Group 3

Liver Function, Electrolytes and Enzymes

LIVER FUNCTION
LIVER FUNCTION
Chief organ of metabolism:
15ml of blood per minute
Functions:
Synthetic Function :
12g Albumin per Day
Conjugation Function:
200-300mg Bilirubin per Day
Detox and Drug:
ammonia , liver enzymes
Storage Function:
vitamins, glycogen
Excretory and Secretory Function
B1 B2
Unconjugated Conjugated
Insoluble Soluble
0.2-0.8mg/dl 0-0.2mg/dl
METHODS
• DIAZO REACTION

– Ehrlich reaction
• Urobilinogen in 2-hour fresh urine
– Van den Berg reaction
• Diazotization of bilirubin to azobilirubin

Malloy-Evelyn Jendreassik-Grof

Coupling accelerator 50% METHANOL Caffeine sodium benzoate

Interferences Not afected by Hb up to

750mg/dl; ascorbic acid
Buffer Sodium acetate

Final reaction Pink to purple azobilirubin Pink to blue

/ Red to reddish purple Azobilirubin
 Clinical Conditions
jaundice Bilirubin >2mg/dl
a. pre hepatic Increased RBC
Hyperbilirubinemia
destruction; B1 B2 Urine Urine
kernicterus Bilirubin urobili
b. hepatic Hepatocyte injury Pre H N NEG H
hepatic
c. post hepatic Failure of bile to
flow to intestine Hepatic H H POS H
Kernicterus Post N H POS D/N
hepatic
Cirrhosis
Biliary obstruction Atresia
Cholesytitis
Cholethasis
Choledolithiasis
 DEFECTIVE BILIRUBIN
METABOLISM
• Gilbert’s Syndrome
• Crigler-Najjar Syndrome
• Dubin-Johnson Syndrome
• Rotor syndrome
• Lucey-Driscoll Syndrome
 ELECTROLYTES
are ions (minerals) capable of carrying an electric charge.
 ELECTROLYTES
CATIONS ANIONS
• Calcium • Chloride
• Potassium • Carbonates
• Sodium • Phosphates
• Magnesium
WATER • Active transport is a
mechanism that requires
BALANCE
energy to move ions across
40-75% average water
cellular membranes.
content of the body

