Glycoproteins and Mucoproteins

Conjugated proteins containing a complex carbohydrate moiety linked to the protein chain are referred to as glyco- and mucoproteins. A large number are known with considerable individual variation in composition and properties. The carbohydrate chain linked to the protein is made up of the hexoses, galactose; the methylpentose, fucose; and a sialic acid, such as N-acetylneuraminic acid. The sialic acid is located at one end of the carbohydrate chain; the other end of the chain is linked by a peptide bond to one of the peptide chains of the protein molecule.

In a useful classification, those carbohydrate-proteins containing less than 4 per cent of hexosamines are termed glycoproteins. They contain from a trace up to about 15 per cent of total carbohydrate. Those with over 4 per cent of hexosamines and containing from 10 to 75 per cent of carbohydrate are termed mucoproteins. The expression mucoids is also used in characterizing those mucoproteins in which the bond linking the carbohydrate chain and the protein is split only with difficulty, in contrast to another group containing acidic mucopolysaccharides, in which the linkage is polar and easily split. The glyco- and mucoproteins differ from the albumins and globulins in that they are not precipitated by perchloric and sulfosalicylic acids.

Mucoproteins of clinical interest are found in both serum and urine. Electrophoretically, most mucoproteins migrate with a mobility associated with alpha-1 globulins, although individual proteins may be found among all the usual electrophoretic groups. They are identified on electrophoretic strips or gels by their capacity to give a positive staining reaction with Schiff's reagent. The characteristic pink color is due to a reaction between the carbohydrate portion of the mucoprotein and the reagent. The mucoid present in serum in greatest amount is orosomucoid; it has a molecular weight of about 40,000 and is quite acidic, possessing an isoelectric point at pH 2.3. A number of other mucoproteins are also present in varying amounts.

Amino Acids and Related Metabolites

Next to urea, amino acids constitute the second largest source of the nonprotein nitrogen (NPN) in serum. In general, serum urea-N and serum NPN parallel each other in serum; as the former rises, the latter increases in about the same proportion.

Amyloid

As a consequence of long term, chronic, suppurative disease and a number of other conditions there is deposited in the blood vessels and matrix of a number of organs a type of abnormal proteinaceous material called amyloid.

Albumin determination by dye binding

All proteins, and especially albumin, tend to react with many chemical species by means of electrostatic and tertiary van der Waal's forces and by virtue of hydrogen bonding. Bilirubin, fatty acids, most hormones, and many drugs are transported about the body bound to albumin. Many colored dyes in the anionic form also possess this protein-binding property. This property has been used in attempts to devise methods by which albumin could be measured directly, without previous precipitation of globulins.

In dye-binding methods, only those dyes or indicators can be used that bind very tightly to the albumin molecule, so that practically 100 per cent of the albumin present is bound to dye. The binding must be unaffected by reasonably small changes in ionic strength and pH. Further, the color of the protein-bound dye should be different from that of the free dye; i.e., there should be a substantial shift in the wavelength of light at which maximum absorption occurs in the two forms. The albumin-dye concentration can then be measured in the presence of excess dye. Finally, dye binding to other protein fractions (globulins) must be negligible if the dye binding is to be the basis for a valid assay of albumin. For practical reasons, the color characteristics of the dye should be such that it can be measured at wavelengths of light where bilirubin and hemoglobin will give negligible or minimal interference. The use of methyl orange was proposed by Bracken and Klotz and Watson and Nankiville; HABA (2-(4'-hydroxyazobenzene)-benzoic acid) by Rutstein, Ingenito, and Reynolds and Martinek, and bromcresol green by Rodkey. The use of bromcresol green is most free of pigment interference. Bilirubin at levels over 5 mg./100 ml. interferes with HABA procedures and to a lesser degree with methyl orange methods.

The use of these dye reagents is now well established, and several manual and AutoAnalyzer procedures using methyl orange and HABA are available; however, there is considerable disagreement over the reliability of the dye-binding methods. Occasionally, albumin values obtained are unusually high as a result of the binding of the dye to proteins other than albumin. As an example of dye-binding technique, a methyl orange procedure will be presented in outline. It is a modification of the method proposed by Wrenn and Feichtmeir and is designed for use with the Coleman spectrophotometer and 19 mm. cuvets.

Assay of Serum Albumin by Methyl Orange Binding
Reagents

1.  Working dye reagent. Prepare by adding 5 to 10 ml. portions of 0.10 per cent (w/v) methyl orange to 1000 ml. of citrate buffer, 0.055 M. pH=3.50 +/- 0.03; check the absorbance against water at 540 nm. after each addition. Continue addition of dye until the absorbance lies between 0.78 and 0.85. Use the reagent at 24 to 27 degree of Celcius, although it is stored in the refrigerator.
2. Standards. These are prepared from crystalline human serum albumin. Bovine albumin should not be used because there are significant differences in dye binding by albumins from different species. It is wise to assay the albumin standard against the biuret total protein method so that the total protein and albumin values will be consistent. Prepare the stock reagent, which has a concentration of 10.00 gm./100 ml., by dissolving the albumin in saline. Prepare the working standards by diluting the stock with saline to give levels of 1.0 through 6.0 gm./100 ml.

