Glycoproteins in saline extracts of tissues of normal adult rats

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Content GLYCOPROTEINS IN SALINE EXTRACTS OF TISSUES OF NORMAL ADULT RATS A Thesis Presented to the Faculty of the Department of Biochemistry and Nutrition University of Southern California In Partial Fulfillment of the Requirements for the Degree Master of Science by Marshall Little March 1953 UMI Number: EP41327 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI EP41327 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 ERRATUM > ■ University of Illinois College of Medicine 1853 West Polk Street Chicago 12, Illinois April 3, 1953 Dr. John Mebl, Chairman Department of Biological Chemistry University of Southern California Los Angeles, California Dear Dr. Mehl: Although you will be joining us here very shortly to attend the Federation Meetings, I wanted to set down in writing my ap preciation to you and the other members of my Committee for your cooperation and assistance with the writing of my thesis. I realize my being so far away made^it-*^difficult for all concerned, but the suggestions and**br$ificisms from you all were none-the-less helpful. Unfortunately, there was one error we all missed, and since it is a major one I felt that I should waste no time in telling you. (I am sorry to say that I didnnot catch this mistake until I compared some recent results of some Oreinol,Carbohydrate determinations with those reported in my thesis.)Jin Figure 1, Page 17, and Table 1, Page 18, I originally recorded my results as "Klett Units"; this was later changed and appeared in the final draft as "Optical Density", the values being divided by 1000. As you of course know Therefore all of the values reported on the graph and in the table under Optical Density mftst be multiplied by 2, or, if simpler, multiplied by 1000 and the column heading changed to read "KLett Units" .| That is the way in which I had originally planned to present the data. I am very sorry I did not catch this sooner, and I hope it does not cause too much inconvenience. P.3. I am still homesick for dear old southern California and the swell crowd at S.C. Please give them my best regards. Optical Density - 2<X Klett Units 1000 Cordially, e,> '5s <Line This thesis, w ritten by MARSHALL LITTLE .............. under the guidance of h%&....Facuity Com m ittee, and approved by a ll its members, has been presented to and accepted by the Council on G raduate Study and Research in p a rtia l fu llfill- ment of the requirements fo r the degree of MASTER OF SCIENCE Date March.. Faculty Committee Chairman TABLE OP CONTENTS PAGE INTRODUCTION............................... .. 1 HISTORICAL REVIEW ..................... 4 MATERIALS AND METHODS •............................... 9 Care and Treatment of R a t s ................ . 9 Procedure for Perfusion of Rats.................. 9 Preparation of Tissues . 11 Chemical Analyses . ............ 12 OBSERVATIONS AND RESULTS . . . . . . ................... 16 DISCUSSION.......................................... 33 SUMMARY................... 38 BIBLIOGRAPHY ................................ 39 LIST OP TABLES TABLE PAGE I, Comparison of Two Orcinol Methods for Determination of Carbohydrate . 18 II. Summary of Data on Comparison of Three Extracts of Normal Liver (Prom a Female Rat Pasted 24 Hours)....................... 21 III. Summary of Data on Long-Evans Strain, Normal Male Rats Which Had Been Fasted for 24 Hours but not Perfused (1-2 animals) . . . 22 IV. Summary of Data on a Normal Long-Evans Strain, Male Rat Which Had Been-Perfused but not Fasted (1 animal)................... 23 V; Summary of Data on Kinosita Strain, Normal Male Rats Which Had Been Perfused and Pasted 24 Hours (5-6 animals).......... . . 24 VI. Summary of Data on Kinosita Strain, Normal Male Rats Which Had Been Perfused but not Pasted (3-4 animals).............. 25 VII.. Summary of Data on Kinosita Strain, Normal Female Rats Whieh Had Been Perfused and Pasted 24 Hours (Tissues Prom Two animals Pooled).......................................26 VIII. Summary of Data on Kinosita Strain, Cancer, Iv TABLE PAGE Female Rats Which Had Been Perfused and Fasted 24 Hours (Tissues From Two Animals Pooled).......................... . 29 IX. Summary of Data on Rat Serum....................31 LIST OF FIGURES FIGURE PAGE 1. A comparison of two orcinol methods for the determination of carbohydrate ...... 17 ACKNOWLEDGEMENTS The writer wishes to express his sincere appreciation to Dr. Henry Weimer for the able direction of the problem, and to Dr. Richard J. Winzler for his helpful advice and criticism. Appreciation is also expressed to the Department of Infectious Diseases at University of California at Los Angeles for providing financial support and laboratory facilities. INTRODUCTION A number of investigators have demonstrated the presence of carbohydrate-rich proteins in plasma and serum. These conjugated proteins are characterized by a relatively high content of hexose and hexosamine and by a relatively low nitrogen content. Such highly soluble and stable preparations have been designated as polypeptides, pep tones, proteose, seromucoid, seroglycoid, mucoidlthnliehe Substanz, Tiergummi, and, mucoprotein by various workers, and may be considered as representatives of the general class, glycoproteins. They have been isolated from human, horse,.beef, rat, chicken, dog, rabbit, and guinea pig plasma and serum. The physiological importance of glycoproteins has been hinted at chiefly through studies dealing with serum polysaccharide levels in