Major ECF : 1/3 of the • Diffusion is the passive

total body water movement of ions (no energy
consumed) across a membrane
Major ICF : 2/3 of the
and depends on both the size
total body water
and charge of the ion being
transported, and on the nature
of the membrane through
which it is passing.
 • Osmolality is a physical
Osmolality property of a solution that
• AVP deficiency: 10- is based on the
20L water excreted concentration of solutes
daily Sweat:
(expressed as millimoles)
50mmol/L sodium;
5mmol/L Potassium per kilogram of solvent
(w/w).
• Balance blood
acidity and
pressure
 SODIUM • Clinical significance
• Function: osmotic regulation
• Major extracellular cation
• Thirst: major defense against HYPERNATREMIA HYPONATREMIA
hyperosmolality and
Diabetes insipidus Renal failure
hypernatremia Renal tubular disorder Nephrotic syndrome
Prolonged diarrhea Aldosterone deficiency
• Hyponatremia : most Profuse sweating Cancer
common electrolyte disorder Severe burns SIADH
Vomiting Hepatic cirrhosis
Fever Primary polydipsia
• NV: 135-145mmol/L Hyperventilation CNS abnormalities
Conn’s disease myxedema
• Convertion factor: 1
• 150-160mEq/L :moderate
water deficit
• >160mEq/L: severe water
deficit
 SODIUM
Specimen: Serum, Plasma, Urine
• Plasma: Lithium Heparin, Ammonium Heparin, Lithium
Oxalate
• Urine: 24hr collection
• Sweat is also suitable for analysis
Methods:
• Chemical methods are outdated
• Flame emission, Spectrophotometry, AAS
• ISE – most routinely used method in clinical laboratories
 POTASSIUM
• Major intracellular cation
• Counterbalance of sodium
• Single most important analyte
Functions:
• Heart contraction
• Neuromuscular
excitability
• Hydrogen ion
concentration
• ICF volume regulation
• Reabsorbed in the ascending
loop of henle
• NV: 3.5 -5.2mmol/L
• Clinical significance
HYPERKALEMIA HYPOKALEMIA
Acute/chronic Gastric
Renal failure Intestinal tumor
Severe dehydration Vomiting and Diarrhea
Addison’s disease Renal loss
Hyperaldosteronin
-Tacrolimus and Cushing syndrome
cyclosporine Leukemia
Bartter’s syndrome
Gitelman’s syndrome
Liddle’s syndome
Malignant hypertension
Alkalosis and Insulin
overdose
 POTASSIUM
• Major intracellular cation
• Counterbalance of sodium
• Single most important analyte
• Functions:
• Heart contraction
• Neuromuscular
excitability
• Hydrogen ion
concentration
• ICF volume regulation
• Reabsorbed in the ascending
loop of henle
• NV: 3.5 -5.2mmol/L
• Collection
– 0.5% RBC hemolysis = increased K +
level by 0.5mmol/L
– Platelet release K during clot
formation
– Tourniquet application should not be
prolonged
– Heparinized plasma preferred
• Methods
– FEP
– ISE (valicomycin gel)
– AAS
– Colorimetry (Lockhead and purcell)
 CHLORIDE
• Major extracellular anion
• Counter ion of sodium • Clinical significance
• Water balance and osmotic
pressure
HYPERCHOLEREMIA HYPOCHLOREMIA
Renal tubular acidosis Prolonged vomiting
• Functions: Diabetes insipidus Aldosterone deficiency
• Maintain osmolality Salicylate intoxication Metabolic alkalosis
Primary Salt-Loving nephritis
• Blood volume
hyperparathyroidism
• Electric neutrality Metabolic acidosis
Prolonged diarrhea
• Sweat Chloride test:
• Pilocarpine iontophoresis
• NV: 98-107 mmol/L
 CHLORIDE
• Methods
METHOD INDICATOR PRODUCT
Mercuric titration Diphenylcarbazone HgCl2
(Shales and Schales) (Blue-violet)
Spectrophotometry a. Mercuric Thiocyanate
a.Whitehorn titration (reddish complex)
b. Colored complex
b.Ferric Perchlorate
Coulometric Spx: Sweat
Amperometric Titration
(Cotlove Chloridometer)
Ion Selective Electrode Tri-n-octylpropylammonium
chloride decanol
 CALCIUM
• Functions:
FACTORS AFFECTING CALCIUM LEVEL
• Coagulation
• Enzyme activity • 1,25 Dihydrocholecalciferol and PTH
• Cardiac and skeletal • Ca reabsoption – Ca from
excitability bones goes back to blood=
• BP maintenance Increase in Ca levels
• NV:
• Calcitonin
Total ionized
• Main hypocalcemic hormone
8.6-10 mg/dl (adult) 4.6-5.3mg/dl (adult) (Ca blood to bone)
8.8-10.8mg/dl 4.8-5.5mg/dl (child)
(child)

• Low albumin=low calcium

 CALCIUM
• DISTRIBUTION: 99% - bone / 1% - blood & other ECF, cytosol is
only little
• In blood: 45% circulates as free Ca ions, 40% bound to protein
mostly albumin, 15% bound to anions such as HCO3, citrate,
and lactate.
CALCIUM
Clinical significance
HYPERCALCEMIA
Primary hyperparathyroidism
Milk-Alkali syndrome
Cancer (lung/breast)
Hyperthyroidism
Iatrogenic causes
Hyperparathyroidism
Sarcoidosis
HYPOCALCEMIA
Primary hypoparathyroidism
Acute pancreatitis
Hypomagnesemia
Malabsorption syndrome
Calcitonin
Hypoparathyroidism
Alkalosis
Renal tubular failure
Vitamin d deficit
 CALCIUM
• Specimen: Serum or Lithium Heparin Plasma
• EDTA or Oxalate are interferences.
• In urine: timed urine collection is preferred.
- Should be acidified with 6mol/L HCl approx. 1ml of the acid for
each 100 ml of urine
CALCIUM
Methods
METHOD REAGENT PRODUCT
Clark Collip Precipitation Oxalic acid (purple)

