A BIOCHEWCAL AND (swam . ANALYSISOF THE f * ~ ’ BRAEHMANN-DE LAINGE SYNDROME f Thesis for the Degree ofth} ' ‘ MECHIGAN‘STATEUNIVERSITY : ;. - , WILLIAM L. DANIEL ' 196:7 ‘ h...— o ‘ r - Mzcmgan it»: .— 2; .. University ; THESIS This is to certify that the thesis entitled A BIOCHEMICAL.AND GENETIC ANALYSIS OF THE BRACHMANN-DE LANGE SYNDROME presented by William L. Daniel has been accepted towards fulfillment of the requirements for PhD. degree in 20010 Wfi/W Major profeéoru Date dug/4) 561 0-169 ABSTRACT A BIOCHEMICAL AND GENETIC ANALYSIS OF THE BRACHMANN-DE LANGE SYNDROME by William L. Daniel This research was designed to define the Brachmann- De Lange Syndrome biochemically and clinically and to attempt the elucidation of the genetic basis of the syndrome. Serum carbohydrates, lipids, amino acids, and keto acids were separated by thin layer chromatography and quantitated either by elution or by the spot area techni- que. Urinary amino acids were assayed by thin layer chromatography and elution. Serum proteins were fraction— ated by Cohn Method 10 and further separated by poly- acrylamide disc electrophoresis (Cohn et_al., 1950). The electropherograms were quantitated with a scanning photo— densitometer using white light. The activities of the serum transaminases were determined by measuring the absorbancy of the keto acid phenylhydrazones. Leukocyte glutamic dehydrogenase activities-were assayed by measur- ing the increase in absorbancy at 3H0 millimicrons. Serum glutamic acid, alpha-keto glutaric acid, and serum glutamic oxalacetic transaminase were elevated in William L. Daniel the patients. Generalized hypoaminoaciduria and hypo- gammaglobulinemia were also present. No leukocyte NAD- linked glutamic dehydrogenase activity could be demon— strated. The hyperglutamicacidemia, hypogammaglobulinemia, and hypoaminoaciduria were previously reported (Ptacek g§_al., 1963). Both parents of each patient had high levels of glutamic acid and somewhat reduced activities of leukocyte NAD—linked glutamic dehydrogenase. Alpha-keto glutaric acid, gammaglobulin, and serum glutamic oxalacetic trans— aminase activity were normal in the parents. Monozygous twin girls (nineteen out of nineteen blood group antigens concordant) were included in this sample. One twin appears to be free of the syndrome. The "normal" twin did not have demonstrable leukocyte NAD—linked glutamic dehydrogenase activity. Both serum glutamic acid and alpha— keto glutaric acid were elevated, but serum glutamic oxal- acetic transaminase activity was normal. Her glutamic acid level was lower and her keto acid level higher than those in her affected sister. No environmental factors which could have caused the syndrome were found in these families. Pedigree data~ failed to reveal any further information of genetic impor- tance. William L. Daniel Cohn, E. J., Gurd, F. R. N., Surgenor, D. M., Barnes, B. A., Brown, R. K., Derouaux, G., Gillespie, J. M., Kahnt, F. W., Lever, W. F., Liu, C. H., Mittelman, D., Mouton, R. F., Schmid, K., and Uroma, E. A System for the Separation of the Components of Human Blood: Quantitative Procedures for the Separation of the Protein Components of Human Plasma, Journal of the American Chemical Society, 72:465-h74, 1950. Ptacek, L. J., Opitz, J. M., Smith, D. W., Gerritsen, T., and Waisman, H. A. The Cornelia de Lange Syndrome, Journal of Pediatrics, 63:1000-1020, 1963. A BIOCHEMICAL AND GENETIC ANALYSIS OF THE BRACHMANN—DE LANGE SYNDROME By AND William LE Daniel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1967 ACKNOWLEDGMENTS I would like to thank my wife, Mary Lou, for her constant encouragement and patience during the course of this research and her technical assistance in the prepa- ration of this thesis. I am grateful for the instruction, advice, and en- couragement given me by Drs. James Higgins, Herman Slatis, R. Gaurth Hansen, Richard.Anderson, and Henretta Band, and appreciate their help in obtaining grants to support this research. The assistance of Drs. C. Stimson and A. Lusis in collecting samples and contacting the families included in this study was instrumental to the success of this research. I would like to thank Mr. John Secord and Mrs. Grace Neitzer for performing the confirmatory enzyme tests and blood typing of the twins included in this study respectively. I am grateful to Dr. H. Waisman for the blood samples from his patients. I extend my sincere gratitude to Mrs. B. Henderson for her patience and cooper- ation in the procurement of the materials for this research. This research was supported by a predoctoral fellowe ship from the National Institute of Mental Health 11 (5—Fl-MH-29,224—O2), Sigma Xi Grant-in-Aid, and Cancer and Biomedical Research Grants from Michigan State Uni- versity. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . vii LIST OF APPENDICES . . . . . . . . . . . viii INTRODUCTION . . . . . . . . . . . . . 1 METHODS AND MATERIALS . 7 Clinical Analyses. . . . . . . . . . 8 Urinary Amino Acids . . . . . . . . . 9 Serum Amino Acids. . . . . . . . . . 9 Serum Keto Acids . . . . . . . . . . 11 Serum Lipids . . . . . . . . . . . 12 Serum Carbohydrates . . . . . . . . . 12 Serum Protein Analyses . . . . . . 13 Leukocyte and Erythrocyte Proteins . . . . 26 Serum Enzyme Activities. . . . . . . . 28 Leukocyte Enzyme Analyses . . . . . . . 30 RESULTS. . . . . . . . . . . . . . . 31 Biochemical Studies . . . . . . . . . 31 Erythrocyte Proteins. . . . . . . . . A2 Clinical Observations . . . . . . . . 5A Family Studies. . . . . . . . . . . 58 Biochemical Studies . . . . . . . . . 62 DISCUSSION. . . . . . . . . . . . . . 67 SUMMARY. 76 BIBLIOGRAPHY . . . . . . . . . . . . . 77 APPENDICES. . . . . . . . . . . . . . 83 Table 10. ll. l2. 13. 14. 15. 16. LIST OF TABLES Page- Stock and Working Solutions for Disc Acrylamide Gels, pH 8.3 . . . . . . 22 Solutions for Disc Acrylamide Electrophoresis pH 4.5 . . . . .‘ . 27 Solutions for Deriving SGOT Standard Curve. 29 General Clinical Tests . . . . . . . 32 Serum Protein Levels: Hycel Precipitation NethOd. o o o o o o o o " o o o 33 Serum Amino Acids . . . . . . . . . 35 Serum Enzyme Activities . . . . . . . 36 Specific Activities of the Leukocyte Glutamic Dehydrogenases . . . . . 38 Serum Keto Acids . . . . . . . . . 39 Serum Carbohydrates . . . . . . . . Al Erythrocyte Protein Concentrations . . . uu Disc Profiles of Total Serum Proteins . . A7 Variations in Fraction II and Fraction III-1’2 o o o o o o o o o o o 5“ Age, Weight, and Height of BDL Patients. . 62 Glutamic Acid, Alpha—Keto Glutaric Acid, SGOT, NAD-GD, and Protein Levels in the Parents and Siblings . . . . . . . 63 Serum Glutamate, Alpha—Keto Glutarate, and SGOT . . . . . . . . . . . 65 Table 17. Comparison of Control, Patient, and. Family Means . . . . II-l. Enzyme, Protein, Amino and Keto Acids in Two Fringe Cases. . . . . . II-2. Urinalysis of Creatinine and Total Amino Acids . . . . . . . . . . vi Page 66 92 93 Figure .1:- \OCDNQU‘I 10. ll. 12. 13. 1A. 15. 16. 17. LIST OF FIGURES Slot and Well Cutter for Immunoelectro- phoresis . . . . . . . . . . Immunoelectrophoresis Apparatus Polyacrylamide Gel Columns . Disc Acrylamide Electrophoresis Apparatus Profiles of Erythrocyte Proteins, pH 8.3 Profiles of Total Serum Proteins, pH 8.3 Cohn Fraction V. . . . . Cohn Fraction IV-6+7 Cohn Fraction IV-l. Cohn Fraction II . . . . Cohn Fraction III—O Cohn Fraction III-1,2. Cohn Fraction I+III-3. . Profile of Brachmann-De Lange (BDL) Patient TG O O O O O O O O O 0 O 0 Total Protein Profiles of BDL Family. . . Fraction III—1,2 Electrophoresis of Leukocyte Extracts vii Page- 14 15 21 2A “3 A6 A9 A9 50 50 51 51 52 52 89 89 91 Appendix I. II. LIST OF APPENDICES Page 8A Pedigree of M. B. Pedigree of T. G. Pedigree of D. S. Pedigree of L. Bu. 9O Electrophoresis of Leukocyte Proteins- Brachmann-De Lange Fringe Cases Urinalysis of Brachmann-De Lange Patients viii INTRODUCTION Brachmann described the first case of a syndrome characterized by profound growth and mental retardation and congenital malformations in 1916 (Brachmann, 1916). Cornelia De Lange, a Dutch pediatrician, followed with a second case report in 1933 (De Lange, 1933). De Lange assigned the name "Typus Degenerativus Amsteldamensis" to the syndrome. Later workers found this name cumbersome. and called the condition the "De Lange Syndrome" or the "Cornelia De Lange Syndrome" (Jervis and Stimson, 1963; Ptacek gt_al., 1963). Ptacek rediscovered the Brachmann paper and appended Brachmann's name to complete the pre- sently accepted form. The Brachmann-De Lange Syndrome (BDL) displays a variety of congenital abnormalities which include pro- nounced hirsutism, bone defects, heart abnormalities, and severe retardation of mental and physical development. The hirsutism is manifest in bushy eyebrows meeting in the midline (synophris), long eyelashes, excessive hair growth on the lower lumbar region of the back, and generalized infantile lanugo. Bone involvement is largely restricted to the extremities, and its severity ranges from slightly incurved fifth fingers (clinodactyly) and proximally located thumbs