FINALS CLINICAL CHEMISTRY a.

globular: relatively symmetrical with compactly folded and

coiled polypeptide chains
CHAPTER 7: PROTEINS b. fibrous: more elongated and asymmetrical and have a
PROTEINS higher viscosity
● Biochemical essential for the body
2. CONJUGATED
STRUCTURE AND PHYSIOLOGY ● Protein (apoprotein) + nonprotein (prosthetic group)
● Proteins are MACROMOLECULES; they have a large ● Has 3 chemical composition of protein
structure as they are composed of linked polymers of ● Lipid content is present
amino acids
● Covalently linked polymers of amino acids Prosthetic group (3 chemical composition of protein):
● Amino acids are linked via PEPTIDE BOND (formed - Lipid (lipoprotein)
upon removal of a water molecule) - Carbohydrate (glycoprotein)
● Amphoteric - Metals (metalloprotein)
- Can be polar or nonpolar
- NonPolar: no charge FUNCTIONS
- Polar: Acidic (negative charge) or Basic - Repair body tissues
(positive charge) - Important in blood coagulation and immunologic
- Polar Neutral: Can either be positive or function
negative - For transport of metabolic substances
● Synthesized in the liver & secreted by the hepatocyte - Maintenance of osmotic pressure
into the circulation EXCEPT immunoglobulins - Maintenance of blood pH (buffers)
(plasma cells) - Biocatalysts: Induce metabolism, CATABOLISM
● Provide 12 – 20% of the total daily body energy specifically
requirement
● Composes 50 – 70% of the cell’s dry weight *In the formation of blood, coagulation proteins are highly
needed
AMINO ACIDS METABOLISM
- Secreted by HEPATOCYTES (cells in the liver) 1. Dietary Intake
- Synthesized in the LIVER 2. Absorption
- There are 20 ESSENTIAL amino acids 3. Production
4. Storage
PROTEIN VS. POLYPEPTIDE 5. Destruction
- Protein and Polypeptide can be interchangeable
MAJOR PLASMA PROTEINS
4 STRUCTURES 1. PRE-ALBUMIN (Transthyretin)
1. PRIMARY STRUCTURE ● Migrates ahead of albumin
● Sequence of the amino acids in the polypeptide ● Rich in tryptophan and contain 0.5% carbohydrate
chain ● Serves as transport protein for T3, T4 and retinol
● Analytical processes: chromatography, (Vit. A)
electrophoresis, dye binding and light absorbance RETINOL
depend on this sequence ● Purest form of Vitamin A

2. SECONDARY STRUCTURE RETINOID

● Arises from the interaction among the different ● Type of retinol
segments of a polypeptide chain
Electrophoresis
Three structures: - Involves anodes (+) and cathodes (-)
a. alpha-helix – chain forms a regular helix ; coil resembling a - Protein is a cathode, therefore it migrates in the
spring anode
b. beta-pleated sheets – in fully extended structures; flat, - Follows the principle of: Opposite Attracts
corrugated structure
Increased: alcoholism, chronic renal failure, steroid
c. random coils – no apparent pattern
treatment
Decreased: poor nutrition, liver disorder, malignancy
3. TERTIARY STRUCTURE
● actual 3-dimensional structure or folding pattern of
the protein 2. ALBUMIN
● Largest plasma protein fraction (52-62%)
4. QUATERNARY STRUCTURE ● Synthesized in the liver at a rate that is dependent on
● association of several polypeptide chains into larger protein intake
“oligomeric” aggregate unit ● Serves as circulating reservoir of amino acids
● Stable complexes: dimers, trimers, tetramers ● Regulator of osmotic pressure
● Transport protein because of ease of binding with
Classification blood components
1. SIMPLE ● “negative acute phase reactant”
● Contain peptide chains that on hydrolysis yield only ● Sensitive & highly prognostic marker in cases of
AA cystic fibrosis
● AMINO ACID ONLY! Reference values: 3.5 – 5.0 g/dL (35 – 50 g/L)

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 ● Increased albumin (Hyperalbuminemia):
- Hemoconcentration
- Dehydration
- Excessive albumin infusion
● Decreased albumin (Hypoalbuminemia):
- Decreased synthesis (liver impairment)
- Malabsorption or malnutrition
- Nephrotic syndrome (renal loss)
- Severe burns
* Albumin levels depends on the diet intake
3. GLOBULINS
● Heterogenous complex mixture of protein molecules
(α1, α2, β and γ fractions)
● Elevated concentration of globulin in early cirrhosis
(occurs in the liver) will balance loss of albumin
resulting to normal levels of total protein
- normal A/G ratio : 1.3 – 3 : 1
- low A/G ratio – liver diseases, infectious diseases, multiple
myeloma, nephritis
*Albumin/Globulin (A/G) A- 1.3 G- 3:1
Alpha1-globulins
a. α-1-antitrypsin
● 90% of the α1-globulin band
● Acute phase reactant
● Neutralize trypsin-like enzymes
● Major inhibitor of protease activity; inhibit lysosomal
elastase released from PMNs during their response to
particles & inhaled bacteria
● Major liver involvement
Increased: inflammation, pregnancy and contraceptive use
Decrease: associated with emphysematous pulmonary
disease & juvenile hepatic cirrhosis
b. α-1-fetoprotein
● Synthesized initially by the fetal yolk sac & then the
parenchymal cells of the liver
● Used as a tumor marker (hepatic & gonadal CA)
● Fetal involvement
● Screening test for any fetal conditions, increase
passage of fetal proteins into the amniotic fluid;
detects neural tube defects
Increased: neural tube defects (spina bifida), atresia of the
GIT, fetal distress, ataxia telangiectasia, tyrosinosis,
hemolytic disease of the newborn (HDN)
Decreased: Down's syndrome
c. α-1-Acid Glycoprotein (orosomucoid)
● Contains high percentage of CHO and sialic acid
(45% CHO + 11-12% Sialic acid)
● Synthesized both by the liver & by granulocytes and
monocytes (WBCs)
● Inhibits the phagocytic activity of neutrophils &
inhibits platelet aggregation
● May inactivate progesterone
● Presence of this affects blood coagulation
Increased: pregnancy, cancer, pneumonia, rheumatoid
arthritis (RA), cell proliferation
Decreased: nephrotic syndrome
d. α-1-Antichromotrypsin
Alpha2-globulins
A. HAPTOGLOBIN
BSMLS-2 GERRYN OLGA ABAN
● An acute phase reactant
● Synthesized in the hepatocytes & cells of the RES
● Binds free hemoglobin by its α- chain
● One of the proteins used to evaluate rheumatic
diseases
Increased: inflammation, nephrotic syndrome, burns,
trauma
Decreased: intravascular hemolysis and liver disease
B. CERULOPLASMIN
● Copper-containing protein BUT does NOT transport
copper; synthesized in the liver, where 6-8 copper
atoms are attached
● Serves as an antioxidant to prevent lipid peroxidation
& cellular damage
● Increased in inflammation and pregnancy
● Indicator for Wilson's disease (0.1 g/L)
● COMPLEX
● Does not transport copper, it only syntheize it
Decreased: Wilson’s disease, malnutrition, malabsorption,
nephrotic disease, Menke’s disease (kinky hair syndrome)
a. α-2-Macroglobulin
● Largest major non-immunoglobulin protein in plasma
● Found principally in the intravascular spaces; does
not diffuse from the plasma space
● Lower amounts can also be found in CSF
● Inhibits proteases such as trypsin, pepsin and
plasmin (3) Beta-globulins
Beta Globulins
a. Transferrin (Siderophilin)
● Major component of the β2-globulin fraction
● A glycoprotein synthesized in the liver
● Tansports oxidized iron (Fe+3) to its storage sites
● Prevents loss of iron through the kidney
Increased: hemochromatosis (bronze skin)
Decreased: liver disease, malnutrition, nephrotic syndrome
b. β2-Microglobulin
● Light chain component of the major human leukocyte
antigen (HLA)
● Found on the surface of most nucleated cells
● Present in high concentration on lymphocytes
● Filtered by the renal glomerulus but reabsorbed
LYMPHOCYTES
- Indicator for viral infections
- ↑ Viral infection ↑ B2-Microglobulin
Increased: RA, systemic lupus erythematosus (SLE), HIV
5 IMMUNOGLOBULINS (GAMED)
1. IgG
2. IgA
3. IgM
4. IgE
5. IgD
4. GAMMA-GLOBULINS
Increased in
- chronic inflammation
- cirrhosis or viral hepatitis
- collagen diseases
CC-1
 - paraproteins (monoclonal bands, gammopathies)
Decreased in
- congenital or acquired immunodeficiency
- Immunoglobulins – synthesized in the plasma cells
- not produced to any extent by the neonate
5. FIBRINOGEN
● One of the largest proteins in the blood plasma
● Synthesized in the liver
● Most abundant of the coagulation factors
● An acute phase reactant; markedly increased during
inflammatory process
● May serve as a marker for long-term prognosis of
cardiovascular disease
● Distinct band between β and γ-globulins on
electrophoresis
● Forms a fibrin clot when activated by thrombin
● Last protein to be activated to have a stable clot
● AKA FACTOR 1
FIBRIN
- Activated fibrinogen
THROMBIN
- Activates fibrinogen to become fibrin
Increased: pregnancy and use of birth control pills
Decreased: extensive coagulation
Reference values: 200 – 400 mg/dL (2.0 -4.0 g/L)
6. LIPOPROTEIN
● Complexes of proteins & lipids (LDL, HDL, VLDL,
Chylomicrons)
● Transports cholesterol, triglyceride and phospholipids
● Transports protein in general
Low Density Lipoprotein (LDL)
- Bad cholesterol in lipid profile test
- Clogs or blocks normal passage of blood in the heart
which causes a heart attack
High Density Lipoprotein (HDL)
- Good cholesterol in lipid profile test
Very Low Density Lipoprotein (VLDL)
*In the lipid profile test these are tested:
- Cholesterol
- HDL
- LDL
- Triglyceride
7. COMPLEMENT
● Participates in the immune reaction and serve as a
link to the inflammatory response
● Circulates as non-functional precursors
Increased: inflammatory states
Decreased: SLE, DIC, malnutrition
8. C-REACTIVE PROTEIN
● Appears in blood of patients with diverse
inflammatory diseases
● Undetectable in healthy individuals
● Precipitates with the C substance, a polysaccharide of
pneumococci
● General scavenger molecule; gamma-migrating
protein
BSMLS-2 GERRYN OLGA ABAN
Increased: acute rheumatic fever, MI, RA, gout, bacterial &
viral infections
MISCELLANEOUS PROTEINS
1. MYOGLOBIN
● A heme protein or oxygen-binding transport protein
found in skeletal and cardiac muscles
● Approximately 2% of the total muscle protein
● Marker for chest pain (angina) and early detection of
acute myocardial infarction (AMI)
● In AMI, the onset is 1-3 hours, peak level 5-12 hours,
normalize in 18-30 hours
● Useful marker for monitoring the success or failure of
reperfusion
Increased: AMI, angina, rhabdomyolysis, muscle trauma,
extraneous exercise, IM injection
2.TROPONINS
● A complex of 3 proteins that bind to the thin filaments
of striated muscle (cardiac & skeletal)
● Diagnostic marker for identifying cardiac injury in the
presence of a skeletal muscle damage
● Levels in blood may elevate after AMI in the absence
of CK-MB elevations
TnT, TnI, TnC = muscle contraction
3.AMYLOID
● A pathological extracellular deposit associated with a
group of disorders collectively called amyloidosis.
MEASUREMENT OF PROTEINS
1. KJELDAHL METHOD
● Determination of protein nitrogen derived from
constituent amino acids
● Nitrogen in pff is converted to ammonia using H2SO4
◾
⦿ The nitrogen in ammonia may be measured using:
◾
◾
Nesslerization
Berthelot reaction
Titration method
2. BIURET METHOD
● Copper binds to the peptide bond structure of the
protein, forming a purple-colored chromogen
3. FOLIN-CIOCALTEU METHOD
● Tyrosine & tryptophan in proteins reduce PT-PMA
reagent (folin-ciocalteu) to give a blue color
● Detects proteins in concentrations as low as 10 – 60
μg/mL
● Widely used in research to measure tissue & enzyme
proteins
4. Absorption of UV light at 280 nm
● Chiefly the result of the high electron density of
aromatic rings of tyrosine & tryptophan in solution (pH
8)
5. Dye-Binding Methods
● Based on the ability of proteins to bind certain dyes
● Bromocresol Green Method (BCG) for Albumin
● Used to stain protein bands after electrophoresis (e.g.
Coomassie Brilliant Blue)
6. PROTEIN ELECTROPHORESIS
CC-1
 ● Direction of migration of proteins in an electrical field 1. Amino Group/ Amine Group: Contains Nitrogen
determined by surface charge & MW of protein 2. Carbon Structure
● Urine protein electrophoresis exactly same as serum 3. Carbonyl group: COOH
except it must be concentrated before application : Also called as Carboxyl Acid
● Support media include cellulose acetate, agarose gel
and starch gel Amine + Carboxyl Acid = Amino Acid
STAINS: Amino acid + Amino acid + Amino acid = Set of amino acid
- amido black chain
- ponceau S and coomassie brilliant blue ● Connected through the end of amino group and
another end of amino group
SIGNIFICANT FINDINGS: ● When 2 amino acids are linked or joined together,
❖ Beta-gamma bridging effect: due to IgA (in serum); there is a removal of water, thus creates PEPTIDE
Cirrhosis BONDS
❖ Monoclonal band (gamma-globulin): Monoclonal ● PEPTIDE BONDS: link that binds 2 amino acids
gammopathy (Multiple myeloma) ● If there are many amino acids that are linked together
❖ Polyclonal band: Chronic inflammation it is called a POLYPEPTIDE BOND (PROTEIN)
❖ Increase in α-2-macroglobulin: nephrotic syndrome ● Protein and Polypeptide are used interchangeably
❖ α-1-globulin flat curve: deficiency in α-1-antitrypsin; ● There are 20 essential amino acids
Juvenile cirrhosis ● Amino acids can be classified according to their
FUNCTION
AMINO ACIDURIA
1. OVERFLOW GLUCOGENIC AMINO ACID
● Plasma level above renal threshold as a result of 1. Alanine
metabolic disorder 2. Arginine
3. Aspartate
a. Phenylketonuria (PKU) ➔ Involved in Krebs Cycle
● Enzyme deficiency (phenylalanine hydrolase) which ➔ Involved in carbohydrate metabolism
leads to :
- increased phenylalanine in the blood KETOGENIC AMINO ACID
- phenylpyruvic acid (prime metabolite) is 1. Leucine
present in both blood and urine in elevated 2. Lysine
concentration ➔ Generates KETONE BODIES
b. Branched chain ketoaciduria - FRUITY ODOR if Ketonuria is present
● Maple syrup urine disease - Acetoacetic Acid
● Caused by markedly reduced or absence of α-keto - Beta Hydroxybutyric Acid
acid decarboxylase - Acetone
● Increased branched chain amino acids (leucine, - These 3 are found in the urine indicating
isoleucine & valine) in blood, urine and CSF Ketonuria

