Liver Enzymes CC2 Lab PDF

CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa LIVER ENZYMES Liver Function Test  The conjugation function of your liver is the ability of...

CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa LIVER ENZYMES Liver Function Test  The conjugation function of your liver is the ability of your liver to metabolize bilirubin or  Tests Measuring Hepatic Synthetic Function urobilinogen o TPAG  Excretion is the ability of the liver to produce bile o PT  Dili pagawason ni liver si ammonia kundi iya ra I  Tests Measuring conjugation and Excretion detoxify and convert into urea ( nontoxic form) o Bilirubin  You can then use ammonia as a detoxification o Urobilinogen function test  Tests for Detoxification Function o An increase ammonia in the blood would o Enzyme mean there is a problem in the liver  ALP  AMINOTRANSFERASES Enzyme Tests  5’N  GGT  Used to assess the extent of liver damage  LDH  Cytolysis and Necrosis  Any injury in the liver can  Differentiate hepatocellular from obstructive lead to the liberation of disease the enzymes  Common liver enzymes: o Ammonia o ALP (alkaline phosphatase)  Is a protein catabolism result but o AMINOTRANSFERASES is very toxic which is why ammonia  AST (aspartate is converted to urea aminotransferase)  Urea is nontoxic substance and  ALT (alanine aminotransferase) can easily be eliminated through o 5’N the kidney via the urine o GGT (gamma-glutamyl transferase) o LDH (lactate dehydrogenase) Note: o OCT – not commonly performed  Liver is the gatekeeper to determine whether a Note: particular substance can be release on the circulation or out on the trash  Remember that enzymes are large molecules so  Functions of the liver: they are bound inside the cell. In a situation 1. Storage where there is cytolysis or necrosis, they would 2. Synthetic function be liberated (mang gawas na sila kay namatay naman ang cell)  Liver is able to produce o This then results into their freedom unto  Proteins the circulation  Enzymes  Which can cause an increase of  Carbohydrates enzyme in blood  Clotting factors  Hepatocellular – there is a problem in the  The synthetic function of the liver is assessed hepatocytes (cells of the liver) through performing  Hepatocellular pertains to functional disorder  total protein albumin (TPAG) o Problems on the hepatocytes (liver cells)  Prothrombin Time (PT)  Obstructive pertains to mechanical disorder o Has clotting factors which are synthesized in the liver CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa o asically obstructive means naa bay nag Note: bara. This is in the form of  ALT – ALANINE AMINOTRANSFERASE  Gallstones  Tumor  Old name SGPT – Serum glutamic pyruvic  Etc. transaminase  General Classification – Transferases/ Transaminase ALT (SGPT)  Catalyzes same catalytic reaction with AST (EC 2.6.1.2) o Transfers amino group  The difference between AST and ALT with regards to their catalytic reaction is.. o AST – Aspartate o ALT – Alanine  CATALYTIC REACTION o Transfer of amino group from your alanine to your α-keto-glutarate which producecs pyruvate acid and glutamate acid.  Higher levels of ALT and AST are seen in hepatocellular disorders rather than intra or extra  Co-factor hepatic obstruction o Pyridoxal Phosphate  The only screening test we do for blood donors are  Major Tissue Source o Transfusion transmittable infection o Liver  HIV  Specific enzyme  Hepa C,A  Because majority is  Syphilis found on the liver  Malari  Minor Tissue source  In US, ALT is included in the screening of blood o Kidney donors as mandated by the IFCC because o Pancreas o ALT is a good marker for post transfusion o RBC hepatitis o Heart  If ALT is increase, the patient o Lungs doesn’t qualify o Skeletal muscle  Significance o Evaluation of hepatic disorders  All liver disease even in acute Methods of Determination phase, ALT and AST elevates  Karmen Method  Mas taas tho si ALT  Remain elevated – 2-6 weeks  In acute inflammatory condition of the liver, ALT increase  Reitman-Frankel o Used to screen blood donors  Not practiced in our country  Reference value: 6-37 U/L CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa Note: Laboratory Considerations  Karmen method  Serum or heparinized plasma free from o Coupled enzymatic reaction hemolysis o On the 1st reaction  Aminotransferases are present in human  It catalyzes first the transfer of o Plasma amino group from alanine and o Bile ketoglutarate with ALT o