.~«;Li.‘-u s . to ~31 a .‘ \ ‘ ‘ t ‘V .' ~ ‘.' ,‘ .- ‘N-‘AJH‘Q'? .r’I-F'u. 5-3. '- “‘9" 3"" COMPARISON OF THE ELECTROPHORETIO PATTERNS OF NORMAL CANINE SERUM AND PLASMA AND CHANGES IN THE SERUM AND PLASMA 0F HEMOLYZED SPECIMENS Dissertation for the Degree of M. S.- MICHIGAN STATE UNIVERSITY VlDA M. AMOS 1 9 7.3 LIB RA RY Michigan State University 5. v {J H 5' g DIN‘BING BY HUAB & SllllS’ 800K_B_|_K0ERY,_‘ND ‘ ! Iltl'i'ii“_‘§;; ~' — COMPAR I :~ CA:- Comparison c serum revealed a due to the preset canine blood was beta globuliu pe were increases 1 haPtog 10b in_hem0 ABSTRACT COMPARISON OF THE ELECTROPHORETIC PATTERNS OF NORMAL CANINE SERUM AND PLASMA AND CHANGES IN THE SERUM AND PLASMA OF HEMOLYZED SPECIMENS By Vida M. Amog Comparison of electrophoretic patterns of normal canine plasma and serum revealed a greater concentration of the beta-3 fraction in plasma due to the presence of fibrinogen. When serum or plasma of hemolyzed canine blood was analyzed electrophoretically, there was slurring of the beta globulin peaks due to the presence of free hemoglobin and there were increases in alphan globulins due to the formation of the haptoglobin-hemoglobin complex. COMPARISON OF THE ELECTROPHORETIC PATTERNS OF NORMAL CANINE SERUM AND PLASMA AND CHANGES IN THE SERUM AND PLASMA OF HEMOLYZED SPECIMENS BY 5} ‘t I 4, Vida Mi? Amog A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1973 J A n .. {X [Al ' \ To my mother, Mrs. Florentina M. Amog, and the loving memory of my father, Mr. Benjamin M. Amog ii I wish I Acting Chair: Merrill, for: of stud)“ I am tha excellent C0” unlimited pat My SinC€3 his guidance a Appreciat MMASCP), for To the st Medicine, Vete and to the num appreciation f of specimens f I My Special Dimlap, HMASCI COOperation. Above all, indebted for th ACKNOWLEDGMENTS I wish to eXpress my sincere gratitude to Dr. R. F. Langham, Acting Chairman of the Department of Pathology, and to Dr. C. C. Morrill, former Chairman, for his advice and support during my course of study. I am thankful to Dr. R. L. Michel, my major professor, for his excellent counsel and guidance in the preparation of this thesis and unlimited patience and unselfish gifts of many hours of his time. My sincere gratitude to Dr. R. W. Bull, my project advisor, for his guidance and assistance throughout my research project. Appreciation is also extended to Ms. M. T. Thomas, M.S., MT(ASCP), for serving on my guidance committee. To the staff of the Immunology Laboratory, Department of Human Medicine, Veterinary Clinic Small Animal Surgery and Medicine faculty and to the numerous veterinary students, I wish to eXpress my sincere appreciation for provision of facilities and cooperation in collection of specimens for the experiments. My special thanks to Ms. P. J. Rosenberger, MT(ASCP), Ms. E. Dunlap, MT(ASCP) and Mr. S. Iwamoto, M.S., for their assistance and cooperation. Above all, to my mother, brothers and sisters, I am deeply indebted for their encouragement, assistance and support in all my endeavors. iii EJRODUCIION LITERAICRE RE Histor Princi Suppor Preser Princi Experi. EXperin TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . . . . . . 3 Historical Background . . . . . . . . . . . . . . . . . . . 3 Principle of Electrophoresis. . . . . . . . . . . . . . . . 4 Supporting Media. . . . . . . . . . . . . . . . . . . . . . 5 Preservation and Storage of Samples . . . . . . . . . . . . 6 Principal Fractions of Serum Proteins . . . . . . . . . . . 7 Albumin. . . . . . . . . . . . . . . . . . . . . . . 7 Alpha (a) Globulins. . . . . . . . . . . . . . . . . 8 Beta (8) Globulins . . . . . . . . . . . . . . . . . 9 HemOgJ-Obin. O O O O O O O O O O O O O O O O O 10 Fibrinogen. . . . . . . . . . . . . . . . . . lO Gamma (y) Globulins. . . . . . . . . . . . . . . . . 11 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . . . 12 Experiment 1. Comparison of the ElectrOphoretic Patterns of Serum and Plasma . . . . . . . . . . . . 13 Source of Specimens. . . . . . . . . . . . . . . . . 13 Determination of Fibrinogen Concentration. . . . . . l4 Isolation and Purification of Fibrinogen . . . . . . 14 Method . . . . . . . . . . . . . . . . . . . . . . . 14 Total Protein. . . . . . . . . . . . . . . . . . . . 14 Experiment 2. Comparison of Normal Canine Serum and Serum from Partially Hemolyzed Samples . . . . . . . 15 Source of Hemolyzed Specimens. . . . . . . . . . . . 15 iv Serum Haptoglobin. Haptoglobin Isolation and Purification . Hemoglobin Isolation . Method . Experiment 3. Source of Specimens. RESULTS. 0 O O O O O O 0 Experiment 1. Patterns of Serum and Plasma . Experiment 2. Partially Hemolyzed Specimens. . Experiment 3. Patterns of Normal Plasma and of Partially Hemolyzed Samples. . DISCUSSION 0 O O O O O O 0 SUMMARY AND CONCLUSIONS. . REFERENCES . . . . . . . APPENDICES O O O O O I O O A Reagents . . . . . . . . B Calibration of Standard Curve. C Calculation Formula. . . v ITA O O O O O O O O O O 0 Comparison of the Electrophoretic Comparison of the Electrophoretic Patterns of Normal Serum and Serum from Comparison of the Electrophoretic Comparison of Normal Canine Plasma Plasma from Partially Hemolyzed Samples. Plasma from Page 15 15 l6 l6 l6 16 18 18 18 24 35 39 41 46 46 48 52 53 hr l- Table LIST OF TABLES Page Comparison of means, ranges and standard deviations of concentrations of plasma and serum proteins of normal specimens for 24 dogs . . . . . . . . . . . . . . . . . . . 