2. RENAL BOTH KETOGENIC AND GLUCOGENIC AMINO ACID

● Normal plasma level but decreased renal threshold or 1. Isoleucine
reabsorption 2. Phenylalanine
cystinuria – increased cystine, lysine, ornithine, arginine in 3. Tryptophan
urine 4. Tyrosine
5. Threonine
METHODS
1. Screening tests ESSENTIAL AMINO ACID
a. TLC with ninhydrin 1. Arginine
b. urine color tests 2. Histidine
c. Guthrie microbiological tests : PKU 3. Isoleucine POLAR BASIC AMINO ACID
4. Leucine
2. Quantitative tests 5. Lysine
a. Ion-exchange chromatography 6. Methionine
b. HPLC 7. Phenylalanine
c. GC-MS 8. Threonine
9. Tryptophan
SHAPE OF PROTEINS 10. Valine
1. Fibrous: Elongated ➔ Not produced in the body
2. Globular: Symmetrical ➔ Supplemented in by the diet/ food intake

CHEMICAL COMPOSITION OF PROTEINS NON ESSENTIAL AMINO ACID

1. Simple: Has amino acids 1. Alanine
2. Conjugated/ Complex: Protein+NonProtein 2. Asparagine
3. Aspartic Acid POLAR ACIDIC AMINO ACID
AMINO ACIDS 4. Cysteine
● Building blocks of protein 5. Glutamic Acid
6. Glutamine
3 COMPONENTS OF AMINO ACIDS 7. Glycine

BSMLS-2 GERRYN OLGA ABAN CC-1

 8. Proline ● Absorbed during digestion
9. Serine
10. Tyrosine Glutamic Acid
● Present in variety of foods (Umami)
COMMON AMINO ACID ● Transports Potassium into the spinal fluid
1. Glutamine ● Potassium transport
2. Leucine
Glutamine
ESSENTIAL AMINO ACIDS ● Transports ammonia to urea
Arginine
● Production of Urea Glycine
● Simplest amino acid
Histidine ● Synthesized from SERINE
● Direct precursor of histamine
● Helps grow and repair body tissues Proline
● Derived from GLUTAMINE
Isoleucine
● Formation of Hemoglobin Serine
● Regulation of glucose
● Component of myelin sheath
Leucine ● Derived from 3-PHOSPHATE IN GLYCOLYSIS
● Helps in nitrogen balance in adults ● Involved in lipid and fatty acid metabolism: creatine &
● Helps in the optimal growth in infants porphyrins
● SECOND MOST COMMON AMINO ACID
Tyrosine
Lysine ● Produced from PHENYLALANINE
● Production of antibodies ● Precursor for hormones
● Formation of collagen ● ↓Phenylalanine= ↓ Tyrosine
● Lowers triglyceride levels ● Precursor of T4 (Thyroxine)
- Hormone in the thyroid gland
Methionine
● Initiates translation of mRNA DIAGNOSTICALLY SIGNIFICANT SERUM
● Helps in the breakdown of fats PROTEINS
Phenylalanine PROTEINS
● Production of norepinephrine ● Linear polymers of amino acids
● Used to treat arthritis and depression ● Perform diverse functions
● Component of natural sweetener ASPARTAME ○ Regulate metabolism
- Aspartame: artificial sweetener made up of ○ Facilitate contraction in the muscle
two amino acids ○ Provide structural framework
1. ASPARTIC ACID ○ Shuttle molecules in the bloodstream
2. PHENYLALANINE ○ Component of the immune system

Threonine
● Formation of collagen, elastin, and tooth enamel

Tryptophan
● Treatment of migraine headaches
● Controls hyperactivity in children

Valine PROTEIN STRUCTURE

● Treatment for insomnia and anxiety
● Helps in nitrogen balance in adults
NON ESSENTIAL AMINO ACIDS
Alanine
● Transfer protein of Nitrogen from tissues to liver
● Product of the breakdown of DNA
Asparagine
● Production of ammonia
● Important role in the synthesis of ammonia
Aspartic Acid
● Involvement in gluconeogenesis
● Metabolite in the citric acid cycle and the urea cycle
Cysteine
1. PRIMARY STRUCTURE
● determined by amino acid sequence
2. SECONDARY STRUCTURE
● folding of short segments of polypeptide into
geometrically ordered units (e.g. alpha-helix,
beta-sheet)
3.TERTIARY STRUCTURE
● overall 3-dimensional shape of the protein (globular
vs fibrous)
4. QUATERNARY STRUCTURE
● number and types of polypeptide units of oligomeric
proteins and their spatial arrangement
SYNTHESIS OF SERUM PROTEINS
● Most serum proteins are synthesized in the liver and
secreted by the hepatocyte into the circulation.
- Except Immunoglobulins and vWF