normal and pathological states. When if, by the usual blood sugar determination methods, the reducing power of blood or serum is determined before and after acid hydrolysis, there is an increase in reducing substances following hydrolysis. This fact has been known since 1855 (Grevenstuk and Laquer, 1925) and has formed the rationale of many studies of serum in normal and pathological states. These increases were 2 traced by most authors to the presence of carbohydrates bound to plasma proteins. Thus the increase in serum polysaccharide levels in patients with cancer, with myo cardial infarctions, pneumonia, tuberculosis and some other pathological states reflects primarily an increase in the level Of plasma glycoproteins. The source of plasma glycoproteins is unknown at • * ■ * . t the present time. While it has been generally accepted that the liver is the chief site of formation of certain of the plasma proteins, notably albumin, prothrombin, and fibrinogen, little - definitive evidence is available regarding the site of formation of the plasma globulins. The presence of glycoproteins in tissues has been demon strated by several investigators employing eytochemical procedures, and neutral mucopolysaccharide fractions have been isolated from many animal tissues. Recent investigators in this field have presented evidence which suggests that in most animal tissues there are polysacchar ides of the type found in plasma and blood group substances. The purpose of the present investigation has been to extend these observations and to determine the relative concentrations of glycoproteins in various tissues of normal rats. The methods used were adapted from those applied to the isolation and determination of plasma________ 3 glycoproteins, and were based on the solubility properties of these substances; consequently, there would be obtained some evidence of a similarity between tissue glycoproteins and those found in plasma. Subsequent investigations of tissue glycoproteins in disease might well show that alterations of plasma glycoproteins reflect alterations of those found in tissue. HISTORICAL REVIEW Plasma glycoprotein was first described by Freund (1892). Later Investigators have studied the material more thoroughly. All of the earlier work was limited t primarily to chemical analyses and solubility studies of crude fractions. It was on the basis of these properties that classification of glycoproteins was attempted. Such chemical properties provide no indication of the homo geneity of the isolated material, since a contaminant may be present in constant amounts. The classification of proteins that is most generally accepted was originally formulated and recommended by the American Physiological Society and The American Society of Biochemists (Committee on Protein Nomenclature, 1908). In this classification the carbohydrate-rich proteins of plasma would be included in the subclassification, glycoproteins, under the general class, conjugated proteins. Glycoproteins are by definition, ‘ 'compounds of the protein molecule with a substance or substances containing a carbohydrate group « other than nucleic acid." At the present time no further definition exists for this class of compounds and there is no accepted nomenclature. As Meyer (1945) stated, "A reviewer of mucopolysaccharides, mucoids, and 5 glycoproteins is faced with the problem of giving his own definitions and classification, since in this field, unlike the general field of proteins, there is no accepted terminology.”, t The classification of glycoproteins proposed by Stacey (1946) has found rather wide acceptance. As one class of glycoproteins, he defines mucoproteins as ”protein-carbohydrate compounds with relatively high protein or peptide content, the chemical reactions of which are predominantly protein. In general, they do not coagulate on being heated in aqueous solutions. All mucoproteins contain a hexosamine constituent,w He distinguishes mucoproteins from mucopolysaccharides which he describes as having ”a low but significant protein content and give reactions which are predominantly carbohydrate. Their aqueous solutions diow a high viscosity and they tend to become insoluble in water after intensive dehydration.” Plasma and tissue glycoproteins have been studied by a large number of investigators from several points of view. Their work has been recently reviewed by Meyer (1945) and by Stacey (1946). There is considerable evidence to indicate that plasma polysaccharide consists of mannose, galactose, glucosamine, and fucose (Rimington, 6 1929,19l4-°» Riuiington, and Van den Ende, 19^+0; Hewitt, 1937; Sorensen, and Haugaard, 1933; Waldron and Woodhouse, 1950; Werner and Odin, 19^9; Dische and Osnos, 1952). Evidence that other uncharacterized sugars may be present has been advanced (Gottschalk, 1951; Gottschalk, 1952, Odin, 1952). The importance and clinical significance of studies of plasma polysaccharide was realized to a great extent through studies of the polysaccharide content of sera from patients with various pathological conditions. Within certain limits, healthy individuals maintain a constant amount of plasma polysaccharide. However, in such conditions as pneumonia, tuberculosis, neoplasia, rheumatic fever, and sterile"infarcts, significant devi ations from this normal have been observed. (e. g., West, 1936; West and Clarke, 1938; Lustig and Langer, 1931; Friedmann and Sutliff, 1939; Hilsson, 1937; Blix, et. al., 19lA; Seibert, et. al., 19i|-7; Kendall, 1937; Seibert, et. al., 19ij.8; Shetlar, et. al., 19k9&; Shetlar, et. al., 19i | . 