22 Comparison of means, ranges and standard deviations of concentrations of serum proteins of normal specimens and partially hemolyzed specimens for 24 dogs . . . . . . . . . 30 Comparison of means, ranges and standard deviations of concentrations of plasma proteins of normal specimens and partially hemolyzed specimens for 24 dogs . . . . . . . . . 34 vi Figure 10 ll 12 13 14 N U Traci repre Traci Traci: Traci: fibrir Compar plasma 24 dog Tracin Iracin; partiai Tracin; With f: Tracing With f] Tracing Tracing hemogh Tracing Comple) comparj of hEmc Traci“g Figure 10 11 12 13 14 15 16 17 LIST OF FIGURES Tracing of electrophoretic analysis of serum of a representative normal dog (Dog 12). . . . . . . . . . . . Tracing of electrophoretic analysis of plasma of Dog 12 . Tracing of electrophoretic analysis of plasma of Dog 12 . Tracing of electrOphoresis of isolated, concentrated fibrinogen of pooled dog plasma . . . . . . . . . . . . . Comparison of electrophoretic analyses of serum and plasma. Means and ranges of protein concentrations for 24 dogs 0 O O O O O O O O O O 0 O O O O O O O O O O O Tracing of electrophoretic analysis of serum of Dog 18. Tracing of electrophoretic analysis of serum from partially hemolyzed blood of Dog 18 . . . . . . . . . . . Tracing of electrophoretic analysis of plasma of Dog 5 with free hemoglobin concentration of 44 mg./100 ml. Tracing of electrophoretic analysis of serum of Dog 15 with free hemoglobin concentration of 60 mg./1OO m1. . Tracing of electrophoretic analysis of serum of Dog 15. . Tracing of electrophoresis of isolated, concentrated hemoglobin of Dog 15. . . . . . . . . . . . . . . . . . . Tracing of electrophoretic analysis of serum of Dog 12. . Tracing of electrophoresis of isolated, concentrated haptoglobin of pooled dog serum . . . . . . . . . . . . . Tracing of electrophoretic analysis of serum of Dog 12. . Tracing of electrOphoresis of haptoglobin-hemoglobin complex and free hemoglobin . . . . . . . . . . . . . . . Comparison of electrophoretic analyses of serum and serum of hemolyzed specimens. Means and ranges of protein concentrations for 24 dogs. . . . . . . . . . . . . . Tracing of electrophoretic analysis of plasma of Dog 11 . vii Page l9 19 20 20 21 23 23 25 25 26 26 27 27 28 28 29 31 figure 13 Traci parti 19 Compé plas: prot4 B-l P1a51 3-2 131381 3-3 Cali trat B-4 Hapt Figure 18 19 3-1 B-2 B—3 Tracing of electrophoretic analysis of plasma from partially hemolyzed blood of Dog 11 . . . . . . . . . . Comparison of electrophoretic analyses of plasma and plasma from hemolyzed specimens. Means and ranges of protein concentrations for 24 dogs. . . . . . . . . . . Plasma hemoglobin standard curve. . . . . . . . . Plasma fibrinogen standard curve. . . . . . . Calibration curve for estimation of haptoglobin concen- tration . . . . . . . . . . Haptoglobin isolation elution curve . . . . . . . viii Page 31 33 48 49 50 51 INTRODUCTION Electrophoresis may be defined as the separation of components in a mixture based on their differing rates of migration in an electric field. The method is particularly valuable for separating labile macro- molecules because it can be carried out in a supported aqueous medium under mild conditions of pH and ionic strength. Electrophoresis was first used only as a means for measuring ionic mobilities and isoelectric points. After Tiselius' important work in 1937, and with later improvements in the optical technique, it became possible to use the method for quantitative analysis of complex mixtures, as well as for the separation of different substances. The analysis of mixtures soon became the most important application of electrophoresis. There have been many reports based on electrophoretic studies of protein distribution in both human beings and animals. However, results have differed because of the differences among the various techniques and species. Hence, it is desirable that each laboratory establish its own standard reference electrophoretic patterns for the species of interest. At times, it is necessary to use plasma for electrophoretic analysis when serum is not available. However, plasma contains fibrinogen which, in human plasma, migrates between the beta and gamma globulins. Thus, a comparison of the electrophoretic patterns of serum and plasma of animals is useful. 2 Hemolysis of blood specimens is a common problem in clinical laboratories. In many cases, it is impossible to obtain specimens completely free of hemolysis. This may be the result of increased erythrocyte fragility or imprOper handling of specimens. If the patient is not available for collection of another sample, it may be necessary to perform the electrophoretic analysis on the hemolyzed specimen. Hemolysis produces certain characteristic changes in serum and plasma reflecting the presence of free hemoglobin and the formation of the haptoglobin—hemoglobin complex. This investigation was designed to compare the electrophoretic patterns of a) serum and plasma, b) serum of partially hemolyzed specimens with serum of specimens free of hemolysis, and c) plasma of partially hemolyzed specimens with plasma of unhemolyzed specimens. LITERATURE REVIEW Historical Background Experiments in electrophoresis started in the early part of the nineteenth century. These studies increased in number into the twentieth century, culminating in Arne Tiselius' important work in 1937 concerning the moving boundary