CLINICAL CHEMISTRY: PRELIMINARIES  Catabolism of body Proteins; Purpose:

Romie Solacito, MLS3C o Synthesis of Proteins (Plasma Proteins,

PROTEINS - CHON
 Amino Acids - building blocks of protein
 Nitrogen – absent in Carbohydrates and Lipids
 Derived from amino acids, essential – external
source and non-essential – internal source of the
body, building blocks of proteins
 Plasma proteins are the most frequently analysed
 Proteins are also present in other body fluids.
Basic Structure
 Contains an Amino (NH2) and Carboxyl (COOH) as its
functional group.
 Differentiated based on their chemical composition
of their side chains. R group
 A bond that links one amino acid to another amino
acid is called Peptide Bond
 If it is a chain, it is known as polypeptide bond.
 Large polypeptide bond constitutes protein.
Intracellular, Extracellular)
o Synthesis of Non-Protein Nitrogen (Purines,
Pyrimidines, Porphyrin, Creatine, Histamine,
Epinephrine, and Coenzyme NAD)
o Provides 12%-20% body energy requirement
 The amino group is removed from the amino acid
thru deamination or transamination, forming a
ketoacid residue will enter into the metabolic
pathway with carbohydrates and fats.
o Glucogenic Amino acid – generates precursor of
glucose such as pyruvate(CAC)
 Alanine when deaminated forms pyruvate,
 Arginine form alpha-ketoglutarate
 Aspartate form oxaloacetate
o Ketogenic Amino Acid - generate ketoacids
(Leucine and isoleucine)
o Degraded to Acetyl CoA or Acetoacetyl CoA
o Some amino acid are both Ketogenic and
Glucogenic
o Ammonium ion from deaminated amino acid is
converted to urea in the liver.

