Clinical-Chemistry-2-Laboratory
Page 1
Alkaline Phosphatase (ALP)
Nonspecific enzyme reacting with various substrates
Functions to release inorganic phosphate from organic phosphate ester
Derived from liver and bone in healthy sera
Elevated in children during growth periods and in adults over 50
Increased ALP in normal pregnancy between 16-20 weeks
Intestinal ALP presence depends on blood group
Higher ALP in B and O blood groups compared to A and AB
Major tissue sources: liver, bone, placenta, and intestine
Diagnostic Significance
Elevated total ALP levels in liver function, especially in obstructive jaundice
Highest elevations in bone disorders like Paget’s disease
Bone ALP isoform B1x studied in chronic kidney disease
Methods like electrophoresis used for ALP analysis
Page 2
ALP Analysis Methods
Electrophoresis for liver and bone ALPs
Heat fractionation test at 56°C
Chemical inhibition test with different solutions
Bowers and McComb method as a specific technique
Notes on ALP
Zinc component in ALP, magnesium as enzyme activator
Factors affecting ALP levels: food ingestion, hemolysis, diet
ALP sensitivity to storage temperature and phosphorous
Conditions with increased ALP levels
Acid Phosphatase (ACP)
Active at pH 5.0, indicates seminal fluid presence
Major sources: prostate, RBCs, platelets, liver, bone
Diagnostic Significance of ACP
Detection of prostatic adenocarcinoma
Forensic use in rape cases investigation
Increased ACP in various conditions like urinary tract obstruction
Page 3
ACP Analysis Methods
Different substrates and end products used in ACP analysis
Specific considerations for ACP testing
Notes on ACP
Stability of serum sample, choice of substrates
Reference ranges for ACP in men and women
Factors affecting ACP levels and stability
ACP in Disease
Increased ACP in conditions like prostatic carcinoma and metastatic bone involvement
Prostatic Acid Phosphatase (PAP)
Used with PSA to monitor recurrence of prostate cancer
PSA more sensitive than PAP in detecting prostate cancer stages
YRDCO || Centro Escolar University- Malolos || 2024
Page 4
Aspartate Aminotransferase (AST)
Transfers amino group between aspartate and α-keto acids
Forms oxaloacetate and glutamate
Predominant in serum as cytoplasmic isoenzyme
Major sources: cardiac tissue, liver, skeletal muscle
Reference value: 5-37 U/L
Diagnostic significance in myocardial infarction, hepatocellular disorders, skeletal muscle involvement
Monitoring therapy with hepatotoxic drugs
Method: Karmen Method at pH 7.5; 340 nm
Alanine Aminotransferase (ALT)
Similar enzymatic activity to AST
Liver-specific enzyme
Reference value: 6-37 U/L
Used to screen blood donors
More sensitive for posttransfusion hepatitis or toxic exposure
Methods: present in plasma, bile, CSF, saliva; requires vitamin B6 as coenzyme
Coupled Enzymatic Reaction: pH 7.5; reading at 340 nm
Page 5
AST/SGOT vs. ALT/SGPT
Major organs affected: Heart (AST), Liver (ALT)
Substrates: Aspartic Alpha Ketoglutaric Acid (AST), Alanine Alpha Ketoglutaric Acid (ALT)
End products: Glutamic Acid + Oxaloacetic Acid (AST), Glutamic Acid + Pyruvic Acid (ALT)
Color developer: 2, 4 DNPH for both
Color intensifier: 0.4N NaOH for both
Methods: Reitman and Frankel for both
Increased Transferase
Various conditions affecting AST and ALT levels
Notes on elevations in acute hepatitis, viral hepatitis, chronic hepatitis, and other conditions
Page 6
Amylase/Alpha-1-4-Glucohydrolase (AMS)
Breaks down starch and glycogen
Pancreatic marker, earliest to rise in acute pancreatitis
Major tissue source: pancreas, salivary glands
Reference value: 60-180 SU/dL; 95-290 U/L
Diagnostic significance in acute pancreatitis
Methods and notes on inhibitors and inhibitors of AMS
Increased Serum Amylase
Conditions like acute pancreatitis and ectopic pregnancy
Notes on amylase increase, urine amylase, and A/C ratio
YRDCO || Centro Escolar University- Malolos || 2024
Page 7:
Lipase (LPS)/Triacylglycerol Acylhydrolase
Enzyme that hydrolyzes fats to produce alcohol and fatty acid
Specific pancreatic marker, secreted exclusively in the pancreas
