1 Clinical Chemistry II Prelims (Bandala)

RENAL ANATOMY & PHYSIOLOGY: OVERVIEW

KIDNEY
- Bean-shaped
- Reddish-brown organ
- 11cm long
- Posterior abdominal wall either side of the vertebral column between T12 and L3
- The right kidney lies slightly lower than the left, as it is restricted superiorly by the large right lobe of the liver
- Each kidney is held to the posterior abdominal wall behind the parietal peritoneum and is partially protected by the lower ribs.

Major parts:
Cortex Renal Functions Renal Functions (Strasinger)
Medulla a. Filters blood a. Renal blood flow
Pelvis
b. Excrete wastes and excess b. Glomerular filtration
Nephron functional unit of the kidney
water thru urine c. Tubular reabsorption
Cortical
c. Regulate blood pressure, d. Tubular secretion
- Found in the renal cortex
volume pH and salt
- Peritubular capillary
- Upper glomerulus
Juxtamedullary
- Vasa recta (straight vessels)
- Lower glomerulus

1. Renal blood flow

How blood is filtered
Track down blood flow into and out of the kidney
a. Renal artery
b. Afferent arteriole
c. Glomerulus
d. Efferent arteriole
e. Peritubular capillaries
f. Vasa Recta
g. Renal vein
2. Glomerular Filtration
- Non-selective filter
o substances having MW of <70k daltons may pass thru and become
part filtrate
- Renin-Angiotensin-Aldosterone System
3. Tubular Reabsorption
- Substances are removed from filtrate and returned to the blood through
cellular transport mechanism
o Active transport o Passive transport
Glucose Water
Amino acids
Urea
Salts
Chloride ions
Sodium
Sodium ions
4. Tubular Secretion
- Involves passage of substances from the blood in the peritubular
capillaries to the tubular filtrates
- 2 major roles
o Elimination of waste products not filtered buy the glomerulus
o Regulation of acid base balance in the body through secretion of
hydrogen ions
RENAL FUNTION TESTS:
NON-PROTEIN NITROGEN COMPOUNDS
o Glomerular Filtration Test (GTFs)
o Tubular Reabsorption Tests
o Tubular Secretion Tests
2 Clinical Chemistry II Prelims (Bandala)
o Renal Blood Flow Tests
A. Glomerular Filtration Tests (GFTs)
a. Clearance test
- Standard test used to measure the filtering capacity of the glomeruli
- Measures the rate at which the kidneys are able to remove a filterable
substance from the blood
o Urea clearance test
o Creatinine clearance test
o Insulin clearance reference method
o Cystatin C
- To ensure accuracy of the test, substance to be analysed must:
o Neither reabsorbed nor secreted
o Stability in the urine during a possible 24-hour collection period
o Consistency of plasma level
o Availability to the body
o Availability of the test for chemical analysis of the substance
B. Tubular Reabsorption Tests
- Often the first function to be affected in renal disease
o Fishberg Test
- Concentration test
- Patients deprived of fluid for
o Tests to determine the ability of the tubules to reabsorb the essential salts and 24 hours prior to measuring
water that have been non-selectively filtered by the glomerulus the specific gravity
Osmolarity
Free water clearance o Mosenthal Test
C. Tubular Secretion Tests & Renal Blood Flow Tests - Compared the volume of day
- To measure the exact amount of blood flowing through the kidney and night urine samples to
- it is necessary to use a substance that is completely removed from the blood evaluate concentrating ability
(peritubular capillaries) rather than being removed when the blood reaches the
glomerulus
o Para-aminohippuric acid (PAH) test
Measures renal plasma flow
o Titratable acidity
o Urinary ammonia ]
Secretion of hydrogen ions & production and
secretion of NH4 by cells in DCT
+

A. GLOMERULAR FILTRATION TESTS

UREA
- Main waste product of nitrogen-containing chemical in the body
- Major excretory product of protein metabolism
- Synthesized in the liver from CO2 and ammonia from the deamination of amino acids (Krebs cycle)
- Widely used as a measure of renal dysfunction but its value as a measure of GFR is not plausible
- Dependent to the rate of urea production and not solely to renal function
o Freely filtered by the glomerulus bit is reabsorbed (approximately 40%) in some parts of nephrons
- Clinical significance
o Azotemia
Increase of all UREA Pre-renal Renal Post-renal nitrogen containing

compound - Congestive heart failure - Acute and chronic renal Urinary tract
3 conditions leading obstruction to azotemia
- Shock, hemorrhage failure
Pre-renal
- Dehydration - Renal disease Renal
Post-renal
- Increased protein catabolism - Glomerular nephritis

o Uremia
- - High-protein diet
- Tubular necrosis Increased urea in blood

Why measure urea?