for which he earned the Nobel Prize in Chemistry in 1948. Since then, many investigators have contributed to the development of these techniques. The books serum Proteins and Dysproteinemias (1964), edited by F. W. Sunderman and F. W. Sunderman, Jr., and Electrophoresis of Proteins and the Chemistry of Cell Surfaces (1964), by H. A. Abramson at al. offer a fairly complete historical background of electrophoresis and topics related to protein chemistry. One of the earliest reports concerning protein electrophoresis in dogs was by Munro and Avery (1946) and concerned the effects of hepa- tectomy on the relative concentrations of plasma proteins. Vesselinovitch (1959) gives an excellent review of paper electro— phoresis in domestic animals, including dogs. He stated that the first extensive study of serum proteins of domestic animals was by Boguth (1953), and the first results of paper electrophoresis of canine serum were published by de Wael and Teunissen (1954). However, de Wael's and Teunissen's studies were restricted to animals with hepatic disorders. Due to differences in techniques and instruments used by various laboratories, the number of bands resolved may range from 5 to as many 3 4 as 90 as reported by Farrow (1972) for human serum. However, only a small number of these proteins have known biological functions. It is probable that the plasma proteins of animals are equally numerous. Principle of Electrophoresis The principle of electrophoresis is simple. An ion or group will migrate towards the positive or negative electrode, depending on its charge, when placed in an electric field. The charged species moves in the electric field at a rate which is a function of its size, shape and charge. After some time, the different charged species in the mix- ture separate into zones detected by suitable techniques (Smith, 1968; Shaw, 1969). The movement of ions is influenced by four factors: 1) Electric field strength. When a charged species is dissolved or suspended in a buffer solution and subjected to a uniform field, the particles will migrate at a constant rate determined by their physical shape, size, and charge. Positively charged species move towards the cathode ( - ) and negatively charged species towards the anode ( + ) (Longsworth, 1959; Wieme, 1965). 2) Ion mobility, which is the distance travelled by the migrating species in relation to the support medium in a given time in a field of unit potential gradient (Longsworth, 1959; Smith, 1968). 3) Buffer pH and ionic strength. The pH of the electrolyte has an influence on electrophoretic behavior because the net charge of most species is dependent on pH. The ionic strength is usually adjusted to 0.05 to 0.1, which is Optimal for a compromise between high mobilities and sharpness of zones. Since conductivity and power consumption are functions of ionic strength, it is desirable to keep the ionic strength low. This minimizes heat production and electrode products formed 5 (Shaw, 1969). 4) Supporting medium. This usually consists of paper, starch gel, agar gel, cellulose acetate or other porous material that is saturated with buffer. Each has its own advantages and disadvantages (Williams and Nixon, 1964; Smith, 1968; Shaw, 1969). Supportinngedia Historically, the most commonly used supporting medium for electro- phoresis has been filter paper. Although excellent reproducibility, low cost and convenience can be achieved with paper electrophoresis, the disadvantages of tailing, blurring and reduced resolution are seen in the electrophoretic pattern. These are due to the fibrous structure of paper and the interaction of ionic sites on the paper and the charged species being separated (Jencks et al., 1955; Peeters, 1959; Yeoman, 1959). Thin, porous cellulose acetate membrane was introduced as a medium for electrophoresis by Kohn in 1957. Due to its regular and fine- textured pores, there is little or no adsorption to the serum. The time required for electrophoretic determination is greatly reduced while accuracy and reproducibility are improved. ‘ The use of gels has been shown to be a rapid means of producing high resolution separation of proteins. Among them is the agar gel which is still widely used today. It forms a firm colloidal gel in concentrations as low as 1%. Compared to other gels, it possesses some outstanding advantages, including ease of handling, excellent trans- parency, stability in the gel state, reusability after re—melting and re-pouring, low cost and rapid separation. R. J. Wieme (1965) has published detailed information on the use of agar gel as a medium for electrophoresis. 6 Starch (Kunkel and Slater, 1952) and polyacrylamide gels1 have also been used for electrophoretic analysis of serum (Raymond and Weintraub, 1959; Smith, 1968). The use of these gels permits the resolution of 20 bands or more (Henry, 1965; Smith, 1968). Polyacrylamide gel is especially useful in molecular weight determinations (Shapiro, 1967). However, for routine clinical purposes, the use of these gels is not generally practical. Preservation and Storage of Samples Fresh specimens are to be preferred (Coles, 1967). Henry (1965) reported that samples can be stored at room temperature for 3 days without any significant change or at refrigerator temperatures (5 to 10 C) for as long as 1 month, although long storage in the refrigerator may result in diminished resolution in the globulin fraction. Damm and King (1965), on the other hand, reported the stability of samples at refrigerator temperatures for at least a week. There are conflicting reports regarding the effects of freezing. Oberman et a2. (1956) suggested freezing samples no longer than 2 days. Engel et al. (1961) generally observed that freezing does not produce notable alterations in starch gel electrophoresis. He noted, however, one instance of diminished resolution and slight alteration in mobility. Henry (1965) reported serum stability for at least 6 months in the frozen state. Repeated freezing and thawing should be avoided, for inactivation and denaturation may occur in some labile proteins (lipo- proteins, purified antibodies and pure ovalbumin). 