reflect changes in health and visible clinical and biochemical parameters when compared with individuals with more natural diet.
Among blood parameters evaluated, liver parameters were ALB and some liver enzymes such as ALT, AST, ALP, and CK. Biochemical parameters differed between groups; while individuals captured in dumps had higher ALP concentrations, individuals from steppes had higher AST, ALT, and CK concentrations. There weren’t ALB differences between groups.
Results showed clear differences in certain parameters between individuals of both groups, which suggests that the foraging location chosen may influence clinical and biochemical values, besides general health status. Vultures captured in landfill had more robust body mass than steppe individuals, suggestive of easy access to food and low energy expenditure. Although biochemical parameter changes related to other physiological functions were reported, biochemical values related to liver status, except for increase of ALP (which has an inconclusive interpretation), didn’t show significant changes. Individuals from steppe areas showed higher concentrations of liver enzymes AST and ALT, which may suggest liver damage related to intoxication by different toxins and pesticides. The high CK values in the steppe individuals were considered difficult to interpret, due to possibility relation to several factors – both due to muscle trauma from competitions for carcasses and due to the stress during capture and blood samples collection.
On a general assessment, both birds residing in landfills and those in steppe areas showed changes in their biochemical parameters related to different causes. Dump group showed an ALP and other biochemical parameters concentration increase (higher values of glycemia, calcium, globulins, uric acid, and hematocrit).
The Steppe areas group captured showed increased values of CK, ALT, AST, and also urea. Some non-standard considered values present in individuals from both sites can be explained alternating habits between both regions – similar to reports in literature, other species usually compensate the lack of carcasses in steppe areas for places with extra supply of food, such as landfills.
Concluding, results showed clinical and biochemical differences resulting from life habits of necrophagous birds in wild and anthropized areas. Despite this, certain birds’ apparent healthy clinical condition can mask real health problems – although individuals living in landfills are more physically robust, they have health issues related to carbohydrate intake. Meanwhile, birds from natural environments that perform expected natural activities and have a more adequate diet may be exposed to human activity toxic substances, and the stress of free-living due to survival struggle appears to have a visible role in these individuals’ clinical conditions. Thus, both groups had some changes in health parameters that may be related to human interference.
4.6 ARTICLE 6
Lavers’s (2019) objective was to understand lethal and sublethal clinicopathological signs of plastic ingestion in Flesh-footed Shearwaters through blood and body condition – biochemistry analytes associated with liver function and injury were AST, GGT, BA, ALB, and CHOL.
No apparent correlation between plastic ingestion and clinical enzymes (AST, GGT), albumin, total plasma proteins, or bile acids was found; however, a positive correlation has been reported between the presence of plastic and serum cholesterol values. Although birds cholesterol concentration increases due to stress and periods of short fasting, concentrations detected in this study were higher when compared to other similar condition juvenile individuals, these without signs of plastic ingestion – suggesting that, as previously reported in marine mammals, plastic ingestion can influence serum cholesterol concentrations and reproduction by altering some mitochondrial membrane transport mechanisms.
4.7 ARTICLE 7
Hung (2019) sought to improve antemortem diagnostic methods understanding and usefulness in parrots with liver disease by assessing the correlation between biochemistry, hematology, radiography, and endoscopic visualization with histopathological liver biopsies abnormalities. 28 clinical reports between2007 and 2016 from a veterinary hospital were reviewed.
Biochemical assessments showed correlation between AST and CK frequent increased concentrations: both enzymes were elevated at the same time (n =10); CK showed normal values with AST values also normal (n = 5). There were situations in which CK had high concentrations without high AST (n = 10). In that study, elevated AST and CK at normal concentrations were not observed. Thus, increased results found appeared to have a greater correlation with venipuncture sampling containment.
SDH showed increased values in three out of five patients, with 2 day intervals between biopsy and blood sample collection and analysis. Therefore, this study demonstrated a low correlation between SDH and liver disease histopathological findings, differently from literature reports. The author, however, points out that the low amount of samples and its 12 hours plasma half-life could explain these results since material analysis exceeded that time. Individuals diagnosed with liver disease through histological evaluation showed abnormal bile acids values only in three of 21 situations. Therefore, this study results strongly disagree with the previous literature, which considers bile acids sensitive and specific for liver disease. Assumed by the author, if the sample collection occurred at an early disease stage, bile acid values possible weren’t changed yet. Plasma biochemistry was considered in this study a non-specific or consistent method compared with histopathological results for liver disease diagnosis in parrots.