o
- Low protein intake
To evaluate renal function
- Severe vomiting and diarrhea To assess hydration

- Liver disease status

To determine nitrogen
- Pregnancy
balance
To aid in the diagnosis of renal disease
3 Clinical Chemistry II Prelims (Bandala)
To verify adequacy of dialysis
Very important conversion factors;
Analytic / Laboratory Methods:
o Urea Nitrogen x 2.14 = Urea (mg/dL)
o Measurements were originally performed on a PFF of whole blood based on
o Urea Nitrogen (mg/dL) x 0.36 Urea (mmol/L)
measurement of nitrogen
o Current: nitrogen concentration
3 methods:
o Conventional Method
o Kinetic Method
o Chemical Method
Isotopic dilution mass spectrometry
Gold standard; Reference method
o Conventional Method
Hydrolysis of Urea by Urease
Quantification of NH4+
2
Nesslers Reagent (yellow)
Berthelots Reagent with nitruprusside (blue)
++CO 3
o Kinetic Method (Coupled-Enzyme) Urea+2 H 2 O Urease 2 NH 4
Urease and Glutamate Dehydrogenase (GLDH)
Measures the disappearance of NADH at 340 nm
Used on common automated machines
Reference method
o Chemical Methods
Diacetyl Monoxime (Direct Method)
Urea reacts directly with diacetyl monixime in
strong acidic condition to form a yellow diazine Principle:
derivative Urea in the sample reacts with ortho-Phthalaldehyde in acid medium
Ferric iron and thiosemicarbazide forming a coloured complex that can be measured by
Stabilizes color spectrophotometry:
intensifies reaction
H + Isoindoline
ortho-Phthalaldehyde Method (Direct Method)
ortho-Phthalaldehyde

Urea+ phthalaldehyde
Naphthylethylenediamine
The intensity of the color formed is proportional to the urea concentration
Specimen Requirement & Interfering Substances of the sample
a.
Plasma
o Not with
ammonium ions
Na citrate
Adult Urea Nitrogen
Na fluoride inhibit urease
Plasma/Serum 6-20 mg/dL (2.1~7.1 mmol urea/day)
o Fasting is NOT required
b. Serum no hemolysis
Urine, 24-h 12~20 g/day (0.43~0.71 mol urea/day)
c. Urine
o Should be refrigerated if cannot be analysed
within an hour
Urea is susceptible to bacterial decomposition

CREATININE
- Synthesized primarily in the liver from
o Arginine
o Glycine
o Methionine
- Transported to tissues and is converted to phosphocreatine
o Serves as a high-energy source
- Endogenous substance
- With a MW of 113 Da
- Waste product of muscle metabolism
o Produced by the muscle from creatine and creatine
phosphate thru non-enzymatic dehydration process
 4 Clinical Chemistry II Prelims (Bandala)
- Released into the circulation at a relative constant rate in
proportional to an individuals muscle mass
Creatine Creatinine
Muscle diseases Associated with abnormal
- Muscular dystrophy renal function
-
-
-
Poliomyelitis
Hyperthyroidism
Trauma
( mgdL ) U ( 24mL
U Cr Cr
hours
)

1.73
mg 1440 minutes A
P (
dL )
- Most widely used marker for GFR
Cr
o Advantages 24 hours
Endogenous substance with constant rate of production
Not bound to plasma proteins Cockroft-Gault formula (mL/minute) (Cockroft, 1976)
Not reabsorbed by the tubules ( [ 140 Age ] [ IBW ] )
Only a small amount is secreted by the tubules ; multiply by 0.85 if female
- Why measure creatinine? (72 SCr)
o To determine sufficiency of kidney function and the severity of IBW = ideal body weight
kidney damage SCr = serum creatinine concentration
IBW is calculated by the ff. formula:
o To monitor progression of kidney disease Males: IBW = 50kg + 2.3 kg for each inch over 5
ft

GLOMERULAR FILTRATION TESTS: CREATININE (cont.)

