CC1: CLINICAL CHEMISTRY 1 (LECTURE)

LECTURE 7: NON-PROTEIN NITROGENS

1st SEMESTER | S.Y. 2024-2025

PROTEINS

CHARACTERISTICS OF PROTEIN
▪ Most abundant macromolecule in the body
▪ The only macromolecule expressed in g/dL, unlike lipids and carbohydrates, which are both
expressed in mg/dL
▪ Contains Carbon, Hydrogen, Oxygen and Nitrogen
▪ Mostly synthesized in the liver, except for some proteins (Immunoglobulins- which are produced
by plasma cells)
▪ Amino acids are the building blocks of proteins
▪ Peptide bonds hold amino acid together

STRUCTURE OF PROTEINS
a) Primary- refers to the sequence of amino acids in a peptide or protein
b) Secondary- may include the alpha-helix, beta-sheet, beta-turn or random structure of proteins
c) Tertiary- folding of proteins into a three-dimensional shape; denaturation of proteins refers to
unfolding that occurs when temperature change or in the presence of organic solvents, detergents
or reagents that may change the tertiary structure
d) Quaternary- incorporation of two or more polypeptide chains or subunits into a larger unit;
example include creatine kinase with two subunits as well as LDH and hemoglobin having four
subunits

PROTEINS CAN BE DIFFERENTIATED BASED ON

- Differential solubility- proteins are affected by pH, ionic strength, temperature and dielectric
constant. Isoelectric point is the pH where proteins have no net charge
- Molecular size- can be accomplished through ultracentrifugation or dialysis
- Molecular mass- can be accomplished through mass spectrometry
- Electrical charge- can be accomplished using ion-exchange chromatography and electrophoresis
- Surface adsorption- can be done using chromatography

PLASMA VS. SERUM PROTEIN: Plasma contains fibrinogen whereas serum does not; an
approximate 4% decrease in total protein content in serum due to absence of fibrinogen

PROTEIN METABOLISM
a) Protein digestion begins in the stomach and completed in the small intestines
b) High acidity denatures the protein making them susceptible to enzyme digestion
c) Pepsin is an enzyme in the stomach responsible for digesting proteins into amino acids

CABAY, J. B. | BMLS3 | PLT COLLEGE, INC.

d) Amino acids are absorbed from the small intestines into the blood and then transported to the
liver

FUNCTIONS OF PROTEIN
- Regulate colloidal oncotic pressure - Form many important intracellular and
- Act as carrier for transport of different extracellular structures
substances - Important for tissue repair (collagen)
- Coagulation cascade - Generate energy through catalysis and electron
- Complement fixation transfer
- Serves as biocatalysts or enzymes - Produce motility through contractile elements
- Maintenance of acid-base balance (proteins - Assemble molecules
act as buffer) - Serve as ion channels and pumps
- Immunologic functions (antibodies and - Serve as receptors, hormones, and cytokines for
complement proteins) intercellular regulation
- Constitute signaling networks for intracellular
regulation

MAJOR PLASMA PROTEIN

FRACTION SPECIFIC PROTEINS
PREALBUMIN Prealbumin
ALBUMIN Albumin
ALPHA1 GLOBULIN Alpha1 antitrypsin, alpha-fetoprotein, alpha-lipoprotein, alpha1-anti-
chymotrypsin
ALPHA2 GLOBULIN Ceruloplasmin, Haptoglobin, Alpha2 macroglobulin
BETA GLOBULIN Transferrin, Hemopexin, Beta2 microglobulin, Complement system,
Fibrinogen, LDL, VLDL, Creactive protein
GAMMA GLOBULIN Immunoglobulins, C-reactive protein

