clinical chemistry protein

1. UNIT 10
PROTEINS
Clinical Chemistry IProteins 1

2. Objective
 At the end of this unit, the student is expected to:
 Describe basic structure and composition of proteins and
amino acids
 Show how protein properties can be used for their
identification and assay
 Describe the physiological functions of proteins
 Describe the pathological aspects of prteins
 Describe the methods used for determination of protein
concentration in blood 2

3.  Introduction
 Classification, Properties and Functions of proteins
 Plasma proteins with clinical significance
 Specific methods for the determination of proteins
 Serum protein electrophoresis
 Samples
 Interpretation
3
Outline

4. Introduction
 Protein is polymer of amino acids linked covalently through
peptide bonds [polypeptide].
 Amino-acid: an organic compound containing both amino and
carboxyl functional groups.
 Simplest units of proteins.
 There are 20 different kinds of amino acids combined indifferent
proportion & arrangements to build all protein molecules.
 Two AA combined by peptide bond are called a dipeptide.
 When amino acids involved in the bond formation become 3, 4,
5 they are named as
 tri-, tetra-, and penta- peptides respectively. 4

5. Amino acids
 Amino acids are organic cpds that combine to form proteins.
 Both Amino acids and proteins are the building blocks of life.
 When proteins are digested or broken down, amino acids are
left.
 The human body uses amino acids to make proteins to and to
help the body:
Break down food, Growth
Repair body tissue
Perform many other body functions
 AA can also be used as a source of energy by the body.

6.  Amino acids are classified into three groups:
Essential amino acids
Nonessential amino acids
Conditional amino acids
 ESSENTIAL AMINO ACIDS
 Essential amino acids cannot be made by the body.
 As a result, they must come from food.
 The 9 essential amino acids are:
 Histidine, methionine, threonine, phenylalanine,
tryptophan, isoleucine, Leucine, lysine, and valine

7. NONESSENTIAL AMINO ACIDS
Our bodies produce these an amino acid, even if we do not get
it from the food we eat.
Nonessential amino acids include:
Alanine, Asparagine, Aspartic Acid, and Glutamic Acid.
 CONDITIONAL AMINO ACIDS
 Conditional amino acids are usually not essential, except in
times of illness and stress.
 Arginine, Cysteine, Glutamine, Tyrosine, Glycine, Ornithine,
Proline, And Serine

8. Proteins
8
 All proteins contain carbon, hydrogen, oxygen, & nitrogen (CHON).
 Some proteins may also contain sulfur phosphorous, copper
, iron,
zinc, iodine, and other elements.
 The presence of nitrogen in all proteins sets them apart from
carbohydrates and lipids.
 The average nitrogen content of proteins is approximately 16%.
 Comprises 50-70% of cell’s dry weight
 Found in cells, as well as in all fluids, secretions, and excretions
 More than 300 different types of plasma proteins have been
discovered

9. Protein classification
 Proteins can be classified based on their composition as:
 Simple proteins (made from aa only)
 e.g. albumin, fibirinogen, globulin
 Complex proteins: (protein + non-protein parts)
 apoproteins (protein parts or outer moiety)
 conjugated proteins (non-protein parts)
e.g. Hemoglobin, ferritin, lipoproteins, glycoprotein
9
 Also can be classified based on their shape as:
 Fibrous proteins, globular proteins
 Fibrous proteins
 They are insoluble in water, acid, base etc
 Have stronger intermolecular force of attraction
 They have thread like structure
 They have helical or sheet structure
[e.g. fibrinogen, troponin, collagen, silk, wool etc]

10.  Globular proteins:
They are soluble in water, acid, base etc
They have week intermolecular hydrogen bonding
They have folded, ball like structure
They have three dimensional shape
e.g. Hgb, enzymes, peptide hormones, plasma proteins
 Retain their biological activities with in narrow range of TO and PH.
 When exposed to high temperatures or extremes of pH causes the
molecules of protein to “denature” and loss their solubility and
biological activities e.g. enzymes lose their catalytic function

11. 11
 Globular proteins have four levels of structure
 Primary = sequence of amino acid units
 Secondary= folds of the protein strand
 α helix = twisted format
 βsheet = zigzag form
 Tertiary = three dimensional assembly
 Quaternary= association of 2 or more polypeptides

12. Properties of protein
 Many properties of proteins are used for their
separation, identification, and assay.
 The following 5 are among them:
1. Molecular size: most proteins have high molecular masses, thus
separated from smaller molecules by
 Dialysis, ultra filtration, molecular gel filtration
chromatography, density gradient and Ultracentrifugation
2. Differential solubility: affected by PH, ionic
strength, temp, and dielectric constant of the solvent.