1Cyanogum 41, a synthetic gel. 7 Principal Fractions of Serum Proteins The number of serum protein fractions which can be identified varies considerably depending on the method used for analysis. However, there are 4 major fractions routinely isolated by electrophoretic methods: albumin, alpha (a) globulin, beta (8) globulin, and gamma (y) globulin (Tiselius, 1937). Each one has its own characteristic rate of migration. Albumin. This is primarily synthesized in the liver. One of its main functions is the maintenance of intravascular osmotic pressure (Coles, 1967; Tietz, 1970; Farrow, 1972). It also acts as a transport agent for drugs, pigments and other substances (Coles, 1967; Farrow, 1972). Albumin comprises approximately one-half of the serum protein concen- tration in both normal man and animals. However, the mean concentration in animals varies from species to species (Coles, 1967; Haurowitz, 1963). Albumin is the fastest moving fraction on electrophoresis at pH 8.6. Human albumin has a molecular weight of approximately 69,000, a sedimentation coefficient of 4.7 S and is isoelectric at pH 4.7 (Sibley and Hendrickson, 1970; Haurowitz, 1963). It is internally crosslinked by many disulfide bonds. Aspartic acid (ASP) is; its N—terminal residue and leucine (LEU) is its C-terminal residue. Its amino acid sequence is not well known and only incomplete data are available (Murayama, 1964). The albumin level is seldom increased above the normal range except in severe dehydration and shock (Coles, 1967). A relative decrease in this fraction is significant and may be due to reduced synthesis, more rapid catabolism or increased globulin concentration (e.g., malnutrition, hepatic disease) (Kaneko and Cornelius, 1970). 8 Alpha (a) Globulins. This comprises a group of proteins which bind a number of substances for transport in the plasma. Included are hapto- globin, a—lipoprotein, transcortin, and ceruloplasmin which bind, respectively, hemoglobin, lipids, corticosteroids, and copper (Farrow, 1972). On electrophoretic patterns at least two a—globulin bands, identi- fied as a1 and a2, are seen. The a1 globulins form a narrow, often barely discernible, band adjacent to albumin. An ionic strength of the buffer of 0.05 or higher permits a good separation of this band from the albumin zone (Wieme, 1965). The a globulins appear as a 2 discrete, compact band. It is in this area where haptoglobin migrates. Haptoglobin, from the Greek word haptein (to fix, seize, or hold fast), is a serum glycoprotein which has a high affinity for globin, whether free or in the form of hemoglobin, with which haptoglobin forms a very stable complex. Haptoglobin is synthesized mainly in the liver and, in complex with hemoglobin, is removed from the circulation by the reticuloendothelial system at the rate of 13 mg. of hemoglobin/100 m1. of plasma/hour (Louderback and Shanbrom, 1968; Blumberg, 1964). Giblett (1961) gives an excellent review of the physiology, chemistry and genetics of haptoglobin. There are 3 main types of haptoglobin in human beings, namely 1—1, 1-2, and 2-2. These are genetically determined by a pair of autosomal genes le and sz, which are responsible for the 3 types le/le, HpZ/sz and sz/le. A fourth haptoglobin type, 0-0 (ahaptoglobinemia), has been shown to occur in 30% of Nigerian Negroes, 5% of American Negroes, 2% of British Caucasians, and 1% of Danish Caucasians. It has also been noted that 90% of all newborn infants lack haptoglobin, but that it appears within 4 to 6 months (Smith, 1968; Miale, 1962, 1972). 9 Although there has been a great deal of investigative work on human haptoglobin, there are relatively few reports on this subject in animals. Only one form (corresponding to human type 1-1) has been detected in animals, including dogs. Shim et a1. (1971) investigated canine serum haptoglobins and found them similar to human type 1—1 with respect to subunit structure, hemoglobin-binding mechanism and binding sites. However, free canine haptoglobin.