BSMLS-2 GERRYN OLGA ABAN CC-1

 ● The nitrogen content of serum proteins is, on ● Transport protein for thyroid hormones; transports
average, approximately 16% vitamin A by forming a complex with retinol-binding
protein
RIBOSOMES:site of protein synthesis within the cell ● A low prealbumin level is a sensitive marker of poor
● Produces proteins nutritional status
● 2 Types of DNA function
1. Replication INCREASED IN: patients receiving steroids, in alcoolism,
2. Protein synthesis and in chronic renal failure
DNA is transcribed into mRNA
↓ DECREASED IN: hepatic damage, acute-phase
DNA is then translated into tRNA inflammatory response, and tissue necrosis
↓
It is then processed to rRNA which completes protein synthesis ALBUMIN
● Protein present in the highest concentration in
IMMUNOGLOBULINS serum
● Not synthesized in the liver Function:
● Produced in the plasma cells - Provide nearly 80% of colloid osmotic
pressure (COP) of intravascular fluid
Von Willebrand Factor (vWF) - Buffers pH
● Essential in coagulation - Binds to various substances in blood (e.g.
● Produced by the: some hormones, drugs, electrolytes,
➢ ENDOTHELIAL CELLS unconjugated bilirubin)
➢ PLATELETS ● Negative acute-phase reactant - marker for
inflammation (ex. C-Reactive Protein (CRP) )
DIAGNOSTICALLY SIGNIFICANT PROTEINS
PROTEIN DESCRIPTION INCREASED: dehydration

Prealbumin (Transthyretin) Indicator of malnutrition; DECREASED: liver disease, malnutrition, malabsorption,

binds thyroid hormones and kidney loss, hemodilution
retinol-binding protein

Albumin Binds bilirubin, steroids, fatty ALPHA-1-ANTITRYPSIN

Alpha-1-antitrypsin
Alpha-1-fetoprotein
acids; major contributor to
oncotic pressure
Protease inhibitor
Principal fetal protein
● Most important function: inhibition of the protease
neutrophil elastase
● Abnormal form of AAT can also accumulate in the
liver and cause cirrhosis
● Major component of a 1-globulin band → deficiency of
AAT seen as lack of an a1-globulin band on SPE

Alpha-1- acid glycoprotein May be related to immune SERPINA1 gene mutation

response
↓
Haptoglobin Binds hemoglobin
AAT Deficiency / Abnormal form of AAT
Ceruloplasmin Transports copper;
peroxidase activity ↓
Uncontrolled activity of neutrophil elastase
Alpha-2-macroglobulin Inhibits thrombin, trypsin and
pepsin ↓
Transferrin Transports iron Destruction of alveoli (due to destruction of elastin)

Hemopexin Binds heme ↓

Emphysema
Complement Immune response

Fibrinogen Precursor of fibrin EMPHYSEMA

➢ AKA: Pink Puffer or Barrel Chest
C-reactive protein Opsonin ● One of the COPDs (chronic obstructive pulmonary
diseases)
● Lungs are inflamed
PREALBUMIN ● Most common cause: smoking
● Aka Transthyretin ● Pathophysiology: excessive inflammation or lack of
● Migrates before albumin in the serum protein AAT leads to destruction of alveolar air sacs → loss of
electrophoresis elastic recoil and collapse of airways during
exhalation → obstruction and air trapping
● Dyspnea, cough with minimal sputum

BSMLS-2 GERRYN OLGA ABAN CC-1

 ● "Pink puffers", "barrel-chest", hypoxemia Manifestations:
HYPOXEMIA
● Low oxygen in the blood Hypoalbuminemia - pitting edema

ALPHA-1-FETOPROTEIN Hypogammaglobulinemia - increased risk of

infection
● Synthesized by the developing embryo and fetus
● Thought to protect the fetus from immunologic attack
by the mother
● No known function in normal adults Hypercoagulable state - due to the loss of
● Elevated AFP: neural tube defects (e.g. spina bifida), antithrombin III
presence of twins
● Low AFP: increased risk for Down syndrome (trisomy Hyperlipidemia and - may result in fatty
21) hypercholesterolemia casts in the urine
● Tumor marker for hepatocellular carcinoma, some
testicular carcinomas
➢ Marker to rule out testicular cancer HAPTOGLOBIN
● Function: bind free hemoglobin to prevent loss of
WILSON'S DISEASE hemoglobin and its constituent, iron, into the urine
● Autosomal recessive ➢ Binds to hemoglobin, so hemoglobin carrying iron is
● Decreased levels of ceruloplasmin not excreted through the urine
● Excess storage of copper in various organs ● Used primarily to help detect and evaluate hemolytic
Liver → hepatic cirrhosis anemia
Brain → neurologic damage
Cornea → Kayser-Fleischer rings

Other laboratory findings:

● Total serum copper decreased
● Free serum copper increased
● Urinary copper increased ● Haptoglobin-Hemoglobin complex removed by
reticuloendothelial cells (mainly disposed in the
ALPHA-2-MACROGLOBULIN spleen)
● Large protein that inhibits proteases such as trypsin, ➢ Free hemoglobin + Haptoglobin= Haptoglobin
thrombin (Blood Coagulation), kallikrein (Blood Hemoglobin
Coagulation), and plasmin→ fibrinolysis (dissolution
of clot) TRANSFERRIN
● Increased in nephrotic syndrome (large size aids in ● Transports two molecules of ferric iron
its retention) ● Negative acute-phase reactant (not acute-phase
reactant)
THROMBIN & KALLIKREIN ● Major component of the beta-globulin fraction
● Blood coagulation/clotting ● Tested to determine cause of anemia (e.g. increased
levels in IDA)
PLASMIN ➢ Has no inflammatory response
● Involved in fibrinolysis ➢ Increased Transferrin indicates iron deficiency
anemia
FIBRINOLYSIS
● Dissolves clot HEMOPEXIN
● Dissolution of clot ● Function: scavenge heme released or lost by the
turnover of heme proteins such as hemoglobin →
Stable clot (FIBRINOGEN) protect body from oxidative damage that free heme
↓ can cause
Fibrin ● Low hemopexin levels are diagnostic of hemolytic
↓ anemia
Clot ➢ It is the indicator of erythrocytic disorders
↓ ➢ Binds with free heme
Plasmin ➢ Protects the body from oxidative damage from free
↓ heme
Clot dissolves
C-REACTIVE PROTEIN
NEPHROTIC SYNDROME ● Precipitates with C substance, a polysaccharide of
● Glomerular disorder characterized by proteinuria pneumococci
(>3.5 g/day) ● Functions in opsonization
Proteinuria: presence of protein in the urine ● One of the first acute-phase proteins to rise in
● Pathophysiology: Disruption of the electrical charges inflammatory disease
that produce the tightly fitting podocyte barrier ● High or increasing amount of CRP suggests an acute
resulting in massive loss of protein and lipids infection or inflammation
➢ Secreted in the urine

BSMLS-2 GERRYN OLGA ABAN CC-1

 FIBRONECTIN ➢ Regulation of salt in the body
● Glycoprotein produced by fetal membranes ➢ Natriuresis: excretion of salt in the urine.
responsible for the cellular adhesiveness of placenta ➢ Diuresis: increased urine output
and membranes to the decidua. - Increase urination
● Fetal fibronectin is produced at the boundary between
the amniotic sac and the decidua (the lining of the ELECTROPHORETIC PATTERNS OF SERUM PROTEINS
uterus) and functions to maintain the adherence of
the placenta to the uterus. a1 a2 B GLOBULINS y
GLOBULINS GLOBULINS GLOB
● Test for assessment of the risk for PRETERM ULINS
DELIVERY in women between 24 to 35 weeks
gestational age. a1-antitrypsin Haptoglobin Pre- B-lipoprotein IgA
➢ Seen in pregnant women a1-fetoprotein Ceruloplasmin B-lipoprotein IgD
➢ Protein that helps in the placenta a1-acid a2-macroglo Transferrin IgE
- PLACENTA: connects the baby and uterus glycoprotein bulin Hemopexin IgG
- PRETERM DELIVERY: childbirth that occurs a1-lipoprotein B2-microglobulin IgM
before 37 weeks of pregnancy 1-antichymotr Complement CRP
- Normal pregnancy lasts up to 40 weeks ypsin Fibrinogen *
inter-a-trypsin CRP*
CROSS-LINKED C-TELOPEPTIDES inhibitor
● Proteolytic fragments of collagen I formed during Gc globulin
bone resorption
● CTX is a biochemical marker of bone resorption that
can be detected in serum and urine. ACUTE PHASE REACTANTS
➢ Not that important as a serum protein 1. The acute phase reactant proteins share the property of
➢ Involvement in the production of collagen showing elevations in concentrations in response to stressful
or inflammatory states that occur with infection, injury, surgery,
TROPONIN trauma, or other tissue necrosis.
● Govern excitation-contraction coupling in muscle 2. They include AAT, orosomucoid, haptoglobin,
● Three subunits: Troponin T (cTnT), Troponin C ceruloplasmin, fibrinogen, serum amyloid A protein, and
(TnC), Troponin I (cTnl) CRP. Others are Factor VIII, ferritin, lipoproteins,
complement proteins, and immunoglobulins.
Clinical Significance: ➢ Protein marker for inflammation
● cTnT or cTnl is used as an AMl Indicator because of
specificity and early rise in serum concentration FACTOR VIII
following AMI ➢ Involved in blood coagulation
- cTnT rises within 3-4 hours, peaks in 10-24
hours, returns to normal in 10-24 days. ACUTE PHASE REACTANTS
- cTnI rises within 3-6 hours, peaks in 14-20 PROTEIN RESPON NORMAL INCREASE FUNCTION
hours, and returns to normal in 5-10 days. SE CONCENT
● Now known as the "gold standard" for diagnosis TIME (hr) RATION
(mg/dL)
of MI
CRP 6-10 0.5 1000x Opsoniza
MYOGLOBIN
tion,comp
● Heme-containing protein that binds oxygen with
lement
cardiac and skeletal muscle
activation
● Levels are related to muscle mass and activity
(reasonable sensitivity but poor specificity)
Serum 3.0 1000x Removal
amyloid A of
Clinical Significance:
cholester
● Increased in skeletal injuries, muscular dystrophy, and
ol
AMI
● Myoglobin is released early in cases of AMI, rising in
1-3 hours and peaks in 5-12 hours, and returns to Alpha 1 24 200-400 2-5x Protease
normal in 18-30 hours. However, it is not tissue -antitrypsin inhibitor
specific. It is better used as a negative predictor in the
first 2-4 hours following chest pain. Fibrinogen 24 110-400 2-5x Clot
formation
BRAIN NATRIURETIC PEPTIDE (BNP) AND
N-TERMINAL-BRAIN NATRIURETIC PEPTIDE (NT-BNP) Haptoglobin 24 40-200 2-10x Binds
● Natriuretic peptides are neurohormones that affect hemoglob
body fluid homeostasis (through natriuresis and in
diuresis) and blood pressure
● NT-proBNP and BNP are found in largest Ceruloplas 48-72 20-40 2x Binds
concentration in the left ventricular myocardium but min copper,
are also detectable in atrial tissue as well as in the oxidizes
myocardium of the right ventricle. iron
● BNP has become a popular marker for congestive
heart failure. С3 48-72 60-140 2x Opsoniza