13. 13
3. Electrical charge:
 proteins are ampholytes; that is, in aqueous solutions they may
have positive and negative charges on the same molecule.
 In pH above or below the isoelectric point, proteins become
ionized
 This property is used to separate protein molecules during
electrophoresis.
 The pH of the solution determines the net charge of the molecule
 The pH at which the net charge on the molecules in solution is
zero is called the isoelectric point (pI).

14. 14
4. Adsorption on freely divided inert materials:
 These materials offer large surface areas for interactions
[hydrophobic, absorptive, ionic, or molecular] with proteins
4. Specific binding to antibodies, coenzymes, or hormone
receptors:
 This is the basis for immunochemical assays,
 Proteins are also separated by affinity chromatography,
in w/c a ligand attached to a solid medium provides
high selectivity.

15. Function of Proteins
15
 Used to construct or build our body
 Catalyze biochemical reactions as an enzyme
 Regulate body metabolism as hormones
 Protect our body from foreign body attack as an antibody and
components of complement
 Maintain osmotic pressure in plasma
 Transport different lipids, minerals, hormones, vitamins etc as
hemoglobin, apolipoprotein, albumin etc
 Assist to arrest bleeding and maintain homeostasis as coagulation
factor

16. 16
Positive APPs
 Proteins increase in plasma concentration in response to
infammation
 include α1- -antitrypsin, α1- -acid, glycoproteins,
haptoglobulin, ceruloplasmin, C3, CRP
Negative APPs
 Proteins decrease in plasma concentration in response to
infammation
 include albumin, transthyretin, and transferrin.
 Most of plasma proteins are synthesized and catabolized in the
liver.
 g-globulins are made by plasma cells

17. Plasma proteins
17
 Many different proteins are present in the blood, and collectively
known as plasma proteins.
 Plasma protiens: Proteins found/present in the blood
 They include
 Acute phase reaction proteins(APR), Carrier proteins,
Fibrinogen & Coagulation factors, complement , Igs, enzyme
inhibitors
 Clinical significance of protein
 The two general causes of alteration of serum total protein are:
 Change in volume of plasma water
 Change in concentration of protein
 The relative hypoproteinemia --hemodilution.
 The relative hyperproteinemia hemoconcentration.

18. Albumin
 Albumin is the most abundant plasma protein of extra
vascular body fluids, including
 CSF
, Interstitial fluid, urine, and amniotic fluid.
 Accounts approximately one-half of the plasma protein mass.
 A globular protein, with molecular mass of 66.3 KD.
 Have no carbohydrate side chain but is highly soluble in
water due to of its high net negative charge at
physiological pH
18

19. Functions of albumin
 Maintaining the colloidal osmotic pressure in both the vascular
and extra vascular space with continuous equilibration.
 Binds and transports isoluble cpds
 Like lipids, metallic ions, drugs, hormones and bilirubin.
 Clinical significance of albumin
 Primarily synthesized by the liver and
 The synthetic rate may be 300% or more in nephrotic
syndrome
 Increased levels of albumin are present only in acute
dehydration and have no clinical significance.
 Decreased levels are seen in a multitude of clinical conditions.
19

20. Decreased levels of albumin
 Decreased levels of albumin is seen in:
 Analbuminemia
 Genetic defect with albumin levels less than 0.5 g/L
clinically related to abnormal lipid transport
 Inflammatory conditions
 Most common cause, resulting from hemodilution,
increased consumption by cells, decreased synthesis
 Hepatic disease
 Mostly result from increased Ig levels, loss in to EVS, direct
inhibition of synthesis by toxins & alcohols; may happen
with the loss of about 95% of function

21.  Urinary loss
 Normal urine may contain up to 20mg albumin per gram of
creatinine.
 Excretion above this level is abnormal.
 Gastrointestinal loss
 Associated with inflammatory disease to GIT
.
 Chronic protein-losing enteropathy (nephrotic syndrome).
 Protein energy malnutrition
 Edema or ascites due to liver diseases
 Secondary to increased vascular permeability, rather than to
hypoalbuminaemia 21

