MICHIGHN STATE UNIVEHS'TY OF AGRICULTURE AND RI'PLIED SUENCE DEPARTMEI'QT CF CHE‘V’HSTRY EAST LANSING, MICHIGAN THE BINDING OF METAL IONS BY PROTEINS IN NORMAL AND ABNORMAL HUMAN BLOOD SERUM By Robert Bastian qu A THESIS Submitted to the School of Advanced.Graduate Studies of Michigan State University of Agriculture and Applied.Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1960 ACKNOWLEDGMENT The author wishes to express appreciation and gratitude to Doctor Hans A. Lillevik for guidance and counsel. Grateful acknowledgment is given to Edward W. Sparrow Hospital, Lansing, Michigan for the use of certain facilities made available dur- ing this investigation. ii To My Wife Jayne iii VITA The author was born June lb, 1928. His secondary education was completed in 19h6 at Cass City High School, Cass City, Michigan. In 1950 he was graduated from Central Michigan University, Mt. Pleasant, Michigan, with the Bachelor of Science Degree. He was admitted to the Graduate School of Michigan State University in September, 1953 and has been in attendance since. The Master of Science Degree was com- pleted in 1955. Employment experience consists of positions as: Medical Tech- nologist from 1950 to 1952 with the Clinical Laboratory at Hurley Hospital, Flint, Michigan; Graduate Teaching Assistant 1953 to 1955 in the Department of Chemistry, Michigan State University; and Tech- nical Director of Laboratories since January 1955 at the Edward w. Sparrow Hospital, Lansing, Michigan. He is a Senior member of the American Chemical Society and a member of the American Association of Clinical Chemists. iv THE BINDING OF‘METAL IONS BY PROTEINS IN NORMAL AND ABNORMAL HUMAN BLOOD SERUM By Robert Bastian qu AN ABSTRACT Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and.Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1960 Approved 3 4/7414 // ”931 31/6 ' Q'Z'g ,f “T \ 1 Robert Bastian Foy AN ABSTRACT Human blood serum contains a variety of proteins and metal ions. The functions of these serum components are varied and numerous. Some of the metals in serum, (e.g. copper, iron, zinc, calcium and magnesium) are considered to be protein bound, at least in part. The determination of serum concentrations of certain metals and proteins is often an aid to diagnosis and treatment in many pathological states. In some cases abnormal changes in the serum content of speci- fic metal-protein complexes have occurred. The study of metal binding by protein fractions of normal and abnormal sera has been accomplished by several means. These include techniques of ultrafiltration, equilibrium and compensation dialysis as well as serum protein electrophoresis after the administration of metal radio-isotopes. Adherence to physiological conditions was not always apparent nor experimentally possible. The present investigation was undertaken to study more suitable methods for the detection and analysis of certain metals among the pro— tein fractions of normal and abnormal human sera. Since some question has been raised regarding the role of copper in multiple sclerosis this disease was included for study as the principal abnormal group. Other pathological states studied included multiple myeloma, liver cirrhosis, glomerulonephritis and severe burns. Paper electrophoresis was utilized for the separation of serum protein fractions. Several organic dyes were employed in the histo- chemical analyses for metals among the various protein fractions. Di- alysis studies aided in the evaluation of these dyes as histochemical Robert Bastian Foy 2 staining reagents. Alizarin red was most useful for detection of calcium on paper electrophoretograms after correction was made for the presence of protein. Calcium was determined to be similarly distributed in all protein fractions of sera from normal subjects and multiple sclerosis patients. However, it was principally located in albumin and the beta and gamma globulins. Iron analyses by use of bathophenanthroline dye showed that this metal was present to some extent in all protein fractions, mainly the alpha and beta globulins. This was true for both normal and abnormal serum proteins. No interference due to the presence of protein with formation of inmrdye complex was apparent. Magnesium and zinc appeared to be distributed among all serum pro— tein fractions. However, the main site of magnesium binding was in albumin, whereas that of zinc was in the beta globulin fraction. A satisfactory reagent was not found for separate identification of these metals on paper electrophoretograms. Albumin and beta globulin contributed most to the binding of cop- per when paper electrophoretograms of normal and multiple sclerosis sera were treated with alizarin blue dye. Protein did not interfer with these results. Sera from other pathological states suggested that calcium, mag- nesium, zinc and copper binding was dependent to some extent upon the amount and kind of abnormal proteins present. vii II. III. IV. INTRODUCTION . HISTORICAL . A. Electrophoresis in Stabilized Media B. Metal Ion—Serum Protein Combinations . TABLE OF CONTENTS C. Multiple Sclerosis . EXPERIMENTAL . Equipment Methods UOUJP DISCUSSION . A. Serum Protein Analysis by Paper Electro- phoresis . B. Serum Metal Distribution on Paper Materials and Reagents . Dialysis Experiments . Electrophoretograms C. Dialysis Experiments . D. Cholesterol Studies SUMMARY BIBLIOGRAPHY . APPENDIX I . viii Page HULKJJ 111 1b 15 19 26 SS Table I. III. IV. VI. VII. VIII. IX. XI. XII. XIII. XIV. XV. XVII. LIST OF TABLES Some Serum Metallo-Enzyme Systems . . . . . . . . Serum Protein Distribution Before and After Deep- Freeze Storage . . . . . . . . . . . . . . . . . . Various Paper Electrophoretic Analyses of Normal Human Serum Proteins . Age and Sex of Normal Subjects and Multiple Sclerosis Patients . . . . . . . . . . . . . . . . . . . . . Effect of Age and Sex upon Serum Protein Distribution Among Normal Subjects and Multiple Sclerosis Patients. Protein Distribution of Normal Sera by Paper Electro- phoresis . Protein Distribution (g./lOO ml.) of Normal Sera . . . Protein Distribution of Multiple Sclerosis Sera by Paper Electrophoresis . . . . . . . . . . . . . . Protein Distribution (g. /lOO ml.) of Multiple Sclerosis Sera . . . . . . . . . . . . Protein Distribution of various Pathological Sera by Paper Electrophoresis . . . . . . . . . . . . . . Protein Distribution (g. /100 ml.) of various Pathological Sera . . . . . . . . . . . Calcium Distribution of Normal Sera by Paper Electro- Electrophoresis . . . . . . . . . . . . . . . . Calcium Distribution of Multiple Sclerosis Sera by Paper Electrophoresis . . . . . . . . . . . . . . . . Individuals in various Ranges of Iron Content Among the Serum Protein Factions . . . . . . . . . . . . Individuals in Various Ranges of Magnesium-Zinc Content Among the Serum Protein Fractions . . . . . . . . . Individuals in Various Ranges of Copper Content Among the Serum Protein Fractions . . . . . . . Metal Distribution Among Serum Proteins of various Pathological Sera From Paper Electrophoretograms . ix Page 27 28 28 29 3O 31 )2 311 3b 35 36 37 38 39 NO Table XVIII. XIX. XX. XXI. XXII. XXIII. XXIV. Page Analytical Results of Pooled Sera Dialysis Study . . . . N1 Protein Distribution of Normal Sera Before and After Dialysis . . . . . . . . . . . . . . . . . . . . . . . . N2 Absorbancies of Calcium-Dye Eluates from Sections of Paper Electrophoretograms Before and After Serum Dialysis Nj Absorbancies of Iron-Dye Eluates from Sections of Paper Electrophoretograms Before and After Serum Dialysis. . . NN Absorbancies of Magnesium-Zinc-Dye Eluates from Sections of Paper Electrophoretograms Before and After Serum Dialysis . . . . . . . . . . . . . . . . . . . . . . . . N5 Absorbancies of Copper-Dye Eluates from Sections of Paper Electrophoretograms Before and After Serum Dialysis. . . N6 Total Serum Cholesterol Levels . . . . . . . . . . . . . N7 LIST OF FIGURES Figure Page 1. Paper Electrophoresis Apparatus . . . . . . . . . . . . . N8 2. Densitometric Tracing of a Protein Electrophoretogram of Serum From a Normal Subject . . . . . . . . . . . . . . N9 3. Densitometric Tracing of a Protein Electrophoretogram of Serum from a Multiple Sclerosis Patient . . . . . . . . 50 N. Densitometric Tracing of a Protein Electrophoretogram of Serum From a Liver Cirrhosis Patient . . . . . . . . . 51 5. Densitometric Tracing of a Protein Electrophoretogram of Serum From a Multiple Myeloma Patient . . . . . . . . . 52 6. Densitometric Tracing of a Protein Electrophoretogram of Serum From a Severely Burned Patient . . . . . . . . . 