Ferro Ham Chloranilate Chloranilate (purple

Precipitation
O-Cresolphthalein Dye: Arzeno II
Complexone Dyes
(colorimetric) Mg+ inhibitor: 8-
hydroxyquinoline (chelator)
EDTA Titration Method
(Bachra, Dawer, and
Sobel)
ISE (Liquid Membrane)
and Flame emission
Photometry
AAS- Reference Method
 MAGNESIUM
• fourth most abundant cation • Clinical significance
• Enzyme activator HYPERMAGNESEMIA HYPOMAGNESEMIA
• Vasodilator
Diabetic coma Acute renal failure
• Life threatening: 5mmol/L
Addison’s disease Malnutrition
• Functions: Chronic renal failure Malabsorption syndrome
• DNA, RNA, Ribosome Chronic alcoholism
structure maintenance Severe diarrhea
• Neuromuscular
transmission
• Cofactor

• NV: 1.2-2.1 mEq/L

MAGNESIUM
• Methods

METHOD PRODUCT
Calmagite (+) reddish violet complex
Formazen dye (+) colored complex
Mg+2 thymol blue (+) colored complex
AAS Reference method
(Mg+2 is unexcitable
Dye lake Titan yellow dye
(clayton yellow or thiazole
yellow)
 PHOSPHATE
• May be absorbed from dietary sources, released from cells
into blood, lost from bone.
• Renal regulation of phosphate: vitamin D, calcitonin, growth
hormone, and acid-base status / PTH is most important factor.
PHOSPHATE
• INVERSE: relationship to • Clinical significance
calcium
• Maximally absorbed in HYPERPHOSPHATEMIA HYPOPHOSPHATEMIA
jejunum
Hypoparathyroidism Alcoholism
• Mostly in INORGANIC Renal Tubular Failure Avataminosis D
FORM Lymphoblastic leukemia Myexedema
Hypervitaminosis D hyperparathyroidism
• NV: 2.4-4.4 mg/dl (adult)
4.5-5.5 mg/dl (child)
1.45-2.91 mg/dl (neonate)
• Types:
• Organic
• Inorganic
PHOSPHATE
• Methods
METHOD REAGENT PRODUCT
Fiske Subbarow method Ammonium- (reduced form)
(ammonium molybdate) phoshomolybdate Blue
complex @600-700nm
@340nm
 ENZYMES
Hasten or catalyze chemical reaction
ENZYMES • Factors affecting enzymatic reactions
• Assay: measured in Enzyme concentration
terms of activity
Substrate concentration
not in terms of
Cofactor
absolute value a. coenzyme
b. activators
• Used as marker for Inhibitors
clinical situration a. competitive
such as in organ b. non-competitive
c. uncompetitive
damage
Isoenzyme
 ENZYMES
• Factors affecting enzymatic reactions • Other fators

Enzyme concentration Temperature

Substrate concentration Power of hydrogen
Cofactor Storage
a. coenzyme
b. activators Hemolysis
Inhibitors Lactescnce or milky
a. competitive specimen
b. non-competitive
c. uncompetitive
Isoenzyme
ENZYMES
• Enzyme classification
CLASS FUNCTION EXAMPLE
oxidoreductase Remove/add electrons CO,LDH,MDH,ICD,G-6-PD