(molecular weight 81,000) had a slightly faster mobility in starch gel electrophoresis than type l-l human haptoglobin (molecular weight 85,000 to 100,000). This was attributable mainly to the slightly lower molecular weight of canine haptoglobin which enables faster migration in the commonly employed molecular sieving starch gel medium. Reduced a—globulin concentrations are seldom seen in domestic animals except in severe, chronic liver disease (Coles, 1967; Farrow, 1972). Haptoglobin concentrations are decreased in conditions charac- terized by intravascular hemolysis. Elevated concentrations of a- globulins (particularly a2) are seen in inflammatory reactions (e.g., bacterial and viral infections, trauma, fever) (Coles, 1967; Kaneko and Cornelius, 1970; Farrow, 1972). Beta (8) Globulins. The main site of biosynthesis of beta (8) globulins is the liver (Farrow, 1972). Included in this group are transferrin (an iron-binding protein), hemopexin (a heme—binding protein) and beta- lipoprotein, which is concerned with the transport of lipids (Wieme, 1965; Smith, 1968; Farrow, 1972). A varying number of beta fractions occur in electrophoretic patterns depending on the method used. Oftentimes, especially in man, only a single 8 peak is identified. In dogs, there may be a single 10 band (Vesselinovitch, 1958; Irfan, 1967), 2 bands (de Wael, 1956; Bulgin et aZ., 1971), or 3 bands (Kozma et aZ., 1967). Detailed characterization of these bands requires sophisticated techniques such as starch gel electrophoresis or immunoelectrophoresis. It is in this fraction that free hemoglobin migrates electrophoretically as shown by several studies (Allison and Rees, 1957; Nyman, 1960; Dacie and Lewis, 1963; Louderback and Shanbrom, 1968). Hemoglobin. Hemoglobin (molecular weight 69,000) is a combination of protein, globin, with heme. Heme is a protOporphyrin consisting of iron chelated with 4 pyrrole groups. In man, there are several known normal and abnormal hemoglobins, each of which has a distinct electro- phoretic mobility (Bromberg et al., 1972). Sydenstricker et al. (1956) reported that canine hemoglobin has an electrophoretic mobility faster than that of Hb S, and that it appears as a homogeneous compound. However, they did not mention the number of dogs used. This agrees well with LeCrone's (1970) observations on 21 adult dogs. It is possible that there are more than one hemoglobin found in dogs. This awaits further research. Fibrinogen. Previous workers have reported that fibrinogen (¢) which is present in plasma but not in serum, migrates between the B and y globulins (Abramson, 1964). It is a glyc0protein which is converted to fibrin in the presence of Ca++ by thrombin. It is also known as Factor 1, playing an important role in blood coagulation. It is synthe- sized in the liver. Fibrinogen is easily precipitated by inorganic salts at high concentrations e.g., 1/2 saturated solution of NaCl, 1/4 saturated solution of (NH 804, and is the only protein reversibly 4)2 precipitated at 56 to 58 C (Hawk, 1965; Henry, 1965; Schalm, 1972). 11 On electrophoresis on agar gel, fibrinogen does not migrate. This is due to its precipitation by the agar (Wieme, 1965). Increased fibrino- gen concentrations may occur in connection with hepatic diseases, acute infections and septicemias. Plasma fibrinogen concentration is decreased in afibrinogenemia, shock, severe burns, and hepatic insufficiency (Hawk, 1965; Coles, 1965; Schalm, 1972). Gamma (y) Globulins. These are normally produced by the cells of the reticuloendothelial tissues (spleen, bone marrow, lymph nodes, and intestinal mucosa) (Tietz, 1970; Farrow, 1972). Immunoglobulins are antibodies produced in response to antigenic stimulation. There are 5 principal types recognized in man: IgG, IgA, IgM, IgD and IgE. Each molecule consists of 2 heavy (H) and 2 light (L) polypeptide chains (Waldenstrom, 1968; Terry and Fahey, 1964; Farrow, 1972). Increased gamma globulin concentrations may be seen in cases of myeloma, severe, chronic infection, chronic, active hepatitis, or advanced neoplasia. Reduced concentrations may result either from congenital conditions (e.g., hypogammaglobulinemia) or acquired conditions such as reticulo— endothelial neoplasia (Coles, 1967; Farrow, 1972; Kaneko and Cornelius, 1970). Electrophoretically, the gamma globulins migrate towards the cathode, behind the application point. This is due to electro-osmosis (Smith, 1968). MATERIALS AND METHODS The experimental studies were divided into 3 parts: 1) comparison of normal canine serum and plasma, 2) comparison of serum from partially hemolyzed blood and unhemolyzed blood, and 3) comparison of plasma from partially hemolyzed blood and unhemolyzed blood. Electrophoresis was carried out by the method of Cawley and Eberhardt (1962) with some modifications. Method: Two electrophoresis strips were prepared for each animal in the following manner: 4 m1. of 1% IonagarR in 1/2 strength Veronal buffer1 were pipetted onto 35mm. film-strips2 6—1/2 inches in length. Serum or plasma (0.006 ml.) was applied to the center of the strips using a Spinco applicator. Eight strips (from 4 dogs) were placed in a Durrum cell containing the Veronal buffer. Electrophoretic fractionation was accomplished using 150 volts and 60 to 80 milliamperes for 1 hour 15 minutes. At the end of the run the strips were fixed in 90% methanol. The strips were then dried in a 95 to 100 C oven for 30 minutes and stained with 0.2% thiazine red3 in 10% acetic acid. The strips were then rinsed in distilled water, decolorized in several washes of 5% 1Barbital-Sodium Barbital Mixture, pH 8.6, ionic strength 0.075, Harleco, 60th and Woodland Avenue, Philadelphia, Pennsylvania 19143. 2E. I. duPont de Nemours, Photo Products Dept., 7415 Melvina, Niles, Illinois 60648. Safety Motion Picture Film, 0.004" thick, unperforated. 3Thiazine Red R, Color Index No. 14780, Harleco, 60th and Woodland Avenue, Philadelphia, Pennsylvania 19143. 