53 7. A Comparative Distribution of Metals Among Protein Fractions From Normal and Multiple Sclerosis Sera . . . . 5N xi I. INTRODUCTION Human blood serum contains numerous proteins as well as a variety of metal ions. Metals such as copper, iron, magnesium, calcium and zinc are thought to exist in serum largely in the form of metal-pro- tein complexes. Some of these serum metals have known biochemical and physiological function. For example, calcium and magnesium are factors in neuromuscular excitability and calcium also has an important role in blood coagulation. Other instances of metal participation in body functions are those of the metal-protein complexes serving as enzymes. Some examples are listed in Table I. TABLE I SOME SERUM—METALLO ENZYME SYSTEMS1 Enzyme Metal Alkaline phosphatase Mg Choline esterase Mg, Ca verdoperoxidase Fe Aldolase Fe, Cu, Zn Carbonic anhydrase Zn 1References, 38, 89. Still another form of metal combination with protein are the serum metalloproteins, such as transferrin (iron) and ceruloplasmin (copper) which are involved in transport functions. Pathological manifestations in humans are often reflected by the biochemistry of the blood. Decreased concentrations of serum iron, zinc and/or copper have been observed in certain anemic states. In some in- Stances specific metal-protein changes have been demonstrated. For 2 example, in Wilson's disease a reduced concentration of serum protein— bound copper was found (22). Multiple sclerosis, a neurological disorder, has not been adequate— 1y evaluated as to etiology, but there are indications that copper may be significant in the pathology of this disease (8, NS). The extent to which a serum metal ion is bound to protein may be estimated by several techniques. These include ultrafiltration, equi- librium dialysis and compensation dialysis. Isolated human serum pro- tein fractions, obtained by salt or alcohol precipitation methods, have been utilized to secure data regarding their relative metal-ion binding capacity. Recently a selective staining method for the location of serum metal~protein complexes upon paper electrophoretograms was described (58). It is suggested that if histochemical techniques could be made accur~ ate and reliable, one would have a simplified approach to the study of serum metal-protein combinations. The purpose of this study was to investigate methods for the loca- tion and distribution of calcium, magnesium, copper, iron and zinc metal ions among the protein fraction(s) of both normal and patholog- ical human blood sera. Special attention was given to multiple sclerosis sera. Methods of study included protein separation and analysis by paper electrophoresis, quantitative chemical analyses, selective histo- chemical staining, dialysis and photometric techniques. II. HISTORICAL In connection with an investigation concerning the binding of cal— cium, magnesium, iron, copper and zinc to human serum protein fractions it would be desirable to review previous methods and findings. Electro- phoresis develOpment and a background for the work with multiple scle- rosis sera will also be considered. A. Electrophoresis in Stabilized Media The entire field of chromatography and subsequent zone electro- phoresis received its impetus from Tswett's (87) original experiments on the separation of plant pigments by adsorbents contained in a column. Other workers brought out different elaborations of this technique, such as new solvents for elution(77) and frontal analysis (5?). Some investigators applied electrical potentials across the column ends (19,86). Integration of previous researches led to the development of zone electrophoresis. This involves the migration of charged components as individual zones upon a supporting medium and gives the advantage of securing more complete resolution. However, mobilities and isoelectric points are not generally evaluated by this method. One of the first reports regarding electrophoresis on paper was given by Kgnig (5N) in 1937. The first application to protein separa- tions appeared in 1939 when von Klobusitzky and Kgnig (N9) described the resolution of a yellow chromoprotein from snake venom. During the decade that followed paper chromatography and various electrochromato- graphic methods appeared to overshadow paper electrophoresis development. The introduction of moving boundary electrophoresis by Tiselius (93) in 1937 competed with the development and acceptance of the paper work. as. N In the late l9NO's various techniques for paper electrophoresis were independently suggested from several laboratories. The simplest apparatus consisted of a circuit which was completed by dipping the ends of an electrolyte moistened paper into electrode vessels. The problems of supporting the paper to prevent "buffer pooling" and sur~ face evaporation were two disadvantages of this setup. Attempts to surmount these difficulties led to three basic con— structions. One method (21, 55) placed the moistened paper strip be— tween two glass plates which were sealed to minimize evaporation. A second construction, known as the gallows type (26, 32) provided support to the strip by suspending it over a glass rod with the paper ends ex— tended downward and outward so as to dip into the electroLyte solution. A third was the box-type apparatus (10, 18) that kept the paper hori- zontal while giving minimum contact to the migration surface. The use of glass rectangles or raised pointed projections provided support. A stretching frame or the application of weights was also utilized to keep the paper in a taut, level position. For control of temperature and surface evaporation, the glass plate method seemed quite effective. However, it was improved by other setups that provided a cover and the optional use of refrigeration. Non— polar immiscible liquids such as chlorobenzene (18), heptane (10), or carbon tetrachloride (21) were used as sealing agents. McDonald (6N) also employed hydrogen and helium as water vapor saturated gas seal— ants. 2V» n u . .u B. Metal-Ion Serum Protein Combinations 1. Calcium.- Rona and Takahashi (79) suggested in 1911 that cal- cium was, in part, bound to serum proteins. Making use of in—vivo com- pensation dialysis in dogs, Greene and Power (39) estimated protein bound calcium to be 35 to N5 per cent of the total serum calcium. By ultrafiltration methods, Nicholas (68) observed that 36 per cent of the calcium in human serum was non-diffusible. Watchorn and McCance (100) using a similar procedure, found a value of N3 per cent for human sera. Todd (9N) concluded from ultrafiltration experiments that about N7 per cent of normal human serum calcium was diffusible. Using alizarin red as a stain for calcium on electrophoretograms of human serum, LeDuc (58) suggested that albumin and gamma-globulin contributed equally in bind- ing two thirds of the total bound calcium. The remainder was distributed in the other globulin fractions. On the other hand, Prasad and Flink (75) reported that albumin bound 50 to 55 per cent of the non-ultra- filterable calcium. The remainder was mainly bound to beta—globulin, while the alpha and gamma globulins contributed only slightly to calcium binding. Prasad and Flink (7N) had previously determined that N3 to 55 per cent of the total serum calcium.was bound by protein. various equilibrium studies with isolated human and equine serum proteins (25, 58, 65, 101) have indicated that both albumin and the globulins bind calcium reversibly, with a greater tendency by the glob- ulins to form an irreversible combination. The amount of circulating calcium, bound to protein, is known to vary with total serum protein concentration and pH of blood (36, 65, 7N). Therefore, discrepancies in the reported levels of protein bound calcium 6 may, in part, be attributable to variations in experimental conditions. 2. I£22,- In 1925 Fontes and Thivolle (3N) demonstrated conclu- sively, by experimentally induced anemias, the presence and importance of iron in equine serum. Two years later Barkan (N) and Warburg and Krebs (99) observed that a portion of the same serum iron was neither dialyzable nor ultrafilterable. Others (35, N8) also noted the similar behavior of serum iron. In 19N5 Holmberg and Laurell (N1) discovered that a component of human serum formed a pink-colored complex with iron. They considered iron to be albumin bound as did Eisler, et_al.