Reference value: 0-1.0 U/mL
Diagnostic significance in acute and chronic pancreatitis
Methods of assay include Cherry Crandal, Tietz and Fiereck, Peroxidase coupling
Lactate dehydrogenase (LD)
Enzyme catalyzing lactic and pyruvic acid interconversion
Found in various tissues, with different isoenzymes
Reference value: 100-225 U/L (forward reaction), 80-280 U/L (reverse reaction)
Diagnostic significance in pernicious anemia, hemolytic disorders, AMI, and various cancers
LD isoenzyme percentages: LD-1 17-27%, LD-2 27-37%, LD-3 18-25%, LD-4 3-8%, LD-5 0-5%
Methods of assay include Wacker Method, Wrobleuski La Due, Wrobleuski Cabaud, Berger Broida
Page 8:
Lactate dehydrogenase (LD) Continued
LD levels rise in AMI, hepatic carcinoma, toxic hepatitis, viral hepatitis, cirrhosis
LD isoenzyme patterns in different conditions
LD-1 > LD-2 in AMI and hemolytic anemia
LD-2, LD-3, LD-4 are LD cancer markers
LD-5 levels in different liver conditions
LD-6 in drug hepatotoxicity and obstructive jaundice
Different LD isoenzymes in various tissues
Methods of assay for LD include Wacker Method, Wrobleuski La Due, Wrobleuski Cabaud, Berger Broida
General:
YRDCO || Centro Escolar University- Malolos || 2024
Information provided by the transcript includes details about Lipase (LPS)/Triacylglycerol Acylhydrolase and Lactate dehydrogenase (LD) enzymes, their diagnostic significance, tissue sources, reference values, and methods of assay.
Page 9
Notes to Remember
Red Blood Cell Characteristics:Red blood cells contain notably high levels of LD.
LD can utilize substrates beyond lactate, such as α-hydroxybutyrate.
α-hydroxybutyrate dehydrogenase (α-HBD) activity represents LD-1 activity.
Elevated α-hydroxybutyrate activity occurs when both LD-1 and LD-2 are increased.
LD activity in pleural fluids aids in distinguishing transudates (low LD) from exudates (high LD).
Total LD levels temporarily increase following blood transfusion but normalize within 24 hours.
Decreased LD values are observed in frozen samples (LD-5 is cold-labile), hence samples should be processed within 24 hours of collection and stored at 25°C.
Conditions with Increased LDH:
Anemias - pernicious, hemolytic, megaloblastic
Myocardial infarction
Leukemia
Renal infarction
Hepatitis and hepatic cancer
Muscular dystrophy
Delirium tremens
Malignancy
Pneumocystis jerovecii pneumonia
Creatine Kinase/ATP-CREATINE-N-PHOSPHOTRANSFERASE (CK)
Catalyzes the transfer of a phosphate group between creatine phosphate and adenosine diphosphate.
Involved in the storage of high-energy creatine phosphate in the muscles.
Dimeric molecule with a small molecular size, composed of a pair of two different monomers called M and B.
Found in small amounts throughout the body, but in high concentrations only in muscle, brain. CK from the brain virtually never crosses the blood-brain barrier to reach plasma.
Major tissue sources:
Brain tissue, smooth and skeletal muscles, and cardiac muscles.
Reference values:
Male: 15-160 U/L
Female: 15-130 U/L
CK-MB: <6% of total CK
Isoenzyme:
CK-BB (brain type), CK-MB (Hybrid type), CK-MM (muscle type)
CK-1 is the most anodal and labile isoenzyme; CK-3 is the least anodal.
CK-BB is the dominant isoenzyme of CK found in the brain, intestine, and smooth muscle.
Adult serum rarely contains CK-BB of brain origin due to its high molecular size; it may be normally present in neonatal sera.
Cardiac tissues contain a significant amount of CK-MB (20%) - myocardium is the only tissue from which CK-MB enters the serum in significant quantities.
CK-MB in serum of a healthy person is < 5 μg/L.
CK-MM is abundantly present in both cardiac and skeletal muscles.
In the sera of healthy persons, CK-MM is the major isoenzyme (94-100%).
Physically well-trained individuals tend to have elevated baseline levels of total CK.
Direct muscle trauma, as seen in contact sports, surgery, strenuous exercise, and intramuscular injections, are common causes of mild elevations of serum CK (up to about 5 to 6 times reference limits).
Intramuscular injections are known to increase CK (<5x URL).