22. Alpha 1-fetoprotein [AFP]
 AFP is one of the first α–globulin appear in mammalian sera
during development of the embryo
 Dominant serum protein in early embryonic life
 Synthesized primarily by the fetal yolk sac and liver.
 Contains approximately 4% carbohydrate with a molecular mass
approximately 70 KD.
22
Clinical significance of AFP
 High AFP levels indicates:
 An open neural tube or abdominal wall defect in fetus.
 Multiple fetuses, fetomaternal bleeding, and incorrect estimation
of gestational age
 Hepatocellular and germ cell carcinomas in childhood and adults

23. C-reactive protein
23
 CRP is one of the first APPs (positive APPS) to become elevated in
inflammatory diseases .
 And also the one exhibiting the most immediate & dramatic
increases in concentrations in inflammatory diseases .
 Consists of five identical subunits and is synthesized primarily by
liver.
 CRP activates the classical complement pathway
 Starting at C1q and initiates opsonization, phagocytosis, and lysis of
invading organisms such as bacteria and viruses.
 CRP can recognize potentially toxic autogenous substances released
from damaged tissue
 To bind them, and then detoxify or clear them from the blood.
 CRP itself is catabolized after opsonization (make some thing
susceptible to destruction).

24. Clinical significance
24
 CRP levels usually rise dramatically after
 Myocardial infraction, stress, trauma, infection, inflammation,
surgery, or neoplastic proliferation.
 The increase begins within 6 to 12 hrs of the infraction & the
level may reach 2000 times normal
 Cord blood normally has low CRP concentration, but in
intrauterian infection, the concentration will be high.
 Determination of CRP is clinically useful for
 Screening for organic disease
 Assessment of the activity of inflammatory diseases
 Detectionof current infectionin systemic lupus erythematosus
(SLE), in leukemia, or after surgery
 Management of neonatal septicemia and meningitis

25. Analysis of proteins
 Methods used to analyze proteins in body fluids include
1. Specific quantitative assays of particular proteins by
immunochemical methods using nephlometery, turbidimetry,
radial immunodifusion, RIA, EIA
2. Detection and identification by electrophoresis
3. Quantitative measurement oftotal protein in serum, urine &
CSF
4. Analysis by mass spectrometry which provides structural and
quantitative information
25

26. Quantitative Measurement of Total Protein in
Body Fluids
26
 When the total protein is measured, two arbitrary
assumptions are made
1. All protein molecules are pure polypeptide
chains, containing 16% by wt of nitrogen
2. Each of the several hundred individual proteins
reacts chemically like every other protein

27. 1. Biuret method
27
 Depends on the presence of peptide bonds
 Peptide bonds react with Cu2+ ions in alkaline solutions to form a
colored product
 The absorbance is measured spectrophotometrically at 540nm.
 The biuret reagent contains sodium potassium tartarate to form a
complex with cupric acid and maintain their solubility in alkaline
solution.
 One copper ion probably is linked to 6 nearby peptide linkage by
coordinate bonds.
 Amino acids and dipeptides do not react,
 But tri-peptides, oligo-peptides, and polypeptides react to yield
pink to reddish- violet products.
 The intensity of the color produced is proportional to the amount of
protein present in the reaction system.

28. 28
 Specimen type and preservation
 Either serum or plasma, but serum is preferred.
 A fasting specimen may be required to decrease the risk of
lipemia.
 Hemolysis should be avoided.
 Serum samples are stable for one month at 2 to 4Oc.
 Specimens that have been frozen and thawed should be mixed
thoroughly before assay.
 Limitations and Sources of Error
 The biuret reaction occurs with other compounds with
structural similarity.
 Hemolysis, Lipemia, ammonium ions interfere
 Sensitivity range in the g/dL so suitable for serum specimens
but not other body fluids

29. 2. Direct photometric methods.
 Absorption of UV light at 200-225 nm and 272–290
nm is used to measure content of biological specimens
 Absorption of UV light at 280 nm depend chiefly on
the aromatic rings of tyrosine and tryptophan at PH 8.
 Peptide bonds are responsible for UV absorption (70%
at A205)
 Specific absorption by proteins at 200 to 225 nm is
some what greater than at 280 nm.
29

30. Limitations and Sources of Error
30
 Accuracy & specificity suffer from
 Uneven distribution of tyrosine and tryptophan among
individual proteins
 The presence of free tyrosine and tryptophan, uric acid, and
bilirubin, which also absorb light near 280nm.
 Interferences from free tyrosine and tryptophan is significant at
200 to 225nm.
 However, a 1:1000 or 1:2000 dilution of serum with sodium
chloride, 0.15mol/l, rduce these interferences.