(27). Barkan and Schales (5) previously suggested the combination to be with globulin proteins, since iron was found with this fraction after one- half saturation of serum with ammonium sulfate. In 19N6 Schade and Caroline (81) reported that a beta protein in human serum was capable of combining with iron. Koechlin (53) in 1952 isolated a beta iron-combining protein from human plasma. Cohn (17) indicated that the protein possessed a chemically specific site for interaction with iron and also showed (16) that iron formed a complex with isolated human beta globulin. By using radioactive iron, several groups of workers (12, NN, 67, 98, 10N) have also concluded that the iron binding protein migrates as a beta-globulin. Neale (67) employed the radioactive isotope, in conjunction with paper electrophoresis, for his study of the iron—binding serum protein. He demonstrated that the abnormal proteins of multiple myeloma, nephrosis and hypogammaglobulin- emias in no way affected the normal iron-binding ability of beta-glob- ulins. Working with isolated human plasma protein fractions Surgenor, etgal (88) indicated that beta-globulin bound, not only iron but, c0pper and zinc as well. out a... 4.» .. 7 Le Duc (58) also showed iron to be associated with beta—globulin of histochemically stained paper electrophoretograms of human serum. 3. Magnesium.- Of the various metal ions involved in serum pro- tein combinations, magnesium has been least investigated. Magnesium is an essential body constituent but little is known regarding its meta- bolic role. Intracellular fluids are rich in this element and perhaps too much has been assumed concerning its metabolism because of its similarity to calcium. Chemical methods for its determination have not been completely satisfactory. In 1931 Greene and Power (39) used in vivo compensation dialysis of canine blood to calculate magnesium binding. Of the total magnesium, they found 35 to N5 per cent to be protein bound. A year later watchorn and Mc Cance (100) showed by ultrafiltration techniques, that only 25 per cent of total human serum magnesium was bound; in contrast to N3 per cent for calcium. According to Cantarow and Schepartz (13) about 15 to 30 per cent of human serum magnesium appeared to be protein bound. They also indicated that this value rose in hyperthyroidism and dropped in hypothyroidism. No further significance has been attached to these changes. In 1955, Lillevik, et_al (60), reported magnesium present in all protein fractions as indicated by paper electr0phoresis of human serum and subsequent staining with titan ye110w. At the same time, LeDuc (58) found that purified human serum albumin retained 20 to 25 per cent of its magnesium after equilibrium dialysis. Copeland and.Sunderman (20) determined the magnesium binding for the serum proteins of 17 human subjects and found that albumin bound 0.0128 mEq./gm while the globulins bound only 0.0081 mEq./gm. 8 Starch block electrophoresis was employed by Prasad and co—workers (76) to separate human serum proteins. Magnesium chloride was added to the individual isolated fractions and the amount of magnesium bound to each was then calculated from ultrafiltration data. Albumin bound 60 to 65 per cent of the magnesium in normal subjects. Alpha—2 globu-~ lin bound magnesium in normal subjects, in patients with multiple myeloma, other hyperglobulinemias and hypoproteinemic patients. They found that beta globulin bound magnesium only in cases of hypoproteinemia. N. .éiEE" By conductivity measurements Pauli and Schon (70) in 192N demonstrated an association of equine albumin with zinc. Cohn and coworkers (16) reported in 1950 that the addition of zinc ions to hu« man plasma aided in fractionation of the proteins. By their respective aforementioned techniques Klotz, gt_al (52) and Tanford (90) demonstrated the ability of bovine albumin to combine with zinc ions. Gurd and Goodman (NO) determined that a totally reversible combin- ation existed between isolated human serum albumin and zinc ions. Cohn (17) indicated that several different human plasma proteins under- went reversible reactions with zinc. Surgenor, 32:31 (88) reported that 3 per cent of human plasma beta-globulin was capable of binding divalent cations, among which was zinc. The first precipitate formed upon adding zinc ions to human serum was found by Ressler, EELEE;(78) to contain albumin and gamma globulin. In 1956 valle (95) stated that 35 per cent of the protein bound zinc in human serum existed as a metallo-globulin. The remaining 65 per cent appeared loosely bound to the other protein fractions. Employing electrophoresis on canine blood serum after the adminis- tration of radioactive zinc, Wolff, et a1 (10N) found zinc—65 principally 9 associated with the albumin and alpha globulins. Little radioactivity was observed in the beta and gamma globulin fractions. An inability of albumin to bind zinc appeared to be the principal factor involved in pathological states related to reduced serum zinc levels. They ob- served this effect in patients with pernicious anemia, chronic hemor- rhage, malignant tumors and anemias caused by chronic infection. By means of paper electrophoresis and histochemical staining techniques, LeDuc (58) suggested in 1956 that zinc was present in the gamma globulin fraction of human serum proteins. 5. Cgppg£3- In 1927 Warburg and Krebs (99) showed that the copper of avian and mammalian serum was loosely bound and that its binding was influenced by pH. They felt that the loosely bound c0pper repre- sented total serum c0pper, whereas Abderhalden and Moller (1) pointed out that serum copper was not dialyzable. Boyden and Potter (11) con- cluded that all bovine serum c0pper was protein bound and further sug- gested that more than one form of organically bound copper existed in such sera. In 1936 Eisler, e£;§l (27), observed the migration of copper dur- ing serum protein electrophoresis. They regarded copper as being bound to albumin. However, Holmberg and Laurell (N2) stated in 19N7 that serum copper coprecipitated with the globulins upon one-half saturation with ammonium sulfate. Polarographic analysis by Tanford (90) demonstrated the formation of a complex between bovine serum albumin and divalent copper. In 1955 Klotz and co-workers (52) substantiated this finding through the use of absorption Spectra analysis. However, a similar complex was not appar- ent to them when human serum albumin was studied. 10 Wolff, gt;al (103), found that immediately after administration of radioactive copper, its distribution was essentially the same for all human serum protein fractions after paper electrophoresis. Bearn and Kunkel (6) studied the localization of orally given radioactive copper in human serum protein fractions, after starch block electrophoresis. Serum samples taken immediately after ingestion showed a rapid uptake of copper by albumin in normal subjects, cirrhotic and Wilson's dis- eased patients. Samples taken after longer time intervals exhibited localization of COpper in the alpha-2 globulins for both the normal and the cirrhotic. Patients with Wilson's disease did not show this shift. Holmberg and Laurell (N3) isolated a copper bearing protein (cerulo- plasmin) from human serum in 19N8. They characterized it as an alpha-2 metallo-globulin. The same year Cohn (15) demonstrated that copper formed a blue-green complex with beta-globulin of human serum, the same protein which binds iron. Koechlin (53) observed that this complex appeared to involve one molecule of protein and one or two atoms of COpper. LeDuc (58) found copper to be iso-migratory with gamma globulin upon staining human serum paper electrophoretograms with diethyldithio- carbamate. Lahey, et_al (56) noted a high degree of correlation between human serum copper levels and the amount of alpha-2 and beta-globulins. There was no significant relationship between serum copper levels and other serum.protein fractions. Thompson and Watson (91) and Cummings, 33:31 (22), employed sodium sulfate fractionation of human serum proteins in their studies upon c0pper binding. They obtained an average per cent distribution of serum copper as follows: albumin 8 to 10 per cent, ‘1‘- ll alpha—globulins 10 to 31 per cent, beta—globulin N2 to 55 per cent and gamma-globulin-19 to 25 per cent. C. Multiple Sclerosis Multiple sclerosis (M.S.) is a disease involving widespread patches of demyelinated fibers of the central nervous system. This nerve cell destruction leads to various neurological manifestations, depending on the site of the lesion. Although the early 19th century findings were misinterpreted by Charcot, Scheinker (82) credited him with the first complete and path- ological account of the disease. Fog (33) reported in 1951 that serum protein levels of M.S. patients were normal whereas Saifer, et_§1 (80) as well as volk and co-workers (97) found that in 23 cases, 85 per cent had lowered serum albumin levels and slightly increased alpha-2 globulin. Jones, _e_t_§_1_ (117) in 195N, studied the albumin levels of 130 patients and likewise reported lowered values. On the other hand, Dobin and Switzer (2N) applied serum.protein studies as well as various liver function tests to 58 M.S. patients and found no significant evidence of hepatic dysfunction. Moving boundary electrophoretic analysis of M.S. serum proteins in 195N by Bernsohn and Cochrane (9) revealed low albumin and elevated alphaa2 globulin content. Other globulin components were not signific- antly abnormal. Total protein average values by Kjeldahl analysis were 7.36 and 7.20 gm/IOO m1 of serum respectively for M.S. patients and normal subjects. Most of the electrophoresis patterns of M.S. patients exhibited a "double peak" effect in the alpha-2 region. 