SERUM PROTEINS

Proteins are the most abundant compounds in your serum ( the rest of your blood when you remove all the cells). They are the basic components of enzymes, many hormones, antibodies, and clotting agents. Proteins act as transport substances for hormones, vitamins, minerals, lipids, and other materials. In addition, proteins help balance the osmotic pressure of the blood and tissue. Osmotic pressure is part of what keeps water inside a particular compartment of your body. Proteins play a major role in maintaining the delicate acid-alkaline balance of your blood. Finally, serum proteins serve as a reserve source of energy of your tissues and muscle when you are not ingesting an adequate amount.
The major measured serum proteins are divided into two groups, albumin, and globulins. There are four major types of globulins, each with specific properties and actions.
A typical blood panel will provide four different measurements - the total protein, albumin, globulins, and the albumin globulin ration.

BLOOD PROTEINS:

1. Albumins = 3-5 g/dL (55%) - Create and maintain oncotic pressure; transport insoluble molecules
2. Globulins = 2-2.5 g/dL (38%) - participate in immune response
3. Fibrinogen = 0.2- 0.45 g/dL (7%) Blood coagulation
4. Regulatory proteins (<1%) Regulation of gene expression
5. Clotting factors (<1%) Conversion of fibrinogen into fibrin

TOTAL PROTEIN

Because the total protein represents the sum of albumins and globulins, it is more important to know which protein fraction is high or low than what is the total protein.

Optimal Range = 7.2 to 8.0 g/dL

Total protein may be elevated due to:

• Chronic infection (including tuberculosis)
• Liver dysfunction
• Collagen vascular disease (Rheumatoid arthritis, Systemic lupus, Scleroderma)
• Hypersensitivity states
• Dehydration (diabetic acidosis, chronic diarrhea etc.)
• Hemolysis
• Cryoglobulinemia
• Alcoholism
• Leukemia (cancers that usually begin in the bone marrow and result in high no. of abnormal WBCs )
• Multiple myeloma (a cancer of plasma cells. Plasma cells develop from B lymphocytes. Their primary function is to produce antibodies. In people with multiple myeloma the malignant plasma cells produce only one type of intact (whole) immunoglobulin in large amounts and/or produce an excess of only one of the light chains, or rarely heavy-chains only types)

Total protein may be decreased due to:

• Malnutrition (insufficient intake and/or digestion of proteins)
• Malabsorption (celiac disease- autoimmune disorder that can occur in genetically predisposed people where the ingestion of gluten leads to damage in the small intestine)
• Liver disease (insufficient production of proteins)
• Diarrhea (loss of proteins through GI tract)
• Severe burns (loss or proteins through the skin)
• Hormone imbalance that favor breakdown of tissue
• Loss through the urine in severe kidney disease (proteinuria)
• Low albumin
• Low globulins
• Dilution of protein due to extra fluid held in the vascular system (during pregnancy)

ALBUMIN:

Albumin is synthesized by the liver using dietary protein. Its presence in the plasma creates an osmotic force that maintains fluid volume within the vascular space. A very strong predictor of health; low albumin is a sign of poor health and a predictor of a bad outcome.

Optimal Range: 3 to 5 g/ 100ml

Albumin levels may be elevated due to:

• Dehydration (actual)
• Congestive heart failure\
• Poor protein utilization
• Glucocorticoid excess (can result from taking medications with cortisone effect, the adrenal gland overproducing cortisol, or a tumor that produces extra cortisol like compounds)
• Congenital

Albumin levels may be decreased in :

• Hypothyroidism
• Chronic debilitating disease (e.g., RA)
• Malnutrition (protein deficiency)
• Dilution by excess water (drinking too much water, termed as 'polydipsia' or excess administration of IV fluids)
• Kidney losses (nephrotic syndrome)
• Protein losing- enteropathy (protein is lost from the GI tract during diarrhea)
• Skin losses (burns, exfoliative dermatitis)
• Liver dysfunction (the body is not synthesizing enough albumin and indicates very poor liver function)
• Insufficient anabolic hormones such as growth hormone, DHEA, testosterone etc.

GLOBULINS:

Globulins are proteins that include gamma globulins (antibodies) and a variety of enzymes and carrier/transport proteins. The specific profile of the globulins is determined by protein electrophoresis (SPEP), which separates the protein according to size and charge. There are four major groups that can be identified : gamma globulins, beta globulins, alpha 2- globulins and alpha- 1 globulins. Once the abnormal group has been identified, further studies can determine the specific protein excess or deficit. Since the gamma fraction usually makes up the largest portion of the globulins, antibody deficiency should always come to mind when the globulin level is low. Antibodies are produced by mature B lymphocytes called plasma cells. While most of the other proteins in the alpha and beta fractions are made in the liver.