1. Direct Jaffe Reaction (1886)
- Creatinine reacts with picric acid (trinitropenol) in alkaline solution to form a red-orange chromogen
Folin-Wu Method (1919)
Nonspecific
Interferences
Acetoacetate
Acetone
Ascorbate
Glucose
Pyruvate
Fullers Earth
PFF + aluminum magnesium silicate
Method of Hare (addition to Lloyds reagent)
PFF + sodium aluminum silicate
Eluted and reacted with alkaline picrate

2. Kinetic Jaffe Reaction

- Performed directly on sample
- Serum is mixed with alkaline picrate and rate of change in absorbance is measured
- Interferences
Alpha-ketoacid
Cephalosporin
3. Coupled-Enzymatic Method
a. Creatininase-CK Method
Creatininase
CK, PK, LDH
NADH to NAD+ (340 nm)
b. Creatininase-Hydrogen Peroxide Method
Creatininase, creatinase
Sarcosine oxidase, Peroxidase
4. Reference Standard
5 Clinical Chemistry II Prelims
Enzymatic Methods (Bandala)
Isotope dilution-mass spectrometry
Creatinine+ H 2 O Creatininase Creatine
Plasma, Serum
o Hemolyzed and icteric
Creatine+ ATP CK Creatine phosphate+ ADP
samples should be

Creatininase-CK
Phosphoenolpyruvate+ ADP PK pyruvate+ ATPavoided

+
+ LD Lactate+ NAD

Pyruvate+ NADH + H
Creatinine+ H 2 O Creatininase Creatine

Creatine H 2 O Creatininase Sarcosine+Urea

Creatininase-H2O2
Sarcosine +O 2 + H 2 O Sarcosine oxidase Glycine+CH 2 O+ H 2 O 2

H 2 O2 +colorless substrate Peroxidase Colored product + H 2 O

Lipemic samples produce erroneous results
o Fasting is NOT required
Urine
o Urine specimen should be refrigerated or frozen if longer than 4 days.
GLOMERULAR FILTRATION TESTS: CREATININE (CONT.)
Interfering substances in urine
Ascorbic acid interfere peroxidase Secretion of hydrogen
Glucose ions & production and
Protein, urea secretion of NH4+ by
Alpha-ketoacids/ketones cells in DCT
Cephalosporins, Dopamine, Lidocaine
o False increase
Hemoglobin and bilirubin
o false decrease
Uric acid
o Decreased in kinetic methods
INULIN
- Polymer of fructose
- Extremely stable substance that is not reabsorbed or secreted by the tubules
- Not a normal body constituent
- Exogenous procedure
BETA2-MICROGLOBULIN
- Has MW of approximately 11,800 Da
- Dissociates from human leukocytes antigens at a constant rate
- Rapidly removed from the plasma by glomerular filtration then reabsorbed completely by the PCT
- Not reliable in patients with immunologic conditions ( production)
Multiple myeloma
Lymphoma
CYSTATIN C
- Has MW of app 13,000 Da
- Inhibitor of cysteine proteinase
- Produced by all nucleated cells at a constant rate
- Freely filtered and completely reabsorbed in the PCT (normally not seen in urine)
- Plasma concentrations appear to be unaffected by gender, race, age and muscle mass
- Measurements are difficult and expensive NOT routinely used

Other GF tests
Beta trace protein
Tryptophan glycoconjugate
Use of Radioisotopes
6 Clinical Chemistry II Prelims (Bandala)
7 Clinical Chemistry II Prelims (Bandala)