12 l3 acetic acid, air dried, and scanned at 505 nm with a densitometer- integrator.l .The densitometer converts the color density pattern into a concentration curve and the integrator measures the relative area under each peak. Total protein concentration was determined by the biuret method of Cornell et al. (1949). Experiment 1 Comparison of the Electrophoretic Patterns of Serum and Plasma Source of Specimens. Blood samples were collected in evacuated glass tubes2 from.24 clinically normal appearing adult dogs. Serum was obtained from clotted blood after centrifugation and plasma from samples with EDTA3 in.the ratio of 9 mg./7 m1. of blood. Hematocrit values and hemoglobin concentrations were immediately determined from EDTA samples. Packed cell volumes were determined using the microhematocrit method with centrifugation at approximately 10,000 g for 5 minutes in a micro- capillary centrifuge.4 Hemoglobin was determined by the cyanmethemo- globin method. The degree of hemolysis of hemolyzed samples was measured by determination of the hemoglobin concentration of serum and plasma using the method of Fielding and Langley (1958), except that a commercial hemoglobin control5 was used in the preparation of the standard calibra- tion curve. This method depends on the peroxidase activity of hemoglobin 1Photovolt Densitometer, Model 425, Photovolt Corp., New York, N.Y. 2Vacutainer, Becton, Dickinson & Company, Rutherford, N.J. 3Tri-Potassium Ethylene Diamine Tetra-Acetate. 4International Equipment Company, Needham Heights, Massachusetts. sHycel Hemoglobin Control, Hycel, Inc., Houston, Texas. 14 which causes oxygen to be released from hydrogen peroxide. The free oxygen then oxidizes orthotolidine to a blue reaction product which is measured photometrically. The reagents required for preparation of the hemoglobin reagent are available commercially combined in tablet form.1 Determination of Fibrinogen Concentration. Fibrinogen.was determined by the method of Loeb and Mackey (1972). The method is based on the heat precipitation of fibrinogen and quantitation of the precipitate by the biuret method. The concentration was derived from a standard curve prepared by using crystallized bovine albumin. Isolation and Purification of Fibrinogen. To determine in which fraction fibrinogen migrates, it was isolated and purified. Electrophoresis was then performed on the concentrated, purified fibrinogen obtained. Method.. Fibrinogen was isolated by the method of Atencio et al. (1965). This method involves salt fractionation coupled with a cold insoluble protein precipitation. The protein fraction thus isolated, after being redissolved in 0.005 M citrate solution, was verified as relatively pure fibrinogen by demonstration of the fact that it was 94.4% clottable by addition of bovine thrombin.2 Total Protein. Total protein concentrations of all serum and plasma samples were determined by the method of Gornall et al. (1949). 1Hematest TabletR, Ames Company, Inc., Elkhart, Indiana. 2Thrombin, Topical (bovine origin), Parke, Davis & Company, Detroit, Michigan. 15 Experiment 2 Comparison of Normal Canine Serum and Serum from Partially Hemolyzed Samples Source of Hemolyzed Specimens. Blood was taken from 24 normal-appearing, adult dogs. A portion of the serum was pipetted off from the tube after centrifugation and the remainder of the specimen subjected to mechanical agitation to obtain hemolyzed samples. The degree of hemolysis of each sample was determined by determination of serum hemoglobin concentra- tions as previously described. Electrophoresis and total protein determinations were then carried out on each of the 24 samples and compared. The following determinations were carried out. Serum Haptoglobin. Serum haptoglobin was measured using the method of Owen et a1. (1960). This is a simple colorimetric method based on the peroxidase activity of haptoglobin—methemoglobin complexes. The concentration of haptoglobin was obtained from a calibration curve and was eXpressed in terms of bound methemoglobin. All tests were done at room temperature (24 to 25 C). Haptoglobin Isolation and Purification. To determine in which fraction haptoglobin migrates, it was isolated and purified according to the method of Connell and Smithies (1959). Dowex 2x-10 (200-400 mesh) anionic exchange resin, as the chloride, was used to adsorb the hapto- globins from dialyzed serum at pH 4.2. The resin was set up in a column after the adsorption and was washed with water to remove the soluble proteins. The haptoglobins were eluted with 0.05 M NaCl. All steps were carried out at room temperature except the dialysis, which was done in the cold room, The isolated haptoglobin was concentrated by vacuum dialysis and concentration determined by the method of Owen et a2. 