BSMLS-2 GERRYN OLGA ABAN CC-1

 tion, lysis
ALBUMIN MEASUREMENT
Mannose-bi ? 0.15-1.0 ? Complem METHOD PRINCIPLE COMMENT
nding ent
protein activation Salt precipitation Globulins are Labor intensive
precipitated in
high salt
TOTAL PROTEIN ABNORMALITIES
concentrations:
1. Hypoproteinemia: albumin in
a. Total protein level less than the reference interval supernatant is
b. occurs in any condition where a negative nitrogen quantitated by
balance exists (excessive loss, decreased intake, biuret reaction
decreased synthesis, accelerated catabolism)
DYE BINDING
2. Hyperproteinemia:
a. Increase in total plasma proteins
Methyl orange Albumin binds to Nonspecific for
b. Not an actual disease state but is the result of
dye; causes shift albumin
dehydration
in absorption
c. When excess water is lost from the vascular system,
maximum
the proteins, because of their size, remain within the
blood vessels
HABA [2,4'- Albumin binds to Many
d. May also be cause by excessive production, primarily
hydroxyazobenz dye; causes shift interferences
of gamma-globulins e.g. multiple myeloma
ene)- in absorption (salicylates,
benzoic acid] maximum bilirubin)

BCG Albumin binds to Sensitive,

(bromcresol dye; causes shift overestimates low
green) in absorption albumin levels;
maximum most commonly
used dye

BCP Albumin binds to Specific,

(bromcresol dye; causes shift sensitive, precise
purple) in absorption
maximum
TOTAL PROTEIN ANALYSIS
Electrophoresis Proteins Accurate; gives
METHOD PRINCIPLE COMMENT separated based overview of
on electric charge relative changes
Kjeldahl Digestion of Reference in different protein
protein; method; assume fractions
measurement of average nitrogen
nitrogen content content of 16%
ELECTROPHORESIS
➢ Used widely for albumin measurement
Refractometry Measurement of Rapid and simple; ➢ Most accurate and precise method
refractive index assume ➢ Separates protein molecules by its charged molecules
due to solutes in nonprotein solids
serum are present in
SERUM PROTEIN ELECTROPHORESIS
same
● Performed when an abnormality in the total protein or
concentration as
albumin is found
in the calibrating
● Principle: separation of proteins based on their charge
serum
density
● Regions are stained using: Coomasie blue, Amido
Biuret Formation of Routine method;
Black, Ponceau S
violet-colored requires at least
chelate between two peptide bonds
ELECTROPHORETIC PATTERN
Cu2+ ions and and an alkaline
peptide bonds medium

Dye binding Protein binds to Research use

dye and causes a
spectral shift in
the absorbance
maximum of the
dye

BSMLS-2 GERRYN OLGA ABAN CC-1

 CHAPTER 8 : NON PROTEIN COMPOUNDS ● synthesized in the liver from CO2 and ammonia that
arises from the deamination of amino acids in the
FUNCTIONS of the KIDNEYS: reaction of the urea cycle
1. Elimination of excess body water
2. Elimination of waste products of metabolism (urea and STRUCTURE: NH2- C = O
creatinine) I
3. Elimination of foreign substances like drugs NH2
4. Retention of substances necessary for normal body function
(proteins & amino acids, glucose) MW = 60 g/mole where:
5. Regulation of electrolyte balance and osmotic pressure of C = 1 x 12 = 12
the body fluids) H=4x1=4
6. Endocrine function: O = 1 x 16 = 16
Primary: production of rennin, prostaglandin and N = 2 x 14 = 28
erythropoietin
Secondary: degradation of insulin, glucagon and aldosterone Conversion factors:
-From urea mass units to urea nitrogen (28/60) = 0.467
Clinically Significant NPN compounds: -From urea nitrogen to urea mass units (60/28) = 2.14
1. urea – 45%
2. amino acids – 20% Renal handling:
3. uric acid – 20%
- 90% excreted through the kidneys
4. creatinine – 5 %
- 10% excreted through the skin and GIT
5. creatine – 1-2 %
- 40 – 70% reabsorbed in the renal tubules by passive diffusion
6. ammonia – 0.2%
Urea concentration depends on the following :
TOTAL NPN METHODOLOGY: 1. renal function and perfusion
TWO STEPS: 2. protein content of the diet
1. KJELDAHL DIGESTION 3. amount of protein catabolism
● The nitrogen in a pff of the specimen is converted to
ammonia using hot conc. H2SO4 with copper sulfate, METHODS FOR UREA DETERMINATION
mercuric sulfate or selenium oxide as the catalysts. I. INDIRECT METHOD / ENZYMATIC
● measures Blood Urea Nitrogen
NPN + H2SO4 —----> NH4HSO4 - Based on the preliminary hydrolysis of urea with urease
followed by some process that quantitates the ammonium ion
NH4HSO4 + NaOH —--------> Na2SO4 + NH3 + H2O 1. Berthelot reaction
2. Nessler’s reaction
3. GLDH-coupled enzymatic method (Dupont ACA
Analyzer)
2. MEASUREMENT OF AMMONIA FORMED ● Decrease in absorbance of NAD at 340 nm
A. NESSLERIZATION 4. Conductometric method: Beckman BUN Analyzer
Nessler’s reagent – double iodide salt of potassium & ● Based on the measurement of the conductivity
mercury generated from the reaction of urease on urea
Gum ghatti – colloidal stabilizer producing ammonium ions & bicarbonates
Dimercuric ammonium iodide – yellow to orange brown
product 5. Urograph or Urastrat strip
● Physical principle: based on chromatography
NH3 + HgI2.2KI —-------> NH2Hg2I2 + KI + NH4I ● Chemical principle: Conway Microdiffusion method

B. BERTHELOT METHOD 6. Indicator dye (uriol): Kodak Ectachem Analyzer

Reagent: phenol and alkaline hypochlorite ➢ Dye is added to NH4 ions from urea hydrolysis & the
Catalyst: sodium nitroprusside color change is measured
Product: indophenol blu ➢ Used in multilayer film reagents, dry reagent strips
and automated systems
NH3 + NaOCl + Phenol —-------> Indophenol + NaCl + H2O
II. DIRECT METHODS
1. Diacetyl Monoxime (Fearon)
C. MONITORING CONSUMPTION OF AMMONIA (Kaplan,
➢ Direct condensation reaction
Manoukian – Fawaz; Kallet – Cook Reaction)
➢ Diacetyl – very toxic

NH3 + α – ketoglutarate + NADH + H —----> Glutamate +
NAD
Catalyst: Glutamate dehydrogenase
● Measure a decrease in the absorbance at 340 nm
UREA
BIOCHEMISTRY
● most abundant NPN compound in plasma
● major excretory product of protein metabolism
2. ortho – phthalaldehyde: adapted by automated methods
Urea + OP —--> Isoindoline
Isoindoline + Naphthylethylenediamine —---> colored
compound
Disease Correlations:
AZOTEMIA – a biochemical abnormality pertaining to
increase NPN compounds especially

BSMLS-2 GERRYN OLGA ABAN CC-1

creatinine and urea defining GFR defect

Three categories:

a. prerenal b. renal c. postrenal

– due to reduced – decreased renal – obstruction of

renal blood flow function urine flow
> hemorrhage, > kidney diseases: > calculi, tumors of
dehydration, glomerulonephritis bladder or prostate
increased protein
catabolism

Uremia/ Uremic syndrome – a clinical syndrome RENAL HANDLING of CREATININE:

characterized by increased BUN accompanying renal failure 1. Glomerular filtration
seen in metabolic acidosis, hyperkalemia and edema 2. Excreted without being reabsorbed. Thus, excretion is
relatively constant. Creatinine output is sometimes used to
3. Decreased Urea: measure the completeness of a 24-hour urine sample
- decreased protein intake collection
- severe vomiting 3. When serum creatinine is elevated, it is secreted in the renal
- diarrhea tubules