Procedure

1.  Pipet 10.0 ml. of working dye reagent into a series of cuvets. One cuvet will be needed for the albumin blank, one each for the six standards, and one for each of the unknowns. Transfer 0.20 ml. each, of saline, standards, and sera, to the respective cuvets. After mixing by gentle inversion, permit the cuvets to stand for 20 minutes.
2. Read the absorbances of the standard and serum specimens against the blank at 540 nm. If the specimens are slightly lipemic, icteric, or hemolyzed, pigment color corrections will have to be made. For this purpose, add 0.20 ml. of the test serum to 10.0 ml. of the citrate buffer and read the absorbance against water (or citrate buffer) at 540 nm. Subtract these readings from the previous readings.
3. Convert the absorbance readings to albumin concentration by use of the calibration curve, which follows Beer's law up to concentrations of about 4 to 5 gm./100 ml. Prepare new calibration curves daily.

 Comments
Normal globulins and most abnormal globulins have no effect on the albumin values. Occasionally paraproteins and macroglobulins will interfere by giving high results. Dye binding, being a dissociation-association equilibrium phenomenon, is influenced by changes in temperature; a rise in temperature increases the dye-albumin dissociation and thus gives lower results. Therefore, standards and sera should be run under identical conditions. Heparinized plasma should not be used because heparin, for reasons not understood, enhances dye binding by albumin. Serum albumin levels obtained by methyl orange dye binding techniques tend to be 0.1 to 0.2 gm./100 ml. higher than those obtained by salt-fractionation or electrophoresis, although some chemists have reported results as much as 0.5 gm./100 ml. higher.

Qualitative tests for urine protein

In devising qualitative tests for urine proteins, it is desirable that the test be negative whenever protein is present in normal concentrations, but that the test be positive whenever protein concentration is greater than 20 to 25 mg./100 ml. The test should not be sensitive to mucins and other proteins of nonrenal origin and it should be capable of rough quantitation. A large number of such tests are available. The older tests are based either on precipitating the proteins by heat or by reaction with anionic protein precipitants. A more recent test depends on a change in the color of an indicator in the presence of protein.

Qualitative tests are best done on fresh morning specimens of urine. Such specimens are usually fairly concentrated, thus making possible the detection of proteinuria early in disease. They will also be free of the orthostatic effect discussed previously. If urines must be held for examination at a more convenient time, and if timed specimens are to be examined, they should be refrigerated and examined within 48 hours. Layering with toluene may be used to minimize microbial growth. Bacterially contaminated specimens (pyelonephritis, lower genitourinary tract infections) should be examined only when fresh.

Heat Coagulation Test

The pH of the urine is checked with bromcresol green pH indicator paper, (pH range, 3.8 to 5.5) and if above pH 5.0, acetic acid, 30 per cent (w/v) is added dropwise until the pH is between 4.0 and 4.6. If any turbidity or insoluble matter is present or is formed (mucoproteins may precipitate on adding the acid), it is removed by centrifugation. About 7 to 10 ml. of clear urine are placed in a 15 x 120 mm. test tube and the upper one-third is gently heated over a Bunsen flame until boiling just begins. The heated part is compared in ordinary light with the lower unheated portion of the specimen. Any turbidity, cloudiness, or precipitate demonstrates the presence of protein. The degree of the reaction is customarily graded 0 to ++++. False positive results may occur and are discussed in the section on interpretation of results.

Sulfosalicylic Acid Test
Into a 15 x 120 mm. test tube, place 5 ml. of clarified urine (pH 4.5 to 6.5). Then 0.50 to 0.80 ml. of 20 per cent (w/v) sulfosalicylic acid is added carefully down the sides of the tube to layer underneath the urine. After 1 minute the interface is examined for the presence or absence of turbidity or cloudiness. A barely evident turbidity (5 to 10 mg./100 ml) is graded as +/-, and increasing degrees of turbidity, cloudiness, or precipitation are graded + to ++++. False positives, due to nonspecific precipitation, may be encountered and are discussed in the section on interpretation of results.

The reagent is prepared by dissolving 100 gm. of reagent grade sulfosalicylic acid (2-hydroxybenzoic, 5-sulfonic acid dihydrate) in about 350 ml. of water with the aid of heat. The warm solution is filtered through two layers of filter paper and the total filtrate diluted to 500 ml. when cool. The solution should be crystal clear and colorless. It is discarded when turbidity or darkening develops.