5. RESULTS AND DISCUSSION
5.1. Bile acids (BA)
In bibliographic reports by Lumeij (2008) and Harr (2002), BA are consiered the best liver function measurers, with high sensitivity and specificity (coming from a sample of a correctly fasting animal).
In article 3, experimental infection increased BA concentration, resulting from liver functional loss in face of severe infection. In article 2, however, BA measurement was minimally altered by induced physical injury. As for the article 6, BA do not seem to be affected by plastic ingestion, whereas the article 7 registered an increase in only 3 of 21 situations where the measurement was requested for animals with liver disease proven by other methods.
Many factors can be related to the assumption inconsistency for this compound, but we believe that the main one is the liver functional resistance. As mentioned by Jaensch (2000), the liver is an extremely important organ, and its high regeneration capability and functional resistance may delay detection of certain diseases, since it will keep the physiological activity stable until limit. This would explain the absence of abnormalities in the research performed on animals in real clinical situations (articles 6 and 7) and in the one rela- ted to induced injury (article 2). On the contrary, the article 3 seems to prove BA ability as a liver function indicator in severe infections directly damaging liver tissue. Thus, despite being highly sensitive and specific for liver function, abnormal BA values appear to be a late indicator of liver disease.
5.2. Albumin (ALB)
Thrall (2012) described albumin as one of the smaller and yet the most abundant proteins made by hepatic synthesis, summed to its main function as molecule transport and blood oncotic pressure maintenance. Literature by Harr (2002) and Jones & Chitty (2020) describe plasma electrophoresis as the best method to quantify and analyze albumins and globulins, which, combined with other exams, may be useful for detecting and monitoring inflammatory diseases.
In articles 3 and 4, ALB decreased during viral diseases, and liver function loss was then interpreted based on protein synthesis. In articles 5 and 6, however, there were no differences in albumin levels on the studied groups. As described by Grunkemeyer (2010), hypoalbuminemia is mainly attributed to conditions of poor digestion and absorption, which may or may not involve enteropathies, nephropathies, and liver failure. As a negative acute phase protein, serum albumin concentrations decrease in the event of liver function loss, giving rise to acute phase proteins production, mostly globulins. Campelli and Crosta (2013) similarly mentioned differentiation between species in terms of protein migration and sensitivity given the conformation variability and surface charge distributions between protein molecules, a possible reason why human values and standards are not effective using biuret method. Despite being involved in several activities, albumin seems important hepatic functionality assessment, especially when globulins are also evaluated and A/G ratio is analyzed, enabling a better interpretation of the values, the reby facilitating clinical diagnosis.
5.3. Alanine aminotransferase (ALT)
According to theoretical framework, ALT is a cytoplasmic free enzyme, which means it overflows when the cell is damaged – despite its magnitude not being related to the severity of the tissue damage, as stated by Thrall (2012). As described by Harr (2002), Lumeij, (2008) and Grunkemeyer (2010), ALT presence in several tissues and mainly in liver and muscle cytosol makes it nonspecific, difficulting interpretation.
ALT showed good reliability in relation to acute infection by P. juxtanucleare in article 1, being considered by authors as a good indicator of liver dysfunction alongside AST. Similarly, for article 4, enzymatic variations were consistent with the histological and macroscopic findings. In article 2, however, both ALT and AST increases were related to muscle injuries. Article 5 results, a complete interpretation of the analytes was not possible due to the limitations related to the analysis of wild animals, so the increase in ALT was only suggestive of intoxication.
Despite its Non specificity, ALT is still measured in several laboratories. Current studies however have not shown greater benefits in relation to use of other methods. According to Thrall (2012), ALT can be useful in detecting liver diseases in carnivorous birds, with a considerable plasma increase in significant liver or muscle damage. If ALT measurement is desired, the use of other correlated analytes (such as CK and AST) is indicated for comparison and better interpretation.
5.4. Aspartate aminotransferase (AST)
Theoretical framework considers AST non-specific to indicate hepatocellular damage, but its considerable sensitivity makes it eligible for comparative analysis when in conjunction with other extra-hepatic enzymes such as creatine kinase (CK). Thus, although not being known as best analyte for avian liver injury, it can still be solicited in clinical laboratories.
In article 3 experimental adenovirus infection, AST significant increase combined with other liver parameters and compared to the control group suggested liver degeneration in agreement with later histological and macroscopic findings. In article 4, successive increases and decreases in AST and ALT were suggestive for liver injury due to acute infection. In article 1, AST and ALT appear to be capable of marking physiological stress, and ALT was more responsive to detect infection peaks than AST, an unexpected result for a less specific enzyme. Article 5 also states that increases in AST and ALT may be related to intoxication, if not related to muscle injury found by increase in CK. Other reviewed authors have inconclusive results for AST. In article 2, increased concentrations of AST were linked to muscle damage from physical injury, since other extrahepatic enzymes were also increased. Article 6 found no apparent correlation between plastic intake and clinical enzymology. Finally, article 7 did not find moments of AST increase without correlated CK increase, which indicates that, in all recorded moments, AST increase is probably related to muscle injury, possibly due to physical restraint.