PLASMA PROTEINS
1. PREALBUMIN
- It migrates ahead of albumin
- It has a half-life of only 2 days
- It is rich in tryptophan
- It has considerable β-pleated sheet conformation
- It serves as transport protein for T4 retinol (Vitamin A)- by complexing with retinol-binding
protein
- It is used to detect malnutrition and individual’s response to dietary supplementation
- It is used as landmark to confirm that the specimen is really CSF- it crosses more easily into
the CSF than other proteins
- Increased- alcoholism, chronic renal failure, steroid treatment
- Decreased- poor nutrition
- Reference value- 18-45 mg/dL
2. ALBUMIN
- It is the protein present in highest concentration in plasma
- It is synthesized in the liver
- A general transport protein (binds various substances in the blood)
- It maintains osmotic pressure
- It is an indicator of nutritional status
- It serves as circulating reservoir of amino acids
- It is a sensitive and highly prognostic marker in cases of cystic fibrosis
- It is a “negative acute phase reactant”- it decreases in acute inflammatory processes
- Lowest plasma albumin levels are seen in active nephrotic syndrome
- Reference value- 3.5-5.0 g/dL

3. GLOBULIN
a. α1- Antitrypsin
- It is an acute-phase reactant; a glycoprotein
- It neutralizes trypsin-like enzymes (like neutrophil elastase)- this enzyme is released
from WBCs
- It is major inhibitor of protease activity- prevents self-destruction of tissues
- Increased- inflammation, pregnancy and contraceptive use
- Deficiency- emphysematous pulmonary disease and juvenile hepatic cirrhosis
- Reference value- 145-270 mg/dL
b. α1- Fetoprotein
- A glycoprotein; migrates between albumin and α1 globulin band
- It is synthesized initially by the fetal yolk sac and then by the fetal parenchymal cells of
the liver
- It is the most abundant protein in fetal serum
- It peaks in the fetus at 13 weeks of gestation
- It is detectable in the maternal blood up to the 7th or 8th month of pregnancy
- Maternal serum AFP is increased in the presence of twins (transmitted across the
placenta)
- It has no known function in adults
- Specimen- maternal serum, amniotic fluid, serum (for cancer screening)
- Diagnostic significance
✓ Maternal serum AFP is used as a screening test for any fetal conditions- it detects
neural tube defects and Down syndrome
✓ Maternal AFP screening for NTD and DS is done between 15- and 20-weeks
gestational age when the maternal serum increases gradually
✓ AFP is also used as a tumor marker (hepatic and gonadal cancer)
✓ Elevated serum AFP levels postnatal occur only with conditions of abnormal cell
multiplication such as cancer
c. α1- Acid Glycoprotein/ Orosomucoid
- It has the greatest affinity for progesterone; binds quinidine
- It is a useful diagnostic tool in neonates with bacterial infections
- Its amino acid sequence is similar with immunoglobulin
- Increased- pregnancy, cancer, pneumonia, rheumatoid arthritis and cell proliferation
- Reference value- 55-140 mg/dL
d. α1- Antichymotrypsin
- It is a serine proteinase with cathepsin G; acute phase reactant
- It migrates between the α1 and α2 zones; synthesized in the liver
- It binds and inactivates prostate-specific antigen
- It is the major form of PSA found in human sera
- It is associated with the pathogenesis of Alzheimer’s disease
- Increased- infection, malignancy, burn, AMI, and Alzheimer’s disease
- Deficiency- liver disease
- Reference value- 30-60 mg/dL
e. Hemopexin
- It binds heme released by degradation of hemoglobin- has the strongest affinity for heme
- It helps in the diagnosis of early hemolysis
- It migrates in the β region during electrophoresis
- Reference value- 50-115 mg/dL
f. Group specific component globulin
- It exhibits affinity with Vitamin D and actin
- It migrates in the alpha-1 and alpha-2 interzone during electrophoresis
- Method- radial immunodiffusion
- Reference value- 20-55 mg/dL
g. Haptoglobin
- Is an alpha 2 glycoprotein: an acute phase reactant
- It is synthesized in the hepatocytes
- It has 2 heavy chains and 2 light chains linked by disulfide bonds in analogy to the basic
