Since the majority of proteins found in plasma are synthesized by the liver from amino acids, including serine protease coagulation factors, prothrombin time, serum albumin, and serum protein electrophoresis results can be used to indicate declining liver function. For example, serum albumin decreases and prothrombin time becomes prolonged with liver failure. Protein levels also reflect other disorders, such as those in which essential amino acids are not provided by the diet or in which proteins are lost by the kidneys or gastrointestinal tract.
Proteins and amino acids have unique structures that allow them to participate in some specific types of chemical reactions. The significant amino acids are found in a table in the Appendix. Proteins are polymers consisting of amino acid units. Amino acid units within the protein are joined by peptide bonds, giving the primary structure of proteins. The amino acid unit on the carboxyl end of the protein contains a free carboxyl group that does not participate in peptide bond formation. The amino acid unit on the amino end of the protein contains a free amino group that does not participate in peptide bond formation. These characteristics of proteins, the amino and carboxyl ends, and peptide bonding play a role in the methods of analysis for total serum proteins.
Proteins are ampholytes, and in aqueous solutions they may have positive and negative charges on the same molecule. This property is used to separate protein molecules during electrophoresis. The pH of the solution determines the net charge of the molecule. At different pH environments, hydrogen ions will be gained or lost from the carboxyl and amine ends and from functional groups of residues of the amino acids. Since proteins are composed of different amino acids, different proteins will gain or lose hydrogen ions at different pH environments.
In addition to their properties as ampholytes, proteins also have other representative structural properties based on their polymer makeup and bonding. Fibrous proteins are stringlike in configuration and usually function as structural components of the body, such as fibrinogen and collagen. Most plasma proteins and enzymes are globular proteins, which are spherical in configuration.
Serum contains a large variety of proteins and a large amount of total protein, averaging 7.0 g/dL in the adult. In contrast, protein levels in serum and urine are normally in the microgram or milligram per deciliter range. Methods for measuring proteins in body fluids are based upon the unique structural properties as well as their relative concentration in the body fluids. Total serum protein test methodology is described in Test Methodology 7-7. There are two main types of proteins, albumin and globulins. They are grouped into five classes as determined by their electrophoretic separation: albumin, alpha1 globulins, alpha2 globulins, beta glob-
ulins, and gamma globulins. Serum albumin, at around 4 g/dL, makes up roughly half of the total serum proteins. A simple way to assess the balance between the patient's albumin and globulins in serum is to calculate the albumin:globulin, or A/G, ratio. Globulins (G) are calculated as albumin (A) subtracted from total serum proteins. Albumin is then divided by globulin.
Although there are many classic and reference methods for quantification of serum protein, the biuret reaction has become the most commonly used method in the clinical laboratory. The peptide bonds of proteins react with biuret reagent containing Cu2+ ions in an alkaline solution to form a violet color measured at 540 nm. Sodium potassium tartrate is a component of the reagent and functions to maintain copper in the correct valence state and in an alkaline solution, while potassium iodide is present as an antioxidant.
Proteins + biuret reagent ^ violet-colored product (540-nm absorbance) Interferences
Marked hyperbilirubinemia and lipemia cause interference unless corrected with a serum blank. Marked lipemia should be removed with acetone-pretreated samples. Ambulatory patients exhibit a slight hemoconcentration causing a physiological increase in serum protein by 0.3 g/dL.
Serum, free from lipemia and collected without prolonged tourniquet use, may be used. Plasma can be used, but the reference range must be adjusted upward to account for fibrinogen. Plasma should not be used for protein electrophoresis.
Adult (ambulatory) 6.4-8.3 g/dL
Total serum protein levels are affected by not only changes in one or more of the individual protein levels, but also by changes in plasma water. A variety of conditions cause hyperproteinemia, or increased serum protein. Hemoconcentration, or decreased plasma water volume, will cause total serum protein levels to be increased. Dehydration is the usual cause of hemoconcentration, which is secondary to a variety of conditions including diarrhea, severe vomiting, and water deprivation. Increased total serum protein levels can also occur when there is an increase in a variety of immunoglobulins following inflammation or infection or a monoclonal increase in immunoglobulins, such as in multiple myeloma. More information about multiple myeloma, a malignancy of the bone marrow, is found in Chapter 13. Increased total protein can also result from measuring an unexpected protein such as fibrinogen. Serum is derived from clotted whole blood in which fibrinogen is removed in the clotting process. However, if incomplete clotting occurs before centrifugation, some fibrinogen can remain behind in the serum specimen.
Hypoproteinemia, or decreased protein levels in the blood, is often due to hypo-albuminemia, since albumin is the most abundant single protein. Typical causes of hemoconcentration - relative increase in blood cells due to decrease in plasma water volume
hypoproteinemia are starvation or nutritional deficiency of essential amino acids, renal loss such as in nephritic syndrome, gastrointestinal loss such as in enteropathy, or hepatic failure in which the liver is unable to synthesize proteins.2
Serum protein electrophoresis results can also indicate inflammatory states of the liver in which there are elevated gamma globulin protein fractions, especially immunoglobulin A (IgA) and IgM levels. In cirrhosis, protein electrophoresis results show that fast-moving gamma globulins often migrate in the beta to gamma region, causing a beta-gamma bridge. Serum protein electrophoresis principles are discussed in more details in Chapter 13.
Figure 7-4 shows the typical beta-gamma bridge in the electrophoretic pattern, and Table 7-5 shows the protein electrophoresis results. The beta-gamma bridge can be seen as a lack of separation of the beta globulins from the gamma globulins. Notice that the beta and gamma globulin results are not reported in separate categories since they migrate in one region.
electrophoresis - a separation technique of different charged molecules in solution in an electrical field of varying potential
Cerebrospinal fluid (CSF) is an ultrafiltrate of plasma formed in the choroid plexuses and ventricles of the brain. CSF contains much less protein than serum and cannot be analyzed by biuret reactions or other methods used in measurement of total serum proteins. The proportion of proteins found in CSF is also different than that of serum, with a predominance of low molecular weight proteins such as
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