March 01, 2025 • Liver secretes plasma proteins, carbohydrates, lipids,

lipoproteins, clotting factors, ketone bodies, enzymes, and

LIVER FUNCTION TEST xenobiotics.
• During carbohydrate metabolism (the liver can do 3 things)
OUTLINE 1. Use the glucose for its own cellular energy
1. ANATOMY requirement
I. Gross Anatomy 2. Circulate the glucose for use at peripheral
II. Microscopic Anatomy tissues.
2. BIOCHEMICAL FUNCTION 3. Store excess glucose as glycogen
3. LIVER FUNCTION ALTERATION DURING DISEASE • The greatest source of cholesterol in the body comes from what the
I. Jaundice liver produce not by dietary.
II. Cirrhosis • Approximately 70% of cholesterol is produced daily by the liver and
III. Tumors the rest is from the diet
IV. Reye's Syndrome • All protein are synthesized in the liver is the albumin.
V. Drug and Alcohol Related Disorders • Normal liver produces albumin for about 12 g/day.
4. ASSESSMENT OF LIVER FUNCTION IV. Detoxification and Drug Metabolism
• FIRST PASS MECHANISM
o It can allow important substances to reach the
systemic circulation and can serve as a barrier to
prevent toxic or harmful substances from reaching
the systemic circulation.
o LIVER acts as the gatekeeper.
• 2 MECHANISM (Inactivation and Excretion)
i. INACTIVATION
• Either bind the material reversibly so
as to inactivate the compound
ii. EXCRETION
• Chemically modify the compound so
• Right lobe is 6x larger than left lobe. it can be excreted
• Two lobes are separated by Falciform ligament. V. Storage Function
o Storage site for all fat-soluble and water soluble vitamins.
Gross Anatomy of the LIVER o Liver is also the storage depot for glycogen, which is released
• The liver is located in the upper right-hand portion of the abdominal cavity, when glucose is depleted.
is protected by the lower rib cage, and is held in place by ligamentous
attachment. BILIRUBIN METABOLISM
• The liver is large and complex organ weighing approximately 1.2 to 1.5 • Bilirubin – break down of RBC
kg. • Approximately 120 days, RBC will undergo destruction either extra or
• Chronic Cirrhosis - >10kg in weight intracellular
• After that RBC will release hemoglobin
PORTAL TRIAD
1. Hepatic artery – 25% (oxygenated blood)
2. Portal vein – 75% (nutrient-rich blood)
3. Common bile duct – drainage of waste, bile, and bilirubin.

MICROSCOPIC ANATOMY OF THE LIVER

• The liver is the chief metabolic organ weighing 1.2 to 1.5 kg in the body.
• It receives 1500 mL of blood per minute.
• It is composed of 2 types of cells: HEPATOCYTES and KUPFER CELLS
(Phagocytes).

TWO TYPES:
1. Hepatocytes
• 80% of the total volume
• Major hepatic function HEMOGLOBIN (3 major components)
• Responsible for REGENERATIVE property 1) Iron (carried by transferring back to the liver)
2. Kupfer Cells 2) Globin (binalik ni amino acid sa liver)
• Has phagocytic role 3) Heme (will be converted to Bilirubin within 2-3 hours
• Has hepatic macrophages that engulf organisms and toxins. • Bilirubin will bind to Albumin then transport to the liver then pag na transport
▪ For the liver to not function, 80% must be destroyed/abolished. na it is now a UNCONJUGATED BILIRUBIN (water insoluble) (kaya dae
▪ LOBULES – anatomic unit/functional unit of LIVER siya makaka travel sa blood stream kaya need niya mag undergo nine
conjugation)
FUNCTIONS OF THE LIVER: • UDP-glucuronyl transferase – converts unconjugated to conjugated para
Biochemical Functions of the LIVER makadaloy sa bloodstream
I. Excretory and Secretion Function • SER (smooth endoplasmic reticulum) – site of Conjugation
• Excretion of bile involves the elimination of bile acids or salts, • From conjugated magigi siyang uribilinogen (80% is oxidized to about 50 –
pigments, and cholesterol. 250 mg of the urobilinogen will be excreted kasama ng feces.
• Bile acids (cholic acids and chenodeoxycholic acid are • Then the 20% of urobilinogen will be absorbed and recirculated to the liver
conjugated with amino acids glycine and taurine form bile salts) and small portion of urobilinogen enters circulation and is excreted in the
o Produced in liver, stored in gallbladder urine
o Body produces 3 L of bile/day
o 1 L is excreted UNCONJUGATED VS. CONJUGATED BILIRUBIN
▪ Bilirubin is the principal pigment in
the liver BILIRUBIN 1 BILIRUBIN 2
II. Conjugation Function Unconjugated Bilirubin Conjugated Bilirubin
• Involves in Bilirubin Metabolism
Water insoluble Water soluble
• 200 – 300 mg of bilirubin is produced daily in healthy adult
III. Synthetic and Metabolic Function
 Non-polar Polar • Total absence of B2 production
Indirect reacting / Indirect Bilirubin Direct reacting / Direct Bilirubin • (+) Kernicterus; bile is colorless

Hemobilirubin Cholebilirubin TYPE 2 CRIGLER-NAJJAR SYNDROME

Slow reacting One-minute prompt bilirubin • It is characterized by partial deficiency of UDPGT
• Small amount of B2 is produced
Pre hepatic bilirubin Hepatic / Post hepatic / Obstructive and
regulative bilirubin 3) DUBIN JOHNSON SYNDROME & ROTOR SYNDROME
• B1 is indirect bilirubin because upon addition of DIAZO REAGENT, B1 will • Blockage of the excretion of bilirubin into the canaliculi, due to deficiency
not react immediately compared to B2 which will react immediately of canalicular multidrug resistance/multispecific organic anionic transporter
protein (MDR2/cMOAT)
REFERENCE VALUE • It is observed with mild jaundice and the total bilirubin level is not exceeding
Conjugated Bilirubin – 0-0.2 mg/dL (0-3 umol/L) 5mg/dL
Unconjugated Bilirubin – 0.2-0.8 mg/dL (3-14 umol/L) • Distinct feature: Intense dark pigmentation of the liver due to accumulation
Total Bilirubin – 0.2-1.0 mg/dL (3-17 umol/L) of lipofuscin pigment
• Elevated B2
Conversion factor: x 17.1
Dubin Johnson Rotor
DELTA BILIRUBIN Dark pigmentation of liver No pigmentation
• It is a conjugated bilirubin tightly bound to albumin Abnormal GB function Normal GB function
• It has longer half life for about 12-14 days, while other form of bilirubin has
only 2-4 hours. 4) LUCEY-DRISCOLL SYNDROME
• It has formed due to prolonged elevation of conjugated bilirubin in biliary • It is a familial form of unconjugated hyperbilirubinemia and may be caused
obstruction by a circulating inhibitor of bilirubin conjugation in the baby’s or mother’s
• Monitoring of declining serum bilirubin following surgical removal of blood
gallstones • It may also be due to Genetic change in the UGT1A1 gene enhancing
• It is computed by using this formula: hyperbilirubinemia
• TB – DB + IB = DELTA BILIRUBIN • Elevated B1
• It is not calculated on neonatal patients (<14 days)
• Reference value: 0.2 mg/dL (<3umol/L) CIRRHOSIS
• A clinical condition in which scar tissue replaces normal, healthy liver tissue
JAUNDICE • Most common cause of cirrhosis is chronic alcoholism and chronic
• It is also called icterus or Hyperbilirubinemia hepatitis C virus infection
• It is used to describe the yellow discoloration of the skin, eyes, and mucous • Other causes:
membranes. o Hepatitis B & D
• It is clinically evident when bilirubin levels exceed 3mg/dL (Bishop) (other o Autoimmune hepatitis
resources; 2mg/dL) o Wilson disease
• French word “jaune” which means yellow o Hemochromatosis
• Serum/plasma – icteric/icterus o Galactosemia
• Treatment:
PRE-HEPATIC JAUNDICE o Abstinence from alcohol
• Prior to liver o Interferon for viral hepatitis
• Cause: Too much destruction of RBC o Corticosteroids for autoimmune hepatitis
• Elevated INDIRECT BILIRUBIN
• High B1 – BRAIN => Kernicterus
• Too much destruction of RBC – caused by hemolytic anemia

POST-HEPATIC JAUNDICE
• Cause: Failure of bile to flow in the intestine/impaired bilirubin Excretion
• Elevated DIRECT BILIRUBIN
• High B2
• Biliary obstruction
• Cholelithiasis (gallstone)
• Pancreatic tumor
• Parasitism