transferase Transfer chemical group CK,AST,ALT

other than hydrogen from
one substrate to another
hydrolase Spilt bond by water addition Esterases
Peptidases
Glycosidases
lyase Removal of groups w/o GLDH, pyruvate,
hydrolysis decaboxylase, aldolase
isomerase Intermolecular arrangement Glucose PO4 isomerase and
of substrate compound ribose PO4 isomerase
ligases Joining two substrate; synthase
breaking of pyroPO4 bond in
ATP or similar compound
ENZYMES
ENZYME THEORY
Emil Fisher’s / lock and key
Kochland / induced fit
ENZYME REACTION
Zero-Order reaction
First-Order reaction
Shape of substrate must fit
into the enzyme
Based on substrate binding
to the active site of enzyme
Depends on ENZYME
concentration
Directly proportional to
substrate concentration
ALKALINE PHOSPHATASE
(ALP) • ALP Method
Liver ALP – Eletrophoresis
Bone ALP – Heat fractionization/stability test
Placental ALP • Most heat stable
• Most heat labile
Intestinal ALP
– Chemical inhibition test
pH=9.0 L-Phenylalanine placental, intestinal
ALP, Regan and Nagao
3M Urea Bone ALP
Levamisole Liver and Bone ALP
Homoarginine Liver Bone
L-Leucine Nagao
– Bowers and McComb
• Most specific
ALKALINE PHOSPHATASE
(ALP)

METHODS SUBSTRATE END PRODUCT

a. Bodansky Beta-glyceroPO4 Inorganic PO4 + Glycerol
b. Shinowara
c. Brock Bessy Lowry P- nitrophenylPO4 P-nitrophenol or yellow
d. Bowers McComb (PNPP) nitrophenoxide ion
e. King-Armstrong Phenylphosphate End Point requires
protein removal
ALKALINE PHOSPHATASE
(ALP)
• Clinical significance
– Osteitis deformans
– Obstructive jaundice
– Osteomalacia
– Rickets
– Osteoblastic bone tumors
– Sprue
– Hyperparathyroidism
– Hepatitis and cirrhosis
ACID PHOSPHATASE
(ALP) – Diagnostic significance
• Tissue Sources • Prostatic carcinoma

• Prostate
– pH : 5.0
• RBC
• Platelets
• Liver
• bone
ACID PHOSPHATASE
(ACP)

METHODS SUBSTRATE END PRODUCT

a. Gutman and Gutman phenyl PO4 Inorganic PO4
b. Shinowara PNPP P-nitrophenol
c. Babson, Read and Alpha NaphtylPO4 Alpha-naphtol
Philips
d. Roy and Hillman Thymolphthalein Free thymophthalein
monoPO$
ACID PHOSPHATASE
(ACP)
• Clinical significance
– prostatic carcinoma
– Breast, lung and thyroid carcinoma
– Gaucher’s disease
– Niemann Pick Disease
ASPARTATE ALANINE
AMINOTRANFERASE AMINOTRANFERASE
• Tissue Sources • Tissue Sources
• Cardiac • Liver
• Liver • METHOD
• Skeletal muscles • Reitman Frankel
• Products reaction
• 7.5
• Oxaloacetate • 340nm
• Glutamate
• Methods
– Karmen Method
• Ph:7.5
• 340nm
• Pyridoxal
• Malate dehydrogenase
 Summary of AST AND ALT
AST/SGOT ALT/SGPT

Major organ affected Heart Liver

Substrate Aspartic alpha- Alanine alpha –

ketoglutarate ketoglutarate
End product Glutamate + oxalate Glutamate + Pyruvate

Color developer 2,4 DNPH 2,4 DNPH

Color intensifier 0.4 N NaOH 0.4 NaOH

Methods Reitman and Frankel Reitman and Frankel

CLINICAL SIGNIFICANCE
AST AND ALT
AST ALT
 AMYLASE
• Earliest Pancreatic Marker • Methods
• Major tissue source
Saccharogenic
• Acinar cells
Amyloclastic
• Salivary glands
Chromogenic
• Most anodal Coupled-enzyme
• Salivary amylase

• Normal AMS:

• creatinine ratio =
1.4%
AMYLASE
• Clinical significance
INCREASED SERUM OTHER PANCREATIC
AMYLASE MARKER
Acute pancreatitis Lipase
Ectopic Pregnancy Trypsin
Peptic ulcer Chymotrypsin
Alcoholism Elastase 1
Mumps