structure of immunoglobulins
- It binds free hemoglobin by it alpha chain
- It prevents the loss of hemoglobin and its constituent iron into the urine
- It is a useful measurement for serially monitoring patients who have a slow but steady
rate of red cell breakdown such as mechanical heart valves, hemoglobinopathies, or
exercise-associated trauma
- It evaluates degree of intravascular hemolysis
- Its plasma level is slightly decreased after blood transfusion
- It is also one of the proteins used to evaluate rheumatic diseases
- Increased- stressful conditions, myoglobinuria
- Decreased- intravascular hemolysis and hemoglobinuria
- Reference value- 26-185 mg/dL
h. Ceruloplasmin
- Is a copper-binding alpha-2 glycoprotein that has enzymatic activities
- It is synthesized in the liver, where 6 to 8 atoms of copper are attached
- It imparts a blue color to protein
- It is a marker for Wilson’s disease
- Clinical features of Wilson’s disease- deposition of copper in skin, liver, brain, and cornea
(Kayser-Fleischer rings)
- Increased- inflammation, cancer, pregnancy
- Decreased- wilson’s disease, malnutrition, malabsorption, nephritic disease and Menkes’
kinky hair syndrome
- Method- copper oxidase activity
- Reference value- 18-45 mg/dL
i. α2- Macroglobulin
- Is the largest major non-immunoglobulin protein in plasma
- It is found principally in the intravascular spaces; it does not diffuse from the plasma
space; lower amounts can also be found in CSF
- It inhibits proteases such as trypsin, pepsin, and plasmin
- It increases 10X in nephrosis- the loss of AMG into urine is prevented by its large size
- It forms a complex with prostate-specific antigen
- Increased- nephrotic syndrome, diabetes and liver disease
- Reference value- 150-420 mg/dL
j. β2-Microglobulin
- Is a light chain component of the major human leukocyte antigen
- It is found on the surface of most nucleated cells; present in high concentration on
lymphocytes
- It is needed in the production of cd8 cells
- It is freely filtered at the glomerulus, and then is reabsorbed and metabolized completely
by the proximal tubule- elevated plasma levels are the result of impaired clearance by the
kidneys or over production of proteins as seen in inflammatory diseases
- Increased- renal failure, multiple myeloma, rheumatoid arthritis, SLE, and HIV
- Reference value- 0.2-0.8 µg/dL
k. Transferrin
- It is a major component of the β2 globulin fraction
- It is a glycoprotein, synthesized in the liver; also a negative acute phase reactant
- It transports iron to its storage sites; CSF contains small amount
- It is used to determine the cause of anemia, to measure iron metabolism and determine
the iron-carrying capacity of the blood
- It prevents loss of iron through the kidneys
- Increased- hemochromatosis, IDA
- Decreased- liver disease, malnutrition, nephrotic syndrome
- Reference value- Male= 215-365 mg/dL; Female= 250-380 mg/dL
 l. Immunoglobulins
- Are synthesized in the plasma cells
- Increased- hepatic disease, infections, toxoplasmosis, cytomegalovirus infections
m. Lipoprotein: It binds with proteins and lipids forming HDL, LDL, VLDL and chylomicrons
n. Fibrinogen
- Is one of the largest proteins in the blood
- It is synthesized in the liver; classified as glycoprotein
- It is the most abundant of the coagulation factor- it forms a fibrin clot when activated by
thrombin
- Is one of the acute- phase reactants
- Increased- inflammatory disorders, pregnancy, and use of birth controls
- Decreased- extensive coagulation
- Reference value- 200-400 mg/dL
- Complement
- Is one of the natural defense mechanisms that protects the body from infection
- It participates in the immune reaction and serve as a link to the inflammatory response
- Increased- inflammatory conditions
- Decreased- DIC, hemolytic anemia and malnutrition
o. C-reactive protein
- Is a general scavenger molecule; gamma migrating protein
- It may be undetectable in the blood of healthy individuals
- It appears in blood of patients with diverse inflammatory diseases
- Increased- acute rheumatic fever, MI, RA, gout, bacterial and viral infection