12 In 1958, Vlad, et_al (96) published data relating serum protein levels to the state of activity in lesions in the M.S. patient. During a quiescent phase they noted normal protein concentrations. When alpha~2 or beta globulin levels were elevated this predicted the onset of a demyelinizing process whereas when elevated gamma—globulin con» tent was observed the patient was regarded as being in a sclerosing stage. Serum cholesterol and other lipids have been studied in M.S. patients, but, as with the proteins, no conclusive agreement has been reached. Fog (33), Wilmont and Swank (102), Bernsohn and Cochrane (9) and Chiavacci and Sperry (1N) all concluded that blood cholesterols in M.S. patients were normal. However, Dobin and Switzer (2N), Pichler and Reisner (73) and Frisch (37), reported that levels of both total and esterified blood cholesterol were high. A recent report by Persson (71) indicated that in male M.S. patients, blood lipids were higher than normal, whereas only a few female patients had elevated values. He found no correlation between lipid levels and patient age or stage of the disease. Some consideration has been given to the possibility of a trace metal deficiency being involved in the pathogenefis of M.S. A naturally occurring demyelinating disease of young lambs was related to a copper deficiency in the Australian pasture (7). Bennetts and Chapman (8) found in 1937 that copper levels of the blood, milk and liver of the diseased lambs were low. Feeding copper to the lambs arrested the ataxic condition. Prevention was aided by dietaby administration of COpper to the ewes prior to dropping. A similar disease of lambs Occurs in many parts of Great Britain. Abnormal assimilation was 13 thought to be involved, since tissue levels in pregnant ewes and ataxic lambs were low, while normal amounts of pasture copper were found (N5, 8N). Additional dietary c0pper produced similar therapeutic results as in Australian sheep. In 19N8 Mandelbrote and co-workers (62) analyzed the blood and urine of 26 M.S. patients for copper content. They were unable to find conclusive abnormalities. Cumings and associates (22) found that the percent of total cop~ per distributed among the globulins of normal serum was as follows: alpha- 31 per cent, beta - N2 per cent and gamma - 19 per cent. For M.S. patients the distribution shifted to : alpha - 19 per cent, beta - 60 per cent and gamma globulin - 16 per cent. III. EXPERIMENTAL A. Equipment Electrophoresis Apparatus.- The Durrum-hanging strip, inverted v-type cell, including a power supply unit, was obtained from the Spinco Division of Beckman Instruments Company, Palo Alto, California. The entire cell and cover were constructed of lucite and utilized platinum wire electrodes. Schleicher and Schull No. 20N3A filter paper strips (3.0 x 30.6 cm) served as supporting media. Schleicher and Schull filter paper No. N70 (5.1 x 31.8 cm) served as buffer wicks. Figure 1 illustrates the apparatus. Analytrol Scanner.- Dyed paper electrophoretograms were analyzed by means of a Spinco recording densitometer. Solution absorbancy measurements were made with either a Coleman Model-1N or a Beckman Model-DU Spectrophotometer. Oven.- Electrophoresis paper strips were dried in a Despatch convection oven (Minneapolis, Minnesota). Analytical Balance.— Chemicals and dyes were weighed on a Voland semi-automatic balance (New Rochelle, New York). Dialysis Apparatus.- A rotating, multiple specimen dialyzer with Visking dialysis bags was obtained from Oxford Laboratories, San Fran- cisco, California. 15 B. Materials and Reagents Serum - Ten to twenty ml. of blood was collected by venipuncture from each subject.* Following blood clotting at room temperature the serum was pipetted off and placed in sterile glass tubes. Samples were stored in the frozen state at ~50C. Before analysis they were thawed by a 5 to 10 minute immersion in a 37°C. water-bath. Electrophoresis Buffer.- veronal buffer of pH 8.6 and 0.075 molar« ity was prepared by dissolving 5.52 g. diethyl barbituric acid (N.F.- Merck) and 30.8 g. sodium diethyl barbiturate (N.F.-Fisher) in suffic- ient distilled water to make two liters of solution. Protein Stain.— The dye bath was composed of 100 mg. bromphenol blue (Harleco No. 859) dissolved in sufficient absolute methanol (Mal- linckrodt) to yield one liter of solution. Acetic Acid 5 per cent (vzv) Rinse Solution.- This was obtained by diluting 100 ml. of glacial acetic acid (Baker) to two liters with distilled.water. Protein Analysis.— Reagents for the biuret method were prepared as directed by Ferro and Ham (31). Standard protein solution (7.2 g./100 ml.) from human serum.was obtained from Dade Reagents, Inc., Miami, Florida. .Biuret reagent was made by dissolving 3.0 g. crystalline cupric sulfate pentahydrate (C.P.-Mallinckrodt), 12.0 g. sodium potassium *we are grateful to Dr. Gabriel Steiner at the Detroit Multiple Sclerosis Center for providing us with sera from his patients. l6 tartrate (C.P.-Baker) and 2.0 g. potassium iodide (C.P.-Mallinckrodt) in about one liter of distilled water. Six hundred ml. of 10 per cent (w/v) carbonate-free sodium hydroxide was added; the solution was then diluted to 2 liters with distilled water and stored in a polyethylene bottle. Sodium sulfate-22.6 per cent (w/v) was prepared by dissolving 226 g. of the anhydrous chemical (C.P.—Mallinckrodt) in warm distilled water and adjusting the volume to one liter. The solution was kept at 37°C. Cholesterol Analysis.- Total serum cholesterol was estimated with Kiliani's reagent according to the method of Shibata and Hasegawa (85). Stock reagent was made by dissolving 10 g. ferric chloride-hexa~ hydrate (C.P.-Cenco) in 100 ml. glacial acetic acid. Before use, 0.25 ml. was diluted with 25 ml. of concentrated sulfuric acid (C.P.-Baker). Standard cholesterol solution contained 200 mg. recrystallized cholesterol (Matheson-5177) in 100 ml. glacial acetic acid. Calcium Analysis.- Total serum calcium levels were quantitatively determined by the method of Bachra, gt_al (2). Potassium hydroxide (1.25N) was prepared by appropriate dilution of a saturated solution of the chemical (Mallinckrodt). A 0.012M stock solution of disodium etbylene-diamine tetraacetate was obtained by dissolving N.5 g. Eastman No. 635N salt in distilled water and diluting to one liter. Before use 5 ml. of stock was diluted to 100 ml. with water. Cal—Red indicator was composed of 25 mg. 2—hydroxy-l-(2-hydroxy- N-sulfo-l-napthylazo)-3 napthdic acid (Scientific Service Laboratories, Dallas, Texas) dissolved in water to a volume of 25 ml. It was stored 17 in a polyethylene container and kept under refrigeration. Standard calcium solution (10.0 mg/100 ml.) prepared from human serum was se» cured from Dade Reagents, Miami, Florida. Iron Analysis.- Bathophenanthroline reagent (0.001 M) was made by dissolving 332 mg. of N,7 diphenyl~1~10-phenanthroline (G. F. Smith No. 108) in absolute i30propanol (Shell Corporation) and diluting to 1 liter with the same solvent. Thioglycolic acid solution, 80 per cent (v/v) was obtained by dilution of 80 ml. of the chemical (Fisher-No. A319) with 20 m1. dis- tilled water. Sodium acetate, saturated. This reagent was prepared by adding 60 g. sodium acetate, trihydrate (Baker-C.P.) to 100 ml. water. After shaking and allowing to stand overnight the supernatant fluid was col- lected. Hydrochloric acid, 0.2N. Dilution of 20 ml. 1 N hydrochloric acid with 80 ml. absolute isopropanol provided this reagent. Trichloroacetic acid, 30 per cent (w/v) was obtained by adding sufficient distilled water to 30 g. of the Mallinckrodt A.R. chemical to yield 100 ml. of solution. Iron standard (2 ug./m1.) was prepared by dilution of 10 ml. of stock iron standard (Harleco No. 2572) to 500 ml. with distilled water. Total Serum Magnesium Analysis.