Serum Enzymes in Medicine

• Serum enzymes are studied in third-year medicine, academic year 2024-2025.
• Dr. R. Bougahgouh, a medical biochemistry assistant professor, presents the topic.

Plan Overview

• The presentation will cover the introduction to serum enzymes.
• Addresses the enzyme origins in plasma.
• Discusses enzyme selection as markers.
• Explains enzymatic activity measurement.
• Will define isoenzymes.
• Will detail the main enzymes of clinical interest.
• Reviews biomarkers of the muscle diseases.
• Includes the conclusion.

Introduction

• Enzymes are biological catalysts that accelerate reactions without being altered themselves during the process.
• Chemical reaction speed is influenced by several factors: temperature, pH, activators, and inhibitors.
• Enzymatic activity is expressed in International Units (IU) or units per liter (U/I).
• These enzymes are normally in plasma in low concentration at specific rates. When an organ or tissue is injured, it releases its own enzymes into the general circulation.
• Plasmatic enzymes determination in clinical biochemistry has multiple benefits.
• Enzymes in plasma can indicate disease before clinical symptoms appear.
• Enzymes in plasma can pinpoint the affected organ or tissue.
• Some enzymes serve as good markers for disease progression.
• Enzyme detection and confirmation in metabolic inherited diseases is possible.

Origins of Plasma Enzymes

• Enzymes present in the plasma originate either specifically or non-specifically from the plasma.
• Specifically plasma enzymes have a function in the plasma of ceruloplasmin, lipoprotein lipase, coagulation enzymes, and fibrinolysis enzymes.
• Non-specifically plasma enzymes lack an evident plasma function, are usually present at low levels, are divided into excretion enzymes, pancreatic lipase and amylase, and prostatic acid phosphatase enzymes.
• Enzymes normally exert their functions in the cells originating from tissues such as liver, heart, or muscles.
• Enzymes are released into the plasma of a healthy person on a cellular renewal basis and, its concentration depends on the equilibrium between cellular release and enzymatic protein catabolism.
• Plasma half-life is variable between enzymes.
• Increased enzymatic activity can be caused by cellular damage resulting from altered membrane permeability (cytolysis) or necrosis.
• Proliferation and activity increase in cells synthesizing enzymes.
• Tumor pathologies can cause certain enzymatic activities to be elaborated by tumor cells and released into circulation.
• Enzymes occur at different levels within the cell, for instance the plasma membranes contains PAL, 5'NU and γGT, whereas the lysosomes contain PAC.

Enzyme Selection as a Marker

• Each tissue features a set of enzymatic equipment tailored to its functions.
• Ideally, each organ would have a unique enzyme.
• The same enzyme can be found in various tissues, requiring a enzymatic profile using multiple isoenzymes to identify the origin of the lesion.

Measurement of Enzymatic Activity

• In measuring enzymatic activity, the reaction catalyzed by the enzyme is assessed by determining either the quantity of the product formed or the quantity of substrate consumed over a specified time.
• UV enzymatic methods using a nicotinic coenzyme can be employed in a direct reaction [LDH, ASAT (couple NAD+,NADH.H)] or the couple NADP, NADPH, H for (CK).
• NADH strongly absorbs at 340 nm, while NAD+ does not.

Isoenzymes

• Isoenzymes represent different forms of the same enzyme catalyzing the same reaction but differ in their physical and chemical properties due to variations in their amino acid sequences and spatial configurations.
• Differences in isoenzymes result in electrophoretic mobility, thermal stability, and sensitivity to certain effectors.
• Isoenzymes can be produced by a single or multiple genes.
• Various isoenzymes can be specific to an organ or tissue.
• Methods for measuring isoenzymes include separation techniques such as electrophoresis, chromatography, and thermostability.

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, releasing phosphoric acid, with optimal activity between pH 7.5 and 9.6.
• Organs of origin are bones, intestine, kidney, liver, and leukocytes.
• Usual values for adults are 30-125 U/I, and for children, 110-400 U/I, higher in children due to rapid osteogenesis with isoenzymes of osseous fraction (50-70%).
• Isoenzymes include a osseous fraction (50-70%), hepatic fraction (30-50%), intestinal fraction (0-20%), and placental origin isoenzyme.
• Pathologies include liver conditions: increased alkaline phosphatases indicate cholestasis.
• Extrahepatic cholestasis is associated with biliary lithiasis and tumors, pancreatic cancers, and significant increases in PAL, bilirubin, γGT, and 5' nucleotidase.
• Intrahepatic cholestasis is associates with cholestatic hepatitis, primary biliary cirrhosis, hepatocellular damage, steatosis, hepatoma and hepatic metastases
• Normal PAL levels indicate cirrhosis, hepatitis, and hemolysis.
• Elevated PAL levels on the other hand denote cholestasis due to primitive cancers of the liver or bile duct stones.
• Disease of the bone is affiliated with elevated levels of PAL and hyperparathyroidism, osteoporosis, rickets, bone tumors, bone metastases, and myelomas.