• Remember
– Wheat Germ Lectin
• Inhibits salivary AMS
 LIPASE
• PRODUCT • Methods
• Alcohol + fatty acids METHOD SUBSTRATE PRODUCT
• Major tissue source Cherry Olive oil (old) Fatty acids
• pancreas Crandal Triolein
(new)
• Most specific Pancreatic
Tietz and
Marker Fiereck
Peroxidase
coupling
 LACTATE DEHYDROGENASE
(LDH)
• PRODUCT
• COLLECTION
• Alcohol + fatty acids
• Tissue source – LACTATE
• More specific substrate compared to
• LD-1 & LD-2
pyruvate
• Heart
• RBC
– LD1
• Forward reaction is preferred
• Kidney
• LD-3 – LD5
• Lungs • Reverse reaction is preferred
• Spleen – Stable at RT for 48 hours
• Pancreas
– Do not store in freezing
• LD-4 temperature
• Skeletal muscles
• Liver
• intestine
 LACTATE DEHYDROGENASE
(LDH)
Methods
• Wacker method (pH 8.8)
• Wrobleuski La Due (pH 7.2)
• Wrobleuski Cabaud
• Berger broida
Clinical significance
• Anemia
• Myocardial infarction
• Leukemia
• Renal infarction
• Hepatitis and hepatic cancer
• Muscular dystrophy
• Delirium tremens
• Malignancy and P. jerovecci
pneumonia
CREATININE
KINASE
• 3 isoenzyme • COLLECTION
• CK-1 – CK is inactivated by light
• CK -BB – Liver cell and RBC do not contain CK
• CK-2 – Hemolysis of >320mg/dl, adenylate
kinase interferes with CK assay
• CK-MB
• CK-3
• CK-MM • METHODS
– Tanzer- Gilbarg Assay (forward/direct)
• pH 9.0
• 340nm
– Oliver – Rosalki (reverse/indirect)
• pH 8.0
• 340nm
CREATININE KINASE
Clinical significance
PRONOUNCED ELEVATION MILD OR MODERATE
ELEVATION
Duchenne’s muscular dystrophy Severe exercise
Polymyositis Trauma
Dermatomyositis Surgery
Myocardial infarction Intramuscular injection

Delirium tremens
Alcoholic myopathy
Severe ischemic injury

Pulmonary infarction
Edema

Hypothyroidism

Acute agitated psychoses

 ALDOLASE
• Isoenzymes • TEST
• Aldolase A – SIBLEY LEHNINGER
•Skeletal muscles • Increased elevation in muscular
• Aldolase B dystrophy
•WBC, Liver, Kidney
• Aldolase C • 5’ nucleotidase
•Brain tissue
– Marker for HEPATOBILIARY
DISEASE
• NV: 0-1.6 Units
– Method
• Dixon & Purdon
• Campbell
• Belfield & goldberg
 GAMMA GLUTAMYL TRANFERASE
• LOCATION: • Elevated in
• canaliculi of hepatic cells – Warfarin
• EC lining biliary ductules – Phenobarbital
• Kidney – Phenytoin therapies
• Prostate
• Pancreas
• Method
– Szass
– Rosalki & Tarrow
• SUBSTRATE:
– Orlowski
• Gamma glutamyl-p-
nitroanilide • Sensitive indicator of chronic
• NV: alcoholism
• Female: 5-30 U/L
• Male : 6-45 U/L
PSEUDOCHOLINESTERASE
Marker for organophosphate poisoning

• Tissue source
• liver
• myocardium
• pancreas

• Method:
• ELLMAN Technique
• Potentiometric
ANGIOTENSIN CONVERTING ENZYME

• Tissue sources
• lungs
• testes
• macrophages
• pancreas
• Possible indicator of neuronal dysfunction
• alzheimer’s disease
• Increased
• sarcoidosis
• MS
• addison’s
• bronchitis
• HIV
• leprosy