— Trichloracetic acid, 10 per cent (W/y) was prepared by adding an amount of water to 10 g. of the Mallin- ckrodt (A.R.) chemical necessary to provide 100 ml. of solution. Dilute magnesium standard consisted of 1 ml. of 0.1 N stock mag- neSium standard (Harleco No. 3633) and 10 ml. of 0.02 M calcium chloride 18 diluted to 100 ml. with 10 per cent trichloracetic acid. Titan yellow 0.05 per cent (w/v) was made by dissolving 0.05 g. (Harleco No. 1835) of the solid in sufficient distilled.water to yeild 100 ml. of solution. Sodium hydroxide, 2.5 N, was obtained by appropriate dilution and titration of a saturated solution. Calcium Stain.- The stock solution for calcium staining of paper electrophoresis strips contained 1.0 g. Alizarin redeS (Fisher-Na—Nl8) per liter of aqueous solution. The working stain was made by diluting 20 ml. stock with distilled water, adding 0.5 ml. 1 N hydrochloric acid and finally diluting to 1 liter with distilled water. For elution 0.N N sodium hydroxide was prepared from a carbonate— free saturated solution. Magnesium-Zinc Stain.- A stock solution (0.5 per cent w/v) was made by dissolving 500 mg. l-(l-hydroxy, 2-napthylazo)-5-nitro-2- napthol,—N-su1fonic acid, sodium salt (Eastman Kodak No. L636l) in suf— ficient distilled water to give one liter. Elutions were accomplished with 0.N N sodium hydroxide prepared as described in the calcium procedure above. Copper Stain.- Alizarin Blue S (Harleco No. 112) served as the histochemical reagent for c0pper detection. A working solution was Prepared fresh from 0.5 g. of dye dissolved in 500 ml. distilled water. One ml. of l N sodium hydroxide was added and the solution made up to 1 liter with water. Acid for elution was obtained.by dilution of N3 m1. concentrated Hydrochloric acid to one liter with distilled water. 19 Miscellaneous Stains.- A saturated solution of dithizone (East- mand Kodak No. 3092) in 95 per cent ethanol was used during preliminary studies for the location of copper and zinc. Pinkish-stained areas indicated the presence of zinc while pale brown appeared where copper was located. Quinalizarin solution (Eastman Kodak No. 2787), 0.05 per cent (w/v) in 0.1 N sodium hydroxide was employed in the first attempts to locate magnesium. Dark blue areas on a light blue background appeared where magnesium was detected. A 0.1 per cent (w/v) Dithio-oxamide (Eastman Kodak No. N39N) solu- tion in 70 per cent (v/v) ethanol was utilized in the study of paper staining for copper. A pale greenish-black color on a white background indicated the location of copper. C. Methods Paper Electrophoresis and Staining.- Paper wicks were put into place and the strip holder, loaded with 8 paper strips, was assembled on the cell. Nine hundred ml. of veronal buffer were added to the cell. Another 100 ml. of buffer was used to wet the paper strips and.wicks. The ends of each paper strip were gently pressed against the wick with a glass rod. The cell was covered and sealed around the edges with masking tape. Fifteen minutes was allowed for equilibration and then the buffer was brought to the same level in each electrode compartment by tilting the entire assembly to one end for 30 seconds. Six micro- liters of serum.were placed on each paper strip with the aid of a wire capillary applicator. Access to the strips was gained through the top of the cell cover by removing, one at a time, individual masking tape 20 seals. Immediately upon completing sample application, current was applied. The electrophoretic separation proceeded for 16 hours under a constant current of 2.5 milliamperes and a difference of potential ranging from 60 to 70 volts. Runs were carried out at room temperature, which averaged from 20 to 22°C. At the end of a run the filter paper strips were immediately oven dried at 120 to 130°C. for 30 minutes. After numbering, strips 3 and 6 were transferred to a staining rack and immersed in absolute methanol for 30 minutes to eliminate buffer salts. A 30 minute immersion in methanolic bromphenol blue followed by three 6-minute rinses in 5 per cent (v/v) acetic acid com- pleted the staining process for location of proteins. The strips were briefly blotted and then dried at 120 to 130°C. for 15 minutes. The dried strips were exposed for 15 minutes to vapors of concen— trated ammonium hydroxide. A stable, bluish-purple color developed on a white background for the areas containing protein. Each strip was analyzed for zone densities by means of the Analytrol instrument (Re- cording Densitometer). Densitometric curves and integration of the areas under each were simultaneously obtained. Percentage values for each protein fraction were secured by computation of the integration data. Results of duplicate analyses were combined and averaged for each protein fraction. The values for serum protein distribution are re- corded in Tables VI, VIII, and X. Total Serum Protein Determination.- The Ferro and Ham (31) biuret technique for total serum protein was utilized. The procedure is based on the intensity of color developed.between peptide linkages and alka— line COpper sulfate. The determination involved photometric comparison 21 of the protein content of the unknown serum with known serum, which was previously standarized by Kjeldahl analysis. Dilutions of unknown and standard sera were prepared by mixing 0.5 ml. of each with 9.5 m1. of 0.85 per cent (w/v) sodium chloride. Eight ml. of biuret reagent was added to the following: First, 2 ml. sodium chloride solution (blank); second, 2 ml. of unknOWn serum dilu— tion; and third, 2 ml. of standard serum dilution. All tubes were mixed by several inversions. After 30 minutes the absorbancies at 5N0 mu of the unknown and standard were obtained. The equation for the calculation of total serum protein content is given in Appendix I. The results of serum protein analyses are reported in Tables VII, IX, XI, and XVIII. Serum Cholesterol Determinations.- The method of Shibata and Hasegawa (85) was followed for the estimation of total serum cholesterol. Duplicate 0.1 ml. serum samples were mixed with 8 ml. glacial acetic acid and centrifuged to obtain the protein-free supernatant fluid. Four ml. of clear supernatant were then mixed with 2.0 ml. Kiliani's working solution and allowed to stand for 15 minutes. A Cholesterol solution standardized by the method of Schoenheimer and Sperry (83) was treated in a similar manner. The absorbancies of unknown and standard at 550 mu were obtained. A brown-violet color developed and its in- tensity followed Beer's law up to concentrations of N00 mg./100 m1. An equation for the calculation of total cholesterol appears in Appendix I. The results of the analyses are recorded in Table XXIV. Tetal Serum Calcium Analysis.- The method of Bachra, et a1 (2) was followed for the determination of total serum calcium. Duplicate 0.5 ml. 22 senunsamples were placed in separate 10 ml. Erlenmeyer flasks. Two chops of Cal-Red indicator and 0.5 ml. of 1.25 N potassium hydroxide were added immediately before titration. Samples were titrated with CL0006 M disodium ethylene-diamine tetra acetate from a wine-red color to a blue end-point. Standard serum samples were determined in like manner. The method of calculation appears in Appendix I. The results of the analyses are given in Tables XII, XIII, XVII—A and XVIII. Total Serum Iron Determination.- Iron determinations on serum were performed according to the method of Peters Efaél.(72)° Two ml. samples of unknown and iron standard (2 ug./ml.) were pipetted into separate 15 ml. centrifuge tubes. A blank containing 2 m1. of water was also prepared. Three m1. of 0.2 N hydrochloric acid and 1 drop of 80 per cent thioglycolic acid were added to each tube. The contents were mixed and the tubes were allowed to stand at room temperature for 30 minutes. One ml. of 30 per cent trichloracetic acid was added to each tube and the contents mixed. After standing at room.temperature for fifteen minutes, the tubes were centrifuged at 2,000 r.p.m. for 10 minutes. Four ml. aliquots of each supernatant were transferred to appro— priately labeled cuvettes. Five-tenths m1. of saturated sodium acetate was added and the contents mixedJ After adding 2 ml. of 0.001 M batho— phenanthroline and.mixing the contents of each tube, they were allowed to develop at room temperature for 10 minutes. The absorbancies were (flouained at 535 mu. An example of the calculation of results from these data may be found in Appendix I. The results are tabulated in Table XVIII. 