Macroglobulins

The majority of plasma globulins have a molecular weight in the range of 150.000 to 200.000. A small proportion, less than 4 to 5 per cent of the total serum proteins, are very large size molecules, with molecular weights in the neighborhood of 1,000,000. These large proteins are referred to as macroglobulins. If plasma is centrifuged at ultrahigh speeds, three classes of proteins can be differentiated as a result of differences in the rate of sedimentation (see the section on ultracentrifuge separations).

The macroglobulins constitute the heaviest, S19. class, and they include 50 to 70 per cent of the alpha-2 globulins and 10 to 30 per cent of the gamma globulins. The normal level present in plasma is about 0.20 gm./100 ml., ranging from 0.07 to 0.43 gm./100 ml. In a very small number of individuals, considerably higher levels may be found. In primary macroglobulinemia, first studied in detail by Waldenstrom, the level of S19 proteins is well over 15 per cent of the total serum proteins. Macroglobulinemia is a metabolic disease, characterized by weight loss, and susceptibility to infection, and involves the entire reticuloendothelial system. The  serum electrophoretic pattern is similar to that found in multiple myeloma: Bence-Jones proteins are occasionally found, and some of the macroglobulins behave like cryoglobulins, with resultant impaired blood flow in the extremities. Secondary macroglobulinemia is more common, and is associated with a large number of different disease entities. The level of macroglobulins is always under 15 per cent. Secondary macroglobulinemia is found in patients with leukemia, lymphosarcoma, and rheumatoid arthritis. In myelomatosis, the concentration of S19 proteins is usually normal, or only slightly elevated.

Macroglobulinemia is best diagnosed by ultracentrifugation of a serum specimen. The peak demonstrating the presence of the high molecular weight IgM globulins shows up quite clearly. Isolation of the macroglobulins can also be achieved by gel filtration on Sephadex-200, using either column or thin-layer chromatography. Immunoelectrophoretic patterns can provide supportive evidence, the increased level of macroglobulins being demonstrated by the presence of heavy precipitant arcs with anti-IgM antisera.

 The Sia test is a simple qualitative test for macroglobulins based on the fact that the IgM globulins have decreased solubility in water that contains a low concentration of salts. In one procedure, 0.10 ml. of serum is added slowly down the sides of a test tube into a solution of 5.0 ml. of 0.01 M phosphate buffer, pH 7.1. If macroglobulins are present at levels over 0.7 gm./100 ml. a flaky, birefringent precipitate is formed, which dissolves on the addition of a spatula tip of NaCl. Normal, myeloma, and arthritis sera give no precipitation, and lipemic sera only a hazy opalescence. Malaria and kala-azar sera produce a positive result.

Serum Protein Fractionation

Only two methods of separating serum protein components are sufficiently simple and convenient to be useful in routine work. Salt fractionation is easy to carry out, involves no special equipment, and, if done carefully, provides very useful clinical information. Commonly, only separation of albumin from the globulins as a group is carried out, but techniques are available for separating and quantitating the chief globulin components, especially gamma globulin. When more precise values of the protein fractions are desired, electrophoretic techniques are used. These require more skill and the use of special equipment, and they are more time consuming, although with cellulose acetate membranes, separations may be made in less than an hour. Ultracentrifugation, requiring an expensive instrument, is not suitable for routine use, but is especially useful in the study of lipoproteins and macroglobulins.

Salt Fractionation; Albumin-Globulin Ratio
Albumins are soluble in water, whereas globulins are not. If an electrolyte solution of low concentration (0.1 M) is added to a globulin, the latter will go into solution by virtue of the phenomenon of salting-in. The cations and anions of the electrolyte bind to reactive groups on the protein molecules and break the bonds holding the protein micelles together, allowing the individual protein molecules to undergo solution. The salt concentration of plasma, approximately 0.15 M, is ideally suited to maintain globulins in solution. As the salt concentration is increased to high levels (of the order of 2 M), the various globulins will again become insoluble and will precipitate by salting-out. The level of electrolyte needed to precipitate the various globulins will vary with the individual protein and electrolyte and will also depend on pH and temperature.

Ammonium sulfate was the first salt to be used and it is still used in protein and enzyme investigations. Those proteins precipitating in one-third saturated ammonium sulfate solution were termed euglobulins (true globulins). As the salt concentration is increased, the pseudoglobulins begin to precipitate out, and at concentrations near 50 per cent saturation, all globulins become insoluble, the proteins still in solution constituting the albumins. The use of ammonium sulfate for the isolation and assay of gamma globulins will be described later. The salt is not used in routine clinical work because it interferes with the biuret reaction and obviously cannot be used with the Kjeldahl nitrogen technique.