 Zwitterion – use for the neutralization of protein pH

 Proteinization – use for the conversion from alkaline
to acidic
 Deproteinization – from acidic to alkaline
 Isoelectric point – average of acidic pH and alkaline
pH
 Peptide bond – bond between amino acid to another
amino acid; polypeptide bond – multiple bond.
Two Classifications of Amino Acids
1. Essential Amino acid -Supplied by the diet in the
form of proteins
a. i.e. Arginine, Methionine, Histidine,
Phenylalanine, Isoleucine, Threonine, Leucine,
Tryptophan, Lysine, Valine
2. Non – essential Amino acid
a. i.e. Alanine, Glycine, Asparagine, Proline,
Aspartic Acid, Serine, Cysteine, Glutamine,
Glutamic Acid, Tyrosine – produced from
phenylalanine
 Branched amino acid- Leucine, Isoleucine and Valine
Source of Amino Acid
Aminoacidopathies – enzyme defect that inhibits the
body’s ability to metabolize certain amino acid.
 Deficiency in Phenylalanine Hydroxylase Enzyme
will lead to accumulation of Phenylalanine and result
in Phenylketonuria (mousy odor urine).
 Deficiency in Tyrosine Aminotransferase Enzyme
will lead to accumulation of Tyrosine and result in
Tyrosinemia.
 Deficiency in Homogentesic Acid Oxidase will lead to
accumulation of Homogentesic acid which results in
Alkaptonuria (blackens urine in alkaline pH)
MS/MS – a highly specific and sensitive method for
aminoacidopathies.
Protein Structure
 Primary- Linear structure
 Secondary- Turning, coiling of structure (alpha helix
& Beta pleated)
 Tertiary- Folding of structure
 Quaternary- Interaction of more than 1 protein
(protein subunits)
Classification of Protein (Based In Their Structure)
 Simple Protein
o 2 forms: Globular and Fibrous
o Globular- water soluble, transporter,can interact
with water (e.g. Albumin,globulin)
o Fibrous – water insoluble, structural protein,(e.g.
Collagen)
 Conjugated protein – protein + non protein
compound
o Metal + protein = metalloprotein
o Lipids + protein = Lipoprotein
o CHO + protein = Glycoprotein
Protein Function
 Part of enzymes, catalyzes biosynthesis inside the
body.
 Structural (e.i. colagen)
 Contractility (e.i. actin and myosin)
 Antibodies
 Transport (haemoglobin – transport oxygen,
lipoproteins – transports lipids, and transferrin –
transports iron)
 Peptide hormone
Protein Types
 Major blood proteins:
o Albumin – fastest and smallest in size.
o Globulin – mostly immunoglobulins
o Fibrinogen – biggest in size
 Blood Panel:
o Total protein
o Albumin
o Globulin
o Albumin Globulin ration – 2:1
 Minor proteins:
o Haptoglobin, transferrin, haemoglobin and etc.
TUNGSTICK ACID – use for deproteinization of Amino
Acid
 Total Plasma Protein: Albumin + Globulin +
Fibrinogen
 Total Serum Protein: Albumin + Globulin (6.5 - 8.3 or
8.5g/dl)
 Albumin: 3.5 - 5.5g/dl
 Albumin Globulin Ratio: 3:1 or 2:1
 Globulin: Total Protein – Albumin
Other Proteins
 Prealbumin/Transthyretin – transport protein for
THYROID HORMONE and Vitamin A
 Alpha 1 Atitrypsin – neutralized trypsin like enzyme
and major inhibitor of protease activity
 Alpha 1 Fetoprotein – synthesized in developing
embryo/fetus. Elevated levels in conditions like spina
bifida, and neural tube defects.
 Alpha 1 Antichymotrypsin – binds and inactivates
Prostate specific antigen
 Hemopexin – helps in the diagnosis of early
hemolysis. Binds heme released by degradation of
