Serum Enzymes

• Serum enzymes are assessed in medicine
• The list of topics to be covered includes the origins of enzymes in plasma, enzyme selection as a marker, enzymatic activity measurement, isoenzymes, common enzymes of clinical interest, muscular disease biomarkers, and a conclusion.

Introduction

• Enzymes are biological catalysts which accelerate reactions without being altered themselves.
• Several factors influence chemical reactions, including temperature, pH, activators, and inhibitors
• Enzymatic activity is expressed in International Units (IU) or units per liter (U/L).
• Enzymes are normally present in plasma in low quantities, at well-defined rates.
• Tissue damage causes enzymes specific to that tissue to be released into the general circulation.
• Plasma enzyme determination in clinical biochemistry is useful for early illness diagnosis, tissue localization, disease progression, and detecting/confirming metabolic deficiencies.

Origins of Plasma Enzymes

• Plasma enzymes are either specifically plasma enzymes, or non-specifically
• Specifically Plasma Enzymes: have a function in the plasma such as ceruloplasmin, lipoprotein lipase, coagulation enzymes and fibrinolysis.
• Non-Specifically Plasma Enzymes: They have no plasma function, and are normally present at low levels
• The non-specifically plasma enzyme group is subdivided into excretion enzymes such as pancreatic lipase and amylase, and prostatic acid phosphatase.
• Cellular Enzymes: normally function within cells of variable tissue origin such as hepatic, cardiac, or muscular
• Cellular enzyme release is related to cellular turnover.
• Plasma enzyme concentration is dependent on cellular release, and enzymatic protein catabolism, the later of which varies across enzymes.
• Increased enzymatic activity can result from cellular damage due to altered membrane permeability (cytolysis) or necrosis.
• Enzyme activity can be increased by proliferation and activity of cells that synthesize it.
• Specific enzyme activities can be produced by tumor cells and released into circulation, which results in tumor pathologies

Enzyme Localization within a Cell

• Plasma membrane: PAL, 5'NU, Y GT
• Cytoplasm: ALAT, LDH, ASAT, CK
• Mitochondria: ASAT, CK
• Lysosomes: PAC
• Microsomes (endoplasmic reticulum): YGT

Enzyme Selection as Marker

• Each tissue has its own enzymatic machinery.
• Ideally, each organ would have a unique enzyme.
• The same enzyme is found in multiple tissues.
• Enzymatic profile determines organ function so iso-enzyme levels determine lesion.

Enzymatic activity measurement

• Enzyme activity is determined by measuring the reaction it catalyzes, either by measuring product formed, or substrate consumed in a specific time: a minute.
• Enzymatic methods in UV use a nicotinic co-enzyme in a direct reaction, like with LDH and ASAT using NAD+ and NADH.H, or with CK using NADP, NADPH, H.
• NADH absorbs strongly at 340 nm
• NAD+ doesnt absorb at 340 nm

Isoenzymes

• Isoenzymes are different forms of the same enzyme. They catalyze the same reaction, but differ in physical-chemical properties such as electrophoretic mobility, thermal stability, and effector sensitivity, due to differences in their amino acid sequence and spatial configurations
• Isoenzymes can be produced by a single gene or multiple genes.
• Various isoenzymes are organ or tissue-specific.
• Techniques for measuring isoenzymes include separation via electrophoresis, chromatography, and thermostability.

Overview of Principal Enzymes of Clinical Interest

• Alkaline phosphatase
• Gamma glutamyl transferase
• 5'nucleotidase
• Aminotransferases
• Creatine kinase
• Lactate dehydrogenase
• Amylase
• Pancreatic lipase
• Aldolase

Alkaline Phosphatase

• Enzymes hydrolyze phosphate esters and release phosphoric acid.
• Optimal activity occurs between pH 7.5 and 9.6.
• It originates in the bones, intestine, kidney, liver, and leukocytes.
• Normal values: 30 to 125 U/I for adults, 110 to 400 U/I for children.
• Higher levels in children indicate faster osteogenesis.

Isoenzymes of PAL

• Bone fraction: 50 to 70%
• Hepatic fraction: 30 to 50%.
• Intestinal fraction: 0 to 20%.
• Placental-origin isoenzyme.

Alkaline Phosphatase related Pathology

• Increased alkaline phosphatases indicate cholestasis.

Extrahepatic cholestasis

• Including gallstones and biliary tumors
• Pancreatic cancers
• PAL↑2 N + BIL↑,γGT et 5' nucleotidase↑

Intrahepatic cholestasis

• Including cholestatic hepatitis, primary biliary cirrhosis, hepatocellular damage, steatosis, hepatoma, and hepatic metastases.
• Normal PAL levels with jaundice indicate cirrhosis, hepatitis, or hemolysis.
• Elevated PAL levels with jaundice indicate retention icterus, primary liver cancers, or bile duct stones (cholestasis).