The only salts whose use has become established in routine clinical chemistry are sodium sulfate and sodium sulfite. The former (Na2SO4) was used as early as 1901, but its large scale use followed the work of Howe in 1921, who recommended the use of 22 per cent (w/v) sodium sulfate to precipitate globulins. Total protein was assayed, and, after removing globulins by filtration, the albumin fraction was measured in the filtrate, globulin being calculated by difference. In 1940 Kingsley introduced the use of ether as a means of separating the precipitated globulins from the soluble albumins, without resorting to the slow, tedious filtration. By carefully dispersing diethyl ether into the mixture of serum plus salt solution, the insoluble globulin micelles would entrap sufficient ether so that on centrifugation, instead of sedimenting as they would normally, the globulins would float above the albumin solution as a compact pellicle. With this type of procedure, serum albumin was found to range from 4.0 to 5.5 gm./100 ml., the globulin from 2.0 to 2.9 gm./100 ml., and the albumin/globulin ratio from 1.5 to 2.5 (average, 2.0).

With the development of Tiselius' moving boundary electrophoresis technique, it was logical to compare the electrophoretic albumin fraction with that separated by the use of Howe's 22 per cent sodium sulfate. It was immediately evident that the Howe technique yielded higher values because it included with the albumins, the alpha-1 and some of the alpha-2 globulins. Studies by Majoor and Milne showed that to free the albumin of all globulin fractions, the concentration of sodium sulfate must be of the order of 26 to 27 per cent (w/v) (1.9 M). Unfortunately, this concentration is beyond the solubility of sodium sulfate at room temperature (25 degree of Celcius), and the salt solution must be kept at 35 to 37 degree of Celcius to avoid crystallization of the salt on storage and during use. With the use of 26 per cent sodium sulfate, the albumin/globulin ratio is of the order of 1.1 to 1.8, averaging about 1.4; this agrees quite well with that obtained by electrophoretic methods. Other salts have also been employed. Campbell and Hanna used sodium sulfite, obtaining complete separation of globulins from albumin at 27 per cent (w/v) (2.2 M). This salt has the advantage of being more soluble than sodium sulfate, and, because it acts as a buffer, better pH control of the precipitation reaction is possible. Reinhold proposed the use of a mixture of sodium sulfate plus sodium sulfite and this procedure will be presented in detail.

By using varying concentrations of sodium sulfate (or sodium sulfite), it is possible to separate all of the main globulin fractions from one another. For example, Kibrick and Blonstein used 15 per cent (w/v) sodium sulfate to precipitate the gamma globulins and 26 per cent salt concentration to precipitate all globulins. The difference between these values gave the sum of alpha and beta globulins. By further use of 19 per cent sodium sulfate, which precipitated the beta- plus gamma globulins, the amount of alpha globulins could also be determined. Such fractionations are tedious, however, and not too satisfactory, especially when abnormal sera are used, and they have never become popular. The electrophoretic separation is simpler and considerably more accurate.

Qualitative tests for globulins in spinal fluids

Pandy Test

The reagent consists of a saturated solution of pure, white-pink phenol in water. About 10 gm. of phenol (free of any brown-black coloration) is suspended in 100 ml. of water and the mixture gently warmed with shaking at 50 degree to 60 degree of Celcius. The material is transferred to a wide-mouth brown bottle and allowed to settle at room temperature (25 degree plus minus 2 degree of Celcius) for 48 to 72 hours before use. The reagent must be clear, colorless, and nonturbid. It is stored at room temperature in a brown bottle away from direct light and away from any cooling drafts.

Procedure

To 1.0 ml. of Pandy reagent in a 10 x 75 mm. test tube, add 0.10 ml. of clear CSF fluid. Mix the tube by inversion and allow to stand for exactly 3 minutes, and then examine it for the presence of any opalescence or turbidity by viewing it against a black background. The turbidity formed indicates the presence of greater than normal amounts of globulins, primarily gamma globulins. Grade the degree of turbidity from negative to ++++. The observed turbidity is scored similarly to the scheme described in Table 5.2 for the heat test for urine protein, but the protein equivalents are different. No consistent relationship between turbidity score or appearance and globulin concentration has been established. A Pandy reading of trace to 1+ will reflect a total protein concentration of 50 to 80 mg./100 ml., with a globulin level of about 20 to 40 mg./100 ml. For a ++ (2+) reading, the respective values are 100 to 160 and 35 to 70 mg./100 ml. In many cases an increase in albumin is accompanied by a proportionate increase in the globulins. In other cases (e.g. poliomyelitis) there is a disproportionate increase in the albumin fraction, giving cause to an increase in total protein without a corresponding Pandy reaction, In multiple sclerosis, in which only gamma globulins are increased. Pandy readings of 1+ or 2+ are obtained even though the total protein is within the normal range.

