Diagnostics Enzymology of Domestic Animals

INTRODUCTION

• Enzyme- Biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis

• Enzymes are found in all tissues and fluids of the body- speed up reaction rates that would be otherwise to slow to support life

• Enzymes activity in serum, plasma, urine or other body fluid can be measured for diagnostics purposes and screen for abnormal organ function

Measuring enzyme activity

• Enzyme activity = measurement of the quantity of active enzyme present

• Perform using automated technique – part of the standard blood test

Enzyme-substrate binding

Enzyme-substrate binding

Enzyme inhibition

• An inhibitor – substances that interferes with the action of an enzyme and slows the rate of reaction

• Can effect enzyme in two ways:

1. reversible- can bind to the enz and subsequently be released – leave the enzyme in its original form

2. irreversible- inactivate the enzyme and the original form cannot be restored

Reversible inhibitors1. Competitive inhibitor• Consist of compound that

structurally very similar to the substrate – can bind to the active site and block substrate from binding

2. Noncompetitive inhibitor• Any inhibitor that binds to the enzyme at

a site other than active site and causes change in structure of the enzyme

• Substrate is still able to bind to the active site, but the enzyme cannot catalyzes the reaction when the inhibitor is bound to it

Enzyme inhibitions in the treatment of AIDS• Development of specific inhibitors

that block the actions of enzymes unique to the HIV

• Key enzymes- reverse transcriptase, integrase and protease

• Protease- essential to the production of new virus particles in infected cells

• Integrase- needed for the virus to copy itself in the host cell

• Reverse transcriptase – needed to convert RNA to DNA inside host genomic

Factors affecting serum enzyme activity1. Organ Mass And Enzyme Tissue

Concentration

• Organs with a high concentration of enzyme have the potential to cause a greater increase in serum activity with disease

• Eg. Intracellular and extracellular concentration gradient of hepatocellular aminotransferase (ALT) is 100, 000: 1

• Therefore, injury to hepatocytes, has the potential of causing markedly increased serum ALT activity

• The higher the concentration gradient of the enzyme, the faster the translocation of significant quantities of the enzyme to the interstitial space

2. Cell location• The location of cellular enzyme relative to blood,

urine or other fluids will determine which fluid it will be found

• Eg. Renal tubular gamma glutamyl transferase (GGT) located on the luminal surface of renal tubular epithelial cells

• Injury to these cells results in release of enz into urine other than blood

Factors affecting serum enzyme activity

Mechanisms release of cytoplasmic enzyme from cells to blood

• Cytoplasmic enzymes are contained within cell membranes

• Healthy plasma are impermeable to macromolecules such as enzyme

• Alterations in the cell membrane is necessary to allow cytoplasmic enzymes to gain access to the blood

• During cell necrosis (death) – perforations and tears of the cell membrane allow the release of cytosolic contents- released the enzymes to the blood

Alternative mechanism (cell survive)

• formation of membrane blebs

• Due to the depletion of the energy in the form of ATP, followed by influx of calcium into the cell, disruption of the cytoskeletal proteins and alteration in lipid membrane content

• Combination of altered cytoskeletal protein, lipid membrane content, and osmotic swelling of the cell lead to the formation of cell bleb, release of these blebs, and resealing of the cell membrane.

• these blebs are ruptured or released as vesicles into the blood where they are broken down, releasing their contents, including cytoplasmic enzymes

• Found in ischemia, shock, viral infections, toxemia, cholestasis

Mechanisms release of cytoplasmic enzyme from cells to blood

Blood clearance rates of enzymes

• The amount of enzyme activity in blood is very dependent on the rate of clearance of the enzyme from the blood following its release from cells

• Can range from minutes to hours to days• They can be filtered through the glomerulus,

degraded or activity may be lost while the protein continues circulating

Enzyme induction

• Changes to serum activity may in some cases reflect changes in enzyme production by the cells, rather than cell injury

• Can be due to the hormonal changes, or can be drug induced

Sources of plasma enzymesa) Plasma derived enzymes: • Act on substrates in plasma and their activity is

higher in plasma than in cells. Eg. Coagulation enzymes

b) Cell-derived enzymes: have a high activity in cells and overflow into the plasma. Further subdivided into:

• Secretory- mainly derived from digestive glands and function in the extracellular space

• Metabolic- fx in the cells and mainly derived from the soluble and microsomal fractions of the cells

The cell-derived enzymes enter the plasma in small amounts as a results of:•Continuous normal ageing of the cells•Owing to diffusion through undamaged cell membranes

Sources of plasma enzymes

Increased release of enzyme

1. Necrosis of cells – due to damage to cells of the tissue. The resultant pattern will depend onnormal enzyme content of the tissue/organon the extent and type of necrosis

2. Increased permeability of cell membrane without necrosis of cellseg. In early stage of viral hepatitis

3. Increased production of the enzyme within cell – may be seen in treatment of patients with protein anabolic drugs- results in increased synthesis of liver cell transaminases

3. An increase in tissue source of enzymes– Increased rate of production in cells – Increase in the number of cells and cell mass -

malignancies

Increased release of enzyme

1) Alanine aminotransferase (ALT)

• Catalyzes the reversible transamination of L-alanine and 2-oxoglutarate to pyruvate and L-glutamate

• Play role in aa catabolism and inter organ nitrogen transport

• Increased serum ALT activity indicates hepatic injury in dogs and cats – indicator for hepatocellular necrosis

• In dogs- can also be found in heart and muscle

• Peak activity between 45 to 60hrs in dogs

2) Aspartate aminotransferase (AST)

• Catalyzes transamination of L-aspartate and 2-oxoglutarate to OAA and glutamate

• Increased serum AST is observed in hepatocellular injury and muscle injury

• Cannot differentiate between hepatocellular or myocyte injury – so further testing is required – such as sorbitol dehydrogenase or creatine kinase

3) Sorbitol dehydrogenase (SDH)• Catalyzes the following reaction

Sorbitol + NAD+ ↔ fructose + NADH

• The highest concentration found in liver followed by kidney

• Considered liver specific in all species

• Half life is short – 3-4hrs for cats and 5hrs for dogs – limit the usefulness of the enzyme- thus less favored for detection in hepatic disease in dogs compared to ALT test

However, there are cases where SDH analysis is useful:

• dogs with traumatic muscle injury, when there is increased serum ALT and CK activity – determination of SDH activity will rule out whether there is concurrent hepatic injury

• determination whether there is persistent hepatocellular injury.