31. 3. Dye-binding methods
31
 Based on the ability of proteins to bind dyes
 such as Amido black 10B and Coomassie Brilliant Blue (CBB).
 The method is simple, easy, and linear up to 150 mg/dL.
 Applicable for assay of total protein in CSF and urine uses CBB
G- 250
 Specimens
 Timed urine and CSF
 Serum or plasma can not be used due to upper limit of linearity.
Because it detects only up to 150 mg/dL.

32. 4. Turbidimetric and nephelometric methods
Principle of the test
 Protein in the sample is precipitated with addition of
sulfosalicylic acid alone.
 Then in combination with sodium sulfateand
trichloroacetic acid (TCA), or with TCA alone to
produce turbidity.
 Degree of turbidity measured with Turbidometeric or
nephelometric methods
 Turbidometeric (transmitted light) or nephelometric
(scattered light) 32

33.  Reference Ranges
 Serum total protein: 6 -8 g/dL
 Interpretation
 Hyperproteinemia:
 Increased serum total protein due to dehydration or
 Increased gamma globulins such as in multiple myeloma
 Hyporproteinemia:
 Decreased protein due to
 burns, renal or intestinal losses, protein energy
malnutrition or sever liver failure. 33

34. Assay Techniques for serum Albumin
1. Dye-binding methods:
 Brom cresol green (BCG) or purple (BCP) dyes
2. Salt fractionation or the 'salting-out' procedure=
 Removal of globulins by salt precipitation followed by
 The quantitation of residual albumin in solution by the
biuret
3. By difference
4. Electrophoresis
5. Immunochemical techniques. 34

35. 35
 Serum Albumin BCP Assay Techniques
 Yellow BCP dye, buffered at pH 5.2 with acetate turns green
when complexed with albumin.
 Absorbance of the green complex is measured at 603 nm.
 Serum Albumin BCG Assay Techniques
 Albumin and BCG are bind at pH 4.2, in succinate buffer
 Absorption of the BCG-albumin complex is measured at 628
nm.
 At pH 4.2, albumin acts as a cation to bind the anionic dye.
 The rxn is extremely fast and finished in only a few seconds.

36. 36
 Specimen For Albumin Testing
 Serum is recommended
 Results tend to be erroneous if the overall
serum protein pattern is abnormal
 Reference Range
 Adult serum albumin: 3.5 - 5.0 g/dl
 In the upright position levels are about 0.3 g/dl
higher because of hemoconcentration.

37. Limitations and Source of Error in serum
Albumin Assay
 Hyperlipemia
 Hyperbilirubinemia
 Hemolysis
 Can generally be eliminated (minimized) by
dilution of serum 1:250
37

38. Methods for the determination of total globulins
38
 Methods for determination of total globulins include
1. Colorimetric method
2. Globulin by difference
3. Electrophoresis:
 Separation of charged molecules (different proteins)
in an electrical field
4. Immunochemical technique

39.  Protein Electrophoresis
 Principle:
 The pH of the solution determines the net charge of the
protein molecules.
 At pH 8.6, hydrogen ions will be lost from the carboxyl
ends and from functional groups of R residues of the
amino acids.
 Since proteins are composed of different amino acids,
when voltage is applied, they migrate to different
positions on the cellulose or agarose media 39

40. Materials and procedures of protein
electrophoresis
 Buffer: barbital with an ionic strength of 0.05 and pH 8.6
 Sample volume: 3 to 5 µl
 Power supply:
 1.5 mA per 2-cm width of cellulose acetate medium
 10 mA per 1-cm width of agarose medium
 Run time: 40 to 60 min producing a 5- 6 cm migration
distance for albumin
40

41. Serum Protein Electrophoresis
 Electrophoresis is widely used in clinical laboratories to study and
measure the protein content of biological fluids- serum, urine or
CSF.
 Screening tool for protein abnormalities
 Electrophoresis techniques include:
Cellulose acetate electrophoresis
Gel and capillary electrophoresis
Specialized techniques termed western blotting,
immunofixation, and two-dimensional electrophoresis

42. Specimen for electrophoresis
 Serum is preferred over plasma
 since plasma contains fibrinogen that makes
interpretation difficult.
 CSF
 Concentrated urine