Serum Cryoglobulins

Clinical Significance

The term cryoglobulins refers to certain abnormal globulins, occasionally encountered in serum, that can precipitate or gel out when the serum is cooled to a low temperature, but that redissolve when the serum is rewarmed to 37 degree of Celcius. Cryoglobulins are not of hepatic origin, but are produced by some part of the reticulo-endothelial system. Often the presence of cryoglobulins is associated with increased levels of plasmacytes or cells resembling plasma cells in bone marrow. Some cryoglobulins are indistinguishable from true immune gamma globulins, whereas others present themselves as separate globulins migrating electrophoretically between the beta and gamma globulins. The molecular weight varies from 100,000 to 1,800,000, and the nitrogen content is of the order of 14 to 16 per cent.

The presence of cryoglobulins is associated with a number of different clinical conditions. They were first found associated with multiple myeloma, but are also encountered in kala-azar, lupus, lymphosarcoma, rheumatoid arthritis, and other autoimmune diseases, and are often associated with vascular sclerosis and platelet defects in blood coagulation. In essential cryoglobulinemia, the cryoglobulins are not associated with any well defined disease states. Clinically, cryoglobulinemia presents the features of Raynaud's syndrome-intolerance to cold, purpura, gangrene of the extremities, and skin sores. The cryoglobulins tend to gel out in the blood when it circulates in the toes and fingers, impairing circulation in these areas. Death may result from blockage of key blood vessels in such vital organs as the kidneys, brain, and lungs. The specific symptoms depend on the amount present and the degree of anti-inflammatory response of the body.

Cryoglobulins are soluble at body temperature (37 degree of Celcius). Occasionally, they will precipitate out at room temperature, but as a rule, sera have to be cooled to 10 degree of Celcius, or lower, before precipitation occurs. The temperature at which gelling will occur depends on the concentration and type of cryoglobulins present. The cryoglobulins in serum may separate out in the form of a gel or as a flocculent precipitate. Rewarming the serum to 37 degree of Celcius, will redissolve the floc or gel, but heating to 57 degree of Celcius, will destroy or modify the cryoglobulins so that they will no longer gel on cooling.

Tests for the detection of Bence-Jones Protein

Bradshaw Test

The urine specimen is treated with 1 or 2 drops of 33 per cent acetic acid to bring the pH down to 4.8 to 5.0, centrifuged clear, and diluted 1:1 with water. The diluted urine is layered carefully over concentrated HCl in a test tube. If Bence-Jones protein is present, it will be precipitated by the HCl and will form a fine or heavy ring at the interface of urine and HCl. The absence of a ring definitely rules out the presence of Bence-Jones protein. A positive test should be confirmed by the heating test because occasionally albumins may also precipitate, if present in large quantity. Protein, as evidenced by a positive sulfosalicylic acid test, must be present. If protein is negative, any HCl interface ring observed in the Bradshaw test is not due to Bence-Jones protein, but probably has formed from the reaction of the HCl with some unknown materials in the urine. The Bradshaw test serves as a useful screening test to determine which specimens should be subjected to the confirmatory heating test.

Heating Test (Jacobson and Milner)

The urine specimen should be fresh or refrigerated under toluene. The pH is checked with pH paper, and adjusted to the pH range 4.8 to 5.0 with 33 per cent acetic acid. The urine is then centrifuged to remove any turbidity (filtration is avoided). If a previous qualitative test for protein was over 3+, the original urine specimen is diluted with normal urine (fresh, free of protein) until it tests 2+ or less. Five to seven ml. of the prepared urine are transferred to a test tube, which is immersed in a beaker of water, warming over a slow-heating hot plate or gas burner. A thermometer is immersed in the urine, and the beaker is heated slowly, with the temperature on the thermometer being watched closely. If Bence-Jones protein is present, turbidity will begin to form in the urine at about 42 degree to 47 degree of Celcius, indicating that coagulation (precipitation) is beginning. The volume of precipitate will increase until the temperature reaches 60 degree of Celcius. If no precipitate is evident at this point, no Bence-Jones protein is present. Any precipitate forming at temperatures above 57 to 60 degree of Celcius is derived from urinary albumins and globulins.

After the Bence-Jones precipitate has formed, the beaker is rapidly heated to 100 degree of Celcius. As the temperature of the urine approaches 100 degree of Celcius the precipitate should dissolve completely or show a diminution in volume. If the precipitate is still present at temperatures of 95 to 98 degree of Celcius, the tube contents should be filtered through a hot funnel. The funnel, filter paper, and receiving tube may be heated either in a drying oven or by passing boiling hot water through them several times. As the filtrate of dissolved Bence-Jones proteins cools to 55 to 60 degree of Celcius, the protein should begin to precipitate out again. If only Bence-Jones protein is present, the test is clear-cut. If albumins are also present, the results of the test may, at times, be equivocal. Low temperature coagulation of the protein is observed, but there is none or only little reprecipitation on cooling.