HEPATOCELLULAR COMBINED JAUNDICE

• Cause: Hepatocytes injury caused by viruses, alcohol and parasites
• Elevated B1 and B2
TUMORS
DERANGEMENT OF BILIRUBIN METABOLISM
1) GILBERT SYNDROME • Classified as: PRIMARY or METASTATIC
• Bilirubin Transport Deficit • 90% - 95% of all hepatic malignancies are classified as metastatic.
• Genetic mutation in the gene (UGT1A1) located on chromosome 2, that • Classified as: BENIGN or MALIGNANT TUMOR
produces UDPGT PRIMARY
• Characterized by impaired cellular uptake of bilirubin • Cancer that begins in the liver cells.
METASTATIC
• LAB RESULT: ELEVATED B1
• Occurs when tumor from other parts of the body spread to the liver
2) CRIGLER-NAJJAR SYNDROME • Colon, lung, breast
• Conjugation Deficit
BENIGN TUMOR
• A syndrome of chronic nonhemolytic unconjugated hyperbilirubinemia
• Hepatocellular adenoma
• Infants are treated by means of phototherapy
• Hemangiomas
• LAB RESULT: ELEVATED B1
• Non-cancerous
TYPE 1 CRIGLER-NAJJAR SYNDROME • Don’t invade nearby tissues
• Deficiency enzyme: UDPGT (Glucoronyl transferase) MALIGNANT TUMOR
 • Hepatocellular carcinoma (HCC) • INDIRECT POSITIVE Van den Berg Reaction
• Hepatocarcinoma • High B1 — hemolytic Jaundice
• Hepatoma
• Cancerous 3 If the serum contains both BI & B2 in high concentration, the purple color is produced
• Can spread to other part of the body immediately which is further intensifed by the addition of alcohol
• BIPHASIC Van den Berg
REYE’S SYNDROME • High B1 and B2 — hepatic jaundice
• Group of disorders caused by infectious, metabolic, toxic, or drug-induced
(Aspirin) disease found almost exclusively in children EVELYN AND MALLOY METHOD
• By conditions: Varicella, Gastroenteritis, Influenza • Coupling accelerator: 50% Methanol
• Encephalopathy, Fatty degeneration of Liver, Transaminase elevation • Diazo reagents:
o DIAZO A-0.1% Sulfanilic Acid + HCl
DRUG AND ALCOHOL RELATED DISORDER o DIAZO B-0.5% Sodium Nitrite
• Most common mechanism of toxicity is via an immune-mediated injury to o DIAZO C-1.5% HCI
the Hepatocytes • Final reaction: PINK TO PURPLE AZOBILIRUBIN AT 560 nm.
• Acetaminophen, ethanol – most common drug
ALCOHOL INDUCED LIVER INJURY JENDRASSIK AND GROF METHOD
• Alcoholic fatty liver, Alcoholic hepatitis, and Alcoholic Cirrhosis • It is the most commonly used method.
• Risk: 30g consumption (3-4 drink/day) • It is a popular technique for the discreet analyzers.
• High risk: >120g (12-16 drink/day) • It is more sensitive compared to Evelyn and Malloy method.
• It is not affected by hemoglobin up to 750 mg/dL and pH changes.
ALCOHOLIC FATTY LIVER • Main reagent: Diazo reagent
• Slight elevations in AST, ALT, and y-glutamyltransferase (GGT), and on • Coupling Accelerator: Caffeine Sodium Benzoate
biopsy, fatty infiltrates are noted in the vacuoles of the liver • Buffer: Sodium acetate
• Ascorbic acid: Terminates the initial reaction and destroys the excess
ALCOHOLIC HEPATITIS diazo reagent.
• Moderately elevated AST, ALT, GGT, and alkaline phosphatase (ALP) and • Alkaline tartrate: Provides an alkaline pH
elevations in total bilirubin greater than 5mg/dLl • Final reaction: Pink to Blue azobilirubin at 600 nm
• Deritis Ratio – AST/ALT ratio greater than 2
NOTES TO REMEMBER
ALCOHOLIC CIRRHOSIS • The purpose of adding a methanol or caffeine benzoate solution is to
• This condition appears to be more common in males than in females. allow indirect bilirubin to react (solubilize)with the color reagent.
• Abnormal liver function tests (AST, ALT, GGT, ALP, and Total Bilirubin), • Caffeine benzoate is preferred over methanol because te latter promotes
decreased albumin, and prolonged prothrombin time protein precipitation and increases turbidity.
• In the measurement of the total bilirubin, the unconjugated fraction
ASSESSMENT OF LIVER FUNCTION produced a color only after the addition of the accelerant.

TEST FOR CONJUGATION INCREASED B1

Sample consideration • Glbert’s Syndrome
• Random serum is utilized in the measurement of bilirubin though a fasting sample • Crigler-Najjar Syndrome
has higher reliability due to the false elevation in the presence of lipemia. • Hemolytic anemia
• Hemolysis should be avoided since it may affect the reaction of bilirubin with the • Hepatocellular disease
diazo reagent, causing false decrease. • Lucey Driscoll Syndrome
• The sample should be protected from light exposure as it would cause • G-6-PD deficiency
insignificant reducation in the actual concentration of bilirubin.
• It should be analyzed within 2-3 hours after collection. INCREASES B2
• Decreased 30-50%/hour • Biliary obstruction (gallstones)
• Serum/plasma is separated from cells; kept in the dark • Pancreatic (head) cancer
• Stable for: • Dubin-Johnson syndrome
o 2 days – Room temperature • Alcoholic and Viral hepatitis
o 1 week – 4°C
• Biliary atresia
o Indefinitely - -20°C
• Hepatocellular disease
• Visible ictresin – occurs when bilirubin is >25 mg/dL
• Septicemia
Methods • Rotor Syndrome
• Accelerants: CAFFEINE and METHANOL are used to measure total
II. BROMSULPHTHALEIN (BSP)
bilirubin
DYE EXCRETION TEST
I. BILIRUBIN ASSAY • It is a test for hepatocellular function and potency of bile duct,
Principle: Van den Berg Reaction: Diazotization of bilirubin to produce however, rarely used.
azobilirubin • It determines the ability of protein albumin to transport the
exogenous dye to the liver where it is excreted in the bile.
Deletion of accelerants • Procedure: BSP dye is administered intravenously.
o B2
o IB = TB – DB MAC DONALD
ROSENTHAL WHITE
(SINGLE
INDICATOR (DOUBLE COLLECTION
Van den Berg(h) Reaction COLLECTION
METHOD)
• Reagent: diazotized sulfonic acid METHOD)
• End color: PURPLE AJOBILIRUBIN product DOSE 5 mg/kg body weight of 2 mg/kg body weight of the
the patient patient
1 Diazotised sulfonic acid reacts with conjugated bilirubin and give purple color SAMPLE After 45 minutes of IV Afte 5 minutes and after 30
Immediately (within 30 seconds) COLLECTION dose minutes of IV dose
• DIRECT POSITIVE VAN DEN BERG REAC NORMAL/ After 45 minutes: +/- After 5 minutes: 50% dye
• High B2 — obstructive jaundice EXPECTED 5% dye retention retention
RESULTS After 30 minutes; 0% dye
2 Diazotised sulfanilic and plus methanol reacts with unconjugated bilirubin and give retention
purple color normally 30 minutes
 III. UROBILINOGEN BERTHELOT REACTION
• Colorless end product of bilirubin metabolism, oxidized by bacteria to form
brown pigment urobilin.
• Either excreted in urine or feces, reabsorbed into portal blood and returned
to the liver.
• Complete biliary obstruction – absence of this substance in urine or stool.
• Specimen: 2 hour freshly collected urine or stool.
GLUTAMATE DEHYDROGENASE (GLDH)
• Method: Erhlich’s method (p-dimethyl aminobenzaldehyde reagent)
• It is currently the most common used method for measurement of ammonia.
• Should be collected 2 hours after meal (preferably noon meal)
• It involves the formation of Schiff base between ammonia and the alpha-
• False increase: presence of drug (methydopa) keto group of alpha-ketoglutarate, then producing glutamine which is
• False decrease: sample was exposed to light and air primary amine at 340 nm in the presence of GLDH.
• Reference range:
o Urine: 0.3-1.0 Ehlich units/2 hour or 0.5 – 4 Ehrlich units/day HEPATITIS
o Stool: 80-280 Ehrlich units/24 hours • Hepatitis implies injury to the liver characterized by presence of
inflammation in the liver tissue.
TEST FOR MEASURING DETOXIFICATION • Infectious causes for the inflammation of liver include viral, bacterial, and
• It involves enzyme and ammonia test. parasitic infections, as well as noninfectious causes, such as radiation,
drugs, chemicals, and autoimmune diseases and toxins.
ENZYME TESTS
• Major hepatitis subtypes include A, B, C, D, and E.
• It is used to assess the extent of liver damage and to differentiate
hepatocellular (functional) from obstructive (mechanical) disease.
• Any injury to the liver that results in cytolysis and necrosis causes the
liberation of various enzymes.
• Enzymes secreted by the liver: ALP, ALT, AST, 5’NT, GGT, OCT, LAP, and
LD