Alkaline Phosphatase related Affections of the bone

• Paget's disease: PAL↑ 20 to 40 N.
• Hyperparathyroidism.
• Osteoporosis: PAL N or slightly ↑.
• Rickets.
• Bone tumors, bone metastases, myelomas.

Gamma Glutamyl Transferase (GGT)

• A heterodimeric glycopeptide
• Cleaves the glutamyl bond of a donor (peptide or glutathione)
• Transfers the glutamyl radical to an acceptor.
• It is a membrane enzyme
• Present in high concentrations in the hepatobiliary system, pancreas, kidneys, intestine, and prostate.
• Normal levels: Less than 61 UI/I for men and less than 36 UI/I for women.

Gamma Glutamyl Transferase Physiological Variations

• Levels increase with age; obesity; hypertriglyceridemia; and alcohol and tobacco use.
• Levels decrease during pregnancy.

Gamma Glutamyl Transferase Pathological Variations

• Cholestasis: Gamma-GT increases 5 to 30 times the normal level, preceding other enzymes like PAL and lasting longer.
• Hepatic steatosis: Gamma-GT increases 2 to 5 times normal level.
• Alcoholic cirrhosis: Isolated increase in Gamma-GT induced by alcohol with return to normal after withdrawal.
• Hepatocellular cytolysis: Moderate increase in Gamma-GT in acute and chronic viral hepatitis.
• Drug-induced: Increase in Gamma-GT due to anticoagulants, antiepileptics, neuroleptics, and oral contraceptives.
• Pancreatic and hepatic conditions: GGT increases in acute pancreatitis and pancreatic cancer.

5'-Nucleotidase

• 5'-ribonucleotide phosphohydrolase functions at alkaline pH.
• A membrane enzyme
• Found in many tissues
• Especially found in the membranes of hepatocytes and epithelial cells of bile canaliculi.
• The 5'N increases in intra- and extrahepatic cholestasis +++.

Transaminases (ASAT and ALAT)

• Enzymes catalyze reversible transfer of an amino group from an amino acid to an alpha-keto acid, in the presence of a coenzyme such as pyridoxal phosphate (PLP) which is derived from vitamin B6.

ASAT

• Aspartate aminotransferase or glutamate oxaloacetate transaminase which catalyzes a reaction such as this one: Ac Aspartique+AC a cétoglutarique — Ac oxaloacétique + Ac glutamique
• Located in the cytosol, and mitochondria it is essentially present in the heart, liver, muscles, kidney, pancreas, spleen, lungs, red blood cells, and brain, and it's half life is 17 hours

ALAT

• Alanine aminotransferase or glutamate pyruvate transaminase which catalyzes this reaction: Alanine + AC a cétoglutarique Acglutamique + Ac pyruvique
• It is a cytosolic enzyme present (100%)
• Mainly in the liver also found in muscles, kidneys, pancreas, spleen, lungs, and red blood cells, and it's half life is 47 hours
• Aminotransferase levels are measured using a kinetic technique which measures the catalytic activity of aminotransferases through a coupled reaction with dehydrogenases.
• Normal values for ASAT: 10-40 UI/L
• Normal values for ALAT: 5-55 UI/L

Pathological Variations

• Increased ALAT indicates the origin is almost always hepatic.
• Increased ASAT indicates hepatic, cardiac (myocardial infarction/pericarditis, cardiac surgery and coronarography), and muscular (prolonged muscular exertion, dermatomyositis, rhabdomyolysis, muscular dystrophy)
• Results should be interpreted considering the simultaneous elevation of AST and ALT activities, their changes over time, and the ASAT/ALAT ratio.

Conditions related to Transaminases

• Cardiac affections: myocardial infarction, and ALAT levels do not increase or increase only moderately, ASAT is proportional to the severity of myocardial damage starting at the 6th hour, peaking at the 36th and normalizing in 4–7 days if it is an increase of 3 to 9N
• Muscular affections: Les rhabdomyolyses Characterized by myoglobin release from destroyed cells, enabling muscular respiration, found in urine (myoglobinuria), with increased of specific enzymes such as CPK and ASAT

Hepatic Conditions

• Increased Cytolysis in hepatic transaminases are translated in higher ALAT levels than AST levels.
• Hepatocellular Necrosis is characterized in a severe case by the release of mitochondrial AST (increasing the AST/ALAT ratio).
• Acute Viral Hepatitis: AST/ALT < 1, and transaminase increase is the only biological sign of anicteric hepatitis, used to monitor evolution of the disease and for detecting eventual relapse. ASAT moderately increases, ALAT increases 15-150N: Normalisation occurs between the 3rd and 6th week.
• Chronic Hepatitis: Caused by Virale: HVB,HVC, Medically/Toxic/Immune Factors, ALAT> 2 to 5 N, ALAT>ASAT, and ASAT/ALAT~1.
• Cirrhosis: Characterized by modest increase in aminotransferases, ASAT>ALAT, ASAT/ALAT> 1.
• Acute Cholestatic HV: increased aminotransferases, reaching plateau, without normalization at 3rd week, alongside with cholestasis signs.
• Alcoholic Hepatitis is the same as toxic hepatitis: increased aminotransferaselevels with ASAT/ALAT>2.