Hemoglobin
 Haptoglobin – binds to free hemoglobin by its alpha
chain and prevents loss of hemoglobin and it
constituent iron into the urine
 Ceruloplasmin – copper binding alpha 2 glycoprotein
that has enzymatic activities
 Alpha 2 Macroglobulin – largest major non
immunoglobulin protein in plasma
 Tranferrin/Siderophilin – transports iron to its
storage site
Protein Regulation and Disorders
 Hypoproteinemia – protein loss in renal disease;
leakage into gastrointestinal tract in chronic disease;
loss of blood, severe burns; dietary
deficiency/malabsorption; liver disease.
 Hyperproteinemia – not as common; dehydration
 Hyperalbuminemia – Chronic liver damage;
malnutrition
 Inverted A/G Ratio – liver disease, kidney disease, ad
chronic infection.
Protein Quantitation
 Albumin – abundant protein
 Serum is added with 22.6% of Na2SO4
 Salt Fractionation – supernatant is analysed
subtraction of globulins.
 Globulin - direct colorimetric method using glyoxylic
acid
 Albumin Globulin Ration – multiple myeloma, Bence
Jones Protein, “tall spike” or “monoclonal peak”
 Fibrinogen – Parfentyev method
 Total Serum Protein
o Serum Protein Electrophoresis
o Microkjeldahl Nesslers Method
o Biuret Method
 Albumin – with Dye Binding Method (methyl orange;
Bromcresol Green; Bromcresol Purple; H-ABA [2,4′-
hydroxyazobenzenebenzoicacid])
Serum Protein Electrophoresis – separation of proteins
in medium
 Buffer – use for maintenance of pH 8.6
 Principle: The separation of protein varies on size,
charge, and support medium: agarose, cellulose
acetate, and polyacrylamide gel
 Strains: Ponceau S, amido black, and Coomassie blue
 Pre-Albumin: 0.01 – 0.04MW – not measured due to
low concentration
Abnormal Serum Protein Electrophoresis
 Gamma Spike – Multiple Myeloma
 Beta Gamma Bridging – Hepatic Cirrhosis
 Alpha-2 Globulin Spike – Nephrotic syndrome
 Alpha-2 Globulin Flat Curve – Juvenile Cirrhosis
Microkjeldahl Nesslers Method
 Reference method but not routinely used
 Measurement of Nitrogen content of protein
 Kjeldahl – digesting agent (H2SO4); end product
(ammonia)
 Nesslerization – Alkaline K2HgI1
 Expressed as Total Protein Nitrogen x 6.25(factor) =
Total Serum Protein
Nitrogent –N2SO4-> NH4  NH3 Gas + K2HgI4 = Yellow
Dimercuric Amino Iodide
Biuret Method
 Most widely used method that measures peptide
bonds
 Requires two peptide bonds and an alkaline medium
 Reagent: alkaline copper citrate, sodium potassium
tartrate NaOH and potassium iodide
 Violet-purplish color is produced when cupric
ions(Cu2+) interact with peptide bonds under alkaline
conditions and the absorbance is read at 545nm
Dye Binding Method – sensitive method to measure
Albumin
 Methyl Orange
 Bromcresol Green – most commonly used
 BCP- most specific dye
 H-ABA
NONPROTEIN NITROGEN
 Important to assess renal function; end product of
protein metabolism; Non protein entities that
contains Nitrogen molecule in their chemical
structure
 Renal Function:
o Filtration – glomerulus
o Reabsorption – tibules
o Secretion – Tubules
o Excretion
 Follows the principle of kidney filtration and/or
reabsorption
 Serum and urine level
 Kidney Function Test
 Clinically Significant NPN – approximately plasma
concentration
 Non Protein Nitrogen:
o Urea - 45-50%
o Amino Acids – 25%
o Uric Acid – 10%
o Creatinine – 5%
o Creatine – 1-2%
o Ammonia – 0.2%