AMMONIA
• It arises from the deamination of amino acids, which occurs mainly through
the action of digestive and bacterial enzymes (bacterial proteases, ureases,
and amine oxidases) on proteins in the intestinal tract.
• Liver is the only organ responsible for ammonia detoxification via the
urea cycle.
• Increased levels: Cirrhosis, hepatitis, Reye’s Syndome, chronic renal
disease, and acetaminophen poisoning
• Reference range; 19-60 ug/dL (11-35 mmol/L)
• Marker of hepatic injury

MEASUREMENT OF AMMONIA (NH3)

• Smokng is a soure of contamination which lead to false elevated
concentrations.
• Prolonged standing of the specimen increases ammonia level due to
enzymatic deamination of labile amides such as glutamine.
• Preferred sample: Arterial blood
• Sample requirement: Heparin or EDTA plasma (sample kept in ice water
immediately)
• Common method: Glutamate Dehydrogenase
• Major interference: Hemoglobin (avoid hemolyzed sample, falsely inceased)

DIGESTION (KJELDAHL METHOD)

• Nitrogen ion in a protein-free filtrate (PFF) of the specimen is converted to
ammonia using hot concentrated sulfuric acid in the presence of catalyst.
• Catalyst: Copper sulfate, mercury, and selenium

NESSLERIZATION REACTION
• YELLOW END COLOR: Nitrogen content in the sample is low to moderate.
• ORANGE BROWN END COLOR: Nitrogen content is high

K2Hg212 – Dimercuric Potassium lodide

Gum Ghati – helps to stabilize the mercury-ammonia complex which is formed during
the reaction
NH2Hg212 – Dimercuric ammonium lodide
ENZYMES • ISOENZYMES
• Highly specialized proteins • STORAGE
• CATALYST — Hastens chemical reactions. • H+ CONCENTRATION
• Normally confined within cells unless membrane permeability allows them • COFACTORS
to enter the blood. • TEMPERATURE
• Measured in terms of their activity. • INHIBITORS
• Do not alter the reaction
• Do not consumed in the reaction COFACTORS
• We measure their activity than the absolute value. • NON-PROTEIN COMPOUNDS
• Organ/cell damage — high level of enzymes (serum/plasma) • need to bind to the enzyme for the reaction to occur.
“CAM”
ENZYMES AS CATALYST 1. Coenzyme
• Have reaction specificity. • ORGANIC COMPOUND/COFACTOR (with carbon)
• It only affects the rate of reaction • ↑ concentration = ↑ velocity of reaction
• Activity affected by temperature and pH • Essential for absolute enzymatic activity
• Contains an active site and allosteric site. • Ex. NAD/NADH, pyridoxal (Vit. B6)

ACTIVE SITE HOLOENZYMES = apoenzyme (enzyme portion) + coenzyme (prosthetic group)

• Is the region within an enzyme that fits the shape of molecules called
substrate. 2. Activator
• The shape and chemical environment inside the site permit a chemical • INORGANIC
reaction o proceed more easily. • Alter the configuration or shape of the enzyme
• (⭐️) Ex: Ca & Cl= AMYLASE, Mg = CK, Zn = LDH
SUBSTRATE
• The substrate of an enzyme are the reactants that are activated by the 3. Metalloenzymes
enzyme. • INORGANIC
• Attached to a molecule
• Ex: Catalase and Cytochrome hydrolase

ISOENZYME – same activity with enzyme

Differ:
• Physical properties
• Tissue distribution
• electrophoretic mobilities
• Heat denaturation

TEMPERATURE
• Active at: 25°C, 30°C, 37°C (optimum temperature for enzymatic activity)
• Except: CK = denatured
• 40 – 50°C = denaturation
• 60 – 65°C – inactivation
• Q10 – every 10°C increased in temperature, there will be twofold increase
in enzymatic activity.

H+ CONCENTRATION and pH
• pH range 7 – 8
• Extreme pH level = denaturation of enzymes

STORAGE
• Lower temp (Refrigeration/freezing) – render enzymes reversibly inactive
• - 20°C = preservation of enzyme
• 2° – 8°C – ideal Storage temp for substrate & coenzyme
• RT = storage of LDH (LD4 and LD5)

INHIBITORS
• are chemicals that reduce the rate of enzymatic reaction.
o COMPETITIVE
o NON-COMPETITIVE
o UNCOMPETITIVE

COMPETITIVE INHIBITOR
• Physically binds to the active site of an enzyme
• Resembles the substrate.
• Both the substrate and inhibitor compete for the same active site of an
enzyme.
Counteracted by:
• Adding more substrate to bind the enzyme
• Dilution (serum/plasma) to reduce the concentration of inhibitor
Chemical reaction – A temperature to molecules move faster
Enzymatic reaction – (⭐️) lower the activation energy NON-COMPETITIVE INHIBITOR
• 108-111X faster • Does not compete with the substrate but look for areas other than the active
site.
FACTORS AFFECTING ENZYMATIC REACTIONS • Increasing the substrate concentration does NOT reverse the inhibition.
• ENZYME CONCENTRATION • When the non-competitive inhibitor binds to the allosteric site, it alters the
• SUBSTRATE CONCENTRATION configuration of the active site and the tertiary structure of proteins.
 • Targets the allosteric site (regulatory site) – determines the conformation 3 Esterases (ACP, ALP, CHS, LPS) Peptidases (trypsin,
Hydrolases
and activity of the enzyme pepsin, LAP) Glycosidase (AMS, galactosidases)
4 Glutamate decarboxylase, pyruvate decarboxylase,
Lyases
UNCOMPETITIVE INHIBITOR tryptophan decarboxylase, aldolase
• The inhibitor binds to the enzyme-substrate (ES) complex. 5 Glucose phosphate, isomerase and ribose phosphate
Isomerases
• Increasing the substrate concentration results in more ES complexes to isomerase
which the inhibitor binds and thereby increases the inhibition. 6 Ligases Synthase
• Increasing substrate concentration results in increased inhibition.
• Targets the ES complex ALT first marker for obstructive jaundice
ALP second marker

ENZYME THEORY
EMIL FISHER'S / LOCK AND KEY THEORY
• It is based on the premise that the shape of the key (substrate) must fit into
the lock (enzyme).

NOMENCLATURE
• Mandated by the Enzyme Commission of the International Union of
Biochemistry (1961).
• Enzymes are classified according to their biochemical functions, indicating
substrate and class of reaction catalyzed, and are designated by individual
identification numbers.
• Coded as CLASS NUMBER. SUBCLASS NUMBER. SUB-SUBCLASS
NUMBER. SERIAL NUMBER
• Ex: Creatinine Kinase
o E.C. 2.7.3.2

Acid phosphatase — E.C. 3.1.3.2

Acetyl-CoA synthetase — E.C. 6.4.1.1 KOSHLAND/INDUCED FIT THEORY
Alkaline phosphatase — E.C. 3.1.3.1 • It is based on the substrate binding to the active site of the enzyme.
Amylase — E.C. 3.2.1.1
• There is a greater range of substrate specificity
Alanine aminotransferase — E.C. 2.6.1.2
Lock (enzyme) — RIGID
Aspartate aminotransferase — E.C. 2.6.1.1
Active site (enzyme) — FLEXIBLE
Aldolase — E.C. 4.1.2.13
Angiotensin-converting enzyme — E.C. 3.4.15.1
ENZYMATIC REACTION
Creatine kinase — E.C. 2.7.3.2.
FIRST-ORDER REACTION
Gamma-glutamyl transferase — E.C. 2.3.2.2
Glucose-6-phosphate dehydrogenase — EC. 1.1.1.49 • The reaction rate is directly proportional to substrate concentration.
Lipase — E.C. 3.1.1.3 • A substrate concentration of > 99 x Km is needed to achieve zero-order
Lactic dehydrogenase — E.C. 1.1.1.27 reaction.
Pseudocholinesterase — E.C. 3.1.1.8 • Depends on substrate concentration
True/Acetylcholinesterase — E.C. 3.1.1.7 ZERO-ORDER REACTION
5' nucleotidase — E.C. 3.1.3.5 • The reaction rate depends only on enzyme concentration.
• Depend on enzyme concentration
CLASSIFICATION OF ENZYMES
EC To measure the extent of enzymatic reactions, two general methods may be used.
Class Functions • STATIC/END-POINT (Fixed time) – measurement of a product or
No.
1 Catalyze the removal or addition of electrons (redox substance produced over a given period of time.
Oxidoreductase • KINETIC (Continous) – measurement of a substrate produced per minute
reaction)
2 Catalyze the transfer of a chemical group other than (or hour) produced constantly over a period of time.
Transferase
hydrogen to form one substrate to another
3 Catalyze hydrolysis or spliting of a bond by the addition Enzyme Activity:
Hydrolases Enzymes are measured based on:
of water (hydrolytic reactions)
4 Catalyze removal of groups from substrate without • Change in substrate concentration
Lyases • Change in product concentration
hydrolysis. The product contains double bond
5 Catalyze the intramolecular arrangement of the • Charge in coenzyme concentration
Isomerases
substrate compound
6 Catalyze the joining of 2 substrate molecules, coupled CAUSES OF ELEVATED PLASMA ENZYME LEVELS
Ligases with breaking of the pyrophosphate bond in ATP or • Tissue necrosis and degeneration.
similar compound • Impaired removal of enzyme from plasma.
• Normal cell turnover.
EC • Increased permeability of cell membrane.
Class EXAMPLES
No. • Increased in the production of enzymes by cells.
1 Oxidoreductase LDH, MDH, G6PD, CO, ICD • Decreased clearance of enzymes from the circulation.
2 Transferase CK, AST, ALT, OCT
METHODS OF MEASUREMENT
 • COLORIMETRIC
• ULTRAVIOLET
o Manometry
o Fluorometry