If ALT is markedly increased, and SDH is not- recovery process may occur

If both are markedly elevated – ongoing liver injury

This sort of interpretation is highly subjective and would require repeated monitoring to be of value

3) Sorbitol dehydrogenase (SDH)

4) Glutamate dehydrogenase (GDH)

• Catalyzes the removal of hydrogen from L-glutamate to form 2-osoglutarate

• The liver has the highest concentration of GDH• In all species, the GDH activity are considered

liver specific• The half life is 14hr in cows – better than SDH• The determination of GDH activity is best done

with other hepatic enzymes

5) Gamma Glutamyltransferase (GGT)

• Catalyzes the transfer of gamma glutamyl groups from gamma glutamyl peptides to other peptides, aa and water

• regulation of intracellular glutathione by hydrolysis of the tripeptide glutathione outside the cell into three components which can be readily be taken up by the cells and be available for glutathione synthesis as needed within the cell

• Highest concentration in kidney for dogs, cattle, goats – useful in detecting tubular injury and nephrotoxicity in dogs

GGT

• Useful clinical indicator of cholestasis in horses and cattle – due to high liver GGT activity compared to dogs and cats

• Can be used in large animals as a screening test for generalized hepatic disease

5) Gamma Glutamyltransferase (GGT)

6) Alkaline phosphatase (ALP)• There are 2 isozymes and several isoforms (posttranslational modification

of isozymes) of ALP

• Major:

1. Liver ALP (L-Alp) – Hepatocytes, epithelium of biliary tract – indicator for hepatobiliary disease

2. Corticosteroid (C-ALP)- in dogs induced by the glucocorticoid treatment

3. Bone ALP (B-ALP) – by osteoblasts – increase due to bone disorders or as a marker in bone formation in young animals

4. Intestinal ALP (I-ALP) – intestinal, renal, mammary, placental tissues – can be due to the tumors in mammary glands

7) Lipase

• Hydrolyzes triglycerides at 1 and 3 positions to produce monoglyceride

• Found in pancreas• Half life 1-3 hours• Used for diagnosis of acute pancreatitis in

dogs

8) Amylase

• Cleaves alpha-D-(1-4) glycan linkage of starch and glycogen

• High concentration in pancreas of dogs and cats

• Can be found in intestinal and liver as well• Half life can range from 1-5 hr• Routinely used as screening test for acute

pancreatitis

9) Trypsin and Trypsinogen• Trypsin- a serine proteinase enzyme produced by

the pancrease in the form of proenzyme trypsinogen

• The pancrease secretes trypsinogen into the intestines – converted to trypsin

• Specific immunoassay to detect trypsin- trypsin like immunoreactivity (TLI)-

• Useful to detect canine exocrine pancreatic insufficiency (EPI)

Isozymes

• Distinct form of the same enzyme but catalyze the same chemical reactions and differ from each other structurally, electrophoretically and immunologically

10) Lactate Dehydrogenase

• Catalyzes the reversible oxidation of lactate to pyruvates

• There are 5 distinct LDH isozymes – LDH-1, LDH-2, LDH-3, LDH-4 and LDH-5

• Each isozyme protein is made up of four polypeptide su- tetramer – Termed H and M

• Different isozymes contain different H and M proportions

LDH Isozymes

Type Polypeptide chains

Tissue rich in isozymes type

LDH-1 H4 – HHHH myocardiumLDH-2 H3M – HHHMLDH-3 H2M2 – HHMMLDH-4 HM3 – HMMMLDH-5 M4 - MMMM liver

LDH isozymes

• After damage to either these tissues, myocardium or liver, total serum LDH is increased and it maybe useful to know the origin of the enzyme

• In normal serum, LDH-2 is the most prominent isoenzyme

• After myocardiac infarction, LDH-1 and LDH-2 predominate

• In acute viral hepatitis – LDH-4 and LDH-5 predominate

LDH Isozymes in malignancy

• An increase in LDH-5 seen in breast carcinoma, malignancies of CNS, and prostatic carcinoma

• In leukemias, rise is more in LDH-2 and LDH-3• Malignant tumor of testes and ovary show rise

of LDH-2, LDH-3 and LDH-4

Creatine-P + ADP → Creatine + ATP

• CK catalyzes the exchange of a phosphate moiety between creatine phosphate and ATP

• found in high concentration in skeletal muscle followed by myocardium and brain but not found at all in liver and kidney

• 2 distinct s/u – Muscle (M) and brain (B) – Can form MM, MB, BB

• Serum value is found to increase after 6hrs, peak level 24-30hrs and returns to normal level in 2-4days

• In domestic species, CK is mainly used as marker of skeletal muscle injury – trauma, nutritional myopathies, exercise-induced muscle injury or congenital myopathies

11) Creatine kinase

Isozymes of Creatine Kinase

Type Polypeptide chain

Tissues found

CK-1 BB Brain

CK-2 MB

CK-3 MM MyocardiumSkeletal muscle