OTHER RENAL FUNCTION TESTS

URIC ACID
- Metabolic waste product of purine metabolism
- Purines (A & G) from the breakdown of ingested nucleic acids or from tissue destruction
- Converted to uric acid in the liver
Filtered by glomerulus
98-100% reabsorbed by the PCT
Small amount are secreted by the DCT
- Present as monosodium urate in the plasma
Relatively insoluble (blood pH)
At concentrations greater than 6.4 mg/dL,
the plasma is saturated
Resulting to urate crystals formations
Precipitation in the tissues and joints
OTHER RENAL TESTS: URIC ACID (CONT)
Why measure?
To assess inherited disorders of purine metabolism
To confirm diagnosis and monitor treatment of gout
To assist in the diagnosis of renal calculi
To prevent uric nephropathy during chemotherapeutic treatment
To detect kidney dysfunction
- Disease Correlation
Elevated
a. Gout
Precipitation of sodium urates in the joints
Cause hyperuricemia
o Overproduction of UA
o Purine rich diet, drugs or alcohols
Prone to renal calculi
Tophi formations (tissues)
b. Increased catabolism of nucleic acids
c. Renal diseases
Chronic - Filtration and secretion are impaired
Increased metabolism of cell nuclei
In patients with proliferative disorders on chemotherapy
o Leukemia
o Lymphoma
o Multiple myeloma
o Polycythemia
Monitoring UA level to avoid nephrotoxicity Lesch Nyhan syndrome
o Hemolytic or megaloblastic anemia
o Glycogen storage disease
X-linked (males) genetic disorder
G-6-PD deficiency Complete deficiency of hypoxanthine
Lesch Nyhan syndrome guanine phosphoribosyltransferase
- Hyperuricemia Synthesis of purines
8 Clinical Chemistry II Prelims (Bandala)
Toxemia of pregnancy and lactic acidosis
Ingestion of purine-rich diet Defective Tubular Reabsorption
Liver o Shellfish o Fanconis Syndrome
Kidney o Legume Disorder in which the proximal tubular function of
Starvation the kidney is impaired
- Hypouricemia
o Chemotherapy
Secondary to liver disease
6-mercaptopurine or azathioprine
Defective Tubular Reabsorption
Decrease DNA & RNA formation
Overtreatment with allopurinol
Treat gout, inhibits xanthine oxidase
9 Clinical Chemistry II Prelims (Bandala)

URIC ACID: ANALYTICAL METHOD

a. CARAWAY METHOD
Based on the oxidation of us in PFF,
with subsequent reduction of Na 2 CO 3 /OH
phosphotungstic acid to tungsten blue
Uric acid + H 3 PW 12 O 40+O2 Sodium carbonate allantn +tung
Sodium carbonate
provides alkaline pH for color development
Lacks specificity

b. URICASE METHOD
Uricase catalyzed the oxidation of UA to allantoin
Measures the differential absorption of uric acid Uric acid+2 H 2 O+O2 Uricase allantoin+CO 2+ H 2 O2

and allantoin at 293 nm
More specific
Interfering substances (negative)
Hemoglobin
Xanthine

c. COUPLE ENZYMATIC METHODS Uric acid+2 H 2 O+O2 Uricase allantoin+CO 2+ H 2

Measures hydrogen peroxide produced as UA is converted to
allantoin
o Peroxidase or catalase H 2 O2 +indicator dye Peroxidase colered compound

Catalyze a chemical indicator reaction or
Color produced is proportional to UA in the specimen
H 2 O2 +CH 3 OH Catalase H 2 CO+2 H 2 O
Bilirubin and ascorbic acid destroy peroxide if sufficient

URIC ACID SPECIMENT REQUIREMENT

Heparinized plasma, serum or urine
o Serum
Should be removed from cells ASAP to prevent dilution by intracellular contents
Fasting is NOT required
Avoid lipemic specimens
Hemolysis decreases value because of glutathione release
High bilirubin concentration
Ascorbic acid
o Falsely decrease results to peroxidase methods
o Addition of potassium ferricyanide and ascorbate oxidase
Drugs
o Salicylates
o Thiazides Falsely increase results

AMMONIA
- Analytical/Lab Methods
Complicated by its
Low concentration
Instability
 Increased Decreased
10 Clinical Chemistry II Prelims
(Bandala)
Ammonium salts Diphenhydramine
Pervasive contamination
Two approaches
Asparginase Lactobacillus
a. Two-step approach in Barbituates acidophilus which ammonia is isolated from the
sample and then Diuretics Lactulose assayed
b. Involves direct measurement of ammonia by an
Ethanol Ledovia
enzymatic method or ion selective electrode
GLUTAMATE Hyperalimentation Several antibiotics
DEHYDROGENASE ASSAY
- Specimen requirement Narcotic analgesics
Whole blood ammonia
concentration increases rapidly following specimen collection because of in vitro amino
acid deamination
Venous blood should be obtained without trauma and placed on ice immediately
Heparin and EDTA
Samples should be centrifuged at 0C to 4C within 20 minutes of
collection and plasma or serum is removed
Hemolysis should be avoided
TOPIC 3 Falciform
LIVER FUNCTION ligament
Liver - separates
Anatomy: right and
Anterior to the gallbladder left lobes
storage of bile
of the liver
o emulsifies fats
o purely for excretion of waste
products
where the excretory waste products
of liver are passed out
Blood supplies:
o Hepatic artery
- contains oxygenated blood
o Portal vein
- Contains nutrient rich blood from
Intestines
Stomach
Spleen
- Unoxygenated blood