Bedridden patients may have decreased CK activity.
Diagnostic Significance
Very sensitive indicator of acute myocardial infarction (AMI) and Duchenne disorder.
Higher elevation of total CK is seen in Duchenne’s muscular dystrophy (50x URL).
CK-MB is found mainly in myocardial tissue - used as a serodiagnostic test for AMI.
Elevated levels of CK-MB, > 6% of the total CK, are considered the most specific indicator of myocardial damage, particularly AMI.
Following AMI, CK-MB levels begin to rise within 4-8 hours, peak at 12-24 hours, normalize within 48-72 hours.
CK-MB is not elevated in angina.
Injury to both cardiac and skeletal muscle accounts for the majority of CK-MM elevation.
Total CK is markedly elevated after trauma to skeletal muscle from crush injury, convulsions, tetany, surgical incision, or intramuscular injections.
Methods
Tanzer-Gilbarg Assay (forward/direct method): pH 9.0; 340 nm.
Oliver-Rosalki (Reverse/indirect method): most commonly used method; faster reaction; pH 6.8.
Notes to Remember
Adenylate kinase (AK) released after red cell lysis interferes with CK assay, particularly with hemolysis of >320 mg/L.
Liver cells and RBC do not contain CK.
To increase both sensitivity and specificity of CK-MB in the diagnosis of acute AMI, serial determinations of MB fraction (3-to-4-hour intervals over a 12-to-16-hour period) are necessary.
Adenosine monophosphate (AMP) is added to inhibit AK in the reverse method.
N-acetylcysteine is added to CK reagent to activate the enzyme and partially reverse the inhibition of oxidized sulfhydryl groups.
Imidazole serves as a buffer; urate and cystine are potent CK inhibitors.
CK is light and pH sensitive; it is also lost with excessive storage.
Cleland’s reagent and glutathione partially restore lost activity of CK.
CK mass units assay is more sensitive than electrophoresis, but electrophoresis is still the reference method for CK.
CK Relative Index (CKI)
Expression of the percentage of the total CK attributed to CK-MB. Helps to understand possible release of CK-MB from non-cardiac tissues when total CK is very high.
Page 11
Increased Creatine kinase:
Duchenne’s muscular dystrophy
Myocardial infarction
Hypothyroidism
Pulmonary infarction
Reye’s syndrome
Strenuous exercise and intramuscular injections
Cerebral vascular accident
Rocky Mountain Spotted Fever
Carbon monoxide poisoning
ALDOLASE/FRUCTOSE 1,6-DIPHOSPHATE ALDOLASE:
Splits fructose-1,6-diphosphate into two triose phosphate molecules
Increased in skeletal muscle disease, leukemia, hemolytic anemia, and hepatic cancer
Isoenzymes: Aldolase A (skeletal muscles), Aldolase B (WBC, liver, kidney), Aldolase C (Brain Tissue)
Other Clinically Significant Enzymes:
5’ NUCLEOTIDASE (5’N): Marker for hepatobiliary diseases, reference value 0-1.6 units
GAMMA GLUTAMYL TRANSAMINE PEPTIDASE/ TRANSFERASE (GGT): Located in hepatic cells, elevated in certain therapies, reference value 5-30 U/L (F) / 6-45 U/L (M)
Diagnostic Significance:
Useful in differentiating ALP level source
Elevated in hepatobiliary disorders, alcoholism, pancreatitis, and prostatic disorders
Page 12
PSEUDOCHOLINESTERASE (PChE):
Secreted by the liver, marker for insecticide/pesticide poisoning
Decreased in acute hepatitis, cirrhosis, carcinoma metastatic to liver, and malnutrition
ANGIOTENSIN-CONVERTING:
Converts angiotensin I to angiotensin II, indicator of neuronal dysfunction
Critical target for lowering blood pressure, diagnostic significance in sarcoidosis
Other Markers:
CERULOPLASMIN: Marker for Wilson’s disease
ORNITHINE CARBAMOYL TRANSFERASE: Marker for hepatobiliary diseases
GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G-6-PD): Maintains NADPH, newborn screening marker
Page 13
INTRODUCTION:
Enzymes are proteins acting as biological catalysts
Classified into Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, and Ligases
PRE LABORATORY DISCUSSION:
Similar function with ALP but works at different pH and environment
Method of Analysis: Shinowara (405 nm)
METHOD, PRINCIPLE, & MATERIALS:
Use of Colorometric Humazym M-Test (Babson and Reed)
Reaction Principle and Materials listed for analysis
YRDCO || Centro Escolar University- Malolos || 2024
Page 14
Sample Preparation
Stabilize samples, AUTOCAL, and Controls by adding stabilizer
Samples stable for 3 days at 2-8°C, 24 hours at 15-25°C
Assay Details
Wavelength: Hg 405nm
Optical path: 1 cm
Temperature: 37°C
Measurement against air
Procedure & Calculation
Warm reagent and cuvettes to 37°C
Pipette Sample 100ul, Working reagent 1000ul into Cuvette
Calculate total acid phosphatase activity using conversion factor
Reference Values
Men: up to 6.6 U/l
Women: up to 5.5 U/l
Page 15
Pre Laboratory Discussion
Known as Serum Glutamic oxaloacetic transaminase (SGOT/GOT)
Diagnostic significance for hepatocellular disorders and skeletal involvement
Method of Assay
Based on Karmen Method
Coupled enzymatic reaction using malate dehydrogenase
Materials & Procedure
Use of LiquiUV Test (KARMEN METHOD)
Reagent preparation and stability details
Procedure for sample start and cuvette preparation
Page 16
Assay Details
Wavelength: Hg 365 nm, 340 nm, or 334 nm
Optical path: 1 cm
Temperature: 25°C, 30°C, 37°C
Calculation of Results
Guidelines for absorbance change per minute
Conversion factor for traditional units to SI units
Dilution instructions if absorbance change exceeds limit
Page 17
Pre Laboratory Discussion
Known as Serum Glutamic Pyruvic Transaminase or (GPT)
Mainly for evaluation of hepatic disorders
Method of Assay
Kinetic method for ALAT activity determination
Coupled enzymatic reaction using LD as the indicator enzyme
Materials & Procedure
Use of LiquiUV Test
Reagent preparation details
Procedure for sample start and cuvette preparation
Page 18
RESULTS AND CALCULATIONS:For ∆ A/min within:
0.06-0.08 (Hg 365 nm)
0.12-0.16 (Hg 334 nm, 340 nm) Use only measurements from the first 2 minutes for calculations (1 minute incubation, 2 min. measurement).
Formula for Calculations: U/l + ∆ A/min x SAMPLE START
Wavelength:
25°C; 30°C; 37°C
Hg 334 nm: 972, 1780
340 nm: 952, 1745
Hg 365: 1765, 3235
Conversion factor from traditional units (U/l) in SI-units (kat/l):
1U/l = 16.67 x 10^-3 ukat/l
1ukat/l = 60 U/l
Reference Values:
Temperature: 25°C, 30°C, 37°C
IFCC* (International Federation of Clinical Chemistry)
Men: up to 22 U/l, 30 U/l, 42 U/l, 45 U/l
Women: up to 17 U/l, 23 U/l, 32 U/l, 34 U/l
with pyridoxalphosphate activation
PRE LABORATORY DISCUSSION-7:
Amylase is the smallest enzyme (50,000-55,000 Da).
It breaks down STARCH and GLYCOGEN.
Amylose (α-1,4-glycosidic bonds)
Amylopectin (α-1,6-glycosidic bonds)
Amylase Isoenzymes:
Ptyalin (S): Salivary Amylase, secreted from the Salivary glands, also present in fallopian tubes and lungs.
Amylopsin (P): Pancreatic amylase, secreted from the ACINAR CELLS OF THE PANCREAS.
Laboratory Analysis:
Affected by Triglycerides in Lipemic samples.
Falsely elevated upon administration of MORPHINE and other opiates as painkillers.
Method of Analysis:
Amyloclastic
Saccharogenic
Chromogenic
Continuous monitoring
METHOD, PRINCIPLE, & MATERIALS-5:
METHOD: Caraway Method, Modified
MATERIALS / REAGENTS:
Amylase Substrate
Test Sera/N - AB controls
Color developer
Deionized water
Test tubes, cuvettes, parafilm, micropipette, pipet tips, centrifuge
PROCEDURE, RESULT, & CALCULATION-5:
Procedure:
Incubate test and control for 5 minutes at 37°C.
Read % T/absorbance at 610/640 nm against water blank.
RESULTS / Calculation of amylase activity:
Factor use: 750 u/l
Calculate the given readings using the formula and interpret the value obtained.
Normal Range: 60-180 U/l
PRE LABORATORY DISCUSSION-8:
Breakdown of Lipids (Triglycerides).
LP is found primarily in the Pancreas, may also be present in the stomach and small intestine.