Fibrinogen

Properties and Clinical Significance

Fibrinogen is one of the plasma protein factors involved in the process of blood coagulation. It is an elongated globulin with a molecular weight of about 350,000, and is synthesized only in the liver. The thrombin formed in the second stage of coagulation acts on fibrinogen to split off a peptide fragment, converting the soluble fibrinogen into insoluble fibrin, which constitutes the clot proper. The fibrino-peptide split-off from the fibrinogen molecule by the trypsin-like action of the thrombin contains some 3 per cent of the original protein nitrogen. The residual fibrinogen fibrils then aggregate and enmesh to form a three-dimensional, cross-linked fibrin gel. A transluscent colorless gel is produced from plasma, but in blood clotting the gel is red because of entrapped blood cells. On standing, the gel contracts (syneresis) to form a tougher mass, extruding serum in the process.

The plasma concentration of fibrinogen is about 200 to 400 mg./100 ml. (0.2 gm. to 0.40 gm./100 ml.); none is present in serum, since fibrinogen is removed as fibrin in the clotting process. Elevations of fibrinogen levels up to 700 mg./100 ml. are encountered in many inflammatory diseases, such as rheumatic fever, pneumonia, septicemias, and tuberculosis. An increase in the erythrocyte sedimentation rate (ESR) is intimately associated with increased plasma fibrinogen levels.

Decreased levels of plasma fibrinogen are rather uncommon. In congenital hypofibrinogenemia, very little fibrinogen is present because of a rare genetic defect that is reflected by the inability of the liver to synthesize fibrinogen. The condition of acquired hypofibrinogenemia is of most interest in clinical work. Low levels of fibrinogen may be observed as a consequence of severe liver disease, which is also associated with low levels of several other clotting factors, such as prothrombin, and Factor VII. However, low levels of fibrinogen are not clinically important in most patients with severe liver disease because liver disease that produces severe hypofibrinogenemia usually leads to rapid decline and death by other causes. The most serious cases of low fibrinogen levels are encountered in certain complications of pregnancy, and it is in connection with these conditions that the laboratory is most often asked to assay fibrinogen levels, frequently on an emergency basis. In premature separation of the placenta (antepartal hemorrhage), the high levels of thromboplastic agents present in the placenta are released into maternal blood, resulting in a rapid conversion of fibrinogen to fibrin in the blood, placenta, and other organs.

Cerebrospinal Fluid Protein

The cerebrospinal fluid (spinal fluid, CSF) is a clear, colorless fluid that fills the nontissue spaces of the brain and spinal cord. The fluid serves to maintain constancy of intracranial pressure and to provide a mechanical, water jacket type of protective coating for the delicate nerve tissue. It is formed as a secretion by cells in the cerebral ventricles. The total volume is about 150 ml. The fluid is a slightly modified ultrafiltrate of plasma. Its composition varies slightly, depending on where it is sampled. There is a gradual change in concentration of its components as the fluid flows down from the brain ventricles to the cisternal space and then to the lumbar area of the spinal cord, where the usual laboratory spinal fluid specimen is obtained by means of a spinal needle puncture.

Normally, a spinal puncture will supply a 6 to 10 ml. specimen. Because of the effort and skill demanded in obtaining good specimens and because of the trauma to the patient, repeat punctures are to be avoided. Spinal fluids are, therefore, very precious specimens; they are handled carefully to avoid loss and every effort is made to get as much information as possible from the volume of sample available. Any leftover portion is stored refrigerated or frozen for 7 to 10 days for possible repeat determinations or for other tests that might be dictated by the clinical status of the patient.

Bence-Jones Protein in Urine

Bence-Jones protein is a type of abnormal protein present in the urine of some 35 to 65 per cent of patients with multiple myeloma. This disease, myelomatosis, is characterized by a large increase in the number of plasma cells, which are probably of an abnormal type. Associated with the plasma cell proliferation is a moderate to large increase in the globulin fraction of serum, the total globulin level at times reaching values of 11 to 12 gm./100 ml. Generally, the increased globulin behaves electrophoretically as gamma globulin. On occasions, a peak, the so-called "M" peak, is seen between the beta and gamma globulin fractions, and even more rarely, in the alpha globulin region. Recent studies have shown that gamma globulins are made up of four peptide chains linked together as a unit. The inner two are the longer and are referred to as H (heavy) chains; the outer two are shorter and are called L (light) chains. In myelomatosis, there appears to exist a considerably increased and unbalanced production of the two forms, so that serum contains a high level of abnormal gamma globulins. These may be either the normal type of gamma globulin, designated as 7S or IgG, or the less common IgA type. The excess of short L forms is excreted and is detected in the urine in the form of those abnormal proteins first detected by Bence-Jones some 100 years ago.