9b.) Hexosamine-containing polysaccharides are not limited to the plasma, for they have been shown to be present in the skin and cartilage, (Kleiner, 19i+5; Meyer and Chaffee, 191+1), cornea (Meyer and Chaffee, 19^0), tendon, sclera, aorta, 7 vitreous humor, synovial fluid, ciliary body, iris, gastric mucosa, and pituitary (Kleiner, 1945; Meyer, 1940, 1941; Meyer and Smyth, 1937; Meyer, et al., 1939; Catehpole, 1949). Little evidence exists concerning the role of these tissue glycoproteins, although an indi cation as to one possible function in a particular tissue is realized through Catchpolls study of rat pituitaries (1949). His evidence strongly suggests that a part of the glycoproteins of the pituitary is the follicle stimulating hormone. A number of workers have speculated that the increased circulating glycoproteins in disease states arise from the ground substance of the connective tissue at the site of invasive growth or degeneration by a process of de- polymerization (Catehpole, 1950; G-ersch. & Catehpole, 1949). However, several of the studies of serum glycoproteins in normal and pathological individuals have shown a rise in the concentration of serum glycoprotein coinciding with tissue proliferation and repair (e.g. Shetlar, et. al. 1949b). Werner (1950) by his studies of certain effects of liver poisons on normal rabbits advanced some evidence that the plasma glycoproteins were formed in the liver. He found that the poisons prevented the rise in plasma hesoxamine produced by withdrawal of large amounts of blood. Thus, it would seem of Interest to determine the relative concentrations of saline-extractable tissue glycoproteins, and to study these substances with the aim of showing their relation to circulating glycoproteins. MATERIALS AND METHODS Care and treatment of rats. The normal animals were of young, adult Kinosita (Japanese, albino) or Long- Evans strains. They were maintained on a normal, stock diet (Purina Laboratory Chow) unless otherwise specified. The tumor-bearing rats studied were of the Kinosita strain. The tumor was the Yoshida sarcoma, first reported by Yoshida, originating in a Japanese albino rat (Yoshida 1946,1947). Histologically, it is an undifferentiated reticulum cell sarcoma (Kinosita, 1948, 1952). Routine transmission was by intraperitoneal or subcutaneous injection of 0.2 ml. of undiluted ascitic fluid into youfcg adult rats. With only few exceptions, the animals were not sacrificed until the developing tumor had reached maximum size. This was usually 7 to 15 days after the injection of the ascitic fluid. Procedure for perfusion of rats, (a) Anesthesia: A solution of nembutal was made up to contain 12 mg./ml. in 0.85 per cent NaCl. This solution was kept at refrigerator temperature until needed. Satisfactory anesthesia was usually obtained by injecting 1.5 ml. of this solution intraperitoneally into a rat weighing 500 to 400 gms. (ca. 6 mg. nembutal per 100 gm. rats) 10 A t (t>) Perfusion: After fairly deep anesthesia was obtained (about 15 minutes after injection of nembutal) the animal was weighed, then tied on its back to an operating board. Each leg was pulled to the side forming an angle of about 45° with the midline, A one-half inch longitudinal incision was made over the left and right jugular and the left and right femoral veins. These vessels were then exposed, using blunt-tip forceps to avoid hemorrhage, When about one-fourth inch of each vessel had been exposed, the incised area was covered with a gauze wet with warm saline, (It was important at this time to avoid as much shock to the animal as possible,) A midline incision was then made into the peritoneal cavity, running longitudinally from the area ventral to the bladder to about one-half inch ventral and posterior to the diaphragm. After the viscera had been carefully displaced to the side and covered with moist gauze, a section of the inferior vena cava was dissected free of connective tissue, and a cotton suture (about 3 inches in length) was inserted' under the vessel. By means of a nee dle connected to a two-way metal stopcock, a sample of blood could be removed before perfusion was started. This * Chiamori and Motrica ( 1 9 4 9 ) . ________________ 11 needle (18 ga., 2 inches) with syringe and perfusion tub ing attached, was inserted into the vena cava immediately posterior to the cotton suture, and a sample of blood slowly withdrawn. The stopcock was then turned and the warm perfusion fluid (Tyrode's solution) allowed to run in at a rate not exceeding 1 ml./minute. After about 2 minutes the cotton suture was securely tied around the needle, and the exposed vessels, the left and right femoral and the left and right jugular veins, were cut longitudinally with assealpel. The rate of perfusion could then be accelerated to 3 ml./minute, which rate was maintained until all observed tissues appeared free * of blood. About 300 to 400 ml. of Tyrodefs solution were usually required. Preparation of tissues. Of the various methods for the extraction of the tissues which.were tried, the fol lowing method was found to be most satisfactory. Immed iately after the animal had been perfused the tissues were removed and placed in mortars. These were then placed in a desiccator under vacuum and stored in a deep freeze at about -30° C. for 18-24 hours. At the end of this period the frozen tissues were lyophilized to dryness in a refrigerated room at -10° G. The dried tissues were _grsuM_JS-Lth_jacmt.ar_an.d_-peatle—to_a_f-ine—powder-.