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Laboratory tests for Liver Disease

Robert Washabau, VMD, PhD, Dipl. ACVIM - 24/08/2014

Laboratory tests for Liver Disease

Robert Washabau, VMD, PhD, Dipl. ACVIM
College of Veterinary Medicine University of Minnesota St Paul, Minnesota, USA

The role of the liver in intermediary metabolism

The liver is involved in many aspects of intermediary metabolism (1). The laboratory testing of liver function usually involves some aspect of the liver’s role in intermediary metabolism.

Carbohydrate metabolism

The liver is at the centre of carbohydrate metabolism through its role in maintaining normoglycaemia.  Carbohydrate stored in the liver as glycogen is hydrolyzed to glucose via glycogenolysis when a need for glucose develops. When the glycogen available is insufficient, glucose is produced from amino acids by gluconeogenesis.

Glucose is also produced from glycerol and intermediates of glycolysis, such as lactic acid and pyruvic acid. With inadequate carbohydrate in the diet, blood glucose is maintained at the expense of body proteins. Body lipid stores are also depleted during starvation, although lipids do not participate in the maintenance of blood glucose other than serving as an alternate source of energy, since glucose cannot be synthesized from fatty acids.

• Glycogen → glycogenolysis → glucose → normoglycaemia

• Amino acids → gluconeogenesis → glucose → normoglycaemia

Clinical relevance - Acute and chronic liver diseases may be accompanied by hypoglycaemia.

Protein metabolism

The liver is an important site of protein metabolism. Amino acids and proteins absorbed from the intestine or produced in the body are delivered to the liver. The liver deaminates amino acids and can convert them to carbohydrates and lipids depending upon nutritional needs. Deamination produces alpha-keto acids, which can be metabolized for energy or used for synthesis of monosaccharides and fatty acids. The liver synthesizes amino acids from intermediates of carbohydrate and lipid metabolism by amination and transamination.

Examples of amino acid transaminations include:

• Alanine + alpha-ketoglutarate ↔ pyruvate + glutamate

• Aspartate + alpha-ketoglutarate ↔ oxaloacetate + glutamate

The liver synthesises many proteins including albumin and fibrinogen, most of the alpha-globulins, some of the beta-globulins, ceruloplasmin, ferritin, and other serum enzyme activities. The urea cycle is involved in the oxidative degradation of amino acids. Ammonia is a primary metabolite of amino acid metabolism. The gastrointestinal tract, and especially the colon, is the most important source, through the action of bacterial urease on endogenous urea that diffuses in the intestine and on degraded dietary amines.

Ammonia produced by the colonic bacteria enters the portal vein and is transported to the liver to be transformed by the urea cycle.

• 2 NH3 + CO2 + 3 ATP + H2O → urea + 2 ADP + 4 Pi + AMP + 2 H

Clinical relevance - Acute and chronic liver diseases may be accompanied by (i) increases in serum aminotransferase activities, (ii) hypoalbuminaemia, (iii) hyperammonaemia, and (iv) decreases in blood urea nitrogen concentration.

Lipid metabolism

The liver is involved in the intermediary metabolism of lipids from (i) triglyceride synthesis and storage to (ii) fatty acid oxidation, and (iii) cholesterol synthesis, storage, secretion and transport (2).

• (i) Synthesis and storage: Acetyl-CoA + malonyl-CoA + NADPH → triacylglycerol + CO2+ NADP + H20

• (ii) Fatty acid oxidation: Triacylglycerol + CoA + NAD + FAD → acetyl-CoA + NADH + FADH

• (iii) Cholesterol: Intestine → cholesterol in chylomicrons → apoprotein B48 → liver
Muscle, connective tissue → cholesterol in HDLs → liver
Liver → cholesterol in VLDLs → serum and bile
Blood → cholesterol in LDLs → apoprotein B100 → liver

Clinical relevance - Acute and chronic liver diseases may be accompanied by hypocholesterolaemia. Biliary obstructive disorders may be accompanied by steatorrhoea.

Coagulation factors

The liver synthesizes plasma clotting factors I (fibrinogen), II (prothrombin), V, VII, VIII, IX, and X. Factors II, VII, IX, and X are vitamin K-dependent clotting factors. The most important factors in liver disease are those with the shortest half-lives, factors VII and VIII.

Clinical relevance - Acute and chronic liver diseases may be accompanied by (i) prolongations in prothrombin and partial thromboplastin times and (ii) associated coagulopathies.