Normal proteins, such as the common albumins and globulins, when heated, do not coagulate and precipitate out of solution until the temperature reaches 56 degree to 70 degree. Bence-Jones proteins coagulate at a much lower temperature, 40 degree to 60 degree of Celcius,; furthermore, unlike normal proteins, the precipitated Bence-Jones proteins redissolve as the temperature of the solution is increased to between 85 degree and 100 degree of Celcius., and often reprecipitate as the temperature is again decreased to 45 degree to 50 degree of Celcius. Because of this peculiar behavior on heating, Bence-Jones proteins have been referred to as pyroglobulins. The Bence-Jones proteins have a molecular weight of the order of 45,000, roughly one-fourth that of complete gamma globulin molecules.

Urine Protein

All urines contain some protein. The protein excreted by healthy individuals is of the order of 50 to 100 or perhaps 150 mg./24 hr. After considerable muscular exertion the value may be as high as 250 mg. Random specimens will contain under 10 mg./100 ml., although in overnight specimens the level may be as high as 15 to 20 mg./100 ml. Proteinuria is said to be present whenever the urinary protein output is greater than that reflected in these normal values. Not all proteinuria is clinically significant, but persistent abnormal levels of protein in the urine is an indicator of the presence of kidney and urinary tract disease. Thus, the detection of urinary protein and the quantitative assessment of the degree of proteinuria are very important laboratory procedures.

In general, the urinary proteins reflect those present in plasma, along with special proteins of renal tubular origin, and those exuded from the tissues that line the genito-urinary tract. The glomerular filtrate contains small amounts of all plasma protein fractions, but most of the filtered protein is reabsorbed in transit through the kidney tubules. Some of the globulins present in urine are of smaller size than their electrophoretic counterparts in serum. The new techniques of immunoelectrophoresis and antibody-antigen diffusion in agar are rapidly changing our knowledge of urine proteins. The old concept that albumin was the only protein present in any significant amount, even in pathological urines, is no longer valid, and therefore the term albuminuria should no longer be used. Some 40 to 60 per cent of the proteins present in normal urines are mucins (glycoproteins) originating from the linings of the lower  genitourinary tract, and are usually of no clinical significance.

Proteinuria is often a manifestation of primary renal disease, although transient proteinuria may occur with fevers, thyroid disorders, and in heart disease, in the absence of renal disease. Proteinuria may be evident very early in the course of various renal disease states. With such conditions as pyelonephritis, reflecting bacterial infection in the kidney, and acute glomerulonephritis, often associated with recent streptococcal infections, the degree of proteinuria is slight, usually amounting to less than 2 gm. per day. In chronic glomerulonephritis and in the nephrotic syndrome, including lipoid nephrosis, and in some forms of hypertensive vascular disease (nephrosclerosis), protein loss may vary from a few grams to as much as 20 to 30 gm. per day. Proteinuria is encountered in certain other disease entities when and if they give rise to kidney lesions. Among these are lupus erythematosus, amyloidosis, toxemia of pregnancy, septicemia, and certain forms of drug and chemical poisoning. In multiple myeloma, not only does one find a modest increase in protein output, but in 40 to 60 per cent of the cases, the urine contains a group of abnormal proteins referred to as Bence-Jones proteins.

In healthy individuals, transitory elevations in urine protein output are encountered after intense exercise or work, and after exposure to cold. Orthostatic proteinuria is benign condition in which protein excretion is normal when the patient is lying down (prone), but is mildly elevated when the patient walks or stands erect for any period of time. Persons subject to orthostatic proteinuria often are embarrassed during medical examinations for insurance because their urines are found to be positive, when tested for protein after the applicants had been on their feet and active for a good part of the day. On rechecking the urine in the morning after a night's rest, the urine is usually found to be negative for protein. 

Plasma and Serum Proteins

CLINICAL SIGNIFICANCE

Blood is made up of particulate cell forms suspended in a fluid medium called plasma, a very complicated mixture of inorganic, and simple and complex organic materials dissolved in water. If the blood is collected without anticoagulant, in vessels with siliconized or non polar surfaces, the fluid separating is referred to as native plasma. This approximates very closely the plasma actually present in circulating blood. It is the practice, however, to use such anticoagulants as oxalate, citrate, EDTA, and heparin to prepare specimens of plasma for study or analysis. This differs from native plasma because it is modified by the presence of the added chemicals and by the partial loss of calcium bound by oxalate, if this is used. If blood is permitted to clot, the fluid separating is referred to as serum. It lacks the protein fibrinogen present in plasma, the fibrinogen having been transformed into insoluble fibrin in the clotting process. The fibrinogen constitutes only some 3 to 6 per cent of the total plasma proteins. It is satisfactory and much more convenient to use serum rather than plasma in clinical chemical studies.