Results found in this review corroborate with the theoretical framework. AST offers consistent results when used in comparison with other hepatic and extrahepatic analytes, but also proves its isolated inefficiency as a liver analyte due to its low specificity. Thus, we suggest again that any biochemical analysis for hepatic profile choose CK and another more sensitive and specific liver enzyme in conjunction with AST.
5.5. Glutamate Dehydrogenase (GLDH)
Grunkemeyer (2010) and Harr (2002) describe GLDH as a mitochondrial enzyme present in epatocytes, which characterizes it as specific for liver damage, despite its low sensitivity and low plasma half-life – consequently making it a late and inconsistent indicator of liver damage, only pointing out serious injuries such as liver necrosis.
Article 2 showed significant variation in one of the groups 24 hours later, which was related to hepatocellular necrosis. Similarly in article 3, there were two groups with abnormal enzymatic concentrations associated with higher mortality and confirmed from findings of irreversible hepatocellular lesion.
Although not being commonly used and directly commercialized in all countries, including Brazil (Rodrigues, 2021), GLDH has been described as a highly specific analyte for hepatocellular damage in birds as described by Thrall (2008) and also Lumeij (2008). Its plasma activity does not seem to suffer direct alteration by extrahepatic injury as shown for AST, ALT and LDH. Despite having a shorter half-life than AST and ALT and only related to advanced stages of the disease, further studies may facilitate its use and popularity in laboratory routine.
5.6. Lactate Dehydrogenase (LDH)
LDH is considered by literature as a low utility analyte for monitoring liver damage, since it has many isoenzymes in tissues (Harr, 2002). Article 2 found a significant increase in LDH after induced liver injury. In this work, the enzyme was considered as extrahepatic, and because it increases together with enzymes in the same category (AST and CK), it corroborates its non-specificity. In the controlled situation of the study, the increase was related to muscle injury from surgical manipulation and physical restraint.
Although having only one research using this analyte in its project, making it difficult to vary the results for analysis in this review, the results of this study corroborate the previous records: despite its sensitivity, its non specificity makes it an analyte of low utility for monitoring function liver disease, specially in routine analyses.
5.7. Alkaline phosphatase (ALP)
The function previously reported for ALP is little related to liver function or injury. Its usefulness is particularly related to osteoblastic activity as registered by Lumeij (2008).
In articles 2 and 5, altered values for ALP did not have a consistent interpretation and do not appear to be directly related to liver function. Thus, ALP activity as a liver marker remains considered inefficient in current reports. The cases of unjustified changes registered in the literature raise an alert to possible need for further studies on its capacity as a marker enzyme of physiological activities. Mohanapriya T. et. al. (2020) is, however, an example of a study in avian clinical biochemistry that found increased values in birds with fractures, agreeing with the theoretical framework.
5.8. Gamma-glutamyltransferase (GGT)
In birds, GGT is considered a highly specific liver enzyme by the theoretical framework. Despite this, its relatively low sensitivity hinders its use in isolation for liver disease diagnosis. When properly measured, this compound appears to suggest cholestatic conditions, hyperplasia and liver disease.
In article 2, its concentration increase cannot be explained by the methodology used, since cholestasis or hyperplastic conditions were not reported or induced by the physical injury. Article 6 found no apparent relationship between plastic intake and changes in GGT or other liver enzymes.
In this review, the scarce reports of GGT measurement became an obstacle to a better interpretation of the results, apart from warning the need for greater popularity regarding this enzyme both in research and in clinical routine. Thereby, we believe that more complete information would be obtained if GGT measurements were possible in articles 5 and 7.
5.9. Sorbitol Dehydrogenase (SDH)
According to Thrall (2012), even though SDH is an extremely short half-life hepatospecific enzyme compared to other analytes, it appears that in Westlak (1983), such as in domestic mammals, it isn’t altered by stress factors or muscular damage like other enzymes. Cray (2008) reports similar reference intervals for SDH in birds to those reported in humans and other mammals.
Article 2 reported an increase in its activity at the first point of analysis (12 hours), suggesting acute hepatocellular lesion. As for the article 7, although few samples were used, the study showed coherence between enzyme measurement and histopathology.