23 Total Serum Magnesium Analysis.- A modification of the Natelson method (66) for serum magnesium was used. Two ml. of serum,2 ml. distilled water and N ml. 10 per cent (w/v) trichloracetic acid were mixed in a 15 ml. centrifuge tube and allowed to stand at room temperature for five minutes. Four m1. of the protein- free supernatant, obtained by centrifugation at 1500 r.p.m. for five minutes, was transferred to a cuvette. A standard was prepared with 2 m1. of dilute magnesium standard solution (2 meq/L) and 2 ml. of water. The blank consisted of 2 ml. water and 2 ml. 10 per cent tri— chloracetic acid. One ml. of 0.05 per cent (w/v) Titan Yellow was added to each tube. At 30 second intervals, 2 ml. of 2.5 N sodium hydroxide was added to each tube and the contents mixed. The absorbancies at 5N0 ml were obtained exactly five minutes after the addition of sodium hydroxide. A sample calculation appears in Appendix I. The analytical results are entered in Table XVIII. Paper Strip Analysis for Metal Ions.- In order to relate metal- ion distribution to the various protein fractions, a method was devised whereby the paper strips were sectioned into areas corresponding to the zone where a given protein fraction was previously identified. This was accomplished by aligning the stained or unstained strip for metal analysis with one of the protein-stained strips. Vertical sections of the strip for metal analysis were then taken; the width in centimeters and order taken for each was recorded. At the same time a section of paper was taken from an area of strip where no serum had been applied and utilized as a control (blank). From an eluate of the latter, a factor was obtained to correct for the amount of dye contributed by the 2N paper alone. Making use of the corrected absorbancies, relative per- centages of dye distribution for each protein—metal area were calculated. The method of percentage computation is described in Appendix I. w.“ The histochemical use of Alizarin Red-S was developed by Dahl (23) in 1952. Strip No. 2 of each run was used for the location of calcium. The strips were pre-rinsed in absolute methanol for thirty minutes and stained in fresh-0.02 per cent (w/v) Alizarin Red-S for ten minutes. Excess dye was removed by rinsing in distilled water; the strips were blotted and dried at room temperature. Red-orange areas on a pale pinkish-white background appeared in those zones bearing calcium. Strip segments corresponding to protein zones were eluted with 5 ml. of 0.N N sodium hydroxide for 20 minutes. After removal of the paper segments the absorbency of each eluate was measured at 550 mil relative to a water blank. The results may be seen in Tables XII, XIII and XVII-A. 13:92.- To locate iron, strip No. N of each run was utilized. In this case the staining and elution was carried out in one step. Four ml. 0.2 N hydrochloric acid and one drop 80 per cent (v/v) thioglycolic acid were added to each strip-segment corresponding to protein zones. The contents of the tubes were mixed and allowed to stand at room tem- perature for sixty minutes after which 0.5 m1. of saturated sodium acetate and 1 m1. bathophenanthroline reagent were added. The tubes were mixed and again allowed to stand ten minutes. The paper sections were removed and the absorbancy at 535 mu of the red ferrous-complex was recorded. A distilled water blank was used. These results are 25 recorded in Table XIV and XVII-B. Magnesium and Zinc.- This method was developed to utilize the colorimetric reagent presented by Liddell and Williams (59). Strip number 7 was used for the location of both magnesium and zinc. Each strip was pre-rinsed in absolute methanol for 30 minutes and then im— mersed in fresh 0.01 per cent (w/v) dye solution for ten minutes. The excess dye was drained off and the paper strip blotted and dried. A pink-red stain on a white background appeared where magnesium and/or zinc was present. Elution of stained segments was accomplished by immersion in 5 ml. of 0.N N sodium hydroxide for 20 minutes. The paper sections were removed and.the absorbanqy at 515 muuwas obtained. These findings are tabulated in Tables XV and XVII-C. Cppp3£.- The dye used by Markowitz, Eiafli (63) was employed for copper detection on paper electrophoretograms. Paper strip No. 5 was utilized in the location of copper. Im- mersion of the strips in methanol for ten minutes preceded the staining and then they were submerged in.A1izarin Blue-S for ten minutes. Ex~ cess dye was removed by rinsing in distilled water. After blotting, the strips were dried at 90°C. for ten minutes. Dark blue areas on a light blue background appeared only where copper was most concentrated. .Strilo segments were each treated with 5 ml. of 0.5 N hydrochloric acid and allowed to stand at room temperature for twenty minutes. After the Paper? segments were removed the absorbancy at 500 mp of each eluate was Obtained. Hydrochloric acid solution (0.5 N) was used as a reagent blank; The results of these analyses are entered in Tables XVI and XVII-D. 26 D. Dialysis Experiments Pooled Serum Study.- Thirty-four ml. of pooled normal human sera were divided in two equal portions. One portion (17 ml.) was dialyzed against four liters of 0.075 M veronal buffer at pH 8.6 and with sodium chloride added to make the total ionic strength 0.15 M. The other por- tion was stored in the refrigerator until the dialysis was completed. Dialysis continued at O to 5°C. for 52 hours, with one change of fresh buffer after 26 hours. Quantitative analyses by the aforementioned methods were performed upon both the non-dialyzed and dialyzed samples for total protein, calcium, iron and magnesium. Paper electrophoretic analyses and histochemical staining of each portion gave the distribu- tion of protein, calcium, iron, magnesium-zinc and copper. These re- sults appear in Tables XVIII, XIX, XX, XXI, XXII, and XXIII. Individual Control Serum Study.- One ml. of serum from each of six control subjects was placed in separate dialysis membranes. These were dialyzed against four liters of 0.075 M veronal buffer at pH 8.6 with sodium chloride added to make a total ionic strength of 0.15 M. Dialysis was allowed to proceed for 18 hours at 0 to 5°C. Paper elec- trqphoretic analysis of each dialyzed serum was performed. Individual paper electrophoresis strips were stained for protein, calcium, iron, magresium-zinc and.copper. The resulting data are recorded in Tables XIX, XX, XXI, XXII, and XXIII. 2? .Hmbou mo pcoouod mm co>wm cam memococaocpoofio coama an cosmetopoQN .mcucoe m on m tom .oowH muses um couopma H.4H o.ma m.m o.a a.» a.c o.m m.m s.mc o.mc coo: m.mH m.oH o.a S.m a.c c.a m.w m.m m.wc m.ma Om c.ma m.ma o.a m.m c.c a.c m.m m.m m.mc m.cc as o.ca c.aa o.oa a.a m.c a.m o.S n.n m.mc c.0a m m.ca o.ca n.0a N.0a a.s S.m S.m c.m o.nc m.ac a m.qa a.na m.oa ®.m c.a s.c s.a m.m m.mc m.~c m a.aa N.NH o.oa m.m m.a m.a 0.4 a.a a.mc o.ac m popwd ocowom copw< ouomom eoum< ouomom touwd oeomom couwd .ouomom assoc seem N-ccea< H-ncaa< nncaascoao Ncaesnaa coonhsm HmmamOHm mNmmmm:dme mmhm< Qz< mmommm ZOHHDmHmHmHQ ZHMHOmm EDmMW HH mqmdw TABLE III VARIOUS PAPER ELECTROPHORETIC ANALYSES OF NORMAL HUMAN SERUM PROTEINSl W Albumin wGlobulins Alpha-l Alpha-2 Beta Gamma (28) 63.7 3.7 10.0 10.5 12.0 (29) 67.0 3.3 6.9 9.1 13.5 (N6) 68.9 2.7 7.3 9.0 12.0 (58) . 