Some 92 to 93 per cent of plasma or serum is solvent water; of the 7 to 8 per cent of total solutes present, the proteins occur in the greatest concentration, roughly 6.8 to 8.8 (6.5 to 8.5) gm./100 ml. of plasma (serum) water. Because of the large molecular weight of proteins, however, their molar concentration calculates to about 0.80 to 1.10 mM/L. In clinical work, the concentration of proteins is given in terms of grams per 100 ml. of serum volume. It would be more precise to express it in terms of serum water. The latter value is 1.07 times the serum volume figure.

Plasma proteins serve a number of different functions in the organism. They play a nutritive role, inasmuch as they constitute a portion of the amino acid pool of the body; thus, the proteins are a form of storage amino acids. If needed, these proteins can be broken down in the liver to produce amino acids for use in building other proteins. Alternatively, they can be deaminated to give keto acids that can be mobilized to provide caloric energy or be transformed into carbohydrates and lipids. Plasma proteins also act as transport agents: many vital metabolites, metal ions, hormones, and lipids are transported about the body, bound to and carried by certain specific proteins. Some proteins have important special functions of their own, such as enzymes, the immune antibodies among the globulins, and the various proteins associated with blood coagulation.

Another important function of proteins is a physicochemical one. The plasma proteins, being large, colloidal molecules, are nondiffusible; i.e., they cannot move through the thin capillary wall membranes as can most other blood solutes. They are thus entrapped in the vascular system and exert a colloidal osmotic pressure, which serves to maintain a normal blood volume, and a normal water content in the interstitial fluid and the tissues. The albumin fraction is most important in maintaining this normal colloidal osmotic or oncotic pressure in blood. If the albumin falls to low levels, water will leave the blood vessels and enter the extracellular fluid and the tissues, thus producing edema.

The maintenance of the acid-base balance in blood also involves the plasma proteins. As amphoteric compounds, they function as buffers to minimize sudden, gross changes in the pH of the blood.

Despite their presence together, the various proteins of plasma do not originate from the same source. The liver is the main organ for the synthesis of albumins and alpha and beta globulins, and perhaps some nonimmune gamma globulins; among these these proteins are included the blood clotting and transport proteins. The cells of the reticuloendothelial system (spleen, bone marrow, lymph nodes) serve as the source of the antibody, immune gamma globulins and perhaps some beta globulins.

The level of total serum proteins found in healthy young and middle-aged adults is 6.0 to 8.2 gm./100 ml. of serum. In plasma, fibrinogen increases this value by an additional 0.2 to 0.4 gm./100 ml. A diurnal variation of 0.5 gm./100 ml. reflects small changes in the ratio of vascular to nonvascular fluid in the course of daily activity. In disease states both the total protein and the ratio of the individual protein fractions may change independently of one another. In states of dehydration, total protein may increase some 10 to 15 per cent, the rise being reflected in all protein fractions. Dehydration may result either from a decrease in water intake, as occurs in frank water deprivation (thirst), or from excessive water loss, as occurs in severe vomiting, diarrhea, Addison's disease, and diabetic acidosis. The absolute quantity of serum proteins is unaltered, but the concentration is increased because of the decreased volume of solvent water. In multiple myeloma, the total protein may increase to over 10 gm./100 ml., the increase being almost entirely due to the presence of markedly elevated levels of myeloma proteins (abnormal forms of gamma globulins). The quantities of other proteins are essentially unaltered.

Hypoproteinemia, characterized by total protein levels below 6.0 gm./100 ml., is encountered in many unrelated disease states. In the nephrotic syndrome large masses of albumin may be lost in the urine as a result of leakage of the albumin molecules through the damaged kidney. In salt retention syndromes, water is held back to dilute out the retained salt, resulting in the dilution of all protein fractions. Large quantities of proteins are lost in patients with severe burns, extensive bleeding, or open wounds. Water is replaced by the body more rapidly than is protein, effecting a decreased total protein concentration. A long period of low intake or deficient absorption of protein may affect the level and composition of serum proteins, as in sprue and in other forms of intestinal malabsorption, as well as in acute protein starvation (kwashiorkor). In these conditions the liver has inadequate raw material to synthesize serum proteins to replace those lost in the normal turnover (wear and tear) of proteins and amino acids.

In general, changes in total proteins may occur in one, several, or all fractions. It is also possible for significant changes to occur in different directions in different fractions, without changes in the total protein concentration.

The physician may occasionally request only a value for total protein in serum; however, more commonly he will ask for total protein, the values for the albumin, and total globulin fractions, and for the albumin-globulin (A/G) ratio. In healthy young and middle-aged adults, the albumin may vary from 3.8 to 4.7 gm./100 ml., and the total globulins from 2.3 to 3.5 gm./100 ml. The range found for the albumin-globulin ratio is 1.1 to 1.8, averaging 1.5. The globulins are usually separated by a salt fractionation procedure, but may be estimated from the sum of electrophoretically separated fractions. Albumins can be estimated separately by virtue of their ability to bind certain dyes such as methyl orange and hydroxyazobenzoic acid. These techniques will be discussed later.