— A 1 -per— 12 cent (by weight) suspension of each tissue was then made up in 0.85 per cent saline. These suspensions were al lowed to stand with frequent agitation for 30 minutes, then centrifuged at 2000 r.p.m. for 30 minutes. The supernatant was filtered through Whatman #1 filter paper and the various chemical determinations were made on the filtrates. Chemical analyses. (a) Carbohydrate determinations. Two methods for carbohydrate determinations were used in the course of this work. Each was based on the orcinol reaction of S8rensen and Haugaard (1933). Method 1 was the procedure of Hewitt (1937). Method 2 was a modifica tion of the procedure of Lustig and Ernst (1937). Since the results of a study of both methods will be presented * later in this thesis each method will be presented for comparison. Method 1, Reagents; 60$ (about 21.6/equivalents per liter) 1.6$ Orcinol in 30$ H2S04 Procedure;. The protein precipitate (see (e) and and (f) below) was dissolved in 1 mi. of water (or 0.1 N NaOH). 7.5 ml. of 60$ HgSO^ were added, followed by 1 ml. of 1.6$ orcinol in 30$ HgSO^. After stirring, the 13 solution was heated at 80° C. for 15 minutes, then « • ' • immersed in an ice bath. In 15 minutes the solutions were read in a Klett at 540myt*. An equlmolar mixture of galactose and mannose was used as standard. This method;will determine carbohydrate in the range of 0.02 to 0.2 mg. Method 2. Reagents: 0.25 N NaOH 72$ BLSO,* (about 27.6 equivalents per 2 * liter) 2$ Orcinol in 25$ H2S04 Procedure: The protein precipitate was dissolved in 0.4 ml. of 0.25 N NaOH. 0.2 ml. of.2$ orcinol in 25$ H2SO4 was added, followed by 1 ml. of water (or standard) and 5 ml. of 72$ HgSO^. After stirring, the . solution was heated in a boiling water bath for ten minutes, cooled for 15 minutes in tap water, and read in the Klett at 540 • An equimolar mixture of galactose and mannose was used as standard. This method will determine carbohydrate in the range of 0,01 to 0.1 mg. (b) Tyrosine determination. A method employing Folin’s pdienol reagent (Folin and Ciocalteu, 1927) as adapted by WInzler, Devor, Mehl and Smyth (1948) was 14 followed. Although the values obtained are reported as ’ ’Tyrosine" it is recognized that this reagent is not specific for tyrosine groups in proteins. Tyrosine in 1/5 saturated NagCO^ was used as a standard. (c) Total protein determinations. Total protein was determined by the biuret method of Weichselbaum (1946). Human plasma albumin and bovine plasma albumin were used as standards. (d) Determination of hexuronic acids. The carbazole method of Dische (1947) was used. Glucuronic acid was" employed as standard. (e) Total polysaccharide determination.' The total polysaccharide was determined by adding 1 ml. of tissue extract (or 1 ml. of a Is10 dilution of serum) to 10 ml. of absolute alcohol. This was allowed to stand 10 minutes, then centrifuged for 10 minutes at 2000 r.p.m. The supernatant was removed by deeantation, the precipitate washed once with 10 ml. of absolute alcohol, recentrifuged, allowed to drain about 10 minutes, and the carbohydrate content of the precipitate determined by one of the methods described above. (f) Determination of total perchloric acid-soluble, phosphotungstic ac'id-insoluble hexose. The method described by 'Winzler, et al. (Winzler, 1948) for___________ 15 determination of serum mucoprotein hexose was used for this analysis. This fraction will be referred to as ”mucoprotein hexose” (MP hexose) or ”the mucoprotein fraction” (MP fraction), although it is recognized that the components of this fraction may differ from one tissue to, the next and may be, to a considerable extent, unlike the mucoprotein fraction of serum. (g) Moisture determination. Moisture was determined by heating the sample to constant weight in a vacuum oven. OBSERVATIONS AND RESULTS In the first series of experiments on normal tissue extracts the carbohydrate method of Hewitt was used for the determination of the total polysaccharide and the mucoprotein hexose. This method was found to be Insuf ficiently sensitive for those tissues which were relatively low in carbohydrate (e.g. muscle, brain). To obtain in creased sensitivity a modification of the method of Lustig and Ernst was tried. In order to show a compari son between the sensitivity of the two methods, standard curves for each method are shown in Figure 1. The values for each point with their standard deviations are given in Table 1. It can be seen from these data that Method 2 Is appreciably more sensitive’ than Method 1. This1sensi tivity can be explained in part by a smaller total volume than Method 1; however, as Method 2 was almost twice as sensitive as the other procedure and had the added conven ience of a boiling water bath rather than an 80° bath, it was selected for the major portion of this work. It can be recognized that many of the analytical methods used in this work were originally designed for studies of plasma proteins (e.g. the biuret method for the determination of total proteins, Winzler*s method for 500 ■Method 2 ■Method 1 3)0 10 IgO ISO 2Q0 M 2 2$0 m 4Q0 u “ ...Galactose-Mannose (Gamma) 'Figure 1. ”A comparison of two orciriol methods for the determination of carbohydrate. (For discussion see page 12). 