COLORIMETRIC
• Needs large amount of substrate
• Longer incubation time
MANOMETRY
• Measures the solution and disappearance of GAS as reaction proceeds
FLUOROMETRY
• Particularly useful in differentiation between oxidized (do not fluorescence)
and reduced (exhibit fluorescence) nucleotidase

UNITS FOR EXPRESSING ENZYMATIC ACTIVITY

• INTERNATIONAL UNIT (IU or U) – one micromole of substrate per minute.
• KATAL UNIT (KU) – one mole of substrate per second
• IU/U = 1 umole/minute
• KATAL (KU) = 1mole/second
CC o Found in lung, breast, ovarian and gynecological cancers.
CLINICALLY SIGNIFICANT ENZYMES • NAGAO ISOENZYMES
o Detected in metastatic carcinoma of pleural surfaces and in
PHOSPHATE adenocarcinoma of the pancreas and bile duct.
• Alkaline Phosphatase (ALP) • KASAHARA ISOENZYMES
o Alkaline Orthophosphoric Monoester Phosphohydrolase o Hepatoma
• Acid Phosphatase (ACP)
o Acid Orthophosphoric Monoester Phosphohydrolase SELECTIVE CHEMICAL INHIBITORS
• PHENYLALANINE
Alkaline Phosphatase (ALP) o Inhibits intestinal and placental ALP and Regan ALP, Nagao
• EC 3.1.3.1 ALP.
• Activity occurs at pH 9.0 • 3M UREA
• Major tissue sources: liver, bone, placenta, and intestinal o Inhibits bone ALP
• In healthy sera, ALP levels are derived from liver and bone (osteoblasts). • LEVAMISOLE Reagent
• Reference range: 30-90 U/L o Inhibits liver and bone ALP
• L-LEUCINE
ALP & ACP – Class 3 — hydrolases o Inhibits Nagao ALP
ACP – ostaclast Nagao — “Pancreas ALP”
Blood pH: 7.35-7.45 = ALP is inactive
Osteoblasts — bone cell
* zinc – cofactor for ALP
*magnesium – activator for ALP
16 – 20 weeks in pregnancy
• ALP can be detected

ALP MAJOR ISOENZYMES

• Bone ALP
• Liver ALP
• Intestinal ALP
• Placental

ELECTROPHORESIS
• Most useful single technique for ALP isoenzyme analysis

ELECTROPHORETIC MOBILITY OF ALP

Fastest to Lowest: Bowers – McComb
• Liver • Reference method
• Bone • Most specific method
• Placental • Currently routine method for ALP
• Intestinal
Bowers and McComb Method
• Electrophoresis
• Distinguish isoenzyme qualitatively
Liver – most anodal
Intestinal – least anodal

• p-nitrophenylphosphate (colorless) is hydrolysed to p-nitrophenol (yellow)

and the increase in absorbance at 405 nm at a pH of 10.15, which is directly
proportional to ALP activity, is measured.
“I Promise to Be Loyal” • Reference method of ALP

• Heat Stability/ Fractionization DIAGNOSTIC SIGNIFICANCE

Placental – most heat stable • BONE DISEASE
- Resist denaturation at 65°C for 30 minutes o Highest elevation occur in PAGET’S DISEASE
- Regan isoenzyme “Living ALP”– most heat stable placental • LIVER DISEASE

HEAT STABILITY OF ALP SOURCES OF ERROR

STABLE • Hemolysis may cause slight elevations because ALP is approximately 6x
Placental more concentrated in RBCs than in serum.
Intestinal • ALP increases at 3% - 10% on standing at 25°C or 4°C for several hours.
Liver • Diet may induce elevations in ALP activity of blood group B and O
Bone individuals who are secretors.
LABILE • Values may be 25% higher following ingestion of high-fat meal.
• ALP is inhibited by phosphorus.
Take note:
➢ ALP is also heat sensitive if stored in low temperature (4°C) CONDITIONS WITH INCREASED ALP
o High ALP in serum/plasma • Osteitis deformans (Paget’s disease) – highest elevation of ALP
➢ Typically, ALP is measured before and after heating the serum in 56°C for • Obstructive jaundice
10 minutes • Rickets – diseased in cjildren caused by lack of Vit. D
➢ = if RA (residual activity) after heating is less than 20% of TA (total acitivity) • Osteomalacia
before heating = BONE ALP
• Osteoblastic bone tumor – osteosarcoma
➢ = if RA (residual activity) after heating is greater than 20% of TA (total
activity) before heating = LIVER ALP • Sprue – digestive enzyme where the small intestine ability to absorbed
nutrients are impaired
ALP ISOENZYMES IN CANCER PATIENTS • Hyperparathyroidism
• REGAN ISOENZYMES • Hepatitis and cirrhosis (slight increased)
 • 20mM L-tartrate – inhibits prostatic ACP
Acid Phosphatase (ACP) • 1mM Cupric sulfate & 2% formaldehyde – inhibit RBC ACP
• EC 3.1.3.2
• Optimally active at pH 5.0 TRANSFERASES/TRANSAMINASES
• Major tissue source: Prostate • Aspartate Aminotransferase (AST)
• Other sources: RBCs, platelets, liver, and bone o Serum Glutamic Oxaloacetic Transaminase (SGOT)
• Prostatic and bone ACP are the clinically significant isoenzymes. • Alanine Aminotransferase (ALT)
• Reference range: o Serum Glutamic Pyruvic Transaminase (SGPT)
o Male: 2.5 – 11.7 U/L (Total ACP)
o Female: 0 – 3.5 ng/mL (Prostatic ACP) Aspartate Aminotransferase (AST)
• SGOT
DIAGNOSTIC SIGNIFICANCE o Serum Glutamic Oxaloacetic Transaminase
ACP as a Tumor Marker: • EC 2.6.1.1
• For detection of prostatic carcinoma • Major tissue sources: Cardiac Tissue, Liver, and Skeletal Muscle
o After a surgical treatment of prostate cancer; ACP levels falls • Other sources: Kidney, Pancreas, RBC
faster than PSA, and plasma levels are expected to be
undetectable following complete removal of tumor. AST: ISOENZYMES
• Medicolegal evaluation of rape. • CYTOPLASM AST – most predominant form in serum
o Vaginal washings are examined for seminal fluid ACP activity, • MITOCHONDRIAL AST
which can persist for up to 4 days.
AST: DIAGNOSTIC SIGNIFICANCE
PSA – Prostate Specific Antigen • Evaluation of MI, hepatocellular disorders and skeletal muscle involvement
• More sensitive than PAP in detecting stages A and B in prostatic cancer • Highest evaluation in ACUTE VIRAL HEPATITIS
Prostatic acid Phosphatase (PAP) together with PSA • It is related to greater degree in chronic disorders of the liver with
• Recurence of prostate cancer progressive damage.