Specimen Requirements and Interfering Substances ANALYTICAL METHODS

1. plasma, serum or urine 1. DIRECT METHOD: JAFFE REACTION
2. plasma : ammonium ions and high concentrations of sodium Creatinine + alkaline picrate —----> Creatinine picrate
citrate and sodium fluoride must (red orange/ yellow)
be avoided 510 nm
3. non fasting sample is acceptable
4. non hemolyzed sample is recommended Alkaline picrate: 1 part 10% NaOH and 5 parts sat. picric acid
5. urine sample guarded against bacterial decomposition of (2,4,6 trinitrophenol)
urea
NOTE:
Reference Interval: ➢ The Jaffe reaction lacks specificity
adult 6-20 mg/dL 2.1-7.1 mmol/L Non-creatinine Jaffe-reacting chromogens:
serum/plasma ● Proteins
● Glucose
● Ascorbic acid
urine, 24hr 2-20 g/day 0.43-0.71 mol/day
● Guanidine
conversion factor : mg/dL ---> mmol/L : 0.357 ● Acetone
● Cephalosporins
CREATININE ● α-ketoacids (acetoacetate and pyruvate)
BIOCHEMISTRY
- principal waste product of muscle metabolism derived mainly 2. INDIRECT / ENZYMATIC METHODS
from Creatine (alpha-methyl guanidinoacetate) a. F. Lim – Creatininase or creatinine iminohydrolase
- Creatine is produced from two enzymatic processes:
● transamination of arginine & lysine forming Creatinine ----------------> N-methylhydantoin + NH3
guanidinoacetic acid in the kidneys, small intestines, (Creatininase)
pancreas and probably the liver
● methylation of guanidinoacetic acid in the liver NH3 + α-ketoglutarate + NADH -----------> glutamate + NAD + H+
(Glutamate DH)
PHOSPHORYLATION OF CREATINE IN SKELETAL
MUSCLES
● Creatine PO4 is the muscles’ energy source
b. G.A. Moss – Creatinine Amidohydrolase
NON-ENZYMATIC CYCLIZATION OF EITHER CREATINE OR
CREATINE PHOSPHATE FORMS CREATININE Creatinine -------------------------------------> Creatine
(Creatinine amidohydrolase)

Creatine + ATP ---------------------> CreatinePO4 + ADP

(Creatine kinase)

ADP + PEP ---------------------> Pyruvate + ATP

(Pyruvate kinase)

BSMLS-2 GERRYN OLGA ABAN CC-1

 ASSOCIATED MYOPATHIES:
Pyruvate + NADH + H+ -----------------> Lactate + NAD ● muscular dystrophy
(Lactate DH) ● familial periodic paralysis
● myasthenia gravis
● dermatomycosis
3. Yatzidis method
➢ Creatinine reacts with alkaline picrate at two different Reference Interval
pH levels (1) plasma/serum
pH 10: protein & other interfering materials will reacts w/ adult female 0.6-1.1 mg/dL (53-97
picrate but creatinine does not μmol/L)
pH 11: both creatinine & proteins react Jaffe
adult male 0.9-1.3 mg/dL (80-115
4. High Performance Liquid Chromatography (HPLC) μmol/L)
Sources of error:
1. ascorbate, glucose, alpha keto acids and uric acid – false
adult female 0.5-0.8 mg/dL (44-71
increase
Enzymatic μmol/L)
2. drugs : cephalosporin and dopamine intake – false increase
: lidocaine intake – false increase
adult male 0.6-1.1 mg/dL (53-97
Specimen Requirements and Interfering Substances
μmol/L)
3. plasma, serum or urine
4. avoid hemolyzed and icteric sample especially for the Jaffe
reaction (2) 24h urine
5. lipemic samples cause errors
adult female 600-1800 mg/day 5.3-15.9 mmol/day
6. non-fasting sample acceptable
7. high protein ingestion may transiently elevate serum
concentrations adult male 800-2000 mg/day 7.1-17.7 mmol/day
8. urine : refrigerated after collection or frozen if longer storage
than 4 days is required URIC ACID
DIAGNOSTIC APPLICATION: BIOCHEMISTRY
● Creatinine Clearance ● major product of the catabolism of purine nucleosides:
3. overestimates but gives a reasonable approximation of adenosine & guanosine
glomerular filtration rate ● formed in the liver & intestinal mucosa from xanthine
Creatinine Clearance = U x V ● The bulk of purines ultimately excreted as uric
P acid in the urine arises from degradation of
4. expressed in mL/minute endogenous nucleic acids.
5. plasma concentration of creatinine is inversely proportional ● Reutilization of the major purine bases (adenine,
to creatinine clearance (elevated hypoxanthine and guanine) is achieved through
creatinine clearance = decreased GFR) “salvage” pathways
6. insensitive marker: >50% renal dysfunction = abnormal - Phosphoribosylation of the free bases
plasma creatinine causes re-synthesis of the respective
● Creatine kinase: used in the diagnosis of muscle nucleotide monophosphates
diseases
RENAL HANDLING of URIC ACID
BUN : CREATININE RATIO
● 75% is excreted through the urine
NORMAL: 10–20:1
● The remainder is secreted into the GIT, where it is
degraded to allantoin & other compounds by bacterial
1. BUN:CREA ratio <10:1
enzymes.
a. Acute tubular necrosis
● Glomerular filtration
b. Low protein intake; starvation
● Tubular reabsorption in the PCT: 98 – 100%
c. Severe liver disease
● Active secretion
d. Repeated dialysis
● Reabsorption in the DCT
e. Severe vomitting or diarrhea
● Net excretion: 10%
2. BUN:CREA ratio >10:1 with normal creatinine
a. Catabolic states w/ tissue breakdown FACTORS:
b. Pre-renal azotemia 1. Diet: legumes, seeds, internal organs
c. High protein intake 2. Age & gender: increase w/ age; higher in males
d. After GIT hemorrhage 3. 2x greater concentration in RBC than in plasma
4. Avoid the use of K oxalate because it forms salts that cause
3. High ratio with elevated creatinine levels turbidity
● Post-renal obstruction 5. UA is stable in serum for several days at RT and longer at
● Pre-renal azotemia superimposed on renal disease ref. temp.
6. Thymol increases its stability
NOTE:
● Creatinine determination is MORE SPECIFIC for METHODS of DETERMINATION:
the diagnosis of renal disease BUN determination is 1. DIRECT METHOD: Phosphotungstic Acid (PTA)
MORE SENSITIVE for the diagnosis of renal disease

BSMLS-2 GERRYN OLGA ABAN CC-1

 4. OTHER METHODS:
Uric Acid + PTA ---- OH----Allantoin + CO2 + Tungsten blue
(710 nm) A. HPLC
B. Amperometric Principle: Polarographic method

Alkaline solution: NaCN: DISEASE CORRELATIONS:

● Folin Na2CO3: Caraway (1) HYPERURICEMIA
● Brown Henry
● Benedict Archibald
● Newton A. Increased Formation B. Decreased Excretion

2. INDIRECT METHODS: ENZYMATIC METHODS

A. Blaunch and Koch (UV test with uricase) Primary: - Idiopathic Primary: - Idiopathic
- Inherited metabolic
Uric Acid —-------> Allantoin + CO2 disorders Secondary: - Renal failure
Secondary: - Excess - Drug therapy: salicylate
● The decrease in the UA concentration is determined
dietary purine intake - Poisons: heavy metal
by measuring the absorbance in the range of 290-300
- Increased nuclear - Pre-eclampsia
nm
breakdown (e.g. Leukemia) - Organic acids
- Psoriasis - Trisomy 21 (Down
B. Trinder – Uricase method - Altered ATP metabolism syndrome)
- Tissue hypoxia
Uric Acid + O2 + 2H2O —-------> Allantoin + CO2 + H2O2 - Pre-eclampsia
- Alcohol
H2O2 + DHBS + PAP —-------->Quinoneimine derivative
(480-550 nm) Hereditary Hyperuricemia:
1. Lesch-Nyhan syndrome : x-linked genetic disorder;
DHBS: 3,5 – dichloro – 2– dihydroxy benzene sulfonic acid deficiency of hypoxanthine guanine phosphoribosyl
PAP: 4 – aminophenazone transferase (muricase)

2. Abnormal phosphoribosyl pyroPO4 synthetase:

C. Uricase – catalase system
prevents reutilization of purine bases in the nucleotide
Uric Acid —--------> Allantoin + CO2 + H2O2 salvage pathway

H2O2 + methanol ---------------> formaldehyde + H2O GOUT: monosodium urate precipitates from supersaturated
(Catalase) body fluids

Formaldehyde + acetylacetone + NH3 —---------> 3H2O + (2) HYPOURICEMIA:

3,5-diacetyl-1,4- dihydro lutidine (410 nm) ● Atrophy of the liver

OR Specimen Requirements and Interfering Substances

H2O2 + ethanol ---------------> Acetaldehyde + H2O
(Catalase)
(Aldehyde DH)
Acetaldehyde + NAD ---------------> Acetate + NADH
(increase in Abs at 340 nm)
● heparinized plasma, serum or urine
● immediate separation from red cells to prevent
dilution by intracellular contents
● non-fasting sample acceptable
● gross lipemia should be avoided
● high bilirubin may cause false decrease
● significant hemolysis will lower results
● drugs : salicylates and thiazides : false increase

3. METHODS based on REDUCING PROPERTY of UA Reference Interval (uricase method)

A. Bittner method
Adult female plasma or serum 2.6-6.0 mg/dL
Cupric ions -----UA—-> Cuprous ions (0.16-0.36 mmol/L)

Cuprous ions + neocuproine —---->copper neocuproine

complex Adult male 2.5-7.2 mg/dL
(yellow to orange) (0.21-0.43 mmol/L)

B. TPTZ Method by Morin Urine, 24h 250-750 mg/day

(1.48-4.43 mmol/day)
Ferric ions -----UA—> Ferrous ions
Child plasma or serum 2.0-5.5 mg/dL
Ferrous ions + TPTZ —--------> blue colored complex (590 (0.12-0.33 mmol/L)
nm)
Conversion factor: 0.059
TPTZ : 2,4,6- tripyridyl – 5 – triazine