Bile secretion

Bile is a slightly alkaline iso-osmotic solution of bile salts, bile pigments, phospholipids, cholesterol, electrolytes, and water. Bile acids and bile salts are the primary component of bile. Bile acids are synthesized from cholesterol and conjugated to an amino acid (usually taurine or glycine) to become a bile salt. They are secreted into the biliary tract where they undergo storage in the gallbladder and are subsequently emptied into the small intestine during feeding. Bile salts carry out the emulsification of ingested lipids to facilitate pancreatic lipase digestion, and the micellarization of free fatty acids to facilitate enterocyte absorption. Ileal re-absorption of bile salts facilitates the return of bile acids to the liver for re-uptake, re-conjugation, and re-secretion.

• Emulsification → pancreatic lipase digestion → β-monoglycerides + free fatty acids + glycerol → micellarization by bile acids → enterocyte absorption → re-esterification → triglyceridemia

Clinical relevance - Biliary obstructive disorders may be accompanied by icterus and steatorrhoea.

Porphyrin metabolism

Porphyrins are intermediates of the heme biosynthetic pathway. In health, the porphyrins are converted into the oxygen-carrying heme compounds of hemoglobin, myoglobin, cytochromes, catalase, and peroxidase. The liver serves as a synthetic as well as excretory pathway for the porphyrins.

• d-aminolevulinic acid → porphobilinogen → protoporphyrin IX → globin + Fe2+ + bilirubin

Clinical relevance - Acute and chronic liver diseases may be accompanied by (i) porphyrin accumulation and the syndrome of porphyria, but more often (ii) bilirubin accumulation and icterus.

Metal metabolism

The liver stores iron which can be toxic in excessive amounts (hemochromatosis). The amount of iron in the body is largely determined by regulation of its absorption in the upper small intestine. Iron is stored intracellularly as ferritin in a number of tissues, with the liver having a large storage capacity. When the capacity of the liver is exceeded, iron accumulates as hemosiderin. The liver incorporates copper into specific copper proteins such as cytochrome c oxidase, mitochondrial monoamine oxidase, and ceruloplasmin. Mobilization of copper from hepatocytes takes place by two mechanisms - ceruloplasmin binding, and biliary secretion.

• Dietary Fe2+ → absorption → ferritin binding → transferrin transport → liver storage → biliary secretion

• Dietary Cu2+ → absorption → albumin binding → albumin transport → liver storage → biliary secretion

Clinical relevance - Cholestatic liver disease may be accompanied by secondary iron and copper retention which may then induce hepatocyte injury through apoptosis and oxygen free radical generation.

Vitamin metabolism

The liver has several major roles in vitamin metabolism. As well as producing bile for absorption of fat-soluble vitamins (A, D, E, K), it is an important site for vitamin storage. Water-soluble vitamins, except for vitamin B12 (cobalamin), are readily absorbed from the small intestine. These vitamins are used primarily as co-enzyme precursors for use in metabolic processes. Large amounts of all water-soluble vitamins, except vitamin C, are stored in the liver.

Clinical relevance - Cholestatic liver disease may be accompanied by steatorrhoea and fat-soluble vitamin malabsorption.

Glutathione metabolism

Glutathione (GSH) is synthesized in most, if not all, mammalian cells; the liver has relatively high levels of GSH. GSH performs a variety of physiologic and metabolic functions including thiol transfer reactions that protect cell membranes and protein and also promotes thiol-disulfide reactions involved in protein synthesis/degradation and catalysis. GSH provides reducing capacity for other reactions, and detoxifies hydrogen peroxide, organic peroxides, free radicals, and foreign compounds.

• Glutamate + cysteine + glycine → glutathione → methylation, sulfuration, aminopropylation reactions

Clinical relevance - Acute and chronic liver diseases may be accompanied by glutathione deficiency and increased apoptosis, oxygen free radical generation, and lipid peroxidation.

Xenobiotic metabolism

Numerous foreign compounds, including drugs, are so hydrophobic that they would remain in the body indefinitely were it not for hepatic biotransformation. The liver is an important site in drug toxicity and oxidative stress because of its proximity and relationship to the gastrointestinal tract. 75-80% of hepatic blood flow comes directly from the gastrointestinal tract and spleen via the main portal vein. Portal blood flow transports nutrients, bacteria and bacterial antigens, drugs, and xenobiotic agents absorbed from the gut to the liver in more concentrated forms. Drug metabolizing enzymes detoxify many xenobiotics but may activate the toxicity of others. The major mechanisms of hepatotoxicity include bile acid induced hepatocyte apoptosis, cytochrome P4502E1-dependent toxicity, peroxynitrite-induced hepatocyte toxicity, adhesion molecules and oxidant stress in inflammatory liver injury, microvesicular and non-alcoholic steatosis.