Gamma Glutamyl Transferase (GGT)

• Gamma-glutamyl transferase is a heterodimeric glycopeptide cleaving the glutamyl bond of a donor (peptide or glutathione) and transferring the glutamyl radical to an acceptor.
• The membrane-bound enzyme is abundant in the hepatobiliary system, pancreas, kidneys, intestine, and prostate.
• The normal values are less than 61 UI/I in men and less than 36 UI/I in women.
• Increased levels are associated with aging, obesity, hypertriglyceridemia, excessive alcohol consumption, and smoking.
• Levels are decreased during pregnancy.
• Pathological include elevated gamma-GT levels, preceding other enzymes such as alkaline phosphatase, can indicate cholestasis (intra- or extra-hepatic).
• Five to thirty times the normal levels can be seen.
• Hepatic steatosis leads to 2-5 times the normal levels, while alcohol-induced cirrhosis elevates levels, returning to normal after cessation.
• Viral hepatitis (acute or chronic) leads to moderate elevation.
• Intoxications, anticoagulants, antiepileptics, neuroleptics, and oral contraceptives will cause elevated levels as well.
• Pancreatic and hepatic conditions like acute pancreatitis and pancreatic cancer cause elevated levels.

5'nucleotidase

• Functions as a 5'-ribonucleotide phosphohydrolase at an alkaline pH.
• This membrane enzyme is mainly in hepatocyte membranes and biliary canaliculus epithelial cells.
• It increases more in intra- and extra-hepatic cholestasis (+++).

Transaminases (ASAT and ALAT)

• Enzymes catalyze the reversible transfer of an amino group (NH2) from an amino acid to an alpha-keto acid, with pyridoxal phosphate (PLP), a vitamin B6 derivative, as a coenzyme.
• Two frequently measured transaminases: ASAT (Aspartate AminoTransferase/TGO) and ALAT (Alanine AminoTransferase/TGP).
• Aspartate aminotransferase catalyses reactions involving aspartic acid and alpha-ketoglutaric acid.
• Alanine aminotransferase catalyses reactions involving alanine and alpha-ketoglutaric acid.
• Aspartate aminotransferase is found in the heart, liver, muscles, kidney, pancreas, spleen, lungs, red blood cells and brain and has a half life of 17 hours.
• Alanine aminotransferase has a half life of 47 hours, presence predominantly in the liver mostly.
• Aminotransferase dosage is performed using a kinetic technique that measures the catalytic activity of aminotransferases through a coupled reaction with dehydrogenases.
• Normal values are ASAT: 10-40 UI/L and ALAT: 5-55 UI/L.
• Increased ALAT levels suggest hepatic origin.
• Increased ASAT levels may relate to hepatic, cardiac (myocardial infarction, acute pericarditis, cardiac surgery, coronarography), or muscular issues (prolonged muscular effort, dermatomyositis, rhabdomyolysis, muscular dystrophy).
• Results interpretation considers concurrent AST and ALT activity elevation with their time variations and ASAT/ALAT ratio value.
• In myocardial infarction, ALAT levels do not increase or increase slightly whereas increased ASAT levels at 3-9 times is proportional to lesion with starts at 6hrs up to 36 hrs then returns to normal after 4–7 days.
• Rhabdomyolysis is marked by myoglobin release and elevated CK/ASAT concentrations which show up in urine.
• Hepatic cytolysis increases transaminases, with ALAT elevation > AST which is highly specific to the liver.
• Severe cases release mitochondrial AST, increasing the ASAT/ALAT ratio.
• Acute viral hepatitis are marked by ASAT/ALAT<1 and increased transaminase levels as sole biological sign of anicteric hepatitis.
• Chronic Hepetitis shows ALAT> 2 to 5 N, ALAT>ASAT, ASAT/ALAT~1 and normalizes within 3-6 weeks.
• Chronic conditions shows causes from viral, medicamentous, toxic or autoimmune hepatitis along with ASAT modulation.
• In cirrhosis, the aminotransferases show modest increases, with ASAT>ALAT and ASAT/ALAT > 1.
• In acute cholestatic HV, aminotransferase levels increase but do not normalize after the third week, accompanied by cholestasis signs.
• Elevated aminotransferases with ASAT/ALAT>2 indicates alcoholic or toxic hepatitis.
• Significantly elevated transaminases (greater than 10 times the upper limit) suggests hepatic cytolysis.
• Elevated but moderate transaminases may indicate muscular origin (check CK levels).
• Other causes, the patient may have obesity, diabetes, hemochromatosis, alpha 1 antitrypsin deficiency, Wilson's disease, or pulmonary embolism.
• Diminished activity often suggests chronic renal failure and a vitamin B6 deficiency.
• Liver enzymes increase differently depending on either a cytolysis or cholestasis syndrome.