18 TABLE I Comparison of Two Orcinol Methods for of Carbohydrate Determination Method 1 (Hewitt) Method 2 (Lustig Modification) ' Galactose- Optical Mannose Density (mgs*) Galactose- Mannose (mgs.) Optical Density .050(8)* .071 t .0059** .024(4)* .075 + .0058** .100(20) .141 ± .0060 .030(6) .081 t .0040 .200(19) .271 t .0066 .032(4) .092 t .0034 .250(4) .345 t .0171 .050(24) .141 t .0047 .400(6) .563 + .0085 .100(40) .271 t .0059 .160(2) .434 t .0035 .200(13) .530 £ .0155 * The values in parenthesis determinations. are the number of individual ** The range of variation is deviation. . ,r-.. «/ I n-1 represented by one standard J s standard devia tion d z deviation from the mean n I Total number of individual deter minations 19 determining plasma,mucoproteins, and the determination of the total polysac charide by precipitating the plasma proteins with alcohol). _ Thus',* if* the methods were to be used in the analysis of water-soluble tissue proteins, it would be highly desirable to extract these proteins with minimum denaturation. The first experiments were designed with this aim in view. The method finally adopted for the preparation of the tissues appeared to solve the problem of denaturation. If the tissues were stored in the deep freeze immediately after removal and were maintained at a temperature below freezing throughout lyophilization, it was possible to obtain the dry tissues with practically no color change or other evidence of denaturation. Furthermore, it is possible' that storing the tissues in the deep freeze at a tempera ture of about -20° C. gave a rate of freezing which was sufficiently slow to aid in the rupture of the tissue ceils and thereby facilitate extraction of the soluble proteins. An indication that the treatment of the tissues was adequate and that release of yater-soluble protein components of the cells had been complete was shown by the following experiment using normal rat liver. Three samples of perfused, dried, liver powder were extracted with (I) distilled water for 24 hours, (II) 0.85$ 20 saline for 2J j . hours^ and (III) 0.85$,saline for 30 minutes (the method already described on page 11). Prom a study of the results of the various chemical analyses of the three extracts (Table II), there appears to be no signifi cant difference between the three methods of extraction. In tables III, IV, V, VI, and VII are shown summarise -H' of the chemical analyses of the normal tissues of the rat. The data of Table III were collected from experiments on tissues of fasted, Long-Evans strain rats which were not perfused. On comparison of the total protein values of Table III with those of Tables IV,V and VT there appears to be a higher total protein extracted from the non-perfused tissues. However, it was found that this difference was due largely to the presence of hemoglobin in the extracts and was not a real protein difference. Hemoglobin was also found to interfere to a lesser extent in the determination of the total polysaccharides. For this reason subsequent experiments were carried out on perfused animals, and corrections were made in the biuret determination for trace amounts of hemoglobin which may have remained in the perfused tissues. With one exception, the spleen, the | effect of trace amounts of hemoglobin on the total poly saccharide determination appeared negligible, and there fore, no corrections were attempted. Due to a relatively TABLE II Summary of Data on Comparison of Three Extracts of Normal Liver (Prom a Female Rat Fasted 24 Hours) Sample No. Total Protein (mg./gm. dry tissue) Total Polysac charide (mg./gm. dry tissue) Mucoprotein Mexose (A)' (mg./gm. dry tissue) Mucoprotein Tyrosine (B) (mg./gm. dry tissue) A B Hexuronic Ac Id (mg./gm. dry tissne I 303 7.0 1.14 1.11 1.03 <0.5* II 294 6.4 1.11 1.06 1.04 <0.5 III 294 6.9 .96 1.01 .96 <0.5 I A 1$ (2) suspension of pulverized tissue in distilled water was allowed to stand with occasional agitation for 24 hours, then treated as described on page 11. II A 1$ suspension of pulverized tissue in-0v85$‘saline was allowed to stand with occasional agitation for 24 hours then treated as I. Ill A 1% suspension of pulverized tissue in 0.85$ saline was treated as described on page 11. * Less than 0.5 mg/gm. tissue not detectable* to H 22 TABLE III Summary of Data oh Long-Evans Strain, Normal Rats Which. Had Been Fasted for 24 Hours hut not Perfused (1-2 animals) Male Tissue Total Protein (mg./gm. dry tissue) Total Polysaccharide. (mg./gm. dry tissue) Mucoprotein hexose (mg./gm. dry tissue) Liver 562 6.4 0.81 Heart 303 3.4 0.49 Lung 680 8.2 0.76 Kidneys 384 5.1 1.00 Spleen 525 8.6 1.64 Skeletal muscle 169 1.6 0.27 Brain 150 2.1 0.44 Testis 308 6.1 1.30 23 . TABLE IV Summary of Data on a Normal Long-Evans Strain, Male Rat Which Had Been Perfused but not Fasted (1 animal) Tissue Total Protein (mg./gm. dry tissue) Total Poly saccharide (Mg./gm. dry tissue) Mucoprotein Hexose (mg./gm. dry tissue) Liver 341 6.1 .39 Diaphragm 142 2.8 .75 Heart 180 2.9 .54 Lung 218 6.5 .90 Kidneys 274 5.8 .99 Spleen 332 8.9 1.17 Skeletal muscle 189 4.1 .27 Brain 132 1.6 .54 Testis 322 7.1 .69 Skin 89 2.2 .69 Omentum 123 4.1 .45 24 TABLE V Summary of Data on Kinosita Strain, Hormal Male Rats Which Had Been Perfused and Fasted 24 Hours (5-6 animals) Tissue Total Protein Total Poly- Mucoprotein (mg./gm. dry saccharide Hexose tissue) (mg./gm.' dry (mg./gm. dry tissue) tissue) Liver 287 4 35* 6.4 4 0.7 1.08 4 0.45* Diaphragm 104 4 24 2.3 t 0.3 1.04 4 0.32 Heart 155 4 11 * 2.8 t 0.5 0.66 4 0.09 Lung 158 4 mm 17 5.3 ± 0.4 1.32 t mm 0.14 Kidneys 263 i 16 5.8 4 0.4 1.19 4 0.22 Spleen 331 t 25 9.8 4 0.6 1.92 4 0.37 Skeletal muscle 157 4 13 2.7 4 1.0 0.46 i 0.10 Brain 91 4 mm 13 1.9 t 0.4 0.54 4 0.08 Testis 307 4 33 7.5 4 1.0 1.17 t 0.24 Skin 46 4 15** 1.9 4 0.0 0.66 4 0.04** Omentum 141 4 61 8.7 4 3.3 2.55 4 mm 2.2 "The range of variation is represented by one standard deviation. (See footnote of Table I). ** This value represents only two animals. (See explana tion page 27). 