to absence of digits and bones-of the lower arm. The proximally located thumb has been found to be due to a short metacarpal bone (Kurlander and DeMeyer, 1967). The lower extremities do not appear to be as severely affected. There is an abnormality of the ankle which appears to displace the arches slightly laterally causing difficulty in walking. X-ray studies have shown a retarded osseous maturation to be responsible for the defect. Other osseous manifestations include elbow ab- normalities which prevent its full extension, micrognatha or small Jaw, and a flattened occipital bone. Investigators have mentioned simian creases, abnormal axial triradii, and lack of dermal ridges on the soles and palms (Ptacek gg_al., 1963; Opitz g£_al., 1965). The simian creases are most common. The patients’ facial features are frequently diagnostic. In addition to the previously mentioned eyebrows and lashes, the nose is foreshortened and the nostrils flared. Their ears are situated at the angle of the Jaw, and their lips are downturned at the ends. The majority of patients have an I. Q. less than 25; however, McIntyre has described one patient who is only mildly retarded (McIntyre and Eisen, 1965). Their oral communication is generally restricted to a low*hoarse cry until the patients are five or older. Their vocabulary never surpasses a few words or sentences. The visceral involvement of the syndrome is striking. The internal organs are usually non-palpable, and, in the majority of cases, the genitalia are immature. Systolic murmurs-are frequent (Jervis gt_al., 1963; Ptacek.gg_al., 1963; McIntyre g£_al., 1965; Schlesinger gt_al., 1963). Autopsies have revealed non-crepitant lungs, immature glomeruli, microcephalic brains, anomalous systemic drain- age, defective heart valves and-gonads in the primitive' streak stage (Ptacek gt_al., 1963; Schlesinger gt_al., 1963; Noe, 196“; Hart gt_al., 1965). Diverse endocrine anomalies have been mentioned, al- though none appear to be common to a majority of the patients examined. Autopsies have demonstrated absence of basophilic cells in the pituitary, hypoplasia of-the thymus and-the adrenal gland, and absence of the Zona Fasciculata of the adrenal gland. Bjorklof and Brundelet (1965) sectioned the hypophysis and discovered a cyst in the mid-section of the pituitary. It was subsequently suggested that the BDL re- sulted from a cyst of Rathke's Cleft. No other data have accumulated to support this hypothesis. Necropsies have exhibited several neural ramifications of the syndrome. Poor myelinization in various regions of the brain is frequently observed. De Lange reported mal- formations in the gyri and absence of the Sulcus of Rolandi (De Lange, 1933). Cortical cerebral atrophy was mentioned by Schlesinger g£_a1. (1963). Hart g£_al. (1965) observed an abnormal sulcal pattern, wide gyri, and general- ized reduction of ganglion cells in the cerebral cortex in one of hinpatients. The developmental histories of the patients include a series of crises which frequently end in early death. The birth weight is usually less than six pounds and fre- quently under five pounds, yet the majority of pregnancies are full term. The patients are cyanotic at birth, have difficulty feeding, and are frequently susceptible to upper respiratory infections. These intestinal obstructions, and peritonitis are the most common causes of death during infancy. The growth rate of the BDL patients is retarded, and the deviation from normalcy increases with increasing age (Ptacek et_al., 1963). The final stature of the patients is that of a dwarf, seldom surpassing fifty—four inches. Biochemical data are highly incomplete. Adrenocortical function and blood levels of cholesterol, protein bound iodine, potassium, sodium, and chloride are normal (Schle- singer et_a1., 1963). There is no elevation of the urinary phenylketones or mucopolysaccharides (Noe, 196A). Blood sugar, calcium, phosphorus, and alkaline phosphatase fall within the normal accepted range (Hart et_a1., 1965). Ptacek e£_a1. (1963) reported hyperglutamicacidemia, general— ized hypoaminoaciduria, and hypogammaglobulinemia in their patients. The hyperglutamicacidemia and hypoaminoaciduria have been confirmed by McIntyre et a1. (1965). Serum glutamic-oxalacetic transaminase was normal in Ptacek's sample. Silver detected a high level of serum lipid, phosphorus in his patient (Silver, 1964). Genetic studies have been fragmentary. Both an autosomal recessive inheritance and an autosomal dominant inheritance with reduced penetrance have