Similar to GLDH, SDH is a relatively new analyte in the market, without much applicability in clinical routine. Despite its short half-life, which reduces its use in chronic cases, it has been reported in literature with excellent liver specificity, together with its cytosolic nature which also makes it a new option for the indication of acute liver injuries (Thrall, 2012). Hence, future studies are necessary to promote and reinforce its applicability.
5.10. Cholesterol (CHOL)
Harr (2002) considers cholesterol metabolism in birds similar to what we know from mammals, which makes it, in theory, a potential analyte to monitor liver function and carbohydrate metabolism as a whole.
In article 2, CHOL little varied after liver injury induction. Meanwhile, in article 6, the abnormal concentrations found in seabirds that had ingested plastic seems to corroborate the increase in this same analyte also found in marine mammals after plastic intake.
Once again, the limited amount of results point out the need for further studies on cholesterol evaluation so as to monitor liver health, especially concerning the integrity of liver function. Results found in article 6, at least, seems to corroborate the capacity of this analyte to reveal valuable information about liver functions and even its communication with agents possibly harmful to the organism, such as plastic. By observing the pattern found in birds that frequented dumps in article 5, we believe that cholesterol evaluation could have offered more concordant and complete information about the health status of these individuals.
5.11. Triglycerides (TG)
TG are synthesized in intestinal mucosa and liver from digestion components and absorption of fatty acids. They are the main form of lipid reserve in birds, by its higher caloric density and low water requirement (Lumeij, 2008).
A very significant increase in the infected group was reported in article 4, related by the authors to a severe process of hepatic steatosis. On the other hand, article 7 found a disagreement between TG variations and the biopsy findings re- ported.
Because of TG subjection to intra and interspecific variations in birds such as age, sex, migratory behaviors and diet, its nature is extremely nonspecific, making it difficult to analyze and differentiate pathological cases described by Jones & Chit- ty (2020) such as diabetes mellitus, hypothyroidism or cardiovascular disease. Further analysis and descriptions of reference values are needed.
5.12. Bilirubin & Biliverdin (BIL & BILV)
According to Harr (2002), Lumeij (2008) and also Thrall (2012), birds in general are not able to convert biliverdin into bilirubin alone on account of the absence of biliverdin reductase in liver tissue. The yellowish color of avian plasma is related to the presence of carotenoids from diet (Lumeij, 2008).
Abnormal plasmatic bilirubin concentrations in article 4 were directly related to viral infection by hepatitis A and parallel to variations of AST and ALT during infection.
Despite being the main bile pigment in birds, biliverdin is only commonly measured in veterinary research laboratories and is not frequently measured in clinical laboratories, as reported by Thrall (2012). Its presence in blood plasma, according to Capitelli (2013), is related to severe hepatobiliary diseases, and is also associated with unfavorable prognosis. Then, biliverdin seems to have a situational participation in the diagnosis of liver diseases in birds. For bilirubin, there are few reports of its use in literature, with Thrall (2012) and Lumeij (2008) suggesting the reason for its presence by means of conversion by bacteria or nonspecific reductase enzymes. Increases in this analyte were found in specific cases of viral hepatitis infection in ducks, Pacheco virus and chlamydiosis in parrots.
5.13. Creatine Kinase (CK) – Extrahepatic analyte
Due to its plasma activity related to muscle tissue injuries, CK is used as a comparison and differentiation analyte between liver and muscle damage as stated by the theoretical framework. In article 2, CK together with other extrahepatic enzymes suggests muscle damage associated with surgical manipulation and physical restraint. In article 5, high values in wild animals also suggest muscle damage, although the exact cause of the injury is unclear. In article 7, CK and AST showed correlations at many times, and at all recorded times the comparison seemed to suggest muscle damage.
The results found in the articles in which CK was used for differentiation with liver analytes or to directly analyze muscle damage suggest reliability, which also corroborates with the theoretical framework.
5.14. GENERAL INTERPRETATIONS
As supported by Cray (2008), most clinical cases demonstrate that the time development of liver diseases is still, in some way, mysterious. Thus, combination of appropriate enzymes measurement, plasma proteins and concentrations of metabolites such as bile acids can be considered a good diagnostic auxiliator for birds.