25 TABLE VI Summary of Data on Kinosita Strain, Hormal Hale Rats Which Had Been Perfused but not Pasted (3-4 animals) Tissue Total Protein (mg./gm. dry tissue) Total Poly saccharide (mg./gm. dry tissue) Mucoprotein Hexose (mg/.gm. dry tissue) Liver 377 4 27 7.3 4 0.7* 0.74 4 0.23* Diaphragm 193 4 39 2.6 4 t m 0.3 0.47 4 0.06 Heart 183 4 8.7 2.7 4 0.4 0.60 4 0.11 Lung 187 4 15 5.8 4 0.4 , 0.95 i 0.23 Kidneys 309 4 21 6.3 4 0.7 1.01 4 0.19 Spleen 329 4 64 9.9 + 2.3 1.44 4 0.40 Skeletal muscle 172 4 24 2.9 4 0.8 0.40 4 0.15 Brain 116 4 5.2 1.7 + 0.2 0.44 4 0.17 Testis 341 4 5.1 8.0 4 0.3 1.07 4 0.28 * The range of variation is represented by one standard deviation. (See footnote of Table I). TABLE VII 26 Summary of Data bri Kinosita Strain, Hormal Female Rats Which Had Been Perfused and Fasted 24 Hours (Tissues From Two Animals Pooled) Tissue Total Protein' (mg./gm. dry tis sue) Total Poly saccharide ‘ (mg./gm, dry tissue) Mucoprotein hexose (mg./gm. dry tissue) Mucoprotein Tyrosine (mg./gm. dry tissue) Liver 345 6.7 1.08 .48 ' Diaphragm 118 2.2 .72 Heart 147 2.5 .75 .33 Lung 161 5.9 1.38 .51 Kidneys 265 6.4 1.56 .84 Spleen 317 8.4 2.04 1.34 Skeletal . - - muscle 161 3.0 .45 .07 Brain 95 1.9 .48 .19 Skin 45 1.3 .60 .057 -Omentum 76 5.7 1.26 .41 27 high concentration of hemoglobin in the spleens of the perfused animals, the total polysaccharide value for this tissue may be slightly higher than the true value. Prom a study of Tables V and VI, it is possible to observe the effect of fasting on the tissues of the Kino sita strain rats. There appears to be a significant decrease in extractable protein when the animals are subjected to a 2I 4 . hour fast; practically no change in the total polysaccharide is observed; and there is possibly a slight increase in the mucoprotein hexose of the fasted animals. Due to the extreme difficulty in pulverizing the dried skin of the animals, the analysis of this tissue was not attempted on all animals, and thevalues reported for skin (Table V) are not strictly comparable to the values reported for the other tissues. The mucoprotein hexose of omentum, although showing considerable variability, agrees well with the value reported by Catehpole (1950). He found 2.3O - O.75 mg. per gram of tissue of water- soluble mucoprotein hexose in connective tissue of mice. Table VTI represents the analysis of two normal female rats of the Kinosita strain which were fasted 2I 4 . hours. Although Table VII represents only a preliminary study and no definite conclusions can be drawn, it can be seen that 28 these values are in close agreement with the normal, fasted males of;the same strain (Table ¥>) with the exception of the total proteins of the liver. In this tissue the values for total protein of the two groups do not agree; however, due to the small number of animals of the female group no statistical evaluation can be made. Another preliminary experiment designed to study the effect of cancer on the tissues of the rat was made on two fasted, Kinosita strain, cancer-bearing female rats. The animals were perfused, the tissues removed and analysed in the usual way. The results (Table WC%)when compared with the values for the fasted normal rats, pre sent several points of interest, although it is recognized that"such a comparison may give, at best, only suggestive evidence. Thus the liver, diaphragm, heart, lung, kidney, spleen and brain of the cancer-bearing animals appear to contain more saline-extractable protein that the corre- , sponding tissues of fasted, normal males, and, with the exception of the liver, females. There also appears to be An increase in the total polysaccharide of the tissues produced by the cancer. This was not so clearly defined, however, except in the liver, diaphragm, lung and, possibly kidney. No effect was apparent on the mucoprotein hexose from the tissues of the cancer-bearing animals._____________ 29 TABLE VIII Summary of Data on Kinosita Strain, Cancer'5 5 ",Female Rats which Had Been Perfused and Fasted 24 Hours (Tissues From Two Animals Pooled) Tissue Total Protein (mg./gm. dry tissue) Total Poly saccharide (mg./gm. dry tissue) Mucoprotein hexose (mg./gm. dry tissue) Liver 341 8.0 1.1 Diaphragm 189 3.6 1.1 Heart 199 3.3 .62 Lung 255 6*8 1.3 Kidneys 293 6.8 1.4 Spleen 379 10.1 2.3 Skeletal muscle 104 3.6 .45 Brain 198 1.8 0 0 «> Skin 85 i.9 .66 Omentum 123 3.4 .81 Tumor 331 11.0 1.4 * These animals were injected with 0.2 cc undiluted ascitic Fluid subcutaneously at age six months.. When tumor mass had reached maximum, size (about 10-15 days) the animals were sacrificed. 50 The chemical analyses of the serum given in Table IX show good agreement with results reported by other workers Catchpole (1950) found 16.9 and 17.7 mgs.% of mucoprotein hexose in the sera of two strains of normal mice. Seibert. et al. (1947), in a study of normal and pathological human sera reported 73-131 m.gs.% of total polysaccharide in normal adult sera, and 91-197 mgs.% in patients with primary to advanced carcinomas. The ranges of total protein in gras.$ reported by Seibert for these two groups were 6.3-8.2 and 5.4-8.1 respectively. Shetlar and his coworkers (1949) also found an increased total polysaccharide in patients with cancer. In a study of 43 normal human sera, he reported a range of 93-127 mgs.$. The range in patients with malignancies he re ported as 106-308 mgs.