been suggested (Ptacek eg_§;., 1963; Opitz a:_§10: 1965). Borghi, Ghiusti, and Bigozzi concluded that the BDL Syndrome was the result of dominant, recessive, and accessory gene interaction (Borghi et_a1., 1954). No environmental factor was apparent in the published cases. No parental or maternal age effect has been observed. The syndrome has been found in most European populations and in Caucasians and Negroes in the United States. Recent studies have re- vealed sibships with several affected children or relatives. Two sets of identical twins have been found to be concordant for the syndrome (Opitz et_a1 , 1965). Nine miscarriages were detected in published family histories (186 pregnancies) (Opitz e£_a1., 1965). No consanguinity for the parents has been detected. Cytogenetic studies have been contradictory. Acentric fragments, a B/G translocation, and partial trisomy for a region on an A group chromosome resulting from a trans- location of that region to a G chromosome have been ob— Served (Jervis gt_a1., 1963; Massimo e§_a1., 196A; Geudeke et a1., 1963; Ford, 196A; Falek et a1., 1966), Opitz criticizes the partial trisomy and suggests that the patients may have a syndrome closely resembling but not identical to the BDL Syndrome (Opitz and Smith, 1966). The majority of karyotypes reported have been normal. This research was conducted to collect data that would permit an unambiguous diagnosis of the syndrome based upon clinical observations and-biochemical criteria. The clinical and biochemical information was also used in an attempt to elucidate the heredity of the syndrome and its probable cause. METHODS AND MATERIALS The state homes for the mentally retarded were screened for patients exhibiting the characteristics of the Brachmann—de Lange Syndrome. The medical records were checked to exclude extraneous syndromes from the sample. The final sample consisted of five Caucasian males and one Negro female. All of them were between ten and sixteen years of age. Two of these patients had been included in the study by Hart eg_g;. (1965). A sixth patient died shortly before the present study was begun; however, the results of the autopsy and other clinical information were available. One of the boys expired during the course of the study; however, an autOpsy was unable to be performed. The parents of each child were contacted via the medical superintendents, and interviewed personally regarding their family histories and the developmental history of their affected child. Permission was obtained to collect blood and urine Specimens from each of their children as well as themselves. The parents were questioned informally about the history of the pregnancy leading to the birth of the affected child. Information was obtained concerning drugs taken prior to or during pregnancy by the mother or by the father preceding conception, possible exposure to radiation, ‘41 and contact of the mother with infectious diseases during the pregnancy. Data were collected pertaining to mis- carriages and stillbirths in the family and among the relatives, possible occurrence of another affected child in the pedigree, and the appearance of individual components of the syndrome in the parents, sibs, and relatives. In— formation regarding the presence of other forms of mental retardation or mental illness was obtained. Further data such as birth order, age of parents, possible consanguinity, and other genetic information were collected. Whenever possible the responses of the parents were verified by other relatives. Controls were selected from within the three insti- tutions in which the patients resided. The controls were chosen at random from patients of similar age and weight who exhibited no known biochemical disorders. Parents of children who were being admitted to the institution and who had no known biochemical disorders were used as controls for the BDL parents. Clinical Analyses Fasting blood and twenty—four hour urine samples were used for the biochemical and clinical tests. The following tests were performed to confirm scattered reports in the literature. Serum glucose was determined by the Nelson— Somogyi method (Nelson, 1944; Somogyi, 19A5). Serum phospholipids were assayed according to Youngburg and Youngburg (1930). Serum cholesterol was measured by the method of Crawford (1958). Total serum amino acid nitrogen was determined according to Danielson (1933). Urinary creatinine was assayed by the method of Folin (191A). Urinary Amino ACids Two hundred fifty microliters of twenty—four hour urine samples