16 (1959). Electrophoresis was then carried out on the isolated, concen- trated solution to determine the migratory characteristics of haptoglobin. Hemoglobin Isolation. Hemoglobin electrophoresis was performed on the hemolysates of each sample to determine in which fraction pure hemoglobin migrates. Method. Hemolysates were prepared according to the method of Dacie and Lewis (1963) with the following modifications: use of 1.5 m1. instead of 1.0 m1. of distilled water per milliliter of packed cells to make a concentration of approximately 10 gm./100 m1. hemoglobin solution; use of chloroform instead of carbon tetrachloride; and freezing and thawing of red cells to insure complete hemolysis before the addition of chloroform. Experiment 3 Comparison of Normal Canine Plasma and Plasma from PartiallyiHemolyzed Samples Source of Specimens. Blood was taken from 24 normal—appearing, adult dogs. A portion of the plasma was pipetted off from the tube after centrifugation and the remainder of the specimen subjected to mechanical agitation to obtain hemolyzed samples. The degree of hemolysis of each sample was determined by determination of plasma hemoglobin concen- trations as previously described. Electrophoresis and the determina— tion of total protein concentration were then carried out on each of the 24 samples and compared. The following determinations were per— formed: 1) fibrinogen determination on each of the 24 samples, 2) fibrinogen isolation as previously described, 3) electrophoresis of purified fibrinogen to determine migratory characteristics of fibrinogen, 17 4) hemoglobin.isolation as previously described, and 5) electrophoresis of hemolysate to determine migratory characteristics of hemoglobin. RESULTS Experiment 1 Comparison of the Electrophoretic Patterns of Serum and Plasma Representative electrophoretic tracings are shown in Figures 1 and 2. The concentration of the B3 fraction of plasma was higher than that of serum due to the presence of fibrinogen (P<0.01). In Figures 3 and 4 it can be seen that isolated, concentrated fibrinogen migrated with the 83 fraction. Fibrinogen determinations were done on plasma samples of each of the 24 dogs, and the concentrations were found to be in the range of 297—695 mg./100 m1., with a mean of 438 mg./100 m1. and a standard deviation of i 22.2. The means, standard deviations and ranges of the concentrations of serum and plasma proteins as determined electrophor— etically are graphically represented in Figure 5 and are shown in Table 1. In Table 1 it can be seen that there were also significant differences in the a and 81 fractions, these being in higher concentrations in plasma 2 than in serum (Pf0.01). In the case of the electrophoretic tracing shown in Figures 1 and 2, there were also higher concentrations of the al and 82 fractions in plasma. However, as can be seen in Table 1, these dif- ferences were not consistent throughout the group. Experiment 2 Comparison of the Electrophoretic Patterns of Normal Serum and Serum from Partially Hemolyzed Specimens Figures 6 and 7 compare representative electrophoretic analyses of normal serum and serum from partially hemolyzed blood in 24 dogs. After hemolysis of the samples, the concentration of the total protein was 18 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 4.00 3.50 3.00 2.50 2.00 J 1 I 1 Figure 1. I 1 Figure 2. 19 albumin application point Tracing of electrophoretic analysis of serum of a repre- sentative normal dog (Dog 12). albumin application point Tracinggof“electrophOretic analysis of plasma of Dog 12. 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 19 application point Figure 1. Tracing of'electrophbretic:analysis of'serum of a repre- sentative normal dog (Dog 12). 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 ‘- v albumin I 1 tr- . 100 i application point Figure 2. Tracing of‘electrophoretic-analysis of plasma of Dog 12. 20 4.00 m 3°50 W albumin 3.00 V 2.50 - 2.00 ‘ 1.50 1.00 application point 0.50 4.00 A 3.50 n 3.00 a 2.50 4 application point 2.00 1.50 1.00 Pr 0.50 - 0 Figure’4.. Tracing of electrophoresis of isolated, concentrated fibrinogen of pooled dog plasma. 22 21 20 19 18 17 16 15 14 13 12 10 O! J-‘UI 21 ‘L Serum Plasma 0- JP d- «i t w tu- n- u- w i‘ I I r £_ 1' b i A. ‘r I . I I L' To. 1- 1' 1. T- . I, 1— , I1 I1. II I!- LIL TP Albumin a1 62 81 82 83 Y A/G ‘Figure 5. Comparison of electrophoretic analyses of serum.and plasma. Means and ranges of protein concentrations for 24 dogs. 22 Table 1. Comparison of means, ranges and standard deviations of concentrations of plasma and serum proteins of normal specimens for 24 dogs Mean Standard Range (gm./100 m1.) Deviation (gm./l00 m1.) Total 6.32* 0.429 5.50 7.22 Protein 6.86I 0.387 5.85 7.10 Albumin 3.85 0.367 3.18 4.99 3.71 0.400 3.14 4.88 Alpharl 0.63 0.182 0.27 1.17 Globulin 0.61 0.172 0.39 1.13 Alpha-2 0.30* 0.113 0.11 0.71 Globulin 0.40+ 0.114 0.19 0.75 Globulin 0.25+ 0.091 0.11 0.52 Globulin 0.46 0.129 0.27 0.80 Beta-3 S 0.50* 0.134 0.28 0.81 Globulin P 0.72+ 0.203 0.40 1.14 Gamma s 0.35 0.121 0.14 0.63 Globulin P 0.35 0.134 0.07 0.63 A/G 1.67% 0.404 1.13 2.88 Ratio 1.38 0.381 0.93 2.79 S - Serum P - Plasma * = Significantly different from i (P<0.01). 1 = Significantly different from 2 (P<0.05). 4.00 " 3.50 w 3.00 v 2.50 n 2.00 1.50 1.00 a application point 0.50 . Figure 6. Tracing of electrophoretie.analysis of serum of Dog 18. 4.00 A 3050 "' albumin 3.00 + 205. "' 2.00 1.50 1.00 ‘ application point 00 so ‘ o 1 Figure 7. ‘Tracing of.electrophoretic analysis of serum from partially 'hemolyzed blood of Dog 18. 