Optimal Range: 2.3 to 2.8 g/dl

Optimal Range (alpha-globulin) : 0.2 to 0.3 g/L

Optimal Range (beta-globulin ): 0.7 to 1 g/L

Globulin levels may be elevated in:

• Chronic infections (parasites, some cases or viral and bacterial infection)
• Liver disease (biliary cirrhosis, obstructive jaundice)
• Carcinoid syndrome
• Rheumatoid Arthritis
• Ulcerative colitis
• Multiple myelomas, leukemias, Wald Enstrom's macroglobulinemia
• Autoimmunity (systemic lupus, collagen disease)
• Kidney dysfunction

Globulin level may be decreased due to :

• Alpha 1 antitrypsin deficiency (emphysema)
• Acute hemolytic anemia
• Liver dysfunction 
• Hypogammaglobulinemia/ Agammaglobulinemia (immune disorder; reduction of types of gamma globulins)

A/G (ALBUMIN/GLOBULIN) RATIO:

The liver can function adequately on 20% of liver tissue, thus early diagnosis by lab methods is difficult. A reversed A/G ratio may be a helpful indicator. With severe liver cell damage, the prolonged prothrombin time will not change with ingestion of vitamin K. The proper albumin to globulin ration is 2:1. When < 1.7, there is maybe a need for increasing stomach acidity. When > 3.5 there may be a need for stomach acidity and pepsin.

Optimal Range: 1.7 to 2.2

A/G Ratio may be elevated in :

• Hypothyroidism
• High protein/ high carbohydrate diet with poor nitrogen retention
• Hypogammaglobulinemia (an immune disorder characterized by a reduction in all types of gamma globulins, therefore low globulins)
• Glucocorticoid excess (can be from taking medications with cortisone effect, the adrenal gland overproducing cortisol, or tumor that produces extra cortisol-like compounds, low globulin)

Decreased A/G Ratio common causes:

• Liver dysfunction
• Overproduction of globulins, such as seen in multiple myeloma or autoimmune diseases
• Increased globulins (without an increase in albumin) due to infection
• Inflammation
• Underproduction of albumins, such as may occur with cirrhosis (scarring of the liver)
• Increased loss of albumin from the circulation, as may occur with kidney disease
• Inflammation (negative acute phase protein-protein whose concentration increase or decrease during inflammation. Albumin, transferrin, transthyretin, retinol-binding protein, antithrombin, transcortin. The decrease of such proteins may be used as markers of inflammation).

With a low total protein that is due to plasma expansion (dilution of the blood), the A/G ratio will typically be normal because both albumin and globulin will be diluted to the same extent.

METHODS OF ESTIMATION :

•  Precipitation method: Precipitate out globulin by addition of 28% sodium sulfite by virtue of their high mol. weight (100- 1000 kDa). The major protein remaining in serum will be albumin which can now be quantitated by biuret. Then subtract albumin from total protein to calculate globulin.
• Enzymatic method: The principal is based on the fact that LDH isoenzyme-5 (LDH-5) binds specifically to Dextran- blue (DB). Albumin inhibits the binding of LDH-5 with DB. The absence of LDH activity in DB fraction after gel filtration indicates the presence of albumin in sample and vice versa.
• Immunoenzymatic Assay: The human albumin assay is a two-site immunoenzymatic assay using as amplified biotin/ streptavidin detection system for maximum sensitivity. Samples containing human albumin are reacted in microtiter strips coated with an affinity purified capture antibody. A second biotinylated anti-HSA antibody is reacted forming a sandwich complex of solid phase antibody- albuminbiotinylated antibody. After a wash step to remove any unbound reactants the strips are then reacted with streptavidin labeled with alkaline phosphatase. Another wash sequence is performed followed by the addition of p-nitrophenyl phosphate (PNPP) substrate. The amount of hydrolyzed substrate is read on a microtiter plate reader and will be directly proportional to the conc. of human albumin present. Accurate quantitation is achieved by comparing the signal of unknowns to HSA standards assayed at the same time.

BCG METHOD:

Bromocresol green (BCG) is a dye which is used as a pH indicator in applications such as growth medium for m/o and titrations.

In acidic pH (below 3.8) BCG will ionize to give the monoanionic form (yellow), that further deprotonates at higher ph (alkaline solution, above pH 5.4) to give the dianionic form (blue).

INTENDED USE: For the determination of albumin conc. in human serum or plasma.

PRINCIPLE OF BCG:

The method is based on the specific binding of BCG, an anionic dye, and the protein at acid pH produce a color change of the indicator from yellow-green to green-blue with the resulting shift in the absorption wavelength of the complex. The intensity of the color formed is proportional to the conc. of albumin in the sample.

Albumin is cationic at pH of 4.3, therefore it interacts with the anionic dye of BCG to form a blue-green colored complex. The conc.is determined by measuring absorbance at 630 nm.