Hepatic vein to
circulation
- Waste Common bile duct
Hepatic lobules
o Functional unit
o 6 sided
o Portal triad
Bile duct
Hepatic artery
Portal vein sinusoids
Hepatocyte
o 80-90%
o Aids in regeneration
Increase proliferation and cell growth
Kupffer cell
responsible for phagocytosis of unwanted
substances
found alongside of hepatocyte
Physiology:
4 general functions
o Excretion and secretion
o Synthesis
o Detoxification
o Storage
11 Clinical Chemistry II Prelims (Bandala)
a. Synthesis
One of the most important function:
Metabolism of carbohydrates
o Use the glucose for its own cellular energy requirements
o Circulate the glucose for use at the peripheral tissues
o Store glucose as glycogen
o Responsible for gathering free fatty acids and breaking
them down to produce acetyl-CoA
o Acetyl coA helps in synthesis of triglyceride and
o Almost all proteins (albumin) are synthesized by the liver
b. Detoxification and drug metabolism
2 mechanisms:
Bind the material reversibly as to inactivate the compound
Chemically modify the compound so it can be excreted

c. Excretion/Secretion
Secretory Functions
Bile
Emulsifying agent
Facilitate fat digestion
Purely of waste product
Has no enzymes
o Bile acids
Cholesterol
o Cholic acids
o Chenodeoxycholic acid
o Bile salts
o Bile pigments
Bilirubin main waste product

bilirubin( B 1) uridyl diphosphate glucoronyl transferase bilirubin diglucoronide( B 2)

2 UDP-glucoronic 2 UDP
acid
Uridyl Diphosphate Glucuronyl Transferase (UDPGT)
enzyme responsible for the conjugation of
B1, forming B2
B1 B2
Unconjugated bilirubin Conjugated bilirubin
Water insoluble Water Soluble
Non-Polar Bilirubin Polar Bilirubin
Indirect reacting Direct reacting
Hemobilirubin Cholebilirubin
Free bilirubin/Slow One-minute/Prompt bilirubin
Pre hepatic bilirubin Post hepatic bilirubin
Bilirubin monoglucoronide Bilirubin diglucoronide

CLINICAL SIGNIFICANCE
JAUNDICE
Icterus term used for sampes
Characterized by yellow discoloration
o Prehepatic jaundice
o Hepatic jaundice
o Post hepatic jaundice

Prehepatic Jaundice
o Excessive amount of bilirubin is presented go the liver for
metabolism
Hemolysis
Hemolytic anemia
Malaria
Increased B1
o Rarely have bilirubin levels that exceeds 5 mg/dL because the
liver is capable of handling the overload
o Unconjugated hyperbilirubinemia

Hepatic Jaundice
12 Clinical Chemistry II Prelims (Bandala)
o Result from impaired cellular uptake of bilirubin, defective
conjugation, abnormal secretion of bilirubin by the liver cell
Hepatic Jaundice:
B1
o Gilberts Syndrome elevated B1
Bilirubin transport deficit Gilberts Syndrome
Characterized by impaired cellular uptake of bilirubin Crigler-Najjar Syndrome
Characterized by a 70-80% reduction in the Lucey-Driscoll Syndrome
glucuronidation activity of the enzyme, UGT1A1
Characterized by intermittent unconjugated B2
hyperbilirubinemia in the absence of hemolysis and Dubin-Johnson Syndrome
underlying liver disease due to a defective Rotors Syndrome
conjugation system
No symptoms but may have mild icterus
o CriglerNajjar Syndrome
Conjugation deficit
Characterized by deficiency of the enzyme UDPGT
Type 1
o Complete absence of UDPGT
o Total absence of B2 Production
Type 2
o Less severe deficience of UDPGT
o Dubin-Johnson syndrome
Bilirubin Excretion Deficit
Transporters:
An autosomalorecessive
MRP2disease which Resistance
Multi-Drug presents shortly
Associated
after birth with an increase of conjugated bilirubin
Protein 2
without elevation of liver enzymes (ALT, AST)
o cMOAT2 - Canalicular Multispecific Organic
Defects in the adenosine triphosphate (ATP)-binding
Anion Transporter 2
cassette (ABC) canalicular multispecific organic anion
o ABCC2 gene that encodes MRP2
transporter, M2/cMOAT/ABCC2
Elevated total bilirubin and B2
o Rotors syndrome
Liver cells are not pigmented
Hypothesized to be due to a reduction in the concentration
or activity of intracellular binding proteins such as
ligandin
o Same symptoms with Dubin-Johnson Syndrome
o Increased B2
o Lucey-Driscoll Syndrome
Familial form of unconjugated hyperbilirubinemia caused
by a circulating inhibitor of bilirubin conjugated
Circulating enzyme inhibitors