CSF producing pyruvate and o saliva glutamate. (refer to the photo above)  Vitamin B6 can hinder the patients who have the o On the 2nd reaction deficiency in vitamin B6  Pyruvate will react with NADH in o Have falsely low or normal ALT result the presence of your indicator o Vitamin B6 is pyridoxal phosphate or a enzyme called LD cofactor  LD – is a redox reaction o Without the cofactor, slow ang reaction  The oxidation reduction of sa enzyme NADH to NAD is the one being  Certain drugs and alcohol may also increase ALT measured activity o Decrease in absorbance of oxidation of  Stability of enzyme activity can be maintained by NADH to NAD is directly proportional to refrigeration of the sample for up to 3 days and the activity of ALT freezing the sample for up to 30 days o The decrease in absorbance on a o Refrigerator – 3 days continuous monitoring technique is o Freezer – 30 days measured o Read at 340 nm in 37 °C  Reitman-Frankel Note: o Coupled enzymatic reaction o On the 1st reaction  No hemolysis as there are minor concentration  Same with Karmen method of ALT found in RBC where there is catalytic transfer of amino group from alanine and AST/SGOT ALT/SGPT ketoglutarate producing Major Organ Heart Liver pyruvate and l-glutamate. Affected o On the 2nd reaction Substrate Aspartic Alpha Alanine Alpha  Pyruvate is made to react with Ketoglutaric Acid Ketoglutaric Acid DNPH in alkaline condition End products Glutamic acid + Glutamic acid + where a colored complex is Oxaloacetic acid Pyruvic acid formed Color Developer 2,4 DNPH 2,4 DNPH o The activity of the colored complex is Color intensifier 0.4N NaOH 0.4N NaOH directly proportional to the ALT activity Methods Reitman and Reitman and Frankel Frankel o Change of absorbance in colored Karmen complex is measured in 500 nm Note: o Read at 500 nm  The major organ affected by AST is the heart which is why it is part of the cardiac markers  ALT is on liver since its liver specific CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa ALP o Hepatobiliary – disorder in (EC 3.1.3.1)  Liver  We have isoenzyme specific for liver  gallbladder  bile duct o bone disorder  such as paget’s disease  ALT also has an isoenzyme for the bone  General Classification - Hydrolases  How to know where the specific problem is  Nonspecific enzyme (liver or bone) because we have 2 specific o Able to react with a variety of substrate enzymes for them?  Magnesium as activator (cofactor) o Run together with the other enzymes to  ALP contains zinc identify if liver or bone ang disorder o People who have a deficiency in zinc will  ALP is more prominent on obstructive jaundice have a falsely low result kaysa sa hepatocellular diseases (hepatitis or  Major sclerosis) o Intestine  AST and ALT mo increase kung hepatocellular o Liver pero normal to slight increase in obstruction o Bone o Placenta  Minor Major Isoenzymes o Kidney o Spleen  Liver ALP  Significance  Bone ALP o Evaluation of hepatobiliary disorder and  Placental ALP Bone disorder (Paget’s disease)  Intestinal ALP  Regan Note: o Lung cancer o Breast cancer  ALP – ALKALINE PHOSPHATASE o Ovarian cancer  Catalytic reaction o colon cancer o Liberate inorganic phosphate from  Nagao organic phosphate producing alcohol o People having pancreatic cancer via hydrolysis (introduction of water) o bile duct cancer  Catalytic reaction happens in the pH 9-10 o Reason why ALP is enzymatically active Note: or physiologically active because its alkaline pH  Liver ALP – is found on biliary ducts  ALP is different with acid phosphatase where it  Bone ALP – found in osteoblast catalyzes same reaction but with an acid pH o For children, alkaline phosphate is  Cofactor: magnesium higher because of osteoblastic activity o In older people, alkaline phosphate is  Contains zinc (people who have deficiency zinc also higher because of osteoblastic naay falsely low ALP) activity  Significance:  CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa  Bone ALP takes part in the transport of calcium urea but but phenyl-  Placental ALP is detected in pregnant women low low alanine from 16 – 20 weeks inhibitio inhibiti o Persists throughout the pregnancy n by L- on by o It will return to normal 3-6 days after phenyl- l- alanine phenyl birth - o Placental ALP is lower than blood group alanin A or AB e  Intestinal ALP is detected on the blood group because of a secretor gene o Blood group B and O has a higher Methods of Determination concentration