60.2 3.7 8.3 12.8 15.0 This work 66.N 3.N 6.N 9.6 1N.2 1Given as per cent of total protein (mean value). TABLE IV AGE AND SEX OF NORMAL.SUBJECTS AND MULTIPLE SCLEROSIS PATIENTS Subjects Patients Age Sex Age Sex 1 38 Female A N7 Female 2 19 Female B 51 Female 3 30 Female C 5N Female N N8 Female D 36 Female 5 20 Female E N3 Female 6 N1 Female F 36 Female 7 39 Female G 35 Female 8 37 Female H 33 Female 9 N3 Female I 3N Female 10 21 Female J N2 Female 11 19 Female K 37 Female 12 23 Female L 29 Female l3 19 Female M NN Female 1N 20 Female N 50 Female 15 21 Female 0 N5 Female 16 18 Female P 33 Male 17 20 Female 0 38 Male 18 28 Male R N7 Male 19 32 Male 3 3N Male 20 32 Male T 33 Male 21 26 Male U 36 Male 22 29 Male v -- Male 23 2N Male 2N 27 Male 25 21 Male 26 25 Male 29 TABLE V EFFECT OF AGE AND SEX UPON SERUM PROTEIN DISTRIBUTION AMONG NORMAL SUBJECTS AND MULTIPLE SCLEROSIS PATIENTS1 Group Albumin Globulins Alpha-l Alpha-2 Beta Gamma Normals Males 66.N 3.N 6.1 10.2 13.9 Females 66.N 3.N 6.6 9.2 1N.N Ages 18-21 ' 66.0 3.6 6.9 9.N 1b.1 Ages 23-30 65.11 3.6 6.5 9.9 111.5 Ages 32-N8 67.7 3.0 5.8 9.N 1N.1 M. S. Males 66.6 2.9 7.6 9.0 13.8 M. S. Females 68.9 2.8 6.3 9.1 12.9 NEiven as Percent of Total Protein. TABLE VI PROTEIN DISTRIBUTION OF NORMAL SERA BY PAPER ELECTROPHORESISl 3O Subject Albumin Globulins ‘_ Alpha-1 Alpha-2 Beta Gamma 1 65.8 3.3 5.9 8.0 17.0 2 70.b 3.6 6.1 9.7 10.2 3 69.0 3.1 7.5 8.2 12.2 N 6N.7 2.8 6.5 11.1 1N.9 5 67.9 2.8 6.7 8.9 13.7 6 69.2 3.1 5.3 6.7 15.7 7 67.8 2.6 5.N 10.2 1N.0 8 70.6 3.3 5.N 9.1 11.6 9 65.N 3.2 7.1 9.8 1N.5 10 61.0 3.N 7.5 9.6 18.5 11 63.2 3.5 5.8 9.7 17.8 12 65.7 N.1 7.2 8.5 1N.5 13 67.0 N.0 6.N 8.9 13.7 lb 68.N 3.3 7.3 8.1 12.9 15 65.1 3.1 7.7 10.9 13.2 16 65.2 3.8 7.3 9.9 13.7 17 66.2 3.5 6.7 8.3 15.3 18 63.N 5.0 6.5 9.9 15.3 19 65.5 3.8 6.2 10.6 13.9 20 72.9 2.3 b.6 9.N 10.8 21 65.6 2.9 7.2 10.2 1h.1 22 66.7 2.9 6.7 9.1 1N.6 23 66.3 3.7 5.6 12.N 12.0 2N 65.5 3.8 6.2 10.9 13.6 25 65.5 N.7 7.0 9.9 12.9 26 65.2 2.5 5.1 9.3 17.8 Range 61 O- 2.3— N.6- 6.7- 10.2- 72 9 N.0 7.8 12.N 17.8 r 66.N 3.N 6.N 9.6 1N.2 g 2.N 0.6 0.9 1.2 2.0 XiS 6N.0- 2.8- 5.5— 8.N- 12.2- 68.8 N.0 7.3 10.8 16.2 HSiven as per cent of total protein. 31 TABLE VII PROTEIN DISTRIBUTION (g./100 ml.) OF NORMAL SERAl Subject Total2 Albumin Globulins Alpha-l Alpha-2 Beta Gamma 1 6.9 N.5N 0.23 0.N1 0.55 1.17 2 7.0 N.93 0.25 0.N3 0.68 0.72 3 7.3 N.75 0.26 0.57 0.68 1.0N N 7.1 N.60 0.20 0.N6 0.79 1.06 5 7.0 N.75 0.20 0.N7 0.62 0.96 6 7.2 N.9l 0.22 0.38 0.N8 1.13 7. 6.9 N.67 0.18 0.37 0.70 0.97 8 6.8 N.81 0.22 0.37 0.62 0.79 9 7.1 N.6N 0.23 0.50 0.70 1.03 10 7.N N.51 0.25 0.55 0.71 1.37 11 7.0 N.N3 0.2N 0.Nl 0.68 1.25 12 6.8 N.N8 0.28 0.N9 0.58 0.99 13 7.0 N.69 0.28 0.N5 0.62 0.96 .lN 7.8 5.33 0.26 0.57 0.63 1.01 15 7.2 N.69 0.22 0.55 0.79 0.95 16 7.2 N.70 0.27 0.53 0.71 0.99 17 7.N N.90 0.26 0.50 0.61 1.13 18 7.1 N.51 0.36 0.N6 0.70 1.09 19 8.1 5.30 0.31 0.50 0.86 1.13 20 8.6 6.26 0.20 0.N0 0.80 0.93 21 7.9 5.18 0.23 0.57 0.81 1.11 22 7.0 N.67 0.20 0.N7 0.6N 1.02 23 7.N N.9l 0.27 0.N1 0.92 0.89 2N 8.0 5.23 0.30 0.50 0.87 1.09 25 7.6 N.97 0.36 0.53 0.75 0.98 26 8.1 5.28 0.20 0.N1 0.75 l.NN Range 6 8- N.N3- 0.18- 0.37- 0.N8- 0.72- 8 6 6.26 0.36 0.57 0.92 l.NN X 7.3 N.8N 0.25 0.N7 0.70 1.0N §, 0.5 0.N3 0.05 0.07 0.11 0.16 XIS 6.8- N.Nl- 0.20- 0.N0- 0.59— 0.88- 7.8 5.27 0.30 0.5N 0.81 1.20 1Calculated from total protein analysis (column 2 above) and electro- :phoresis data (Table VI). ZDeterminedby biuret analysis (31). 32 TABLE VIII PROTEIN DISTRIBUTION OF MULTIPLE SCLEROSIS SERA BY PAPER ELECTROPHORESIS1 Globulins Albumin .Patient Gamma Beta Alpha-2 Alpha-l S7 oooooooooooooooooooooo 1N281258N2N23101935677 7unv7L0,O,O/O/O.JnualnunuRoRuRSC/Qullnv0/7. 111.111 11.111 O/OSSIQOO221UIU25388299226 661Mh5/O/Ol478 7.5958558 9.N/.196 ”(HD.)00n71lnonurDRomoo/IJRonoilLuvtAuRoRunu 23212321233332222h2322 IHIU285785147280/38101U2188 25.18 LLoo.0.2whw1./7.9H 1.8.27.1“1705 7676776866666767766566 ABCDEFGHIJKLMNOPQRSTUV S7 1- 80.5 Range _ LuAqunu 3206 1 ll O.lO../l 9.2/0.1 nwnvnwnw 7192:20/ 9817 2023 VAoDVA NSiven as per cent of total protein. TABLE IX PROTEIN DISTRIBUTION (g./100 ml.) 0F MULTIPLE SCLEROSIS SERA1 J} Patient Total‘2 Albumin Globulins Alpha-1 Alpha-2 Beta Gamma A 7.0 5.07 0.19 0.N2 0.53 0.79 B 7.6 N.96 0.27 0.50 0.78 1.09 C 7.3 5.35 0.17 0.33 0.55 0.91 D 8.1 5.57 0.15 0.36 0.53 l.N8 E 6.6 N.85 0.19 0.36 0.N6 0.7N G 7.3 5.0N 0.20 0.NN 0.N9 1.15 H 7.0 5.6N 0.07 0.29 0.N3 0.57 I 8.5 5.30 0.21 0.61 1.15 1.21 J 7.5 N.85 0.28 0.63 0.78 0.95 K 7.6 N.81 0.23 0.56 0.89 1.12 L 6.8 N.62 0.27 0.35 0.7N 0.83 M 7.2 N.53 0.2N 0. 8 0.78 0.97 N 6.8 N.8N 0.19 0.36 0.59 0.7N 0 7.6 5.23 0.21 0.67 0.67 0.82 P 7.3 5.26 0.15 0.N2 0.6N 0.82 O 7.5 5.77 0.18 0.39 0.NN 0.73 R 7.0 N.29 0.33 0.62 0.8N 0.91 s 8.6 5.27 0.22 0.85 0.96 1.30 T 8.6 N.92 0.33 1.05 0.89 l.N3 U 7.6 N.63 0.21 0.70 0.75 1.32 v 8.5 5.68 0.17 0.56 0.66 1.51 Range 6.6- N.29- 0.07- 0.29— 0.N3— 0.57- 8.6 5.77 0.33 1.05 1.15 1.51 ‘x 7.5 5.09 0.22 0.52 0.68 1.00 s 0.6 0.35 0.06 0.19 0.18 0.27 'th 6.9- N.7N- 0.16— 0.33- 0.50- 0.73- 8.1 5.NN 0.28 0.71 0.86 1.27 1Calculated from total protein analysis (column 2 above) and electro- phoresis data (Table VIII). 2Determined by biuret analysis (31). TABLE X PROTEIN DISTRIBUTION OF VARIOUS PATHOLOGICAL SERA BY PAPER ELECTROPHORESIS1 3N fiflEPatient Albumin Globulins Alpha-1 Alpha-2 Beta Gamma 1. Multiple myeloma(M.M.) 29.2 1.8 3.2 N.9 60.9 2. Multiple myeloma(M.M.) l9.N 3.0 5.0 N.6 68.0 3. Multiple myeloma(M.M.) N.N 1.7 2.0 3.N 88.5 N. Liver cirrhosis(L.C.) 52.5 N.3 5.0 8.5 29.7 5. Severe burns(S.B.) N9.2 9.6 17.0 11.N 12.8 6. Glomerulonephritis(G.N.) 61.6 N.3 11.7 8.N lN.0 7. Mean Control Value(M.C.v.) 66.N 3.N 6.N 9.6 lN.2 1Given as per cent of total protein. TABLE XI PROTEIN DISTRIBUTION (g./100 m1.) OF VARIOUS PATHOLOGICAL SERAl Patient Total2 Albumin Globulins Alphail Alpha-2 Beta Gamma 1. M.M. 9.7 2.83 0.17 0.31 0.N8 5.91 2. M.M. 8.8 1.70 0.26 0.NN 0.N0 6.00 3. M.M. 17.8 0.79 0.30 0.36 0.60 15.76 N. L.C. 6.0 3.15 0.26 0.30 0.50 1.79 5. G.N. 5.1 3.1N 0.22 0.60 0.N3 0.71 6. M.C.V. 7.3 N.8N 0.25 0.N7 0.70 1.0N 1Calculated from total protein analysis (column 2 above) and electro— ;phoresis data (Table X). 2Determinedby biuret analysis (31). TABLE XII CALCIUM DISTRIBUTION OF‘NORMAL SERA BY PAPER ELECTROPHORESIS “A: m -1— __ _' 35 Subject Total1 Albuminz Globulins2 ‘ Alpha-l AIpha-2fi Beta Gamma 1 9.N 52.N 7.9 9.6 11.N 18.7 2 10.3 58.6 6.5 13.8 11.7 9.N 3 9.9 56.N 5.9 12.7 10.5 1N.5 N 10.1 61.5 5.8 10.1 11.0 11.8 5 9.5 N9.2 7.N 10.8 15.2 17.6 6 10.2 50.6 8.5 8.9 1N.2 17.7 7 9.0 50.9 7.N 9.2 13.7 18.9 8 9.N 5N.7 8.0 9.6 13.0 1N.8 9 9.N N5.2 9.8 10.2 11.6 23.2 10 9.9 50.8 7.7 12.1 12.9 16.5 11 10.1 50.7 7.8 10.0 10.0 21.5 12 9.6 N8.7 7.2 11.8 13.5 18.8 13 9.9 60.3 9.7 13.8 11.1 5.1 1N 10.9 N9.7 11.3 11.8 1N.6 12.6 15 9.8 N7.9 10.7 1N.6 1N.6 12.2 16 10.3 N5.7 15.3 15.8 15.8 7.N 17 9.7 NS.N 9.N 12.2 17.N 15.6 18 9.8 50.1 10.9 1N.6 13. 7 10.7 19 10.9 N5.5 1N.O 12.5 IN. 9 13.1 20 10.0 52.0 9.0 13.5 IN. 6 10.9 21 10.N N5. 6 11.3 17.9 1N.6 10.6 22 9.6 N2.1 13.N 16.2 16.9 11.N 23 10.0 N5. 7 13.3 12.3 17.5 11.2 2N 9.7 N8. 5 1N.6 13.6 15.3 8.0 25 10.1 N6.1 13.8 13.8 1N.5 11.8 26 9.5 No. N 13.2 13.2 21.7 11.1 Range 0- N0.N- 5 8- 9.2- 10.0- 5.1- 10 9 61.5 15 3 17.9 21.7 23.2 I 9.9 N9.8 10.0 12.5 1N.0 13.7 ~15. 0.5 5.1 2.8 2.2 2. 5 N.N th 9.N- NN.7- 7.2- 10.3- 11. 5- 9.3- 10.N 5N.9 12.8 1N.7 16. 5 18.1 1Determined by the chelatometric method(2); given in mg./100 ml. serum. RSiven as per cent of total calcium on paper electrophoretogram. TABLE XIII CALCIUM DISTRIBUTION OF MULTIPLE SCLEROSIS SERA BY PAPER ELECTROPHORESIS Patient Totall Albuminz Globulins2 Alpha-1 Alpha—2 Beta Gamma A 10.2 51.0 8.8 11.3 12.2 16.6 B 11.7 5N.2 8.1 10.0 11.2 16.6 C 9.3 50.5 9.5 7.9 13.9 18.3 D 8.3 N6.5 9.7 9.9 11.6 22.2 E 10.6 NN.N 8.N 10.2 16.2 20.2 F 11.8 N6.7 7.8 15.1 13.6 16.8 0 6.N N7.3 9.5 9.8 l3.N 19.9 H 6.7 55.6 5.N 12.0 13.0 1N.0 I -- 37.0 10.0 1N.9 18.1 20.0 J -- 37.2 10.1 17.6 18.2 16.9 K 10.7 NN.2 9.3 11.0 19.1 16.N L 8.0 N6.8 10.0 13.2 13.6 16.3 M 8.0 53.5 10.1 12.0 l3.N 11.1 N 9.7 59.2 8.N 9.8 11.1 11.5 0 -- 5N.3 7.5 13.6 12.0 12.6 P 9.9 57.1 7.5 9.6 13.2 12.5 0 6.7 50.7 9.7 2.0 10.9 16.7 R 8.8 53.5 11.0 10.7 12.N 12.0 S 12.6 N2.2 10.8 1N.9 15.1 17.1 T 11.2 N6.8 10.0 13.1 13.0 17.1 U -- N8.7 8.5 1N.3 12.9 15.7 v 11.3 N3.0 8.5 17.0 12.1 19.N Range 6.N- 37.2- 5.N- 7.9- 10.9- 11.1- 12.6 59.2 11.0 17.6 19.1 22.2 '1? 9.6 N8.7 9.0 12.3 13.6 16.3 §_ 1.8 5.N 1.3 2.5 2.3 3.0 .x:s 7.8- N3.3- 7.7- 9.8- 11.3- 13.3— 11.N 5N.1 10.3 1N.8 15.9 19.3 1Determined by the chelatometric method (2); given in mg/100 ml. serum. Rfiiven as per cent of total calcium on paper electrOphoetogram. TABLE XIV INDIVIDUALS IN VARIOUS RANGES OF IRON CONTENT AMONG THE SERUM A. For Sera of 26 Normal Subjects2 PROTEIN FRACTIONSl Range3 Albumin Globulins Alpha—l Alpha—2 Beta Gamma O - 10 20 2 3 7 2 ll - 20 3 3 S 8 2 21 - 30 3 3 7 6 1 31 - N0 0 7 7 3 0 N1 - 50 0 5 1 1 0 51 —_60 0 N 1 0 1 Over 60 O 2 2 l 0 Mean % 6.5 37.5 31.0 20.0 5.0 B. For Sera of 22 Multiple Sclerosis Patients2 0 — 10 16 N 6 5 22 11 - 20 3 3 6 5 0 21 - 30 2 2 2 N 0 31 - N0 0 3 2 2 0 N1 - 50 0 5 2 2 0 51 - 60 0 N 1 1 0 Over 60 l l 3 3 0 Mean % 8.0 39.0 28.0 25.0 0.0 le.g., 20 out of 26 subjects showed an iron content for their serum albumin fraction to be in the 0 - 10% range. aSiven as the number of individuals in each range. 