BSMLS-2 GERRYN OLGA ABAN CC-1

 AMMONIA CLINICAL SIGNIFICANCE
BIOCHEMISTRY I. PRIMARY OR INHERITED HYPERAMMONEMIA:
● from deamination of amino acids thru the action of A. Enzyme defects in the Krebs Henseleit Cycle
digestive and bacterial enzymes on proteins in the B. Defects in the metabolism of amino acids: Lysine &
GIT Ornithine
● used in the liver for urea production C. Defects in the metabolism of:
● level in circulation is extremely low (15 – 45 μg/dL) - Propionic acid
● increased in concentration in the blood in cases of - Methylmalonic acid
severe liver damage - Isovaleric acid
● most ammonia in the blood exists as ammonium ion
concentration is not dependent on renal function II. ACQUIRED HYPERAMMONEMIA
● high ammonia : neurotoxic --- encephalopathy A. Severe liver disease:
● Acute – toxic or fulminant viral hepatitis &
METHODS for AMMONIA DETERMINATION: Reye’s syndrome
1. Conway and Cook Diffusion Method ● Chronic – cirrhosis
● specimen is alkalinized to convert NH4 ions to NH3 Hepatic encephalopathy among cirrhotic patients is caused
● NH3 is trapped in acid medium of diffusion cell by:
● Quantitated by titration or colorimetry ● GIT bleeding
● Time consuming with poor accuracy and precision ● Excess dietary proteins
● Constipation
2. Forman’s Resin Absorption Method ● Infections
● Uses cation-exchange resin ● Drug effects
● NH3 absorbed by the resin and eluted
● Quantitated by Berthelot reaction or by Nesslerization B. Impaired venous drainage (from intestine to liver by
N.V.: 16 – 33 μmol/L portal vein)
C. Impaired renal excretion —-----> Decreased urine
3. Kunahashi, Ishihora and Euhera Method output Increased BUN reabsorbed —-----> increased
● NH3 is obtained through the use of a Dowax column excretion of urea into intestines converted to
● Assayed using the Berthelot method ammonia

4. Van Anken Enzymatic Method Reference Interval

2-oxoglutarate + NH4+ + NADPH —--------> Glutamate + plasma 19-60 ug/dL 11-35 umol/L
NADP + H2O adult

N.V.: 11 – 35 μmol/L
urine,24h 140-1500 68-136
5. Ion Selective Electrode mg N/day ug/dL
● Based on the diffusion of NH3 through a selective
membrane into NH4 chloride causing pH change
which is determined potentiometrically child 10 days to 10-107 40-80 umol/
● Good precision and accuracy 2y mmol N/day

SOURCES OF AMMONIA CONTAMINATION:

1. Smoking
2. Laboratory atmosphere
● Blood collection & NH3 analysis must be done in a lab
w/ restricted traffic
● Glassware: soaked in hypochlorite solution (52.5g/L)

3. Poor venipuncture technique

● Use of heparin lock
● Probing for a vein
● Partial fill of the evacuated tube
● Drawing blood into a syringe & transferring it into an
anticoagulated tube

4. Metabolism of nitrogenous constituents

Minimized by
● placing the specimen in ice water
● centrifuging w/o delay
● performing the assay immediately

BSMLS-2 GERRYN OLGA ABAN CC-1

 CHAPTER 9: LIPIDS & LIPOPROTEINS
LIPIDS
● Organic substances which are soluble in nonpolar
organic solvents (chloroform and ether) and insoluble
in polar solvents (water)
CHEMICALLY: yield fatty acids on hydrolysis OR complex
alcohols the can combine with fatty acids to
form esters
HUMAN PLASMA LIPIDS:cholesterol, triglycerides,
phospholipids and non-esterified fatty acids
LIPOPROTEINS
● Macromolecule which consists of varying proportions
of protein, cholesterol, triglyceride and
phospholipid
● Water-soluble, thus, it facilitates the transport of the
lipids in the circulation
● Lipids are diverse in terms of their structure and
function
TRIGLYCERIDE/ NEUTRAL FAT
● Secondary energy source
● Serves as a thermal insulator
● Protects tissues from physical trauma
CHOLESTEROL
● Precursor of biological hormones
● Source of bile acids
● Component of the Cell membrane
PHOSPHOLIPIDS
● Component of the Cell membrane
● Associated with vital life processes (e.g. CNS)
BIOCHEMISTRY OF TRIGLYCERIDES
● Also known as triacylglycerol
● Transported by chylomicrons (exogenous) and
VLDL (endogenous)
● Complete Hydrolysis: 3 FA + glycerol
● Partial Hydrolysis: 2 FA + monoglyceride
● Absorption: Glycerol (H2O-soluble) = via the portal
route
● Fatty acids (H2O-insoluble) = via the lymphatic route
● Monoglyceride (H2O-insoluble) = via the lymphatic
● route
● Synthesis of TG from monoglyceride & glycerol
● β-oxidation of fatty acids in the mitochondria:
degradation of the fatty acids by 2C atoms with
subsequent production of Acetyl CoA
BIOCHEMISTRY OF CHOLESTEROL
● Found exclusively in animals and humans
● Contains cyclopentanoperhydrophenanthrene ring
● Contains 27 C atoms
ABSORPTION:
● Cholesterol in the intestines comes from the diet, bile,
intestinal secretions and cells
● To be absorbed, cholesterol has to solubilized by the
formation of mixed micelles containing unesterified
cholesterol, fatty acids, monoglycerides,phospholipids
and conjugated bile acids
SYNTHESIS:
● 90% of body's cholesterol is synthesized by the liver
and gut from simpler molecules like Acetyl CoA
BSMLS-2 GERRYN OLGA ABAN
ESTERIFICATION
● Complexing of cholesterol with a fatty acid; this
process enhances the lipid carrying capacity of
lipoproteins
● Cholesterol is mainly esterified in the vascular
compartment
Catalyzed by:
● LCAT – Lecithin Cholesterol Acyltransferase (in
plasma)
● ACAT – Acyl Cholesterol Acyltransferase
(intracellularly)
TRANSPORT
● Transported by LDL to the cells
● Transported by HDL out of the cells
CATABOLISM
● Cholesterol that reaches the liver is either secreted
unchanged into bile (free cholesterol) or metabolized
to form bile acids
LIPOPROTEINS: MAJOR CLASSES
1. Chylomicron (CM)
- Synthesized & released from the SI (Exogenous
pathway)
- Very rich in triglycerides (80%); transports dietary fat
- Relatively poor in cholesterol, phospholipids and
proteins
- Proteins: Apo B-48, AI, AII, AIV, C (1-2%)
- When present in high levels : milky plasma (floating
creamy layer)
2. Very-Low-Density Lipoprotein (VLDL)
- Synthesized and released from the liver
- Transports cholesterol & triglyceride which are
synthesized in the liver (endogenous pathway)
- Smaller than chylomicrons
- Elevated levels produce a turbid plasma
- Contain : 50% triglycerides, 40% cholesterol and
phospholipid, 10% protein
- Proteins: Apo B-100 & Apo C-II
3. Intermediate Density Lipoprotein (IDL)
- Derived from VLDL hydrolysis by lipoprotein lipase
(LPL)
- Partly depleted of triglyceride
- Contains an almost equal amount of triglyceride &
cholesterol
- Proteins: Apo B and E
4. Low-Density Lipoprotein (LDL)
- Produced from the action of LPL on IDL
- Consists of 50% cholesterol, 25% protein, 20%
phospholipid and 5% of triglycerides
5. High-Density Lipoprotein (HDL)
- Produced & catabolized in the liver & intestines
- Contains Apo AI & Apo AII
- Richest in protein (50% of its weight is protein)
- Involved in reverse cholesterol transport (excess
cholesterol is returned from the tissues to the liver)
ABNORMAL LIPOPROTEINS
1. Lipoprotein(a) or Lp(a)
- similar to LDL in terms of density and overall
composition
CC-1
2. LpX Lipoprotein
- abnormal lipoprotein found in patients with obstructive
biliary disease, and in patients with familial
lecithin:cholesterol acyltransferase (LCAT) deficiency
3. β-VLDL (“floating β” lipoprotein)
- abnormal lipoprotein that accumulates in type
hyperlipoproteinemia
- Richer in cholesterol than VLDL
APOLIPOPROTEINS
- Protein portion of lipoproteins
Apolipoprotein A : major protein of HDL and chylomicron
- originate in the intestine or liver
METHODS: Carr-Drekter, Saifer-Kammer, Leoffler-McDougold
3. THREE STEP METHOD
- Requires extraction w/ petroleum ether followed by
saponification and then colorimetric determination
- Standard method
METHOD: Abell-Kendall
4. FOUR STEP METHOD
- Requires extraction, saponification, purification with digitonin
then colorimetric determination
- Considered as the reference method
METHODS: Schoenheimer-Sperry, Sperry-Webb
B. ENZYMATIC METHODS

Apolipoprotein B : major protein of all lipoproteins except
HDL
- Two forms:
1. Large B or B100 (found in lipoprotein formed in
the liver)
2. Small B or B48 (found in lipoprotein formed in the
S.I.)
Apolipoprotein C : major protein of VLDL and chylomicrons
and a minor protein of HDL and LDL
Apolipoprotein D : function as a transfer protein
Cholesteryl ester + water ----cholesterol esterase----->
cholesterol + FFA
Cholesterol + O2 ----cholesterol oxidase----> cholest-4-en
3-one + H2O2
DETERMINATION OF TRIGLYCERIDES
1. COLORIMETRIC
a. Hantzsch-Lutidine Reaction (most common)
Formaldehyde + acetylacetone + NH4 3,5-diacetyl-1,4-
dihydro lutidine (yellow @ 410 nm)