of intestinal ALP than A or 1. Electrophoresis – You can differentiate AB isoenzyme the use of electrophoresis. Check the o Involved in the transport of lipids mobility from cathode to anode:  Regan and Nagao – carcinoplacental isoenzyme  Liver - fastest o Carcino – isoenzymes occur in  Bone – 2nd neoplasms (active cell division)  Placenta -3rd (common in cancer)  Intestine – slowest o Placental – its activity is close to placental alkaline phosphatase enzyme Note: activity  Liver and Bone medyo close ang difference sa Alkaline Phosphatase Isoenzyme Characteristics electrophoresis maong lisod sila e differentiate Name of Isoenzyme  To improve the separation of liver and Characterist Hepatic Bone Intestinal Placental Other bone isoenzyme: ic  You can add lectin in wheat Heat Stable at Labile: Intermedi Stable at Rega germ Stability 56°C for disapp ate labile: 65°C forn  Or neuraminidase 30 ears at disappear 30 isoen minutes 56 °C s at 56°C minutes zyme 2. Heat Fractionation/Stability Test – 56 °C for 10 within within 15 : minutes 10 minutes most  Placenta – most heat stable minut stabl  It can resist denaturation up to es e 65°C up to 30 minutes Electroph Most Interm Cathodic Migrate Rena  This is also the same with oretic anodic ediate to bone s with l isoenzyme regan and nagao Order fraction hepatic isoen  Intestine or bone zyme  Liver forms : rare but  Bone – most heat labile most 3. Chemical Inhibition Test – inhibit the reaction cath  Phenylalanine: Common inhibitor odic  Inhibit: Chemical Modera Strong Strong  Placenta Inhibition te inhibiti inhibition  Regan inhibitio on by by l-  Nagao n by urea  Intestinal isoenzyme CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa  Synthetic urea Note:  Inhibit:  There are also several tests/measurements that  Bone isoenzyme can be performed for alkaline phosphatase.  Levamisole  Remember that your alkaline  Inhibit: phosphatase is nonspecific, so it can  Liver isoenzyme react to several substrates.  Bone isoenzyme 4. Bowers and Mc Comb Methods Substrate End products Bodansky Beta- Inorganic PO4 + p-nitrophenylphosphate → p-nitrophenol + phosphate Shinowara glycerophosphate Glycerol ion Jones  p-nitrophenylphosphate – Blue Reinhart King and Phenylphosphate Phenol  p-nitrophenol – Yellow Armstrong Bessy, Lowry p-nitro phenyl PO4 p-nitrophenol and Brock or yellow Principle of Bowers and Mccomb: Bowers and p-nitro phenyl PO4 nitrophenoxide McComb ion  Its reaction is based on the hydrolytic reaction Huggins and Phenolphthalein Phenolphthalein  Breakdown of p-nitrophenylphosphate Talalay diphosphate red because of hydrolysis, so there is water Moss Alpha naphthol Alpha - in the reaction. PO4 Naphthol  p-nitrophenylphosphate is colorless and once it Klein, Babson Buffered Free is hydrolysed, it will be liberated and the p- & Read Phenolphthalein Phenolphthalein nitrophenol and phosphate ion will become PO4 color yellow.  The absorbance activity of the yellow color change of the p-nitrophenol will be Laboratory Considerations measured at 405 nm and the increase in 1. Hemolysis the absorbance of the liberated p-  Old samples are prohibited nitrophenol at the 405 nm is directly 2. Serum or heparinized plasma less than 3 hours proportional to your ALP activity. old  The higher the absorbance, the higher  3 hours is the maximum because the the ALP activity. longer it stands, the more the pH will increase, and the more active the  Absorbance measure: 405 nm alkaline phosphatase.  pH: 10.15 3. Anticoagulants that remove calcium and magnesium will prevent product formation  Remember that magnesium is your co- factor.  So, if magnesium is inhibited by the anticoagulant, there will be no activator or no product formation. CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa 4. Lipids, hemoglobin or Bilirubin interferences GGT  Remember that our absorbance (gamma-glutamyl transferase) measurement in Bower and Mccomb is 405. (EC 2.3.2.2)  These substances can absorb light at 405 nm that is why, they can interfere with  It is classified as transaminase or transferase. the result.  Catalytic reaction: Transfers itself to amino  Hemolyzed samples should not be used acids, other peptides or water molecules because of the free hemoglobin.  Distribution 5. Fasting  Kidney  It would be ideal if the patient is fasting  Brain because remember in the intestinal  Prostate alkaline phosphatase isoenzyme, after  Pancreas every meal, it would increase.  