43. Procedure for Protein Electrophoresis
1. Specimen is placed into a sample trough within agarose gel, is
placed in an alkaline buffer solution
2. A standardized voltage is applied and allowed to run for 1hr
3. The agarose gel is processed in acetic acid and alcohol washes to
fix the proteins in the agarose.
4. The protein fractions are stained with Coomassie Brilliant Blue
protein stain.
5. After a second wash, fixed protein bands can be visualized and
quantified with densitometry.
6. In normal serum electrophoresis 5-6 bands are visible:
 Albumin, α1-Globulins, α2-Globulins, β-Globulins, and γ-
Globulins (+ve to -ve charge migration)

44. Electrophoresis Calculations
44
 Total serum protein x % fraction gives quantity in g/dL
 Example: TSP 6.0 g/dL and % albumin of 50% albumin = 6.0 x
50% = 3.0 g/dL
Globulin= TSP – Albumin
 Example TSP 6.5 g/dL and albumin 3.5 g/dL
 Globulins = 6.5 – 3.5 = 3.0 g/dL
 Albumin/ globulin ratio
 Example 3.5/ 3.0 = 1.2

45. Limitations and Sources of Error
45
 Wrong pH or ionic strength of the buffer
 Wrong voltage
 Too long or too short of time
 Excessive heat

46. Normal serum protein electrophoresis pattern
+ -
Albumin 1 2  
46

47. Serum Protein Electrophoresis: agarose medium
47
Cathode:
Anode: + Electrode
- Electrode
 Albumin at bottom
(anodic), than alpha 1,
then alpha 2 then beta 1
and beta 2 then gamma
close to top (cathodic)

48. 48
 Reference Range of total protein
 Serum---------------------------6-8 g/dl
 CSF----------------------------- 8-32 mg/dl
 For electrophoresis
 serum:
 albumin-----------------3.9-5.1 g/dl
 α1-globulin------------0.2-0.4 g/dl
 α2-globulin------------0.4-0.8 g/dl

49. Interpretation of Protein Electrophoresis Results
 Further resolves cause of hyperproteinemia
 Gamma globulin increase
 Hemoconcentration
 Multiple myeloma
 Normal gamma globulins but increased albumin
 Look for individual increases in alpha or beta
globulins 49

50. Quality Control
50
 A normal & abnormal quality control sample should be
analyzed along with patient samples.
 Using Westgard or other quality control rules for acceptance or
rejection of the analytical run.
 Assayed known samples
 Commercially manufactured
 Validate patient results
 Detects analytical errors.
 Documentation of protein Results
 Record patient results in result logbook
 Record QC results in QC logbook
 Retain records for recommended time

51. Summary
51
 Proteins are polymers of amino acids that are linked covalently
through peptide bonds.
 The presence of nitrogen in all proteins sets them apart from
carbohydrates and lipids.
 Proteins are classified based on the number of amino acid
molecules,composition of amino acids.
 Protein have four structural levels;10,20,30,and 40.
 Properties of proteins include molecular size, differential solubility,
electrical charge, adsorption on finely divided inert materials, and
specific binding to antibodies, coenzymes, or hormone receptors

52. 52
 Proteins function includes building our body , serving as
enzymes, as antibody. etc..’
 Major plasma proteins include Albumin, Alpha1Acid
lycoproteins, ceruloplasmin, C-reactive protein, complements,
fibrinogen and immunoglobulins
 Increase level of protein caused by acute dehydration and has
no clinical significance; decreased levels of proteins seen in
edema and ascites, analbuminemia, urinary loss, inflammatory
conditions, gastrointestinal loss, hepatic disease, protein energy
malnutrition.

53. Review Questions
53
1. What properties of proteins allow for electrophoresis?
2. Why are different methods used for measuring total
protein in serum than in urine?
3. What are two causes of hyperproteinemia?
4. Describe how liver disease orkidney disease can affect
serum albumin and protein levels.

54. References
54
1. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
Fundamentals of Clinical Chemistry. WB Saunders, Co.,
Philadelphia, 2001.
2. Arneson, W and J Brickell: Clinical Chemistry: A Laboratory
Perspective 1sted. FA Davis, Philadelphia, 2007.
3. Burtis, Carl A., and Ashwood, Edward R.. Tietz: textbook of
Clinical Chemistry. WB Saunders Co., Philadelphia, 1999.