Creatine Kinase

• Creatine kinase (CK) is a muscle enzyme which produces energy.
• It transforms creatine phosphate + ADP into Creatine + ATP.
• It is a contraction for muscles.
• It is a dimer of subunits M and B, creating isoenzymes with differences tissue distributions, analytic plan, and electrophoretic mobility.
• Isoenzymes have different tissue distributors; CKBB (CK1) = brain, prostate, digestive tract, bladder, uterus, thyroid and placenta, CKMB (CK2) = myocardium, and CKMM(CK3) = Skeltal muscle and myocardium.
• Values for males range 40-300 UI/L, and females 25-200 UI/L.
• Levels will have variations if they affect mass or injected intramuscularly.
• If the patient has myocardial infarctions (IDM), the augmentation of the CKMB has a 10x to 20x normal value and will have masses correlative to tissue or CPK tissue.
• For myopathies such as Duchenne, CPK may show levels 10x to 50 x’s with and affect on CKM and Rhabdomyolyse, and be even more present in CKMM and CKMB.
• Systems such as cardiac, nervous and tissues may be affected as well.

Lactate Dehydrogenases LDH

• Enzymes intervene in the catabolism process of glucose to lactate.
• The location is localized at the cytoplasm.
• Tetrameric enzymes are formed from two types of unterunits with M and H forms.
• LDH1 = 4 Heart
• LDH2=H3M
• LDH3=H2M2
• LDH4=HM3
• LDH5=M4 of the skeletal.
• Normal values are < to 245 UI/L.
• Electrophoretic will have separation of isozymes with the ranges.
• LDH1 : 14-24%
• LDH2 : 29-39%
• LDH3: 20-26%
• LDH4: 8-16%
• LDH5 : 6-16%
• Variations for the test will determine the cause, if infections, hepatitis or issues with pregnancy.
• If the patient has an infarction of the myocardium or is associated with 18 to 24 hour delay in 2 day and then normalizes on the 6th day.
• For Haematologic has two anémies, leucémies, and neoplasms and muscular necrosis caused by trauma.

Alpha Amylase

• Enzymes will degrade the content of the intestinals, dextrines and maltore
• Two isoforms with amylase either saliva or located in the panreas.
• Usual values from Amylasémie are 31-107 UI/L andAmylasurie < 440 UI/L, 24 hours.
• Tests will diagnose pathologica ,afflictions or Syndromes
• Affliction diagnoses in pancreas
• Syndrome diagnoses related abdomen

Lipase

• Enzymes have a huge role to play in digestives that the pancreas have
• Roles will secrete and allow the triglycerides to hydrolyse
• To have a maximal function colpase are needed a protein that origins from the pancreas
• Usual Values: 13-60UI/L- Variations
• In other cases the chronic is a variant of some affliction.

Aldolase

• Enzymes have an important role to play is the biochemical metabolism of glucose and cell levels.
• There will be 3 isoenzymes that may happen because of muscular issues or cerebral issues.
• The isoenzymes will depend, A or C.A being in muscular and erythrocytes and B being mostly for the river and C in cerebral.
• usual Value 2-7’6 UI/L- Variations depending the tissue.
• Pathology will show how the Aldose is having issues and where from to define if it’s the river or muscular.

Muscular

• Markers that define that the muscular structures are fine.
• Issues and enzymes realse important in a muscular way such as muscles. The key Enzymes: (Cpk, ,LDH and ALAT)
• Markers such Myoglobine.
• The way the antibodies and the way pathologies are happening (auto-corps)
• genetic anomalies that may happen along the way

Conclusion

• Measurement of activities in plasmatic ways allows to have a test and to define how the tissular way and its damages
• Test can have issues to define the diagnose of some diseases but in most is it very specific to see what’s happening