Liver  To avoid false increase due to  Significance food ingestion, it is ideal that the  Evaluation of liver damage patient will fast.  This is because in all 6. Temperature sensitive hepatobiliary disorder, GGT will  At 4˚C, it would falsely elevate ALP increase. That is why, it is the activity. most sensitive liver enzyme 7. Zinc deficiency assay in all hepatobiliary  For those who have deficiency in zinc, disorder—either obstruction or they will have a low ALP because zinc is functional. part of the ALP enzyme.  There is also a higher elevation Note: of GGT in obstruction just like in the ALT. Hyphosphatasia (low ALP)  Detection of alcoholism  A genetic disorder wherein there is an  GGT is elevated in alcoholism. abnormal development of the bones and  Monitoring of alcohol intake by patients the teeth.  GGT is used if the patient has Low ALP can also be found in patients after blood already abstained from alcohol transfusion. within 2-3 weeks, GGT will The reaction/testing process of ALP can be normalize. inhibited by phosphorus. Placental ALP is a good tumor marker for germ Note: cell tumor (those in the gonads).  For patients taking enzyme inducing drugs, such  If the patient is pregnant, expect an as: increase of the placental ALP, not  Phenytoin because of the tumor marker.  Phenobarbital  Warfarin  These can lead to an increase in the GGT, not because of damaged liver, but because of the location of the GGT. CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa  GGT is in the canaliculi of the Methods of Determination liver cells, particularly in the smooth endoplasmic reticulum.  Methods  So, any mitochondrial induction  Szass (the mitochondira of the cell),  Rosalki & Tarrow the activity of GGT will be  Orlowski triggered and it will increase.  Substrate  Aside from the liver damage, GGT is also  Gamma-glutamyl-p-nitroanilide elevated in the ACUTE PANCREATITIS  Remember that a substrate is  This is because GGT can also be found in the one that is acted upon an the pancreas. enzyme.  For patients experiencing DIABETES  Once an enzyme is acted upon a MILLETUS, since the injury is in the substrate, the substrate (y- pancreas, GGT will increase. Glutamyl-p- nitroanilide) will be  Patients who are experiencing PROSTATIC converted into Glycylglycine. DISORDER, it will also elevate.  And then, Glycylglycine will be  Patients who are experiencing ACUTE liberated into a para-nitroaniline MYOCARDIAL INFARCTION, GGT will also increase.  Product  However, there is no explanation behind  P-nitroaniline this since there is no heart in the tissue  The activity/appearance of the distribution. p-nitroaniline, which is a  GGT is also useful in differentiating the ALP chromorgenic product, so its increase. absorbance would be measures  Remember that when there is a problem at 405-420 nm, pwede na dayon in the liver and bone (skeletal muscle), ma measure ang kani na ALP will increase. chromogenic product.  In the GGT  The method of measuring can  If there is a problem in the liver, be: it will increase.  END POINT: Remember  If there is a problem in the bone, endpoint, it measures it will be normal. only one which takes  If ALP is increased and GGT is normal, time for testing. then the problem will point in the  KINETIC BONE. So, this will be the diagnostic  CONTINUOS purpose of the GGT.  Reference Value  M: 6-45 U/L  F: 5-30 U/L  As you can observe, females has lower reference value. This is because they have Estrogen and Progesterone and these hormones may suppress the activity of GGT. CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa Laboratory Considerations Other Clinically Significant Enzymes 1. Basically, GGT is STABLE. Acid Phosphatase  It is not affected by hemolysis ACP (EC 3.1.3.2)  You can put this in the refrigerator  You can store this at 4˚C  Catalyze same reaction as ALP but active at pH 5  Same catalytic reaction with ALP wherein inorganic phosphate or 5’ Nucleotidase phosphorus is liberated from the organic (EC 3.1.3.5) and it produces alcohol.  A phosphoric monoester hydrolase  However, the difference here is that:  Predominantly secreted in the liver  ALP has a pH of 9-10.  Diagnostic Significance  ACP has an acidic pH of 5.  Hepatobiliary disease (marker)  Significant levels found in:  RBC Note:  Platelet  In adult men, 50% found in the prostate  It is also a marker for infiltrative lesions of the gland liver.  