$>. In the last two columns of Table IX are shown the results on sera from Kinosita rats which had been'given a tumor which later regressed. Ascitic fluid was InjecteqL subeutaneously into young adult rats. The tumor, which developed to maximum size in 10 to 15 days, would in some cases metastasize to other tissues and eventually kill the animal, while in other cases the tumor would decrease in size, and from all appearances the animal would return to its normal state.. lhe_datu_o.n__s.ara_Q.f-- ■ TABLE IX Summary of Data on Rat Serum Long-Evans Male Normal Kinosita Male Normal Kinosita Female Normal Kinosita' ’ ’ Regressed Cancer" Male Kinosita "Regressed Cancer" Female Total Protein(mg/%) 6.2 t 1.2* 6.4 i 1.2* . # 6.8 I 0.9 6.4 t 0.3* 6.7 t 1.3* Total Polysaccharide (Mg/%) 144 t 18 152. t 15 157 t 5.9 169 ± 14 156 t 16 Mucoprotein Hexose . . : (mg/%) 19.1 t 4.1 18.2 t 3.8 24.4 i 3.0 32.2 t 14 26.5 ±9.0 Number of i&nimals 10-11 9-12 8 5-10 5-6 * The range of variation is represented by one standard deviation, (see footnote of Table I). 52 the latter group (regressed cancer rats) were Investigated for comparison with the data of normal animals and to gather material for subsequent studies of this group. In some cases a tumor mass was still palpable at time of sacrifice, and in a few animals the presence of ascitic fluid was noted* otherwise, the animals appeared healthy and normal at the.time of sacrifice. In general, a close correlation existed between the trend of the data on the sera of these animals and the degree to which the animal had overcome the tumor, i.e., an- imals with a relatively large tumor mass, and/or some ascit ic fluid at the time of sacrifice had relatively low serum protein, high total polysaccharide, and high mucoprotein hexose values. DISCUSSION The modification of the orcinol procedure of Lustig and Ernst promises to be a help in the determination of carbohydrate bound to protein. Since it is both a sensi tive and a relatively simple procedure, it could be used in the routine analyses of small amounts of glyco proteins . The"method presented here for the drying and extrac tion of tissues is of special interest in that it pro vides a means of extracting water-soluble components in an apparently undenatured state. The method also permits a large, number of small tissue samples to be lyophilized to dryness and preserved for jsie/veral months with minimum deterioration. It should also be pointed out that al though the extract does contain water-soluble components of tissues, certain macromolecules are also present, as evidenced by the turbidity of all extracts, with the exception of heart, diaphragm, and skeletal muscle, after the final centrifugation and filtration. This factor presented no difficulties in various colorimetric determinations since the turbidity disappeared immediately on treatment of the extracts with the reagents used in the tests. 34 As there was no increase in the amount of glyco proteins extracted when the pulverized tissue was sub jected to 24 hours suspension in distilled water, as compared to 30 minutes extraction with saline, the pre sent method for the treatment of the tissues appears to be adequate in bringing about rupture of the cell walls. There may remain behind in the cell residues after extraction an appreciable amount of glycoprotein existing, perhaps, in a polymerized state (Gerslhand Catchpole, 1950) and therefore insoluble in water, However, it is conceivable that the glycoproteins of tissues which are most readily soluble in water or saline may be, in some way, related to the circulating glycoproteins. This is supported by the low amounts of hexuronic acid in the water soluble extracts. The results of these tissue analyses have shown the presence, in all tissues investigated, of a protein frac tion which is soluble.in water and perchloric acid, and is precipitated by phosphotungstic acid. Such solubility properties resemble those of serum mucoproteins (Weimer, et. al., 1950). As evidenced by the turbidity mentioned above, the total polysaccharide fraction of the tissues does not consist entirely of water-soluble components; however, the study of this fraction and its relation to 35 the serum total polysaccharide in various diseases should show whether or not the alterations which occur in the serum total polysaccharide in diseases such as tuber culosis, sarcoidosis, and others (Seibert, 1947) reflect alterations of the tissue polysaccharides. Catchpole (1950) has demonstrated an increase in the water-soluble glycoproteins of tumor connective tissue as compared to normal connective tissue in mice. He believes these to arise from a depolymerization of normally water- insoluble glycoproteins. The depolymerization he believed to be the result of the invasive growth and tissue break down. Schlamowitz, et. al., (1950) have demonstrated changes in both plasma and5tissue hexosamine during the development of subcutaneous ’ ’nodules” following a sub cutaneous injection of trypsin. The decrease observed in the total protein fraction of the fasted Kinosita rats when compared to the non fasted group indicates that those proteins which are readily extracted from the tissues are closely related to the catabolic protein processes of the animal. Since it is generally believed that the serum proteins are also important in this way, this result may be an indication that the more readily saline-extractable proteins of tissues are very closely related to the circulating_______ 36 proteins. In the initial stages of this work it was suspected that some glycogen would be removed from such tissues as liver, skeletal muscle, and kidney in the process of extraction and would appear as a fraction of the total polysaccharide. The liver, the principal