Posthepatic Jaundice
Biliary obstructive jaundice
o Tumor
o Gallstones

*Cirrhosis
Scar tissue replaces normal, healthy liver tissue
Blocks the flow of blood through the organ and prevents the liver from
functioning properly
Secondary to chronic alcoholism and chronic hepatitis C virus infection

ANALYSIS OF BILIRUBIN
Ehrlichs reaction (1883)
o Urine bilirubin reacted to diazotized sulfanilic acid
Hydrochloric acid
Sulfanilic acid
o Classic diazo reaction
Van den Bergh (1913)
o Using serum
o Used alcohol accelerator for the coupling of bilirubin to
diazotized sulfanilic acid, solubilizing unconjugated bilirubin
Malloy-Evelyn method (1937)
o Serum bilirubin
o Classic diazo reaction with a 50% methanol solution as an
accelerator
o Final reaction: Pink to purple azobilirubin

Jendrassik and Grof (1938)

13 Clinical Chemistry II Prelims (Bandala)
o Serum bilirubin
o Lesser interference of hemoglobin
o Buffer: sodium acetate
o Accelerator:
Caffeine-benzoate-acetate
Caffein-sodium-benzoate
Ascorbic acid terminates reaction and destroys
excess diazo reagent, stopping the reaction
o Final reaction: Pink to blue azobilirubin
Icterus index
o Yellowishness of the serum or plasma
o 0.01% potassium dichromate
Liver enzymes
o Alkaline phosphatase (ALP)
o Alanine transaminase (ALT) / Aspartate transaminase (AST)
o 5N
o Gamma-glutamyl transferase (GGT)
o Leukocyte Alkaline Phosphatase (LAP)
o Lactate dehydrogenase (LDH)

TEST MEASURING THE HEPATIC SYNTHETIC ABILITY

Total Protein Determination
Prothrombin Time
Albumin

TEST FOR DETOXIFICATION FUNCTION

Enzyme tests
Ammonia
o Nesslerization Reaction
o Ghum gatti enzyme used in reaction principle

SPECIMEN REQUIREMENTS
Serum
o Free from hemolysis and lipemia
o Should be stored in the dark
If left unprotected from light, bilirubin values may
reduce by 30%-50% per hour
o Measure ASAP or within 2-3 hours after collection
 Clinical Chemistry II Prelims (Bandala)

ENZYMES
Enzymes Enzymology
- Biologic catalysts - Study of enzymes
- Hasten chemical reactions o Ac
o Not consumed during the reactions tivi
o Not
ty
undergoi
ng a
chemical
change
after the
reactions
Nomenclature of Enzymes
1. Substrate + -ase 3. Enzyme Commission Nomenclature (E.C.)
Lipid = lipase E.C. 1.1.1.21
Ester = esterase 1st digit = class
Protein = protease 2nd digit = subclass
Lactose = lactase 3rd and 4th = serial number
Starch = amylase

2. Reaction it catalyzes
Oxidase = oxidation
Reductase = reduction
Hydrolase = hydrolysis
Dehydrogenase = remove hydrogen atoms, transferring them to a coenzyme
Decarboxylase = remove carboxyl groups
CLASSIFICATIONS OF ENYZYMES
1. OXIDOREDUCTASES
Oxidation and reduction Classifations of Enzymes
o Oxidation removal of H ion 1. Oxidoreductases
o Reduction accept H ion 2. Transferases
o E.C. 1.1.1.27 : L-lactate
3. Hydrolases
NAD + Oxidoreductase
4. Lyases
Lactate Dehydrogenase
5. Isomerases
6. Ligases