(⭐️) ACP activity is greater than 50 U/L = indicates presence of seminal fluid in the INCREASED AST
sample • AMI
• Used for Forensic Clinical Chemistry investigation of rape • Trichinosis
• Dermatomyositis
OTHER CAUSES OF INCREASED SERUM ACP • Muscular Dystrophy
• Urinary Tract Obstruction • Acute Pancreatitis
• Acute Urinary Retention
• Extensive prostatic massage AST: ACUTE MYOCARDIAL INFARCTION
• Prostatic Inflammation • Rises: 6-8 hours
• Infarction/ischemia • Peak: 24 hours
• Prostatic manipulations (needle biopsy and cytoscopy) • Normalize: within 5 days

INCREASED ACP (with Metastatic Bone Involvement)

• Prostatic carcinoma
• Breast, lung, and thyroid carcinoma
• Gaucher’s disease
• Niemann-Pick Disease
March 16, 2025
ACP METHODS

METHOD SUBSTRATE END PRODUCT

Roy and Thymolphthalein Free thymolphthalein

Hillmann monophosphate
Gutman and Phenyl phosphate Inorganic phosphate
Gutman
Shinowara PNPP (p-nitrophenyl Para-nitrophenol
phosphate)
Babson, Reed, Alpha-naphthyl phosphate Alpha-naphthol
and Phillips

Roy and Hillman AST: KARMEN METHOD

• Substrate: Thymolphthalein monophosphate • Coupled enzymatic reaction
o Specific substrate for PROSTATIC ACP • MD (malate dehydrogenase)
o Substrate of choice for quantitative endpoint reaction o Indicator reaction
• End product: Free thymolphthalein • Absorbance: 340nm
• pH 7.5
Babson, Reed, and Phillips Method
• Heparin inhibit AST activity
• Substrate: Alpha-naphthyl phosphate
• Avoid hemolysis = increased AST 10x the upper reference limit
o Preferred for continous monitoring methods
• End product: Alpha-naphthol AST
• Stable for 3 – 4 days at 4°C
NOTES TO REMEMBER!
• Interference: hemolyzed and interic — high AST
• Serum sample must be free from hemolysis.
• Interference: presence of heavy metals — low AST
• Serum ACP decreases within 1-2 hours if left at room temperature.
• If not assayed immediately, serum should be frozen or acidified to a pH Alanine Aminotransferase (ALT)
lower than 6.5
• Serum Glutamic Pyruvic Transaminase (SGPT)
• With acidification, ACP is stable for 2 days at room temperature.
• EC 2.6.1.2
• Major tissue source: LIVER
 • Other sources: Kidneys, Pancreas, RBC, Heart, Skeletal Muscles, End product Glutamic acid + Glutamic acid + Pyruvic
Lungs Oxaloacetic acid acid
• More liver tissue specific composed to AST Color developer 2,4 – Dinitrophenyl hydrazone
• Through cardiac tissue has ALT activity, it is not included as a marker for Color intensifier O. 4 N NaOH
AMI, since it has low activity in the heart. Wavelength reading 505 nm (495 nm – 535 nm)
• It will not exhibit serum/plasma elevations in the presence of MI unless Note: Maximum incubation is up to 6 minutes at RT and at 37°C
hepatic is abnormally included.

ALT: DIAGNOSTIC SIGNIFICANCE

• Evaluation of hepatic disorders
• Markedly increased in acute inflammatory conditions than AST
• Monitor the course of liver (hepatitis) treatment and the effects of drug
therapy SUMMARY OF LIVER FUNCTION TEST FOR COMMON DISORDER
• More sensitive and specific screening test for post-transfusion hepatitis or
occupational toxic exposure compared to AST
DISORDERS B1 B2 ALBUMIN TOTAL ALT AST ALP
PROTEIN

Biliary Obstruction N H N N N N H

Cirrhosis H H L L N/L N/L N/SI.

Inc
Hepatitis H H N N H H H

* LD (Lactate Dehydrogenase) – indicator

* absorbance 340 nm
* pH 7.5

INCREASED ALT
• Toxic hepatitis
• Wolff-Parkinson White Syndrome
o Condition in which there is an extra electrical pathway in the
heart
▪ Resulting in rapid heart rate “tachycardia”
• Chronic alcoholism
• Hepatic cancer
• Reye’s Syndrome
• Viral hepatitis

NOTES TO REMEMBER!
• ACUTE HEPATITIS
o Highest elevations of transferases
▪ DE RITIS RATIO (AST:ALT) is >1.0
• Normal De Ritis Ratio: approximately 1.15
• Alcohol-induced hepatic injury: AST:ALT ratio is 3:1
• ALT is slightly increased in obstruction jaundice but markedly increased in
necrotic jaundice.
• Moderate elevations of transferases: chronic hepatitis, hepatic cancer, and
infectious mononucleosis
• Minimal increase: hepatic cirrhosis and obstructive jaundice
• (⭐️) severe viral toxic hepatitis
o Increased ALT & AST about 20x the normal limit
• (⭐️) end – stage cirrhosis
o ALT & AST are low due to massive tissue destruction
• Both ALT and AST require pyridoxal phosphate, an essential cofactor that
should always be added in any measurement.
• The use of icteric and lipemic samples may cause a significant interference
with this method.
• Use of hemolyzed sample cause false increase in AST activity while a slight
elevation or non at all may be observed in ALT.

Pyridoxal
• Absence result to diminished the activity of transferases
• Vit B6
• Coenzyme (prosthetic group)

Comparison b/w AST & ALT using Reitman & Frankel Method
AST/SGOT ALT/SGPT
Substrate Aspartic Alpha- Alanine Alpha-
Ketoglutanic acid Ketoglutamic acid
March 23, 2025 • Acute Pancreatitis
• Ectopic pregnancy
AMYLASE • Peptic ulcers
• Alpha-1-4 Glucan-4-Glucohydrolase • Alcoholism
• EC. 3.2.1.1 • Mumps
• It catalyzes the breakdown of starch and glycogen
• It is the smallest enzyme in size NOTES TO REMEMBER
• It is the earliest pancreatic marker • SALIVARY AMYLASE – inhibited by wheat germ lectin.
• MAJOR SOURCES: acinar cells of the pancreas and the salivary glands • MACROAMYLASEMIA – AMS +IgG/IgA
• Other Sources: Fallopian tube, small intestine, skeletal muscles, and • Normal Amylase: Creatinine Ratio: 1% - 4% (0.01 – 0.04)
adipose tissue • Acute Pancreatitis AC ratio: >4 (up to 15%)
• “diastase” • Heparin and TAG may inhibit AMS activity in some methods.
• MW 50,000 – 55, 000 daltons • The administration of morphine and other opiates for pain relief before blood
o Freely filtered by the glomerulus sampling will lead to falsely elevated serum AMS level
o Normally present in urine • AMS is stable in serum and urine specimens for 7 days at room
• Ca and Cl – activator of AMS temperature.
• Major isoenzymes: S-type (ptyalin) and P-type (amylopsin)
• Isoforms of salivary amylase: S1, S2, S3 LIPASE
• Isoforms of pancreatic amylase: P1, P2, P3 • TRIACYGLYCEROL ACYLHYDROLASE
• EC 3.1.1. 3
1. P-type – derived from pancreatic tissue —migrates slower to cathode • It is an enzyme that hydrolyzes the ester linkages of fats to produce alcohol
• High in acute pancreatitis and fatty acid
• P3 = most predominantly isoenzyme • Major tissue source: PANCREAS
• Predominant in urine • MOST SPECIFIC PANCREATIC MARKER
2. S-type – derived from salivary gland — migrates faster to anode • Other sources: Stomach, Liver, Intestine, Adipose Tissues, Breast milk, and
• ⅔ of the AMS activity WBCs.

AMYLASE: METHODS LPS: ISOENZYMES

Amyloclastic Measures the disappearance of starch substrate • Pancreatic LPS – predominant in serum
Saccharogenic Measures the appearance of the product • Intestinal LPS
Chromogenic Measures the increasing color from production of product • Lipoprotein LPS
coupled with chromogenic dye. • Gastric LPS
Continuous monitoring Coupling of several enzyme systems to monitor AMS activity
LPS:ISOFORMS
• L1
1. Amyloclastic • L2 – Sensitive and specific marker
• measures the disappearance of the substrate • L3
2. Saccharogenic
• Reference method expressed in Somogyi units (SU) • LPS is not affected by renal disorders
• No lipase activity appear in salivary gland
• LPS is not present in normal urine
• LPS persist approximately 5 days in acute pancreatitis
• AMS persist approximately 2 – 3 days

LPS: DIAGNOSTIC SIGNIFICANCE

3. Chromogenic 1. ACUTE PANCREATITIS
• High color AMS activity • Rises: 4 – 8 hours (average 6 hours)
4. Continuous monitoring • Peak: 24 hours
• pH 6.9, 340 nm • Remains elevated: 7 days
• Normalize: 8 – 14 days
• Persistent and prolonged elevations of serum lipase more than
2 weeks may indicate the presence of pancreatic cyst.
• In chronic pancreatitis, acinar cell degradation occurs, resulting
in loss of amylase and lipase production.
1. Cherry – Crandall