Apolipoprotein E : plays a significant role in the recognition

& catabolism of chylomicron remnant and IDL via specific b. Van Handel and Zilversmit
receptors in hepatic cells Formaldehyde + H2SO4 + chromotropic acid (CTA) pink
chromophore
BLOOD SAMPLING & STORAGE
1. Biologic variation 2. ENZYMATIC
2. Fasting – ideally fast for 12 hours3. a. WEILAND METHOD
3. Posture- 10% decrease noted in TC, LDL-C, HDL-C
after 20 mins of recumbent position4. Glycerophosphate + NAD ---glycerophosphate DH --->
4. Venous vs capillary samples – capillary levels DHAP + NADH + H
generally lower than venous NADH + tetrazolium dye ---diaphorase---> formazan + NAD
5. Plasma vs serum – either can be used when only (340 nm)
cholesterol, triglycerides and HDL-C are measured
and LDL-C is calculated from these three
b. TRINDER REACTION
measurements
6. Storage – generally, frozen samples can be Glycerophosphate + O2 ---glycerophosphate oxidase--->
satisfactorily analyzed DHAP + H2O2
H202 + reduced chromogen -- Quinoneimine dye
ESTIMATION of PLASMA LIPIDS
A. COLORIMETRIC DETERMINATION of CHOLESTEROL
c. EGGSTEIN UND KREUTZ METHOD
STEPWISE METHODS
1. DIRECT COLORIMETRIC/ ONE STEP ADP + PEP ---pyruvate kinase ---> ATP + pyruvate
- serum is combined with the color reagent pyruvate + NADH + H ---LDH---> lactate + NAD (340 nm)
then incubated to allow color reaction to
occur
HDL-C DETERMINATION
METHODS: Watson’s, Ferro-Ham, Pearson, Zlatkis
- automated homogeneous assays
A. Liebermann-Burchard Reaction
- enzymatic method
Reagents: Acid anhydride, conc. H2SO4
End-product: cholesta polyene sulfonic acid
(emerald green) LDL-C DETERMINATION
B. Salkowski’s Reaction FRIEDEWALD CALCULATION
Reagents: conc. HAc, Ferric ions - for calculation of LDL-c and VLDL-c
End-product: cholesta polyene carbonium ion - cannot be used when TG is >400 mg/dL
(reddish-purple)
LDL-c = TC – HDL-c – plasma TG mg/dL
2. TWO STEP METHOD 5
- Involves extraction step prior to colorimetric reaction
- Eliminates protein interference

BSMLS-2 GERRYN OLGA ABAN CC-1

 VLDL-c = plasma TG mg/dL
5

VLDL-C in mmol/L = plasma TG

2.175

DELONG, 1986
VLDL in mmol/L = plasma TG
2.825

VLDL in mg/dL = plasma TG

6.5

ADULT TREATMENT PANEL III OR ATP III

CLASSIFICATION FOR LDL, TOTAL & HDL CHOLESTEROL
& TRIGLYCERIDE VALUES
LDL CHOLESTEROL (mg/dL)

<100 Optimal
100 – 129 Near optimal/above optimal
130 – 159 Borderline high
160 – 189 High
≥ 190 Very high

TOTAL CHOLESTEROL (mg/dL)

<200 Desirable
200 – 239 Borderline high
≥ 240 High

HDL CHOLESTEROL

<40 Low
≥ 60 High

TRIGLYCERIDES

<150 Normal
150 – 199 Borderline high
200 – 499 High
≥ 500 Very high

DYSLIPIDEMIAS
● Diseases associated with abnormal lipid
concentrations
multifactorial :
● genetic abnormalities
● environmental/lifestyle imbalances
● secondary to other diseases

BSMLS-2 GERRYN OLGA ABAN CC-1

 CHAPTER 10: LIVER FUNCTION TEST
LIVER
REVIEW OF THE ANATOMY & PHYSIOLOGY OF THE
LIVER
● Largest organ (1.2 – 1.5 kg in adult)
● Located in the right upper quadrant of the abdomen

Dual Blood Supply

a. Portal Vein – carries blood from alimentary tract
b. Hepatic Artery – carries oxygenated blood to the
liver

Functions of the Liver:

1. Receives, processes & stores materials absorbed
from the digestive tract
2. Synthesis: multiple plasma proteins METHODS
3. Main organ of detoxification ● Jendrassik-Grof & Evelyn-Malloy procedures:
4. Synthesis of bile acids from cholesterol - Both have acceptable precision
5. Major site of catabolism of hormones - Adapted by many automated instruments
- Most frequently used methods to measure
CLINICAL MANIFESTATIONS OF LIVER DISEASE Bilirubin
1. JAUNDICE – “Icterus”
- yellow discoloration of the plasma, skin, CLINICAL SIGNIFICANCE
sclerae & mucous membranes caused by 1) PRE-HEPATIC JAUNDICE
Bilirubin accumulation - Occurs when the problem causing the jaundice
2. PORTAL HYPERTENSION (>20 mmHg) occurs prior to liver metabolism
Increased: sinusoidal infiltration, scarring or hepatic vein - Caused by an increased amount of bilirubin being
obstruction presented to the liver (e.g. Acute & Chronic Hemolytic
Major Complications: Hepatosplenomegaly, Ascites & Anemia)
Hepatorenal syndrome - Unconjugated hyperbilirubinemia
2) HEPATIC JAUNDICE
- Primary problem causing the jaundice resides in the
3. HEPATIC FAILURE & ENCEPHALOPATHY liver (intrinsic liver disease)
- Accumulation of substances that are
normally metabolized by the liver DISORDERS OF BILIRUBIN METABOLISM
4. ALTERED DRUG METABOLISM A. Gilbert’s Syndrome – Intermittent hyperbilirubinemia;
- Liver plays a major role in enzymatic cause by reduction in the expression of UGT1A1
transformation & disposition of drugs gene (thereby reduced conjugating ability of the liver)
5. ENDOCRINE ABNORMALITIES B. Crigler-Najjar Syndrome
- Hormone imbalance - Chronic unconjugated hyperbilirubinemia;
6. NUTRITIONAL & METABOLIC ABNORMALITIES rare & more severe disorder
7. IMMUNOGLOBULIN ABNORMALITIES - Molecular defect w/in the gene involved with
- The liver efficiently sequesters dimeric IgA & bilirubin conjugation
secretes it into the bile Type I – complete absence of enzymatic bilirubin conjugation
8. DISORDERED HEMOSTASIS Type II – mutation causing a severe deficiency of the enzyme
- Chronic liver disease produces alterations in for conjugation
Hemostasis and a generalized hemorrhagic
tendency
DISORDERS RELATED TO TRANSPORT (TRANSPORT
DEFECTS)
BILIRUBIN METABOLISM
A. Dubin-Johnson Syndrome
DAILY BILIRUBIN PRODUCTION (average 250 – 300 mg) - Removal of conjugated bilirubin from the
● 85%: Derived from degradation heme from liver cell & the excretion into the bile are
hemoglobin defective
● 15%: - Caused by deficiency of the canalicular
FORMS OF BILIRUBIN transporter protein (MDR2/cMOAT)
1. UNCONJUGATED – bound by albumin & transported - Appearance of dark-stained granules on a
to the liver liver biopsy
2. CONJUGATED – formed from the reaction of UCB B. Rotor Syndrome
with glucuronic acid - Hypothesized to be due to a reduction in the
3. DELTA – conjugated bilirubin bound to Albumin concentration or activity of intracellular
binding proteins (e.g. Ligandin
- Liver biopsy does NOT show dark pigmented
granules

BSMLS-2 GERRYN OLGA ABAN CC-1

3) PHYSIOLOGIC JAUNDICE of the NEWBORN PROCEDURE
- Unconjugated hyperbilirubinemia BLANK STANDARD SAMPLE
- Deficiency in the enzyme Glucuronyl transferase
(one of the last liver functions to be activated in
Glucose 1.0 ml 1.0 ml 1.0 ml
prenatal life)
Reagent
- Kernicterus – deposition of bilirubin in the nuclei of
brain & nerve cells
- results in cell damage & death in the newborn Water 10 μL - -

4) POST-HEPATIC JAUNDICE Standard - 10 μL -

- Usually results from biliary obstructive disease (e.g.
gallstones, tumors) Serum - - 10 μL
- Prevents the flow of conjugated bilirubin into the bile 1. Mix, incubate @ 37 C for 5 mins
canaliculi 2. Read absorbance of standard (As) and samples (Ax)
- Clay-colored stool against reagent blank

CC LABORATORY COMPUTATION OF RESULTS

EXP 10: SPECTROPHOTOMETRY
PROCEDURE:
A. Examine the photometer (stat fax) and identify its parts
B. Operation of the Spectrophotometer (Stat fax machine)
1. Plug the line cord into the line power receptacle. SPECIMEN STABILITY
2. Position the power on-off switch at ON. Allow 15 ● Serum
minutes to warm up. ● Plasma
3. For obtaining absorbance readings, press the ABS ● Urine
key then ENTER. ● CSF
4. Using the number keys, select the filter appropriate - Separated and non-hemolyzed samples are stable 8
for the wavelength required for the analysis hours at 25C and 3 days at 2 – 8 C.
5. When “Read blank tube” is shown on the monitor, - Variable stability is observed with longer storage
insert the stat fax tube that contains the blank (water periods. Glycolysis decreases serum glucose by
blank, patient’s blank or reagent blank). approximately 5 –7% in 1 hour (5-10 mg/dL) in
6. After the blank tube, insert the stat fax tube that normal uncentrifuged coagulated blood at room
contains the standard reagent or calibrator. Record temperature.
the absorbance reading. - The rate of in-vitro glycolysis is higher in the presence
7. Remove the standard tube and replace it with a stat of leukocytosis or bacterial contamination.
fax tube containing the sample material. Again, record - Glycolysis can be inhibited and glucose stabilized for
the absorbance reading of the sample material. as long as 3 days at room temperature by adding
Note: Make sure that the outside of the stat fax tube is sodium iodoacetate or sodium fluoride (NaF) to the
properly wiped with gauze before inserting it into the cuvette. specimen.
8. Compute the concentration of the analyte being
measured based on Beer’s formula LINEARITY
- The method is linear for glucose values up to 500
mg/dl.
EXP 11: ELECTROPHORESIS
MATERIALS/EQUIPMENT: Horizontal Gel Electrophoresis
Unit EXP 13: GLYCOSYLATED HEMOGLOBIN
REFERENCE RANGES:
EXP 12: BLOOD GLUCOSE DETERMINATION
REFERENCE RANGES: NGSP IFCC
- Conventional Unit: 70 – 105 mg/dL
- S.I. Unit: mmol/L PREDIABETES 5.7 – 6.4% 39-46 mmol/mol

CONVERSION FACTOR: 0.055 PRESENCE OF >6.5% >48 mmol/mol

METHOD: Trinder/ God DIABETES
PRINCIPLE:
- The enzyme glucose oxidase catalyzes the oxidation TARGET IN <7.0% <53 mmol/mol
of glucose to gluconic acid and H2O2. DIABETES
- The H2O2 reacts with phenol and 4-aminoantipyrine
in the presence of peroxidase to form a quinoneimine
dye. METHOD: Boronate Affinity Assay
- The intensity of the color formed (pink) is proportional PRINCIPLE:
to the glucose concentration and can be measured - The CERA-STAT 4000 is a boronate affinity assay
photometrically between 480 and 520 nm - The CERA-STAT HbA1c Test Kit consists:
● Test device
● R1 reagent
● R2 reagent.