• Oxidation, reduction, hydrolysis, methylation, sulfuration, acetylation, glucuronidation → inactivation

Clinical relevance - Acute and chronic liver diseases may be accompanied by accumulation of xenobiotics as well as endogenous hormones (e.g. glucocorticoids).

Hormone metabolism

Natural and synthetic hormones including mineralocorticoids (aldosterone), glucocorticoids (cortisol, corticosterone), and sex steroids (androgens, estrogens, progesterone) are metabolized in the liver. Liver disease reduces the capacity for metabolic transformation.

Clinical relevance - Acute and chronic liver diseases may be accompanied by accumulation of endogenous hormones.

Immune surveillance

The reticulendothelial system of the liver removes microbes, endotoxins, enterotoxins, and exotoxins from the portal circulation. The liver regulates T-cell homeostasis, induces T-cell tolerance, and supports intrahepatic T-cell responses against hepatotropic pathogens.

Clinical relevance - Acute and chronic liver diseases may be accompanied by portal bacteraemia and predisposition to systemic infection.

Laboratory tests of liver disease

In animals with suspected liver disease, the minimum database should include: i) complete blood count - red blood cell, white blood cell, and platelet counts; ii) serum chemistries - electrolytes, urea nitrogen, creatinine, glucose, cholesterol, albumin, globulins, bilirubin, ALT, AST, and ALP or gamma-glutamyltranspeptidase (GGT) activities; iii) urinalysis; and iv) faecal flotation. Survey abdominal radiographs should be performed as part of the minimum database in any animal suspected as having liver disease. Additional laboratory tests and imaging studies may be considered following development of the minimum database.

(i) Complete blood count

Complete blood count will be useful in the assessment of severity and chronicity of anaemia, as well as in the characterization of inflammatory response and thrombocytopenia.figure 1: erythrocyte dysmorphias (leptocytes and schistocytes) in a young dog with portosystemic shunting Haematology usually reveals only non-specific changes in liver disease such as microcytic anaemia, or normocytic, normochromic non-regenerative anaemia. Erythrocyte dysmorphias, e.g. schistocytes and leptocytes, may be evident in the blood smears of more severely affected animals with liver failure and dyslipidaemia (Figure 1). Severe leukocytosis and neutrophilia may be observed in animals with bacterial, viral, or granulomatous hepatitis, hepatic necrosis, hepatic abscessation, and hepatic neoplasia.

(ii) Serum chemistry

Routine serum biochemical analysis helps identify metabolic causes of disease, including liver disease (increases in serum ALT, AST, ALP, GGT and bilirubin; decreases in serum glucose, albumin and cholesterol), renal disease (increases in BUN, serum creatinine and phosphorus), as well as endocrine disorders such as diabetes mellitus, hyperadrenocorticism and hypoadrenocorticism. An exaggerated BUN/creatinine ratio (often > 50:1) secondary to gastrointesitnal hemorrhage may accompany liver disease. Paraneoplastic changes (e.g. hypercalcaemia, hyperglobulinaemia) associated with hepatic or systemic neoplasia (e.g. lymphoma, extramedullary plasmacytoma) may also be identified by routine serum biochemical analysis.

(iii) Urinalysis

Urinalysis (Figure 2) will prove useful in the determination of hyposthenuria (e.g. urea depletion), haematuria (e.g. coagulopathy), and crystalluria (e.g. ammonium biurate – Figure 3). Urine protein-creatinine ratio determinations may be necessary to exclude protein-losing nephropathy as a cause of hypoalbuminaemia. It may be difficult to obtain a cystocentesis sample in an animal with concurrent ascites, in which case cystocentesis should be via guided ultrasound.

figure 2: urinalysis and the detection of crystalluria may assist the clinician in diagnosis and figure 3: ammonium biurate crystalluria in a young dog with portosystemic shunting

(iv) Faecal examination

Direct faecal smears and faecal flotations should always be part of the initial screening tests even in suspected cases of liver disease. While many parasites and microbes preferentially infect one part of the G.I. tract, others may induce pathology throughout the whole tract (e.g. Salmonella, Campylobacter, Pythium, Histoplasma), including the pancreas, liver, and biliary tract.

Additional diagnostic tests

Further tests may be necessary depending upon the outcome of the initial medical investigation. Liver function tests (e.g. PT and PTT, plasma NH4 +, and serum bile acids) should be performed in any animal in which there is suspicion, but not yet definitive proof, of liver disease. This is particularly true in cases of suspected hepatic cirrhosis in which there may be only mild elevation in serum liver enzyme (ALT, AST, ALP, GGT) activities.