AMS: DIAGNOSTIC SIGNIFICANCE

1. Acute Pancreatitis
• Rises: 2 – 12 hours
• Peak: 24 hours
• Normalize: within 3 – 5 days
2. PAROTITIS (due to Mumps)
• Inflammation of salivary glands
• Enzyme involve: S-type

High AMS = blood accompanied with high urinary excretion

High urine AMS – remains elevated up to 7 days
• Reference method
In renal failure with absence og acute pancreatitis • Substrate: 50% olive oil
High serum AMS • Modified: Triolein (pureform of TAG)
Low urine AMS • Incubation: 24 hours at 37°C
• End product: Fatty Acid
INCREASED AMS 2. Tietz and Fierecks
 • Substrate: Olive Oil • LDH activity in pleural fluids is useful for differentiating transudates (low
• Incubation: 3 hrs LDH) from exudates (high LDH)
• End Product: Fatty Acid • Total LDH increases temporarily after blood transfusion but returns to
• pH of buffer: 8.0 baseline within 48 hours.
3. Peroxidase Coupling • Samples should be processed within 24 hours after collection and stored at
• Most commonly used method 25 degrees celcius
• Substrate: Triolein (pureform of TAG) • Decreased LDH – when samples are frozen (LD5 is cold labile)
• End product: Fatty Acid
INCREASED LDH
LACTATE DEHYDROGENASE (LD/LDH) 1. Anemias (pernicious, hemolytic, and megaloblastic)
• EC 1.1.1.27 2. Myocardial infarction
• Zinc-containing enzyme that is part of glycolytic pathway and Kreb’s cycle 3. Leukemia
4. Renal infarction
• Present in almost all cells in the body
5. Hepatitis and hepatic cancer
• Tetrameric molecules witj 4 subunits of 2 possible forms (H & M)
6. Muscular dystrophy
7. Delirium tremens
Tissue Sources:
8. Malignancy
A. LD1 & LD2 – heart, kidneys, RBC
9. Pneumocystis jerovecii pneumonia
B. LD3 – lungs, pancreas, spleen
C. LD4 & LD5 – skeletal muscles, liver, intestine
Heart and RBC – abundant LDH levels

LDH: ISOENZYMES
LDH subunits Tissue Sources Distribution
LD – 1 (HHHH) Heart and RBCs 17 – 27%
LD – 2 (HHHM) Heart and RBCs 27 – 37%
LD – 3 (HHMM) Lungs, lymphocytes, spleen, 18 – 25%
pancreas
LD – 4 (HMMM) Liver 3 – 8%
LD – 5 (MMMM) Skeletal muscle 0 – 5%

1. LD1 Wacker
• Abundant in cardiac muscle • Most preffered over Wroblewski-Ladue
• Not found in skeletal muscle and liver • Not affected by product inhibition
2. LD2 • pH 8.8
• Major isoenzyme in the sera of a healthy person • 340nm
3. LD3
• Inflammatory enzyme marker Wroblewski-Ladue
4. LD4 • 2x faster than Wacker
• Most abundant in skeletal muscle • Use in dry slide technology
5. LD5 • pH 7.2
• More abundant in skeletal muscle Advantage: small amount of sample
• Most abundant in liver Disadvantage: early loss of linearity
• Almost undetectable in the hearr, RBC, kidney Influenced by LDH inhibitors

OTHER ISOENZYMES
6. LD-6
• Represents the alcohol dehydrogenase enzyme
• 6th band in electrophoresis
• Elevated in drug hepatoxicity and obstructive jaundice
• Responsible for the metabolic conversion of methanol and
ethylene glycol to toxic compounds.

LDH: DIAGNOSTIC SIGNIFICANCE

• Highest LDH serum levels are seen in pernicious anemia and hemolytic
disorders.
• Hepatic carcinoma and toxic hepatitis will have 10-fold increase.
• Viral hepatitis and cirrhosis: slightly increased values (2 - 3x upper
reference limit)
• LD2, LD3, and LD4 are cancer markers (predominantly LD – 3)for acute
leukemia, germ cell tumors, and breast and lung cancers.
• LD-5 is moderately increased in acute viral hepatitis and cirrhosis and
markedly increased in heptic cancer and toxic hepatitis.
• NORMAL PATTER IN SERUM:
o LD2 > LD1 > LD3 > LD4 > LD5
• “FLIPPED PATTERN”
o Seen in myocardial infarction and hemolytic anemia
o LD1 > LD2 > LD3 > LD4 > LD5

LDH: ACUTE MYOCARDIAL INFARCTION CREATININE KINASE (CK)

• Rise: 12 – 24 hours • EC 2.7.3.2
• Peak: 48 – 72 hours • ATP-CREATINE-N-PHOSPHOTRANIFERASE
• Remains elevated: 10 – 14 days • Catalyzes the transfer of a phosphate group between creatine phosphate
and adenosine diphosphate (ADP)
NOTES: • It is involved in the storage of high-energy creatine phosphate in the
muscles
 • Major tissue sources: brain, smooth and skeletal muscles, and cardiac • ALP
muscles • 5’NT
• Very sensitive indicator of Acute Myocardial Infarction and • GGT
Duchenne’s muscular dystrophy • LAP
• HIGHEST ELEVATIONS is seen in Duchenne’s muscular dystrophy Substrate: Gamma-glutamyl-p-nitroanilide
(50x URL)
• Diagnostic marker for nueroleptic malignant • 2nd marker for obstructive jaundice
• Only seen in smooth muscle • Use to determine the cause of elevated ALP

CK: ISOENZYMES Enzyme Liver Bone

1. CK-BB (CK1) – BRAIN TYPE ALP Increase Increase
2. CK-MB (CK2) – HYBRID TYPE GGT Increase N
3. CK-MM (CK3) – MUSCLE TYPE 5’NT Increase N

CK-BB (CK1) GGT: DIAGNOSTIC SIGNIFICANCE

• Most anodal and labile isoenzyme • Elevated in hepatobiliary disorders such as biliary tract obstructions
• Dominant isoenzyme of CK found in brain, intestine, and smooth muscle • Most sensitive marker of acute alcoholic hepatitis
• Serum of adults rarely contains CK-BB of braim origin due to its high • Sensitive indicator of alcoholism (occult alcoholism); however it is
molecular size; it may normally present in neonatal sera. often elevated in alcoholics even without liver disease
• It is found to be elevated in individuals taking high dosage of anti-
CK-MB (CK2) inflammatory and anti-seizure drugs such as acetaminophen,
• Cardiac tissue have significant amount (20%) carbamazepine, and phenytoin
• Serum of healthy person is <5 ug/L
• Utilized as a serodiagnostic test for myocardial infarction GGT METHOD
• Diagnosis of AMI • SZASZ METHOD
• Is not elevated in angina (chest pain) o Measurement: 405-420 nm
Myocardium – only tisues from which CK-MB enters the serum in significant quantities o Measured by the addition of 10% NAOH
Rickettsia ricketsii (wood tick)
5’NUCLEOTIDASE (5’NT)
CK-MM (CK3) • EC 3.1.3.5
• Both abundantly present in cardiac and skeletal muscles • A phosphoric monoester hydrolase; predominantly secreted from the liver.
• In the sera of healthy persons, CK-MM is the major isoenzyme (94 – • Marker for hepatobiliary disease and infiltrative lesions of the liver
100%) • Routinely increased in cholestatic disorder
• Intramuscular injections are known to increase CK (<5x URL) • Methods: Dixon and Purdon, Campbell, Belfield and Golaberg
• Bedridden patients may have decreased CK activity.
CHOLINESTERASE
INCREASED CK • PSEUDOCHOLINESTERASE (PChE)
• Duchenne’s muscular dystrophy o EC 3.1.1.8
• Myocardial infarction o Marker for insecticide/pesticide poisoning (organophosphate
• Hypothyroidism poisoning)
• Pulmonary infarction o Detects acute exposure
• Reye’s Syndrome o Low in pesticide poisoning
• Rhabdomyolysis • ACETYLCHOLINESTERASE (AChE)
• Carbon monoxide poisoning o EC 3.1.1.7
• Rocky Mountain Spotted Fever o Detects chroning exposure
• Cerebral vascular accident (occasional)
2 reasons
ALDOLASE 1. 2 detect pesticide poisoning
2. During post-operative paralysis
• FRUCTOSE 1,6 – DIPHOSPHATE ALDOLASE
Succinylcholine – mucle relaxant used in anesthesia
• It is a glycolytic enzyme that splits fructose-2,6-diphosphate into two triose
Method:
phosphate molecules in the metabolism of glucose
Michael method – measures acetic acid as lowering pH
• It is included in the panel of markers for skeletal muscle injury Ellman method – measure choline
• Increased: Skeletal muscle disease, leukemia, hemolytic anemia, and ANGIOTENSIN-CONVERTING ENZYME (ACE)
hepatic cancer • EC 3.4.15.1
• Aka “Peptidyl-dipeptidase A or Kinase II.
MAJOR TISSUE ENZYMES AND TISSUE SOURCES
• It converts the inactive angiotensin I to its active form, the Angiotensin II,
• ALDOLASE A: SKELETAL MUSCLE
within the lungs.
• ALDOLASE B: LIVER, KIDNEY, and WBC
• Component of the RAAS
• ALDOLASE C: BRAIN TISSUE
• Diagnostic test for sarcoidosis
• Possible indicator of neuronal dysfunction (Alzheimers disease using CSF
sample