Transaminases in Practice

• High transaminase levels ( more that 10 times the normal levels), it means hepatic cytolysis..
• Transaminase muscular origin should be checked and confirmed if the ASAT/ALAT is > 1.
• Moderate increase in transaminases mean a should elevate suspicion of muscular origin and dosing the CK.
• Other non related origins: obesity, diabetes, hemochromatosis, alpha 1 antitrypsin deficiency, Wilson's disease, pulmonary embolism.
• Decreasing activity of the aminotransferases happen in terminal renal failure and vitamin B6 deficiency.

Enzymes for the biological investigation of the liver

• Includes TGO-TGP, PAL, 5'nucleotidase.GGT, and LDH.
• Differently increase depending on cytolysis or cholestasis syndrome

Creatine Kinase

• A muscular enzyme that functions to create energy and muscular contraction.
• The protein is a dimer of subunits M (muscle) and B (brain).
• Combinations of subunits form three isoenzymes.
• These have different tissue distributions and electrophoretic mobility.
• CKBB (CK1) in the brain, prostate, digestive tract, bladder, uterus, thyroid and placenta.
• CKMB (CK2) in the myocardium.
• CKMM (CK3) is in skeletal musculature and myocardium.
• Normal levels:40-300 UI/ L in men, and 25-200 UI/L in women.
• Levels depend on, muscle mass, age, sex, and physical activity, and is increased by intramuscular injections.

Creatine Kinase related Pathology

• Myocardial Infarction causes a 10 to 20 fold increase in CKMB. the level of which correlates with the size of the tissue involved, and parallel increase in the total CPK, which starts at around the 6th hour after the even peaking between 22h to 26h then is back to normal at around 72h
• Myopathies such as Duchenne muscular dystrophy causes ↑ CPK 10 to 50 times the normal with↑ CKMM. and ↑ CKMM+++, ↑ CKMB+ indicating Rhabdomyolysis
• Other potential causes can include central nervous system lesions or surgery, cerebral vascular events or infarct, neoplasmic pathologies and cardiac surgery and hypotyroidism.

Lactate Dehydrogenases

• The enzyme operates in glucose catabolism: Pyruvate + NADH,H+ ---> LACTATE + NAD+
• It is present in the cytoplasm.
• it is made of M subunits or A form, and H form and B form, so it forms these Isoenzymes" in order of tissues (H4) (H3M) (H2M2) (HM3) (M4)
• LDH1=H4 found in the heart, erythrocytes and kidneys
• LDH2 = H3M
• LDH3 = H2M2 found in Leukocytes, platelets, lymphoid tissues
• LDH4 = HM3 found in Skeletal muscle tissue, and liver
• LDH5= M4
• Normal values in adults < 245 UI/L
• After electrophoretic separation of isoenzymes, the percentages are:
• LDH1: 14-26%
• LDH2: 29-39%
• LDH3: 20-26%
• LDH4: 8-16%
• LDH5: 6-16%
• Pathological increased LDH observed in such conditions as myocardial infarction, liver conditions and hematological conditions, and certain infections.
• LDH testing is used to monitor the effects of certain therapies.

Alpha-Amylase

• Amylase degrades starch from diet.
• There are 2 amylases: amylase S from salvia, and amylase P from pancréa.
• Normal amylase:
  • Amylasemia is 31- 107 UI/L
  • Amylasuria <440UI/ 24 hours.
• Pathology when there is an affection happening to:
  • Pancreas in particular when there is cancer, pancreatis or stones in the pancréa
  • Salvia in particular when there is stones parotidite.
  • Perforation of the viscus or the viscure which is a symptom of abdominal pain.

Lipase

• Digestive enzymes primarily secreted by the pancreas to hydrolyze dietary triglycerides.
• Colipase from the pancreas is required for maximum activity.
• The normals is: 13-60UI/L.
• Related pathological affections:
  • Pancréatite when there is parallel Lipase release.
  • Cancer
  • Chronic alcohol and kidney failure.

Aldolase

• It is involved in the glycolytic metabolis
• In all cells where fructose biphosphate is scinded into glycraldhyde.
• There are 3isoenzymes
  • A is on erythrocytes.
  • B: from the liver
  • C in the brain

Aldolase levels

• Normal is (2-7,6 UI/L)
• Increases are seen in muscles affection, and liver damage
• In decrease it indicate hereditary fructose
• At the current time there is no value to dosing aldolase.

Muscle Biomarkers

• Muscle Enzymes: released by muscle in significant muscle injuries include (CPK, LDH, Aldolase, ALAT).
• Other markers include myoglobin, markers for auto-antigens and genetic conditions
• Despite a lack of specificity, plasma enzymatic activity measurement can be used to detect tissue damage and has utility in the ongoing management of conditions such as hepatitis or Paget disease.