24 increased (P<0.01) and the B globulins tended to be slurred and only 2 8 peaks were identifiable. The presence of approximately 50 mg. of free hemoglobin/100 m1. of serum or plasma was sufficient to produce slurring of the B globulins (Figures 8 and 9). Furthermore, there was an elevation of the a2 globulin fraction (P<0.01) as a result of the formation of the haptoglobin-hemoglobin complex (Figure 7). Finally, in some dogs,.there was an apparent retardation in the migration of the 02 globulin fraction resulting in the formation of a distinct valley between it and al. In Figure 11 it can be seen that hemoglobin migrates with the B globulins. By contrast, haptoglobin and the haptoglobin-hemoglobin complex both migrate as 02 globulins (Figures 13 and 15). Haptoglobin determinations were carried out on each of the 24 samples and were found to be from 0-235 mg./100 ml. with a mean of 133 mg./100 ml. and a standard deviation of :_34.03. The means and ranges for normal serum and for serum of partially hemolyzed specimens are graphically illustrated in Figure 16 and are shown in Table 2. In Table 2 it can be seen that albumin concentration was somewhat higher in hemolyzed specimens than that of unhemolyzed specimens. However, as determined by the Student's t test, there were no statisti— cally significant differences between them. Experiment 3 Comparison of the Electrophoretic Patterns of Normal Plasma and of Plasma from Partially Hemolyzed Samples Representative electrophoretic patterns comparing normal plasma and plasma of hemolyzed samples are shown in Figures 17 and 18. As was the case with serum, hemolysis resulted in slurring of the B globulins. There was a tendency of the whole electrophoretic pattern to deviate somewhat 25 4.00 W 3.50 w albumin 3.00 w 2.50?3« /100 2.00 'i U gm 1.50 i T 1.00 application point 0.50 Figure.8.. Tracing of electrophoretic analysis of plasma of Dog 5 with free hemOglobin concentration of 44 mg./100 ml. 4.00 A 3.50 4 ' albumin 3000 ‘F 2.50 .0 m1 2.00%5. ‘7 1.5090" 1.00 4 application point Figure 9. Tracing of electrophoretic analysis of serum.of Dog 15 with free hemoglobin concentration of 60 mg./lOO m1. 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 26 albumin application point Figure 10. Tracing of electrophoretic analysis of serum of Dog 15. A A U 100 m1. a T application point Figure 11. Tracing of electrophoresis of isolated, concentrated hemoglobin of Dog 15. 27 4.00 u 3.50 h albumin 3.09 2. 50 2.00 1.50 1.00 0.50 Figure 12. TraCing of electrophoretic analysis of serum of Dog 12. 4.00 W 3.50 v 3.00 W 2.50 it 2.00 1.50 1.00 w 0-59 T application point 0 A: Figure 13. Tracing of electrophoresis of isolated, concentrated haptogldbin of pooled dog serum. 28 4.00 +1 30 50 ”- albumin 3000 ”- 2.50 w 2.00 1.50 1.00 a 0.50 . application point 0 1 Figure 14. Tracing of electrophoretic analysis of serum of Dog 12. 4.00 w 3050 T 3000 ‘i' 2050 ‘F' 4 2.00 1.50 10 00 ‘F' Hemoglobin 00 50 i" Haptoglobin—hemoglobin application point complex -”- ' ' ' O ' “‘ Figure 15. Tracing of electrophoresis of haptoglobin-hemoglobin complex and free hemoglobin. 29 22 4b 21 4 Serum 20 - Serum from hemolyzed specimens 19 b 18 4' '17 «- 16 u 15 w 14 w 13 t 12 .. _I I 11 A l I 10 I- : 9 w I l e y- Y I 7 - l l 6 - .L .1. 5 4* T' I ' I 4 II- + I l I 3 . L : 2 i “r O T- 1 - ‘P i ‘L ' w. I 1 I: , ‘r 4. 0 , 1.1. IL I 14- I}. TP Albumin 01 a2 81 82 83 Y A/G Figure 16. Comparison of electrophoretic analyses of serum and serum of hemolyzed specimens. Means and ranges of protein concentrations for 24 dogs. 30 Table 2. Comparison of means, ranges and standard deviations of concentrations of serum proteins of normal specimens and partially hemolyzed specimens for 24 dogs Mean Standard Range (gm./100 ml.) Deviation (gm./100 ml.) 1 Total 8 6. 322 0.429 5.50 7.22 Protein HS 8.08 1.289 5.98 12.35 Albumin S 3.85 0.367 3.18 4.99 HS 4.06 0.579 2.97 5.28 Alpha-1 S 0.63 0.182 0.27 1.17 Globulin HS 0.69 0.205 0.19 1.12 Alpha-2 S 0.30: 0.113 0.11 0.71 Globulin HS 0.75 0.354 0.36 1.79 Beta-1 S 0.19 0.066 0.08 0.44 Globulin HS 1.57 1.162 0.53 5.67 Beta-2 0.49 0.270 0.22 1.20 Globulin HS * * Beta-3 S 0.50 0.134 0.28 0.81 Globulin HS 0.58 0.197 0.29 0.95 Gamma S 0.35 0.121 0.14 0.63 Globulin HS 0.43 0.161 0.15 0.84 A/G S 1.67: 0.404 1.13 2.88 Ratio HS 1.10 0.361 0.56 1.91 S - Normal serum HS - Serum from partially hemolyzed specimens - B and 82 fractions were not separately resolved. 1 = Slgnificantly different from 2 (Puao soauounwamo .mlm unawah eHH was «Ha «Ha NHH sea and sea use see sea mes sea mes Ned Hos eoa em as as as en es en as ea Ah.eaz use .Ha eea\.wac nanoseconds no.0 m¢0.0 «0.0 mm0.0 00.0 nN0.0 No.0 mH0.0 H0.0 m00.0 .* 0 unau\.Ha concave cocoa he vouoouuoo “Ham.o .Hae.oc .o>u=o doaudao soaumaoma manoflwounom .eln shaman 51 69H 00 am an ufiWum 60 an oe on 6N 6H uoaaoz «any _ .4 — Aownoawounmnv Amdfiououm canoaomv 8330 802 z no.9 :33? can m.0 n.H APPENDIX C CALCULATION FORMULA LU.) APPENDIX C CALCULATION FORMULA Determination of Clottability of Fibrinogen Isolated dl-d2 + d3 d C = 100 1 where C % clottability d1 = optical density of 0.1 m1. of fibrinogen preparation diluted with 4.0 m1. of 0.005 M citrate solution; read against citrate blank d - optical density of dilution as above with 0.2 ml. of thrombin solution d = citrate blank with 0.2 ml. of thrombin solution. 52 V ITA The author was born in Palauig, Zambales, Philippines, on April 10, 1944. She graduated from Centro Escolar University, Manila, Philippines, with her B.S. in Medical Technology in 1964. After passing the Louisville Civil Service Board Examination in Kentucky, she joined the staff of the clinical chemistry laboratory of Louisville General Hospital in Louisville, Kentucky, in February 1967. In September 1969 she enrolled in a program of Graduate School in Clinical Laboratory Science in.the.Department of Pathology, Michigan State University, with the aid of a full-time job at the clinical .laboratory, Veterinary Clinic, Department of Pathology. 53 M'IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILIII'IIIIIIIs