CERULOPLASMIN
GAMMA-GLUTAMYL TRANSFERASE (GGT)
• EC 1.16.3.1
• EC 2.3.2.2
• A copper-carrying protein with an enzymatic activity
• Catalyzes the transfer of glutamyl groups between peptides or amino acids
through linkage at a gamma-carboxyl group • Marker for Wilson disease (hepatolenticular disease)
• It is located in the canaliculi of the hepatic cells and particularly in the
GLOCOSE-6-PHOSPHATE DEHYDROGENASE (G-6-PD)
epithelial cells lining the biliary duct; also in the kidney, prostate and
pancreas. • EC 1.1.1.49
• Elevated among individuals undergoing warfarin, phenobarbital, and • Newborn screening marker
phenytoin therapies
• Patients with DM may have increase GGT due to impaired pancreas
March 29, 2025
• Increased: Obstructive jaundice, alcoholism, and DM
ELECTROLYTES
Tests for obstructive jaundice:
 • Ions capable of carrying an electric charge Average adult consumed 1000 ml/day

Electroneutrality REGULATION OF BLOOD VOLUME

• Balance of charges where fluid always contains equal numbers of cations
and anions.
• Anion (-) — (+) anode
• Cation (+) — (-) cathode
• Women has lower average water body content than men as a result of high
fat content
• 90% H20

WATER BALANCE AND DISTRIBUTION

• 40 – 75% = average water content of the human body.
• Water is the solvent for all processes in the human body.
• FUNCTIONS:
4. Transports nutrients to cells
5. Determines cell volume by transport into and out of cells
6. Removes waste products by way of urine
7. Acts n body’s coolant by way of sweating.
Low BV = High
1. 30L of fluid passes from the blood to the tissue spaces daily. High BP = High
2. Normal plasma = 93% water, 7% solutes
• Water content is 12% higher than whole blood Kidney (RENIN) — (signal) Liver — Angiotensinogen — (convert using RENIN)
• Edema = retention of 3L of fluid Angiotensin I (inactive form) — (ACE) Angiotensin II
Adrenal gland
WATER COMPARTMENTS IN THE BODY • Aldosterone
• INTRACELLULAR FLUID • 2 functions:
• EXTRACELLULAR FLUID (1)Reabsorb sodium
(2) Excrete potassium
INTRACELLULAR FLUID
• Found inside the cells. PISO ni NIKO
PISO – either intracellular or extracellular
• Approximately 60% (24 liters) or ⅔ of the total body water.
NIKO – aldosterone
• Main electrolytes: K, Mg, PO4
Pituitary gland
• Anti-diuretic hormone (ADH) – reabsorb water
EXTRACELLULAR FLUID
• Found outside the cells. SODIUM
• Approximately 40% (16 liters) or ⅓ of the total body water. • Also known as “natrium”
• Main electrolytes: Na, Cl, HCO3
• Major extracellular cation
• 3 types:
• Major contributor of osmolality
i. Interstial fluid
• Regulates water balance in the body
• Fluid found between the cells and blood
• Its plasma concentration depends greatly on the intake and excretion
vessels
of water
• Approximately 11.2 L (28%)
• Reference range: 135 – 145 mmol/L or 135 – 145 mEq/L
ii. Intravascular fluid
• Fluid inside our vessel
Sodium (Na²+)
• PLASMA — Major fluid
• 90% ECF cation
• Approximately 3.2 L (8%)
• Sodium and Potassium can readily diffuse in and out of the cell.
iii. Transcellular fluid
• Fluid inside the epithelial-lined spaces
• Ex. CSF, Pericardial, Synovial fluid
• Approximately 1.6 L (4%)

SOURCES OF BODY WATER

SOURCE AMOUNT/DAY
Drink 1000 ml
Food 1000 – 1200 ml/day Normal Plasma Osmolality
Fat Metabolism 100 ml/100g • 295 mmol/L with 270 mmol/L being the result of Sodium and associated
Protein Metabolism 44 ml/100g Ions
Carbohydrate Metabolism 60 ml/100g CSF Na2+ Reference range: 135 – 150 mmol/L
Renal Threshold/Sodium Critical Value:
Average adult – looses 2500 ml/day of water through excretion Hypernatremia: 160 mmol/L
1500 mL = urine Hyponatremia: 120 mmol/L
1000 mL = insensible looses (sweating, tears, breathing)
HORMONES AFFECTING SODIUM LEVELS HYPOVOLEMIC HYPONATREMIA
• ALDOSTERONE • Result from sodium loss in excess of water loss.
o Sodium retention by increasing its reabsorption in the DCT and o Renal loss (Na >20 mmol/day)
increases potassium excretion o Extra-renal loss or cellular shift (Na <20 mmol/day)
o High Na – reabsorb
o Low K - excrete CAUSES OF RENAL LOSS
• ANP (Atrial Natriuretic Peptide) • Diuretics
o An endogenous anti-hypertensive agent secreted by the • Potassium depletion
cardiac atria • Aldosterone deficiency
o Blocks aldosterone and renin secretion • Ketonuria (DM)
o Inhibits the action of angiotensin II and vasopressin resulting in • Salt-losting nephropathy (eg. Polycystic kidney disease)
natriuresis.
o Low Na CAUSES OF EXTRA-RENAL LOSS OR CELLULAR SHIFT
o High K • Vomiting
o Natriuresis – excretion of Sodium in urine
• Diarrhea
• Excess fluid loss (burns, sweating, trauma)
SODIUM REGULATION
1. Intake of water in response to thirst (if plasma osmolality is decrease). • Potassium depletion
2. The excretion of water, largely affected by ADH release.
3. Blood volume status, through RAAS and ANP. NORMOVOLEMIC HYPONATREMIA
CAUSES:
SODIUM: CLINICAL APPLICATIONS • SIADH
HYPONATREMIA • Pseudohyponatremia (artefactual hyponatremia)
• Sodium: <135 mmol/L • Severe hypoglycemia
• Most common electrolyte disorder • Excess water intake
• Clinical concern arises when the serum Sodium is <130 mmol/L • Adrenal insufficiency
• Symptoms occurs if the serum level is 125 – 130 mmol/L • Pregnancy
• Serum Sodium level <125 mmol/L may result in severe neuropsychiatric
symptoms. PSEUDOHYPONATREMIA/ARTIFACTUAL HYPONATREMIA
TOP CAUSES: • It is the reduction in serum sodium concentration caused by a systematic
• Overhydration error in measurement.
• Diuretic Abuse • The most common cause is in vitro hemolysis.
• SIADH • Marked hemolysis may cause a decreased of sodium levels due to dilutions
• Adrenal Failure effect.
• Barter’s Syndrome
HYPERVOLEMIC HYPONATREMIA
• Diabetic Hyperosmolarity
• Na >20 mmol/day
o Acute/chronic renal failure
2 Types of Diuretics
• Na <20 mmol/day
1️⃣Thiazide – act on the DCT of our nephron
o Nephrotic syndrome
2️⃣Loop – act on the loop of henle itself o Hepatic Cirrhosis
o Congestive heart failure
Ascending LOH — chloride pump — blocked —Low Cl- — High Na2+
Chloride pump — active transport which transport chloride back to the interstitium HYPERNATREMIA
High Cl- = High Na2+ • Characterized by increased plasma sodium.
• It usually does not occur unless the thirst mechanism is impaired.
SIADH (Syndrome of Inappropriate ADH secretion)
CAUSES
• Excess loss of water relative to sodium loss
• Decreased water intake
• Increased sodium intake

MAIN CAUSES:
DEHYDRATION
• Profuse sweating or breathing
• Diarrhea
• Severe burns
• Conditions that increase water (fever, burns, exposure to heat)
• Noted: Serum Sodium is Elevated and the urine sodium is also high due to
increased renal excretion of NaCl.
DIABETES INSIPIDUS
• Characterized by copious production of dilute urine (3-20 L/day)
• Absence of ADH function results to inadequate water retention
• Increase serum sodium (dilute urinary sodium)
2 TYPES
• Neurogenic Insipidus – decreased ADH secretion
• Nephrogenic Insipidus – decreased renal response
2500 ml
• 1500 – urine
• 1000 – insensible loss
March 30, 2025 HYPERALDOSTERONISM
• ADRENAL HYPERPLASIA – Genetic disorder that results from
CAUSES OF HYPONATREMIA overproduction of hormone like aldosterone and cortisol.
• Hypovolemic Hyponatremia • CUSHING SYNDROME AND DISEASE
• Normovolemic Hyponatremia
• Hypervolemic Hyponatremia
 • HYPERALDOSTERONISM (CONIN’S DISEASE) – primary Hypokalemia due to insulin overdose:
hyperaldosteronism characterized by excessive secretion of aldosterone • Excessive cellular uptake of glucose as facilitated by insulin results to large
POTASSIUM influxes of potassium into cells lowering it in serum.