BSMLS-2 GERRYN OLGA ABAN CC-1

R1 reagent contains the agents that lyse erythrocytes and DETERMINATION OF TOTAL PROTEINS
precipitate hemoglobin specifically, as well as a blue boronic METHOD: Biuret, Modified
acid conjugate that binds cis-diol of glycated hemoglobin. PRINCIPLE:
- When a solution containing proteins is rejected with a
- When blood is added to the R1 reagent, the tartrate-complex cupric ions and alkali, the copper
erythrocytes are lysed and all hemoglobin binds to the peptide bond structure of the proteins,
precipitates. forming a purple colored chromogen
- The boronic acid conjugate binds to the cis-diol
configuration of glycated hemoglobin. COLORIMETRIC PROCEDURE
- An aliquot of the reaction mixture is added to the test
device and all the precipitated hemoglobin, BLANK STANDARD SAMPLE
conjugate-bound and unbound, remains on top of the
filter. Protein 3.0 ml 3.0 ml 3.0 ml
- Any unbound boronate is removed with the R2 Reagent
reagent. The precipitate is evaluated by measuring
the blue (glycated hemoglobin) and the red (total Water 20 μL -
hemoglobin) color intensity respectively with the
CERA-STAT 4000 Analyzer, the ratio between them Standard - 20 μL -
being proportional to the percentage of glycated
hemoglobin in the sample. Serum - 20 μL

PROCEDURE: 1. Mix well by gently inversion. Stand at room

1. Perform a clean capillary puncture. Fill the capillary tube temperature for 5 mins.
provided in the reagent kit with blood. 2. Read absorbance at 550 nm against a reagent blank.
2. Place the filled capillary tube into the vial which contains
reagent 1 (R1). Mix ten (10) times by gentle inversion and STABILITY: 60 mins
allow the vial to stand for two (2) minutes.
3. After gently mixing again the contents of the vial, use an DETERMINATION OF GLOBULIN
automatic pipet to aspirate 25μL of the mixture and transfer Globulin = gms. of total protein - gms. of albumin
this into the reaction area of the cartridge. Allow the sample to
be absorbed into the membrane for at least 10 seconds. DETERMINATION OF A/G RATIO
4. Add 25μL of reagent 2 (R2) into the test device. Again, allow A/G Ratio = gms. of albumin
the sample to be absorbed into the membrane for at least 10 gms. of globulin
seconds.
5. Insert the test device into the tray of the CERA-STATTM NORMAL VALUES AND CONVERSION FACTORS
04000 Analyzer and record the %HbA1c and CONVENTIONAL C.F. S.I. (gms%)
estimated blood sugar level as indicated on the screen. (gms%)

EXP 14: ALBUMIN, TOTAL SERUM PROTEINS,

A/G RATIO Total 6-8 10 60 - 80
proteins
SERUM ALBUMIN DETERMINATION
METHOD: Bromcresol Green (BCG) Method Albumin 3.5 - 5 10 35 – 50
PRINCIPLE:
- Determination of Albumin in serum or plasma is
Globulin 1.8 - 3.2 10 18 - 32
usually based on the binding behavior of the protein
with an anionic dye, bromcresol green forming a
green colored complex. A/G 1.5-2.4 : 1 - -
ratio
COLORIMETRIC PROCEDURE
BLANK STANDARD SAMPLE
EXP 15: BLOOD UREA NITROGEN
Reagent 3.0 ml 3.0 ml 3.0 ml DETERMINATION
REFERENCE RANGES:
Water 20 μL - - - CONVERSION FACTOR: 10-20 mg/dL
- S.I UNIT: 1.7 – 8.3 mmol/L
Standard - 20 μL - METHOD: Coupled enzyme reaction (Urease & Glutamate
DH)
Serum - - 20 μL PRINCIPLE:
3. Mix well by gently inversion and stand at room - The urease enzyme hydrolyzes urea in the sample to
temperature for 1 min. release ammonium ions, which react with
4. Read absorbance at 600 nm against the reagent 2- oxoglutarate and NADH in the presence of
blank. glutamate dehydrogenase to form glutamate and
NAD. The decrease in absorbance is measured at
STABILITY: 60 mins 340 nm.

BSMLS-2 GERRYN OLGA ABAN CC-1

PROCEDURE: PROCEDURE:
BLANK STANDARD SAMPLE BLANK STANDARD SAMPLE

Protein 2.0 ml 2.0 ml 2.0 ml Creatinine 1.0 ml 1.0 ml 1.0 ml

Reagent Working
Reagent
Incubate @ 37 C for 5 mins
Incubate @ 37 C for 5 mins
Water 20 μL - -
Water 100 μL - -
Standard - 20 μL -
Standard - 100 μL -
Serum - 20 μL
Serum - 100 μL
1. Mix; incubate 30 seconds at 37 C, then record
absorbance as A1. After exactly 60 seconds, record 1. Mix;incubate for 60 seconds at 37 C,then record
again absorbance as A2. absorbance as A1. After Exactly 60 Seconds, record
again absorbance as A2.
2. Read the absorbance against reagent blank at 510
COMPUTATION OF RESULTS: nm.
Urea mg/dL = A2 – A1 (sample) x50 (standard value)
A2 – A1 (standard)
COMPUTATION OF RESULTS:
Creatinine mg/dL = A2 – A1 (sample) x 2 (standard value)
LINEARITY: method for urea nitrogen is linear to values up to A2 – A1 (standard)
300 mg/dL

INTERFERENCES: SPECIMEN
- Plasma collected in anticoagulant containing either ● Serum
ammonium or fluoride salts cannot be used. ● Plasma
- No interference was observed by the presence of: ● Urine
Hemoglobin ≤500 mg/dL STABILITY:
Creatinine is stable 24 hours at 2 – 8 C. Freeze samples for
Bilirubin ≤4.4 mg/dL prolonged storage

Lipids ≤600 mg/dL LINEARITY: the method is linear for creatinine values up to 20
mg/dl.
EXP 16: CREATININE DETERMINATION INTERFERENCES
- No interference was observed by the presence of:
REFERENCE RANGES:
Hemoglobin ≤200 mg/dL
MALE 0.7 – 1.2 mg/dL (62-105μmol/L)
Bilirubin ≤7 mg/dL
FEMALE 0.6 – 1.1 mg/dL (53 - 97μmol/L)
Lipids ≤600 mg/dL
CONVERSION FACTOR: 88.4
METHOD: Modified Jaffe Reaction
PRINCIPLE: EXP 17: BLOOD URIC ACID DETERMINATION
- Creatinine reacts with picric acid in an alkaline
environment to form a red-colored product (creatinine REFERENCE CONVENTIONAL S.I UNIT
picrate). The intensity of the red color is directly RANGES
proportional to creatinine concentration which can be
measured at 500-520 nm. The use of a surfactant MALE 3.5 – 7.2 mg/dL 0.21 - 0.42 mmol/L
and sodium tetraborate keeps interferences at
minimum
FEMALE 2.6 – 6.0 mg/dL 0.15 - 0.35 mmol/L

CONVERSION FACTOR: 0.059

METHOD: Modified Trinder-Uricase/ POD
PRINCIPLE:
- Uric acid in the sample is oxidized by uricase to
allantoin and hydrogen peroxide. Hydrogen peroxide
reacts with ADPS and 4-aminoantipyrine in the
presence of peroxidase to yield quinoneimine dye.
- The intensity of the dye is measured photometrically
(510-560) and is directly proportional to the
concentration of uric acid in the sample.

BSMLS-2 GERRYN OLGA ABAN CC-1

PROCEDURE:
BLANK STANDARD SAMPLE

Reagent 1.0 ml 1.0 ml 1.0 ml

Water 25 μL - -

Standard - 25 μL -

Serum - - 25 μL
1. Mix; incubate at 37 C for 5 minutes.
2. Read absorbance of standard (As) and sample (Ax)
against reagent blank.

COMPUTATION OF RESULTS:

SPECIMEN:
● Serum or heparinized plasma.
● Oxalate, citrate and fluoride could yield a small
decrease in uric acid.

STABILITY: Uric acid in serum is stable for 5 days at 4C

LINEARITY: Method is linear to uric acid values up to 30

mg/dl.

INTERFERENCES:
No interference was observed by the presence of:

Hemoglobin ≤50 mg/dL

Bilirubin ≤33 mg/dL

Lipids ≤1,200 mg/dL

The following substances interfere in very high doses:

● a-methyldopa
● L-dopa
● 3,4 dihydroxyphenylacetic acid
● gentisic acid

BSMLS-2 GERRYN OLGA ABAN CC-1