That is why, one of its diagnostic  5’N also increases in Cholestatic disorders value is detecting prostatic  Stop/minimizes the production of the cancer or recurrence of the bile prostatic cancer together with  This will help in differentiating the liver and the the prostatic antigen test. bone problems in the alkaline phosphatase.  Isoenzymes fractioned into 5 bands  The same with GGT, this would increase  Band 1 (B1) in the liver, but normal in the bone.  Found in the PROSTATE - ACP is significant for detecting  Methods prostate cancer.  Dixon & Purdon  Band 2 (B2)  Campbell  Found in the WBC  Goldber (granulocytes)  Band 3 (B3)  Reference Value  Found in the:  0-1.6 units  Platelet - ACP increases if there is thrombocytopenia.  RBC - ACP increases when there is hemolytic anemia.  Monocyte  Band 4 (B4)  Found in the WBC (granulocytes) CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa  Band 5 (B5) Laboratory Considerations  Found in the Osteoclast (bone) - ACP increases if there are  Free from hemolysis problems in the osteoclastic  Decreases when left at Room Temp activity.  If left at RT for 1-2 hours, decrease enzymatic activity  Aid in detecting:  pH will tend to rise  Metastatic CA  It will become alkaline  Other types of cancer and bone disease  The activity will decrease because it is  Forensic (rape cases) not physiologically active in an alkaline  Significant in rape cases because pH. ACP is found in the semen  Serum should be frozen or acidify  ACP is one of the proteolytic  If not run immediately, it should be enzymes that is part of the frozen or acidify semen.  Prostatic ACP is inhibited by L-tartrate ions  ACP can persist or its level can  Remember that Prostatic ACP is Band 1 still be detected until 4 days. isoenzyme  From vaginal washing.  RBC ACP is inhibited by formaldehyde  Remember that RBC ACP is Band 3 isoenzyme Methods of Determination  TRAP Methods Substrate End Product  Tartrate-Resistant Acid Phosphatase Gutman and Phenyl PO4 Inorganic PO4  Cannot be affected by L-tartrate ions Gutman inhibition Shinowara PNPP p-nitrophenol  This is present in people having: Babson, Read Alpha naphthyl Alpha-  Leukemia & Phillips PO4 napthol  Lymphoma Roy and Thymolphthalein Free  Elevated Serum Bilirubin Hillman monophosphate Thymolph  Cause a low value of TRAP activity Note:  TRAP is affected by bilirubin  Somehow same with the ALP because it is NON- Other Minor Liver Enzymes SPECIFIC enzyme.  Different substrates are present 1. ALDOLASE (4.1.1.13)  But, we will be using Kinetic/ Endpoint  Splits fructose testing.  Actually has 3 isoenzymes  ENDPOINT  ALD A  The substrate of the test will be:  Skeletal Muscle Thymolpthalein monophosphate.  ALD B:  KINETIC (continuous monitoring)  White Blood Cell  The substrate of the test is Alpha  Liver naphthyl PO4.  Kidney  Different substrate, different end product.  ALD C:  Brain tissue CC2 Lab – Prelims – PART 3 Ma’am Michelle Mabasa  A Lyases enzyme (4) 4. Ceruloplasmin  Its catalytic reaction is to breakdown  Copper-carrying protein fructose.  Marker for Wilson’s disease  Clinical Significance:  A genetic disorder wherein  Increase in problems in the: there is an excess storage of  Skeletal muscle copper which can lodge in the  Hepatic diseases eyes and brain.  Hemolytic anemia 5. Ornithine Carbamoyl Transferase  Leukemia (OCT) 2. Pseudocholinesterase (EC 3.1.1.8)  Marker for hepatobiliary diseases  A hydrolytic enzyme  Majority is in the liver, but it is not part 6. G-6-PD (EC 1.1.1.49) of the liver enzyme  An oxidoreductase  Because it reflects more on the  Functions to maintain or suppress the synthetic function rather than activity of NADPH in the RBC detoxification function.  Part of newborn screening because…  Remember, liver enzymes assess  In some genetic disorder, if they detoxification function. fail to produce the enzyme: G-  This is more of a marker in the: 6-PD, then it indicates that  Insecticide or rodenticide there is a problem in the RBC. poisoning  This is because G-6-PD can be  It will decrease in: found in the:  Acute hepatitis  Adrenal cortex  Cirrhosis  Spleen  Carcinoma  RBC  Malnutrition  Lymph nodes  Tissue Sources:  Deficiency of this enzyme will  Liver lead to hemolysis.  Myocardium  G-6-PD will increase in:  Pancreas  Myocardial infarction  Megaloblastic anemia 3. ACE (EC 3.4.15.1)  “Angiotensin-converting enzyme”  Converts angiotensin 1 into angiotensin 2  A hydrolase  Diagnostic Significance:  Diagnosis and monitoring of sarcoidosis  Abnormal collection of inflammatory cells.