Coagulation... In addition to coagulation factor synthesis, the liver is involved in the clearance of activated clotting factors and products of fibrinolysis. Therefore assessment of prothrombin (PT) and activated partial thromboplastin (APTT) times are assays of liver function, and should always be assessed prior to invasive procedures such as liver biopsy. The APTT and PT are both likely to be prolonged in severe, acute hepatic necrosis or parenchymal collapse, while only the APTT tends to be prolonged in dogs with congenital portosystemic shunts (3).

Plasma ammonia... Ammonia is primarily a byproduct of intestinal bacterial metabolism that is normally transported to the liver via the portal vein, and further metabolized to urea by hepatocytes in the Krebs-Henseleit cycle. With portosystemic shunting or severe liver disease, ammonia accumulates in the systemic circulation and subsequently the brain where it rapidly saturates transformation capacity giving rise to the syndrome of hepatoencephalopathy. Fasting plasma ammonia should be measured before oral tolerance testing; a markedly elevated level should obviate any further need for tolerance testing. If findings are equivocal, plasma concentrations can be measured before and after the administration of ammonium chloride (100 mg/kg) orally or per rectum. Blood is collected into ammonia-free heparinised tubes, placed on ice, and assays should be performed within 20 minutes of collection (4).

Serum bile acids... Total serum bile acid concentrations will be increased when pathology alters the enterohepatic recirculation. The uptake and resecretion of bile acids into bile is diminished in many forms of primary liver disease, from inflammation to infection, malignancy, and portosystemic shunting. The finding of a fasting and/or postprandial serum bile acid concentration greater than 15 μmol/L in cats and 25 μmol/L in dogs is supportive of a diagnosis of liver pathology or portosystemic shunt (5).

Heavy metal analysis... Normal hepatic copper concentrations in the dog are maintained at approximately 200-400 μg/g dry weight liver (6). Cholestatic liver diseases are often accompanied by heavy metal (Cu2+, Fe2+) retention. Copper retention in particular produces hepatocyte injury primarily through generation of oxygen free radicals, lipid peroxidation, and apoptosis. Tissues assays of copper content are readily available through many toxicology laboratories.

Imaging studies

The diagnosis of liver disease should never rely solely on laboratory testing (7). Survey abdominal radiographs should be obtained as part of the minimum database in all cases of suspected liver disease. figure 4: ultrasonography is an invaluable diagnostic aid when investigating liver diseaseNegative or equivocal radiographic findings should be further investigated with other imaging studies such as abdominal ultrasound. Ultrasonography (Figure 4) permits the assessment of size, shape, and density of parenchymatous organs, and is therefore a more useful tool in the further delineation of hepatic, renal, splenic, pancreatic, and mesenteric disease. Confirmation and definitive diagnosis of liver disease may require tissue biopsy by percutaneous or direct surgical sampling. Percutaneous ultrasound-guided liver biopsy may be difficult or challenging in some cases and laparoscopy or open surgical biopsy may offer a safer approach in these patients.

Conclusion

The complexity of liver function (Table 1) and the diversity of liver disease in dogs and cats are such that a veterinarian cannot place reliability on any one test or indeed one group of tests in reaching a definitive diagnosis. A conscientious clinician will take a holistic approach in assessing potential hepatic patients and beware the many pitfalls that can trap the unwary, and in particular the possibility that normal results do not necessarily rule out a liver problem.

 

This article was kindly provided by Royal Canin, makers of both wet and dry Hepatic diets for dogs and cats.  For the full range please visit www.RoyalCanin.co.uk or speak to your Veterinary Business Manager:

Royal Canin Hepatic

 

References

1. Lehninger AL, Nelson DL, Cox MM. Principles of biochemistry. 4th ed. New York: Worth Publishing, 2004.

2. Goldstein J, Brown M. From cholesterol homeostasis to new paradigms in membrane biology. Trends in Cell Biology 2003; 10: 534-539.

3. Kummeling A, Teske E, Rothuizen J, et al. Coagulation profiles in dogs with congenital portosystemic shunts before and after surgical attenuation. J Vet Intern Med 2006; 20: 1319-1326.

4. Rothuizen J, Van den Ingh TS. Rectal ammonia tolerance test in the evaluation of portal circulation in dogs with liver disease. Res Vet Sci 1982; 33: 22-25.

5. Center SA, Joseph SA. Measurement of serum bile acids concentrations for diagnosis of hepatobiliary disease in cats. J Am Vet Med Assoc 1995; 207: 1048-1054.

6. Schultheiss PC, Bedwell CL, Hamar DW, et al. Canine liver iron, copper, and zinc concentrations and association with histologic lesions. J Vet Diagn Invest 2002; 14: 396.

7. WSAVA Liver Standardization Group. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Diseases. Philadelphia: Saunders Elsevier, 2006.

This article was first published in 2011.

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