MISCELLANEOUS PROTEINS
• Bence-Jones protein: Are proteins identical light chains excreted in the urine of patients with
multiple myeloma. BJP characteristically precipitates at 40-60ºC but dissolves at 100ºC
• Myoglobin:
- Is a heme containing protein found in the striated skeletal and cardiac muscles. It can be used
as a marker of AMI
- Rises at 1-3 hrs, peak at 5-12 hrs, returns to normal in 18-30 hrs
- It is not specific for AMI since it can also be elevated in muscle-related disorders
• Troponin
- Are complex of three proteins that bind to the thin filaments of cardiac muscles
- Are regulators of actin and myosin

METHODOLOGIES OF PROTEIN DETERMINATION

▪ Direct optical method: Absorbance of UV light at 200-225nm (or 210) and 270-290nm (280) have
been used to measure protein concentrations and is applied to monitor chromatographic
separations of peptides and proteins
▪ Kjeldahl method
- Is based on the digestion of protein with consequent measurement of nitrogen content
- Considered as the reference method; assumes average nitrogen content of proteins is 16%.
Actual nitrogen content of serum protein ranges from 15.1-16.8%. some references prefer the
use 6.54, which is the average nitrogen content of different proteins
- Interferences: NPNs like urea and amino acids can cause an increase- therefore precipitation
test is required
▪ Biuret reaction
- Is based on the formation of violet colored chelate between Copper ions and requires at least
2 peptide bonds, measured at 540nm
- Biuret is a molecule that forms when urea, the end product of protein metabolism is heated at
180ºC
- Reagents include:
✓ Alkaline copper sulfate
✓ Sodium hydroxide
✓ Rochelle salt
✓ Potassium iodide
- Interferences in Biuret reaction
✓ High bilirubin, elevated lipids, hemolyzed sample- false increased
✓ High blood ammonia level- false decreased
▪ Lowry (Folin-ciocalteu) method
- Specimen are mixed with an alkaline copper solution followed by addition of Folin-Ciocalteu
reagent
- Phosphotungstomolybdic acid are reduced to tungsten blue by copper complex with peptide
and by tyrosine and tryptophan
- Absorbance products are measured at 650-700nm; dependent on phenolic groups
▪ Dyes
- Bromcresol purple- most sensitive, specific and precise among the dye-binding assays
- Bromcresol green- most commonly used; measure absorbance
- Tetrabromphenol blue- used in urine reagent strip, sensitive to albumin
- Ninhydrin- for amino acids
- Methyl orange
- Hydroxybenzeneazobenzoic acid
- Coomasie brilliant blue
- Pyrogallol red- used for analysis of fluids with lower protein concentrations such as urine and
CSF
▪ Refractometry
- Rapidly estimates protein at high concentrations
- Often used in urine specimen
▪ Salt precipitation test- used for urine and CSF
▪ Electrophoresis:
✓ Movement of proteins during alkaline electrophoresis, from origin towards the anode:
- Gamma, beta, alpha 2, alpha 1, albumin, Prealbumin
✓ Gamma globulins tend to migrate towards the cathode instead of moving towards the anode
during electrophoresis. This is known as electroendosmosis
✓ Serum is electrophoresed at an alkaline pH (8.6). Since the pH is alkaline, proteins in serum
become negatively charged, causing them to be attracted towards the positive pole or
electrode known as the anode
✓ After separation, regions are stained using Coomassie Brilliant blue, Amido black and
Ponceau S
✓ After staining, stained bands are quantified using densitometer
✓ Electrophoretic patterns:
- Beta-gamma bridging effect- seen in patients with liver cirrhosis caused by an increase in
IgA which is found between the beta and gamma region during electrophoresis
- Gamma spike- seen in cases of monoclonal gammopathy (multiple myeloma and
Waldenstrom’s macroglobulinemia)
- Alpha-2 macroglobulin elevation with albumin decrease- seen in patients with nephrotic
syndrome
- Alpha-1 antitrypsin deficiency- seen in patients with emphysema
- Increased beta region- use of plasma instead of serum during electrophoresis
- Low albumin, high alpha-1 and alpha-2 region- acute inflammation

VALUES TO REMEMBER
▪ Reference range
- Total protein- 6.5 to 8.3 g/dL
- Albumin- 3.5-5.5 g/dL
▪ Globulin= total protein minus albumin
▪ Conversion factor- g/dL to g/L= 10