2. TRANSFERASES
Transfer of functional groups other than hydrogen from one substrate to another
o E.C. 2.6.1.1 :
L-Aspartate: 2-Oxaloglutarate Aminotransferase
Aspartate Aminotransferase

3. HYDROLASES
Hydrolysis of various bonds
Addition of water to a bond resulting in bond breakage
 Clinical Chemistry II Prelims (Bandala)
o E.C. 3.2.1.1 : 1,4-D-GlucanGlucanohydrolase
Alpha-Amylase

4. LYASES
Addition of a group to a double bond or the removal of a group to form a double
bond
o Carbonic Anhydrase
o Citrate Lyase

5. ISOMERASES
Rearrange the functional groups within a molecule and catalyze the
conversion of one isomer into another
o Phosphoglycerate mutase

Prosthetic Organic compounds Heme

Group Tighly bound to the apoenzyme Flavine adenine dinucleotide (FAD)
Cofactor Organic cofactor Flavin mononucleotide (DMN)
Their association with the apoenzyme Thiamine pyrophosphate (TPP)
is only transient Nicotinamide adenine dinucleotide
Usually occurs during the course of (NAD)
catalysis Lipoic acid
Metal Ions A number of enzymes require metal Zn2+
ions for their activity Cu2+
Mg2+

6. LIGASES
Catalyze a reaction in which C-C, C-S, C-O, or C-N bond is made or broken
Accompanied by an ATP-ADP interconversion

TERMS
Substrate Acted upon by the enzyme; Specific
Isoenzyme Different form but with the same action
Cofactor Non-protein molecule
 Clinical Chemistry II Prelims (Bandala)

Activator Inorganic cofactor

Coenzyme Organic cofactor
Apoenzyme Polypeptide portion
Holoenzyme An active enzyme; Apoenzyme + Coenzyme
Proenyzme (Zymogen)
Inactive form of enzyme
It is then converted, usually by proteolysis, to the active form when it
has reached the site of its activity
ENZYME KINETICS
Michaelis Menten Theory

INDUCED FIT THEORY

o Daniel Koshland, 1958

LOCK AND KEY THEORY

o Emil Fischer, 1890

Factors That Influence Enzymatic Reactions

 Clinical Chemistry II Prelims (Bandala)
1. Substrate Concentration
First-order Reaction
Second-order Reaction
Zero-order Reaction
a. First-order Reactions
Proceed at a rate concentration of one reactant

Rate of reaction Rate of disappearance of A

Manganese or the appearance of P
b. Second-order
Zinc Reactions
Are those in which the rate is proportional to the product of the
Potassium ions
concentration of two reactants

Rate of reaction Rate of disappearance of A

or the appearance of P
c. Zero-order Reactions
The reactions are zero-order with respect to the reactants
Rate of reaction depends on the concentration of catalysts or on
some factor other than the concentration of the molecular species
undergoing reaction
2. Enzyme Concentration
The higher the enzyme level, the faster the reaction will proceed
3. pH
pH = 7.0 8.0
Changes in pH may denature the enzyme
Protein in nature
4. Temperature
37 degrees Celsius (normal)
Denaturation at 40-50 degrees Celsius
Assay temperatures:
o At 25, 30, or 37 degrees C
5. Cofactors
Nonprotein entities that must bind to particular
Activators
enzymes before a reaction occurs Proper substrate binding
Metallic or nonmetallic Linking substrate to the enzyme or
Calcium
coenzyme
Ferrous
Magnesium Undergoing oxidation or reduction

6. Inhibitors
Interfere with enzyme reactions
o Competitive
o Noncompetitive
o Uncompetitive
 Clinical Chemistry II Prelims (Bandala)

Enzymes Of Clinical Significance

Aminotransferases
Aspartate Aminotransferase (AST)
o Serum Glutamate Oxaloacetate Transaminase (SGOT)
Heart, Liver
Alanine Aminotransferase (ALT)
o Serum Glutamate Pyruvate Transaminase (SGPT)
Liver, Heart

Phosphatases
Alkaline Phosphatase (ALP)
o Bone
o Liver
Acid Phosphatase (ACP)
o Prostate Gland
o Red Blood Cells
Clinical Chemistry II Prelims (Bandala)