Urea
 Most abundant non-protein nitrogen: 45-75% of
circulating NPN
 Source: Protein Catabolism  Amino Acid
Catabolism  Nitrogen  Ammonia  Urea (Urea
cycle in Liver)
 Equal in the ICS and ECS
 Chief by product of protein metabolism – amino
acids to ammonia to urea (liver)
 Waste product removed by kidneys
o 90% excreted and 10% reabsorbed
 Factor: 2.14
 CLINICAL SIGNIFICANCE:
o Rough estimate of renal function: needs a 50%
decrease of glomerular function
o Urea in the blood is influenced by: dietary
protein and renal excretory function
o Uremia – abnormally high nitrogen in the plasma
accompanied by kidney disease
o Azotemia - an elevated concentration of urea in
the blood and increase levels of other NPN
 SPECIMEN
o Serum
o Heparinized Plasma
o Urine and other body fluids
o Consideration:
 Anticoagulant
 Bacterial Action
 Preservation: 72 hours refrigeration
 QUANTITATIVE DETERMINATION:
o Microkjeldahl Nesslerization (indirect) method
heparinized plasma – measures Urea by nitrogen
content (multiplied by factor 2.14 to get urea)
o Rosenthal (direct) method – diacetyl monoxine
(DAM Method) – direct method for urea
measurement
o Enzymatic method – urease enzyme or Berthelot
Method (Berthelot Reagent/Na Nitroprusside
Phenol = Indophenol Blue)
o Glutamate Dehydrogenase – most commonly
used method; the decrease of absorbance of
NADPH is measured at 340nm
o Urostrat/Urograph – paper chromatography
o Urease – urea amidohydrolase
o Isotope Dilution Mass Spectrometry – reference
method
 REFERENCE VALUE:
o 8 – 23mg/dL
o 2.9 – 8.2 mmol/L
o Conversion Factor: 0.357
Creatine
 By product of muscle activity – creatine and creatine
phosphate
 Creatine is formed from Arginine, Glycine and
Methionine (Liver)
 CK is storage form of energy
 99% is excreted in urine as waste product
 Produced in constant rate
 Mostly excreted + produced in constant rate + not
secreted or reabsorbed by tubule=reason why it is
preferred over urea.
 Dependent in muscle mass.
 Preferred over urea since:
o Not influenced by protein intake
o Least variable (blood flow)
 Output is influenced by:
o Muscle mass
o Exercise
o Body Size
 CLINICAL SIGNIFICANCE:
o Kidney Function Test
o Crea Ratio:
 10 to 20:1
 Significantly Low: tubular necrosis, low
protein intake, starvation, sever liver disease
 High Ratio with Normal Creatinine: tissue
breakdown pre-renal azotemia, high protein
intake
 High Ration with Increased Creatinine:
post-renal obstruction or pre-renal azotemia
associated with renal disease
 QUANTITATIVE DETERMINATION
o Jaffe Reaction
 Crea. reacts with alkaline picrate to form an
orange – red solution that is measured
spectrophotometrically; most commonly
used method but is subjected to
interferences (Carbohydrates, Protein,
Ascorbic Acid, Pyruvate, Lipemic and Icteric
sample)
 Interferences: protein, carbohydrates,
ascorbic acid, and pyruvate
 Solution: acidification step, Loyd’s reagent
o Enzymatic Methods – sensitive method for
creatinine
 Creatinine amidohydrolase (creatininase)
 Creatine amidohydrolase (creatinase)
 SPECIMEN:
o Serum
o Heparinized plasma
 Cl: ammonium heparin tube
o Diluted urine (1:100 or 1:200)
o Consideration:
 Separate cells – prevent hemolysis and
ammonic production
 Preservation – 1 week refrigeration
 CREATININE CLEARANCE
o mL of plasma clearance of creatinine by the
kidneys/minute
o dependent of patient’s body surface area (1.73)
o assesses glomerular filtration (indirect)
o Sample:
 24hour urine collection (refrigeration)
 Blood (12th hour)
 CREATINE (REFERENCE VALUE)
o 0.8 – 2.0 mg/dL
o 70 – 180 µmol/L
o Factor: 88.4
 Not a kidney function test
 Difference between creatinine?
o Elevated creatinine – renal disease
o Elevated creatine – muscle disease
Uric Acid
 By product of purine nucleoside metabolism
 Relatively insoluble (pH: 7) in plasma: in high
concentration can be deposited in joints causing
painful inflammation
 Major disease associations:
o Gout
o Increased catabolism of nucleic acids
o Renal disease
 Not a Kidney Function Test, 90% reabsorbed
 CLINICAL SIGNIFICANCE:
o Assessment of inherited disorders of purine
metabolism
o Confirmation of diagnosis and monitoring of
gout
o Assistance in diagnosis of renal calculi
o Prevention of uric acid nephropathy in
chemotherapy
o Detection of kidney disease
o Lesch Nyhan Syndrome – lacks Hypoxanthine
guanine phosphoribosyl transferase
o Increased: increased catabolism of nucleic acid,
chronic renal disease, and megaloblastic anemia
o Decreased: Liver disease
 SPECIMEN:
o Serum
 o Heparinized plasm  Measures GFR
o Urine  Unlike Creatinine, it is not dependent on body mass
o Should be removed from cells  Detects mild to moderate filtration loss
o Stable once Red Blood Cells is removed
o Refrigeration – 3 to 5 days
 QUANTITATIVE DETERMINATION:
o Enzymatic Method – uricase/urate oxidase –
measures differential absorption of uricase and
Allantoin at 282–292nm; Catalyse oxidation of
uric acid to allantoin
o Chemical methods/Caraway – phosphotungstic
acid (PTA); most commonly used method; Blue
reaction as Phosphotungstic Acid is reduced by
Urate in alkaline medium.
o Isotope dilution Mass Spectrometry – Reference
method
 REFERENCE VALUE:
o Male: 3 – 7mg/dL or 0.2 – 0.5mmol/L
o Female: 2 -6mg/dL or 0.16 – 0.43mmol/L
o Factor: 0.059

Ammonia/NH3
 By product of Amino Acids
 High levels are neurotoxic
 Major disease associations:
o Hepatic failure (most common)
o Reye’s syndrome
o Inherited enzyme deficiencies
 Not a Kidney Function Test
 SPECIMEN:
o Whole blood ammonia – increase in nitro rapidly
o Heparinized
o EDTA Tube
o Centrifuged at 0 – 4C within 20 min
 QUANTITATIVE DETERMINATION
o ISE – measures changes in pH of ammonium
chloride as ammonia diffuses across a semi-
permeable membrane
o Enzymatic Method – Glutamate dehydrogenase
 REFERENCE VALUE:
o 19 – 60 ug/dL
o 11 – 35 umol/L

Cystatine C
 Protein synthesized by all nucleated cells
 Has a constant production rate in the body