After finding answers to the first two questions of this review through isolated discussion of all analytes, we propose an answer to the third question by (b) Infectious hepatitis: GGT, GLDH, CHOL, BIL, ALB, BA, AST with or wi-means of a suggestion, based on the findings of this articles review, regarding a requestable hepatic profile with biochemical analytes suspected of liver related pathologies (find marked in bold the five highest priority analytes for each case, when there is no possibility of measuring all recommended markers):
(a) Routine checkups on clinically healthy birds or nutritional/infectious problems, such as steatosis: GGT, GLDH, CHOL, TG, ALB, BA, AST, with or without ALT and CK; thout ALT and CK; (c) Suspected poisoning with potential for liver damage: GGT, AST with or without ALT, CK, ALB, CHOL, BA and GLDH.
Based on primary studies analyzed, the response pattern of these analytes for birds is variable, and can be contradictory. Apparently, some analysis patterns inspired by clinical pathology of domestic mammals are still rooted in clinical pathology of wild and exotic animals nowadays, which results in the choice of inappropriate analytes for both single clinical cases and a whole chain of biochemical routine analyses. Thus, finding repetitive patterns in this study suggests that, in addition to inherent need for better understanding the liver health and its markers, an improvement in clinical knowledge of professionals involved is highly important, since the choice of inappropriate analytes, lack of knowledge or devaluation of plasma half-life and the neglect or lack of technique for preanalytical and analytical sample handling are examples of skills with a decisive role in the evaluation of biochemical results, difficulting or omitting important information.
Clinical biochemistry, like all science subjects, continues to evolve by introducing new methods for the measurement of analytes already known or newly discovered. In this review, we observed a greater assertiveness and understanding of values and changes in cases of liver disease of certain species, in addition to greater attention to their habitat, their diet and seasonal behavior, especially with free-living animals – which indicates that the existing theoretical framework for this theme played a crucial role in the matured development of recent research. We estimate that, within more 10 years, there will be more concrete findings regarding analytes that indicate liver injury or function, as well as their viability for clinical use, specially for those which our review could not totally assure.
- 6. CONCLUSION
This literature review compiled different approaches used in avian hepatic profile for over the past 10 years, condensing information on monitoring liver health through a more recent view, as well as establishing new questions to be solved in the future. In view of the importance that biochemical methods have as to support diagnosis, from routine examinations in veterinary clinical laboratories to the consequences of anthropological actions in ecosystems involving birds, it is important to keep further investigations regarding reference values and pathological changes – specially because among the causes of illness in birds, liver diseases are really challenging clinically as stated before.
The growing development of technology, veterinary medicine and relevance of the One Health concept and growth of emotional value for birds raised as pets are reasons why we believe that studies related to avian medicine, mainly in
7. ACKNOWLEDGEMENTS biochemical analysis and clinical pathology field will be more frequent, reaching a greater understanding to current doubts and contradictions in literature. The authors acknowledge and thank the opportunity granted by the National Council for Scientific and Technological Development (CNPq) and Professor Humberto Fernandes for all support, opportunity and knowledge given throughout the production of this article and for encouraging the development of scientific writing through the course “Scientific English Writing Workshop”. We also appreciate our supervisors Antonio Peixoto Albernaz and André Lacerda de Abreu Oliveira for the ideas and support given to the development of this study.
Finally, we also thank our colleague Thiago Xisto de Oliveira for helping with the revision of our writing and providing alternative unbiased inputs.
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This article has been developed at the 1st Scientific English workshop, a partnership between the Pro-Rectory of Postgraduate and Research (ProPPG-UENF), the International and Institutional Af- fairs Advisory (ASSAII-UENF), and the Program of Scientific and Technological Initiation (PIBi).
Title: Avian Biochemical Analytes for Hepatic Function and Injury Interpretation: A Re- view
Type of publication: Original Research Article
Name: Beatriz Garcia Sousa & Gustavo D’Anunciação Braga
Publishing name: SOUSA, B.G. & BRAGA, G.D.
Field of Study: Avian Clinical Pathology
Institution and Term: Universidade Estadual do Norte Fluminense Darcy Ribeiro /
Center of Agricultural and Livestock Sciences and Technology
About the author
Undergraduate student in Veterinary Medici- ne at UENF, with course conclusion expected for mid-2024. Scientific Initiation scholar since 2018, focusing on animal experimentation aiming innovations in the area of Clinic and Surgery of companion animals. Gustavo was advised by Prof. André Lacerda de Abreu Oliveira.
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About the author
I joined the course of Veterinary Medicine at UENF in 2019 and I am currently carrying out Scientific Initiation in the area of Clinical Biochemistry of Birds by the Clinical Pathology sector of the Laboratory of Animal Clinic and Surgery (LCCA), more specifically with free-living raptors supported by the Center for Studies and Rese- arch in Wild Animals of UENF (NEPAS). Beatriz was advised by Prof. Antonio Peixoto Albernaz.
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