Alkalosis
• Red blood cells are excellent buffers, they can exchange potassium for
hydrogen ions. Thus in alkalosis, hydrogen ions leave the red cells to
neutralize excess base while K+ ions enter the red cells.

Hypokalemia due to Vomiting

• This results to the depletion of both hydrogen and potassium ions from the
stomach.

• Also known as “kalium”

• MAJOR INTRACELLULAR CATION
• It is the chief counterbalance of sodium
• It is the single most electrolyte wherein any abnormality is life threatening
• RBC concentration: 105 mmol/L (23x its concentration in plasma)
• REFERENCE RANGE: 3.5 -5.2 mmol/L

REGULATION OF POTASSIUM
• Filtered at the glomeruli and is mostly (70% - 80%) reabsorbed by active
and passive mechanisms in the proximal tubule
• In the Ascending loop of henle, potassium is reabsorbed together with
sodium and chloride by the sodium potassium chloride transporter
Diarrhea
POTASSIUM FUNCTIONS • Most common cause of extrarenal loss of K+
• Regulation of neuromuscular excitability • Direct K+ losses in the stool
• Contraction of the heart
• Regulates ICF volume and hydrogen ion concentration Vomiting
• K+ loss in urine
RMP • Cause metabolic alkalosis
• RESTING MEMBRANE POTENTIAL
Renal Tubular Acidosis (RTA)
Increase K = decrease RMP = increase muscle excitability – muscle weakness • Hydrogen ions cannot be excreted into the urine because of the pathologic
Decrease K = increase RMP = decrease muscle excitability – paralysis/arrhythmia impermeability of the DCT membrane to it
• Hydrogen ions (H*) build up in the blood acidosis!
Hormones affecting Plasma Potassium
• Urine is alkaline
• Aldosterone
• Hypokalemia results from the increased excretion of potassium to
• Epinephrine - provides channel for cellular entry of K+ compensate for the inability to excrete H
• Insulin

SPECIMEN CONSIDERATIONS Hyperaldosteronism

• Increased action of aldosterone in the DCT will excessively reabsorbed
sodium resulting to the excretion of increased amount of K+

Pseudohypokalemia
• Leukocytosis can cause falsely decrease K* levels – because K+ is taken
• Slight hemolysis (50 mg/dk hemoglobin): K+ is increased by 0.5 mmol/L up by the WBC, like active leukemic cells, if sample is left at room temp.
or 3%
• Severe hemolysis (>500 mg/dL hemoglobin): K+ is increased by 30% Hyperkalemia
• Muscular activity such as exercise and prolonged standing: K+ increased • an abnormal physiological state resulting from high concentrations of
by 10%-20% potassium
• Prolonged contact of serum and red cell: K+ false increased
• Prolonged application of tourniquet: K+ false increased Causes of Hyperkalemia:
• Forearm exercise and fist clenching prior to blood sample collection: K+ 1. Reduce aldosterone / aldosterone responsiveness
false increased 2. Impaired renal excretion in renal failure
• Release of platelets into serum during clot formation: K+ false increased 3. Reduce distal delivery of sodium
(plasma K+ levels are lower by 0.1-0.7 mmol/L compare to serum)
Hyporeninemic hypoaldosteronism
Hypokalemia • Most common cause of chronic hyperkalemia
• most commonly caused by impaired renal function
Hyperkalemia
Causes of Hypokalemia: • Hypoaldosteronism
1. Insulin overdose • Acidosis
2. Alkalosis • Cellular injury
3. Vomiting • Artifactual
4. Renal tubular acidosis
5. Hyperaldosteronism Hypoaldosteronism
• Decreased excretion of potassium resulting to increased serum level.
3.0-3.4 mmol/L = mild hypokalemia • E.g. Addison’s disease.
<3.0 mmol/L = critical hypokalemia symptom
Acidosis • 1,25 Dihydroxycholecalciferol (1,25-(OH)2-D3)
• hydrogen ions enter the cell in exchange for potassium ions. • Parathyroid hormone
• Calitonin
Cellular injury
• Any kind of cell damage can cause an increase in potassium level, basically (1,25 DIHYDROXYCHOLECALCIFEROL (1,25-(OH)2 – D₂)
because it is highly found inside the cells. • it increases intestinal absorption of calcium
• Popular causes are rhabdomyolysis and hemolysis. • it increases reabsorption in the kidneys
• it increases mobilization of calcium from bones
Acidosis - plasma K increases by 0.2-1.7 mmol/L for each unit reduction of pH
PARATHYROID HORMONE (PTH)
Alkalosis – plasma K decreases by 0.4 mm01/2 per 0.1 unit ptt rise. • It conserves calcium by increasing reabsorption in the kidneys
• It increases the level by mobilizing bone calcium
Artifactual (Pseudohyperkalemia) • It activates the process of bone resorption
• Hemolyzed sample • It suppresses urinary loss of calcium
• Prolonged tourniquet application • It stimulates the conversion of inactive vit. D to active Vit D, in the kidneys
• Excessive fist clenching
• Blood stored in ice CALCITONIN
• High blast counts in leukemias • hypocalcemic hormone
• Thrombocytosis and severe leukocytosis • secreted by the parafollicular C cells of the thyroid gland
• Use of EDTA as anticoagulant • Inhibits PTH and Vitamin D3
• Inhibits bone resorption
CHLORIDE • Promotes urinary excretion of calcium
• Major extracellular anion
• It is the chief counterion of sodium
• Works with Na” in the maintenance of water balance and osmolality
• Has a close relationship with HCO, in the maintenance of acid-base balance
• The only anion to serve as an enzyme activator
• Reference range: 98 – 107 mmol/L or 98 – 107 mEq/L

Vit. D – inactive
Vit. D3 – active — kidney

HYPOCALCEΜΙΑ
• Alkalosis
• Vitamin D deficiency
• Primary hypoparathyroidism
CAUSES OF HYPERCHLOREMIA • Acute pancreatitis
• Renal Tubular Acidosis • Hypomagnesemia
• Diabetes insipidus • Malabsorption syndrome
• Salicylate intoxication • Renal tubular failure
• Primary hyperthyroidism
• Metabolic acidosis HYPERCALCEMIA
• Prolonged diarrhea • Primary hyperparathyroidism – main cause
• Cancer (lung and mammary)
CAUSES OF HYPOCHLOREMIA • Acidosis
• Prolonged Vomiting • Increased Vitamin D
• Aldosterone deficiency • multiple myeloma
• Metabolic alkalosis • Sarcoidosis
• Salt-losing nephropathy • Hyperthyroidism
• Milk-alkali syndrome
CALCIUM
• It is present almost exclusively in the plasma. SAMPLE PREPARATION
• 99% is part of the bones and 1% is in the blood and ECF. • Best sample for total calcium: Serum (heparinized plasma, alternate
• Involved in blood coagulation, enzyme activity, excitability of skeletal and sample)
cardiac muscle and maintenance of BP • Best sample for ionized calcium: Heparinized plasma (serum, alternate
• Ca2+ cofactor in blood coagulation sample)
• Preferred anticoagulant: Calcium titrated heparin
FORMS OF CALCIUM
• Sample collection and transport: Should be anaerobically, transported
1. Ionized (active) calcium - 50%
on ice
• Circulates freely as calcium ions
• If there is delayed in the analysis: Store at 4 degrees celsius (to prevent
• MOST SENSITIVE and SPECIFIC FOR CA2+ glycolysis and loss of CO2)
2. Protein-bound calcium - 40%
• calcium bound to albumin
• 1g/dL ↓ serum albumin = 0.8 mg/dL ↓ total Ca2+
3. Complexed with anions - 10%
• complexed w/ bicarbonate, citrate, lactate

CALCIUM REGULATION