3Range is given as per cent of total iron on the paper electrophor- etogram. TABLE XV INDIVIDUALS IN VARIOUS RANGES 0F MAGNESIUM—ZINC CONTENT AMONG THE SERUM PROTEIN FRACTIONSl A. For Sera of 26 Normal Subjectsz Range3 Albumin Globulins Alpha-l Alpha-2 Beta Gamma 0 - 10 7 l 0 1 2 11 - 2O 8 17 IN 9 ll 21 - 3O 10 6 11 15 8 31-N0 1 2 1 1 N N1 - 50 0 0 0 0 1 Over 50 0 0 0 0 0 Mean % 18.0 18.0 21.0 21.0 22.0 B. For Sera of 22 Multiple Sclerosis Patients2 0 - 10 N O 0 0 0 ll - 20 9 IN 10 3 6 21 - 30 N 3 7 IN 7 Over 30 0 0 0 0 0 Mean % 15.0 18.0 20.0 2N.0 23.0 le.g., 7 out of 26 subjects showed a magnesium-zinc content for their serum albumin fraction to be in the 0 - 10 % range. BGiven as the number of individuals in each range. 3Range is given as per cent of total magnesium plus zinc on the paper electrophoretogram. 39 TABLE XVI INDIVIDUALS IN VARIOUS RANGES OF COPPER CONTENT AMONG THE SERUM PROTEIN FRACTIONSl A. For Sera of 26 Normal Subjects2 Range3 Albumin Globulins Alpha-l Alpha-2 Beta Gamma 0 - 10 0 9 12 5 9 ll - 2O 1 15 9 8 12 21 - 30 5 2 N 8 2 31 - N0 8 0 1 N 2 N1 - 50 6 O O 1 l 51 -- 60 2 O O O 0 Over 60 N 0 0 0 0 Mean % N2.0 12.0 11.0 20.0 15.0 B. For Sera of 22 Multiple Sclerosis Patients2 0 - 104 6 11 8 9 15 11 - 20 O 3 7 3 3 21 - 30 N N 2 N 0 31 - N0 3 2 2 2 1 N1 - 50 3 0 O 1 l 51 - 60 0 0 0 0 0 Over 60 N 0 1 l 0 Mean.% 38.0 1N.O 20.0 20.0 8.0 § le.g., none of the 26 subjects showed a c0pper content for their serum albumin fraction to be in the 0 - 10 % range. SEiven as the number of individuals in each range. SRange is given as per cent of total copper on the paper electrophor- etogram. 4Three patients had no detectable copper in any fraction. N0 TABLE XVII METAL DISTRIBUTION AMONG SERUM PROTEINS OF VARIOUS PATHOLOGICAL SERA FROM PAPER ELECTROPHORETOGRAMS A. Calciuml Patientz Albumin Globulins Alpha-1 Alpha-2 Beta Gamma l M.M. 28.6 5.N 9.7 9.6 N6.7 2 M.M. 23.3 6.6 10.7 10.0 N9.N 3 M.M. 9.0 9.9 5.7 6.N 69.0 N L.C. 31.9 6.9 12.8 16.2 32.2 5 S.B. 51.0 10.8 20.2 10.1 7.9 6 G.N. 55.2 7.8 13.1 11.8 12.1 7 M.C.V. N9.8 10.0 12.5 1N.0 13.7 B. Iron1 1 M.M. 15.3 30.6 25.8 23.6 N.7 2 M.M. 15.N- 33.N 20.N 30.8 0.0 3 M.M. 8.N 27.7 27.7 19.3 16.9 N L.C. 12.3 32.3 20.0 15.N 20.0 5 S.B. 18.1 25.3 10.9 27.6 18.1 6 G.N. 10.7 N6.5 17.8 21.N 3.6 7 M.C.V. 6.5 37.5 31.0 20.0 5.0 C. Magnesium-Zincl 1. M.M. 28.0 10.0 7.0 10.0 N5.0 2. M.M. 25.5 5.7 9.3 10.5 N9.0 3. M.M. 12.5 0 0 12.5 75.0 N. L.C. 18.5 16.7 18.5 9.3 37.0 5. S.B. 27.7 17.0 2N.1 16.3 1N.9 6. G.N. 28.8 15.6 21.N 17.5 16.7 7. M.C.V. 18.0 18.0 21.0 21.0 22.0 lGiven as per cent of total metal upon the paper electrophoretogram. 2The abbreviations represent the patients described in Table X. N1 TABLE XVII (Cont.) D. Copperl PatientZ Albumin Globulins Alpha-l Alpha-2’ ’Beta Gamma l M.M. 0 31.N 27.N Nl.2 0 2 M.M. 23.5 29.5 23.5 23.5 0 3 M.M. 0 37.6 2N.8 37.6 0 N L.C. O 75.6 12.2 12.2 0 5 S.B. 28.0 9.N N0.0 22.6 0 6 G.N. 0 0 0 0 0 7 M.C.V N2.0 12.0 11.0 20.0 15.0 NEiven as per cent of total metal upon the paper electrophoretogram. 2The abbreviations represent the patients described in Table X. TABLE XVIII ANALYTICAL RESULTS OF‘POOLED SERA DIALYSIS STUDY Before After Total Protein (g./100 ml.) 6.10 5.30 Calcium (meq./L.) _ N.NO 0.00 Iron (meq./L.) 0.067 0.056 Magnesium (meq./L.) 2.58 0.00 N2 .cwoooea Hmpou mo peso boa mm co>wo~ .mesoc omumm Eoum cowem> mafia mwmxflmmea a.na a.ma a.w m.oa m.a c.s m.m m.m N.Nc N.mc can: m.aa m.6a c.m S.a S.m a.S N.N m.m c.ma a.ms 0N a.aa «.ma a.a m.w a.m s.c ®.N m.m m.6a N.cc as a.ma c.aa c.m H.m m.m S.m m.m m.m m.0s c.0s m o.ma 6.4a m.s m.oa 6.N S.m S.m 6.N w.ac m.ac a o.ma s.mH s.m m.m m.c N.6 N.N w.m N.NS m.sc m S.aa N.NA o.a N.® a.c m.a m.m a.m S.ac o.ac m S.aa a.ma m.a c.ma m.m a.a m.a «.4 S.Sc N.ac Hooa eopm< oeomom topmd oeomom umpmd ouomom suave ouomum umpwd upomom oases seem N-ncaa< H-ccaa< nocaaanoao Ncaasnaa booaeam amHmsdHQ maid Q72 mmommm Sim Adzmoz mo ZOHHDmHEmHQ szhomnH XHX 592. TABLE XX ABSORBANCIES 0E CALCIUM-DYE ELUATES FROM SECTIONS OF PAPER ELECTROPHORETOGRAM’S BEFORE AND AFTER SERUM DIALYSISl 03 Subject Albumin Globulins Alpha—1 Alpha-2 Beta Gamma Pool Before 0.100 0.017 0.027 0.031 0.039 After 0.087 0.011 0.020 0.020 0.031 3 Before 0.280 0.029 0.063 0.052 0.072 After 0.122 0.021 0.023 0.018 0.015 5 Before 0.187 0.028 0.000 0.058 0.067 After 0.105 0.019 0.026 0.036 0.033 7 Before 0.265 0.037 0.006 0.069 0.095 After 0.102 0.010 0.020 0.025 0.028 8 Before 0.273 0.000 0.008 0.065 0.070 After 0.170 0.020 0.020 0.032 0.030 17 Before 0.170 0.036 0.007 0.067 0.060 After 0.231 0.037 0.037 0.002 0.050 20 Before 0.202 0.002 0.063 0.068 0.050 After 0.200 0.039 0.007 0.009 0.035 Mean Before 0.222 0.033 0.000 0.059 0.065 After 0.157 0.023 0.029 0.032 0.032 Difference of mean320.050 0.008 0.012 0.020 0.030 Mean % Calcium3 00.3 6.0 9.7 19.0 20.2 lWave length 550 mu. 2Corrected for dilution. 3Calculated from difference of means. TABLE XXI ABSORBANCIES OF IRON-DYE ELUATES FROM SECTIONS OF PAPER ELECIROPHORETOGRAMS BEFORE AND AFTER SERUM DIALYSIS1 Subject Albumin Globulins Alpha-l Alpha-2 Beta Gamma Pool Before 0.017 0.050 0.051 0.033 0.019 After 0.009 0.029 0.030 0.023 0.013 3 Before 0.027 0.010 0.025 0.036 0.000 After 0.000 0.021 0.009 0.005 0.000 5 Before 0.000 0.022 0.010 0.018 0.001 After 0.007 0.025 0.015 0.020 0.010 7 Before 0.000 0.003 0.037 0.028 0.006 After 0.007 0.030 0.008 0.007 0.000 8 Before 0.007 0.016 0.002 0.000 0.000 After 0.013 0.033 0.020 0.025 0.010 17 Before 0.000 0.002 0.000 0.052 0.000 After 0.010 0.021 0.020 0.022 0.016 20 Before 0.032 0.070 0.003 0.007 0.020 After 0.017 0.023 0.021 0.019 0.000 Mean Before 0.012 0.031 0.025 0.031 0.007 After 0.010 0.026 0.018 0.018 0.007 lWavelength 535 mu. TABLE XXII ABSORBANCIES OF MAGNESIUM-ZINC-DYE ELUATES FROM SECTIONS OF PAPER ELECTROPHORETOGRAMS BEFORE AND AFTER SERUM DIALYSISl Subject Albumin Globulins Alpha-l Alpha-2 Beta Gamma Pool Before 0.051 0.018 0.030 0.000 0.070 After 0.057 0.022 0.007 0.000 0.080 3 Before 0.033 0.023 0.027 0.032 0.018 After 0.070 0.039 0.005 0.009 0.000 5 Before 0.072 0.038 0.050 0.003 0.008 After 0.078 0.050 0.056 0.009 0.053 7 Before 0.030 0.030 0.000 0.036 0.030 After 0.073 0.016 0.021 0.039 0.032 8 Before 0.017 0.022 0.036 0.036 0.035 After 0.003 0.016 0.017 0.030 0.021 17 Before 0.010 0.009 0.016 0.009 0.005 After 0.090 0.030 0.002 0.050 0.058 20 Before 0.007 0.018 0.025 0.023 0.001 After 0.090 0.027 0.030 0.052 0.002 Mean Before 0.031 0.023 0.030 0.031 0.036 After 0.073 0.029 0.037 0.005 0.039 lWavelength 515 mu. TABLE XXIII ABSORBANCIES OF COPPER-DYE ELUATES FROM SECTIONS OF PAPER ELECTROPHORETOGRAMS BEFORE AND AFTER SERUM DIALYSISl Subject Albumin Globulins Alpha-l Alpha-2 Beta Gamma Pool Before 0.006 0.022 0.032 0.022 0.012 After 0.036 0.022 0.023 0.020 0.011 3 Before 0.036 0.003 0.013 0.007 0.007 After 0.012 0.002 0.002 0.000 0.000 5 Before 0.013 0.000 0.001 0.013 0.011 After 0.020 0.000 0.006 0.010 0.000 7 Before 0.013 0.000 0.000 0.000 0.005 After 0.000 0.000 0.002 0.000 0.000 8 Before 0.002 0.017 0.016 0.026 0.020 After 0.003 0.005 0.016 0.010 0.003 17 Before 0.000 0.001 0.000 0 000 0.000 After 0.012 0.000 0.019 0.011 0.000 20 Before 0.012 0.008 0.003 0.013 0.003 After 0.030 0.010 0.017 0.023 0.002 Mean Before 0.020 0.007 0.009 0.012 0.009 After 0.028 0.008 0.015 0.013 0.003 ¥ lWavelength 500 mp. TABLE XXIV TOTAL SERUM CHOLESTEROL LEVELSl W I Normal Patients Subjects 1 160 A 350 2 150 B 260 3 190 C 270 0 250 D 260 S 185 E 200 6 165 F 250 7 170 G 250 8 185 R 235 9 205 I 255 10 195 J 275 ‘11 170 K 310 12 215 L 225 13 265 M 350 10 210 N 300 15 195 P 230 16 195 Q 205 17 200 R 195 18 195 s 260 19 285 T 300 20 155 V 215 21 200 22 225 23 208 20 200 25 228 26 258 Range 150 - 285 195 - 350 X 200 206 s 33 08 RSiven as mg./100 m1. serum. 07 1.2-00.41 .Ord( OJil * 00:} £25.03 1 .... . 1 IHWREZ DENSITOMETRIC TRACING OF A PROTEIN ELECTROPHORETOGRAM OF SERUM FROM A NORMAL SUBJECT - -~‘ H- i Globulins . Albumin Y '13 laz'al Origin 09 FIGURE 3 DENSITOMETRIC TRACING OF A PROTEIN ELECTROPHORETOGRAM OF SERUM FROM A MULTIPLE SCLEROSIS PATIENT ; Globulins lrl I...2 lal Origin Albumin 50 «< o -r‘. '1er ‘2: ‘xt a (m--‘-- DENSITOMETRIC TRACING OF A PROTEIN ELECTROPHORETOGRAM OF FIGURE 0 SERUM FROM A CIRRHOSIS OF LIVER PATIENT Globulins Blaz 1 Albumin L 51 FIGURE 5 DENSITOMETRIC TBACING OF A PROTEIN ELECTROPHORETO0RAM OF SERUM FROM A MULTIPLE MYELOMA PATIENT (Case #2) Globulins ' Albumin 1 i Y I P I 02 I 01 F9 Origin 52 2‘7“.” FIGURE 6 DENSITOMETRIC TRACING OF‘PROTEIN ELECTROPHORETOGRAM OF SERUM FROM A SEVERELY BURNED PATIENT Globulins Albumin l 13 E512 3‘11! Y +—€> Origin 53 S0 ZHSDmA< mZHMDmOAO ZHQqud mZHMDmOAO ZHEqud mZH @040 _ mmmmoo EDHUAdU ZHMHomm Ow Hocucoo Hm80021m00< pmmao 3 883m 0333218: £8 ozHN-§Hmmzo§ 5%. mm» 8. mo R 0mm: HH