METHOD, PRINCIPLE, & MATERIALS-6:
METHOD USE: Enzymatic Colorimetric Test for the Quantitative Determination of Lipase
REACTION PRINCIPLE:
Lipase catalyzes the reaction
MATERIALS:
BUFFER/ENZYME REAGENT
SUBSTRATE
CALIBRATOR AUTOCAL / SERODOS
PROCEDURE, RESULT, & CALCULATION-6:
PROCEDURE: Pipetting scheme
RESULT CALCULATION:
Reference Value: ≤ 60 U/l
Important Procedural Notes:
Carry over should be avoided by cleaning the glassware thoroughly.
Discard SUB if it becomes red. Precipitation may appear on storage, slightly rotate the vial to resuspend before analysis.
Sodium azide is a preservative, avoid swallowing and contact with skin and mucous membranes.
Page 22
Pre-Laboratory Discussion
Highest LDH activity in heart, liver, skeletal muscle, kidney, and erythrocytes.
Less activity found in lung, smooth muscle, and brain.
LDH isoenzymes: LD-1, LD-2, LD-3, LD-4, LD-5.
Analysis methods: electrophoresis, flourometric or colorimetric detection, immunoinhibition, chemical inhibition.
Reaction types: forward (Lactate) and reverse (Pyruvate).
Method, Principle, & Materials
LiquiUV Test using Wroblewski Ladue method.
Reagent preparation: mixing substrate and buffer.
Specimen: serum or heparinized plasma, avoid hemolysis.
Assay parameters: wavelength, optical path, temperature.
Procedure, Result, & Calculation
Procedure involves mixing sample with working reagent and measuring absorbance.
Calculation of LDH activity using mean absorbance change per minute.
Conversion factors for traditional units to SI-units.
Conversion factor for IFCC recommended method.
Page 23
Results / Calculations
Calculate LDH activity in the sample using absorbance readings.
Factors for converting absorbance to LDH activity units.
Conversion factor for traditional units to SI-units.
Factor for converting results to IFCC recommended method.
Page 24
Pre-Laboratory Discussion
Creatine Kinase (CK) widely distributed in tissues.
Higher in skeletal muscle, heart muscle, and brain tissue.
CK isoenzymes: CK-MM, CK-MB, CK-BB.
Analysis methods: forward and reverse reactions coupled with different systems.
Method, Principle, & Materials
CK-MB NAC Activated method using immunoinhibition.
Reagent preparation involves reconstituting enzyme with buffer.
Specimen: serum, heparinized plasma, or EDTA plasma.
Assay parameters: wavelength, optical path, temperature.
Page 25
Procedure, Result, & Calculation
Reagent preparation involves reconstituting enzyme with buffer.
Specimen considerations and assay parameters.
Important procedural notes for accurate results.
Calculation of CK-MB activity using absorbance change per minute.
Results / Calculations
Calculation of CK-MB activity using factors for different wavelengths.
Conversion factors for traditional units to SI-units.
Reference range for myocardial infarction diagnosis.
Page 26
Control sera with CK-MB values determined by the method can be used.
Only control sera with human CK can be employed for quality control.
Page 27:
Electrolytes
Ions with electric charge (anion or cations)
Crucial in various body processes
Influence water distribution intracellularly or extracellularly
Act as osmoregulators on cell membrane
Pre Laboratory Discussion-11
Sodium as major extracellular cation
Sodium-Potassium ATPase Pump
Methods of analysis: Ion Selective/Specific Electrode, Atomic Absorption Spectrophotometry, Chemical Method
Method, Principle, & Materials-9
Method Use
Photometric Determination of Serum Sodium Mg-Uranylacetate Method
Principle of the Test
Sodium precipitated with Mg-uranyl acetate
Uranyl ions form a yellow-brown complex with thioglycolic acid
Materials
Reagents: Precipitating solution, Colour reagent, Standard
Specimen: Serum/Plasma, urine
Assay details: Wavelength, optical path, temperature
Page 28:
Procedure, Result, & Calculation-9
Procedure
Pipetting scheme
Mixing, standing, shaking, centrifuging steps
Result/Calculation
Conversion of mval/l to mmol/l
Normal ranges for Serum/Plasma, Urine, Cerebrospinal fluid
Important Notes
Use semi-micro procedure with efficient centrifuge
Store PREC. protected from light
Rinse equipment to avoid sodium contamination
Disposable plastic tubes recommended
**YRDCO || Centro Escolar University-