tissue involved In the storage of glycogen, did show a significant de crease in the total polysaccharide of the fasted group as compared with the non-fasted, and slight drops were also noted In the other tissues mentioned; however, more data would be necessary to determine if these latter differences are significant. The rise In the tissue mucoprotein fraction which was s ■ • observed in all tissues when the animals were subjected to a 24 hour fast was not‘Significant except in the dia phragm, where greater than a two-fold Increase in the mucoprotein fraction was noted. On comparing the results of the tissue studies to those of the sera in normal animals, it was of particular interest to find that the fraction of the tissue total polysaccharide which was MmueoproteinM was in the same range as was found in the total polysaccharides of sera. This was a surprising coincidence and may very well be evidence of a close kinship of the tissue glycoproteins 37 to those of serum. As there have been several investigations of the effect of various diseases such as cancer, pneumonia and tuberculosis on the plasma glycoproteins (references previously cited), it would seem'highly desirable to further the work reported in this thesis by a study of the effect of such diseases on the saline-extractable glycoproteins of tissues. Such a study might well give evidence as to the origin and function of glycoproteins in various diseases as well as show the effect of the disease on the various organs. It is conceivable that many tissues in such diseases as cancer, although appar ently remote from the main body of malignant cells, are intimately involved in the progress of the disease and such progress is reflected by alterations of the tissue glycoproteins. It would also be of interest to study the chemical composition and electrophoretic mobilities of the tissue glycoproteins in order further to demonstrate the relation of these glycoproteins to those of plasma. SUMMARY A method has been described for obtaining rat tissues, in a dry, powdered, state, which show no signs of denatura tion and which can be stored for several months at room temperature. These tissue powders have been extracted "\ with 0,85 per cent saline and the extracts analysed for total protein, total polysaccharide, and perchloric acid (PA)-soluble, phosphotungstic acid (PTA)-insoluble hexose. In all tissues studied glycoproteins were found to be present, as well as a fraction which was soluble in water and in perchloric acid and insoluble in phosphotungstic acid. A study has also- been made on the effects of fasting on the tissue glycoproteins. In general, there was a decrease in the total protein and total polysaccharide and a slight, but not significant, rise in the PA-soltjble, PTA-insoluble fraction; A sensitive method for the.determination of carbohydrat bound to protein has been described. This method is a modification of the method of Lustig and Ernst (1937). e BIBLIOGRAPHY >0 Blix, G., A. Tiselius, and H. Svens3on. J. Biol. Chem., 137, i-l-85, ( 1 9 iA )‘ Catchpole, H. R. J. Endocrinol. 6, 218, (191$). Catchpole, H. R. Proc. Soc. Exp. Biol. & Med., 221> (1950). Chiamori, G. and S. Notrica, Personal Communication, 19$. Committee on Protein nomenclature, J. Biol. Chem., 1^, xlviii, (1908)4 Dische, Z., and M. Gsnos, Federation Proc., 9, 165> (1950). Dische, 2., and M. Osnos, Federation Proc., 11, 202, (1952). Folin, .0., and V. Ciocalteu, J. Biol. Chem., 73. 627, (1927). Freund, E. Zentr. Physiol. Lepz. u. Wien., 6, 3^5, (1892). Friedmann, T. E., and W. E. Sutliff. Science, £0, 335. (1939). : Gersch, I.,'and H. R. Catchpole. Am. J. Anat. 85, 1+57, < 1914. 9}. Gottschalk, A, Nature 167, 8I 4 . 5, (195D- Gottschalk, A. Nature. 170, 662 (1952). Grevenstuk; A., and E. Laquer. Ergeb. Physiol. 23. II Abt. 1-267, (1925). Harter, B. T. Anat. Record, 102, 3ip9, (19l[*8). Hewitt, L. F., Biochem. li,.360, (1937). Kendall,.F. E. J. Clin. Invest., 16, 921, (1937). Kinosita, R. Paper read at the Annual Meeting of the Ameri can Cancer Society in N.Y., November 8, 19$. Kinosita, R. Pending publication 1952. • . . ip. Kleiner, J., "Human Biochemistry", St. Louis, C. V. Mosby Co., (19k$), Pp 90, ll£, 118. Lustig, B., and A. Langer. Biochem. Z. 2ij2, 320, (1931)* Lustig, B., and T. Ernst. Biochem. Z. 289. 365, (1937). Meyer, K;,E. M. Smyth, and M. H. Dawson. J. Biol. 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Cancer: Res. 9, 515, (19i|9). Shetlar, M. ,R., R. S. Bryan, J. V. Foster, C. L. Shetlar, and M. R. Everett. Proc. Soe. Exper. Biol, and Med., 22, 2 9 1 * . , (1949a) Sdrensen, M., and G. Haugaard. Biochem. Z., 260. 2 1 4 . 7, (1933). Stacey, M. "Advances in Carbohydrate Chemistry", The Academic Press, N. Y., 2, 161, (191^6). Waldron, D. M.,. and D. Woodhouse. Nature, 166. 186, (1930 Weichselbamn, T. E. 1m. J. Clin. Path, 16. I 4 . O , (I9I 4 . 6). Weimer, H. E., J.- W. Mehl, and R. J. Winzler. J. Biol. Chem. 183. 361, (1930). Werner. I. and L; Odin. Upsala Lak&refdren. Forh. N. F., 34 Hflft 1-2, 69, (1949). Werner, I., Acta Physiol. Scand. 1£, 27 (1949). West, R., Trans. Assoc. Am. Physicians £1,'23O, (1936). West, R., and D. H. Clarke. J. Clin. Invest., 17, 173, (1938). Winzler, R. J., A. W. Devor, J. W. Mehl, and I4 1 M. Smythe J. Clin. Invest. 27. 609 (1948). Yoshida, T. Trans. Japan. Path. Soc., 33. 31, (1946). Yoshida, T. Trans. Japan. Path. Soc., 33. 33, (1946). ) .

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Core Title Glycoproteins in saline extracts of tissues of normal adult rats

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