were chromatographed on Whatman 3mm paper and developed in two dimensions using n-butanol: methanol: acetic acid: water (500:500:3:250) and n-butanol: acetone: diethylamine: 29% ammonium hydroxide: water (A50:A50:45: 0.75:228) respectively. The dried chromatograms were sprayed with 0.5% ninhydrin in n—butanol, heated at 1000 C., and the individual spots eluted with one m1. of 50% aqueous ethanol. The optical density was determined spectrophoto— metrically at 5A0 millimicrons. Urinary amino acids were expressed as milligrams of amino acid nitrogen per mg. creatinine. Serum Amino Acids Serum proteins were precipitated from 1 m1. of serum with 1 m1. of 10% trichloroacetic acid and centri— fuged. The supernatant was heated in a boiling water bath for fifteen minutes to reduce the acidity. Standard 0.1% solutions of the serum amino acids were treated similarly. One hundred microliters of serum extract were Spotted on a 0.3 mm. layer of Whatman CC-Al cellulose powder (avail- able from Reeve Angel Corporation, Clifton, New Jersey). 10 The powder slurry (10 g.: 20 ml. water) was layered on 20 cm. x 20 cm. glass plates with a calibrated spreading de- vice (A. H. Thomas, Philadelphia) and allowed to dry at room temperature. Ten microliters of each standard were spotted on each of two plates. One plate was developed unidimensionally in chloroform: methanol: 4% NHMOH (2:2:1) and the other in n-butanol: ethanol: 0.5 N ammonium hydrox- ide (3:1:1). The serum extracts were chromatographed two dimensionally in these solvents and the plates were sprayed with a ninhydrin solution consisting of the following: Solution A 50 ml. 0.2% ninhydrin in anhydrous ethanol 10 m1. glacial acetic acid 2 m1. collidine Solution B 1% cupric nitrate in anhydrous ethanol Fifty parts of A were mixed with three parts of B immediately before use (Moffat et_§1., 1965). The plates were heated over a hot plate at 1000 C., and the spots circled. The amino acids were scraped from the plates, eluted from the powder with one m1. of 50% aqueous ethanol, and centrifuged. The supernatant was decanted into a cuvette, and the Optical density read at 540 millimicrons in a spectrophotometer. A blank was prepared by scraping a similar quantity of powder from a region of the plate over which the solvents 11 had passed and treating it similarly. Standard curves were prepared using known amounts of amino acids. Serum Keto Acids Serum alpha-keto acids were isolated as their 2,4-dinitrophenylhydrazones by a method modified from that of Seligson and Kvamme (Seligson et_a1., 1952; Kvamme et_a1., 1954). Two and one-half milliliters of serum were used, and 2.5 ml. of 10% trichloroacetic acid were substituted for meta—phosphoric acid. The supernatant was diluted ten times with distilled water, and 0.2 m1. of 0.5% 2,4—dinitropheny1- hydrazine in 6N HCl was added. The reaction was allowed to proceed for thirty minutes. The reaction mixture was ex— tracted three times with 1.5 m1. of chloroform: ethanol (4:1). The combined solvent layers were extracted with 1.5 ml. of 1N Na2003, and the solvent was discarded. The Na2CO3 solution of the dinitrophenylhydrazones was washed with 1.0 m1. of CHCl :EtOH (4:1) and acidified at 0—40 C. with 3 0.5 ml. of 6N HCl. The solution was extracted three times with CH0132EtOH (4:1) using 1.0, 0.5 and 0.5 ml. respectively. The aqueous layer was discarded and the combined extracts were evaporated to dryness at 0 to 4 degrees in a gentle steam of air. The dinitrophenylhydrazones were redissolved in 0.1 ml. of CHCl32EtOH (4:1) and spotted on plates coated with a 0.3 mm. layer of cellulose powder slurry (Whatman CC—4l, 1 part powder to 2 parts water, available from Reeve Angel Corporation, Clifton, New Jersey). The chromatogram l2 was developed with n—butanol: ethanol: 0.5N ammonium hydrox— ide (70:10:20). An ultraviolet lamp was used to locate the spots (sensitivity 0.01—0.l micrograms). The spots were circled, scraped from the plates, treated with 1.0 m1. of 1N NaOH, and shaken-for ten minutes. After centrifuging, the supernatant was placed in a cuvette and read at 455 millimicrons (sensitivity 0.2 micromoles). Serum Lipids Serum lipids were extracted with chloroform: methanol (4:1). One hundred—fifty microliters of the extract were spotted on plates coated with a 0.3 mm. layer of SG—4l (Reeve Angel) and chromatographed with chloroform: methanol: water (65:24:4) (Wagner et_al., 1961). The plates were sprayed with 50% H2304 and heated over a hot plate set at 100° C. Serum Carbohydrates Serum sugars from the trichloroacetic acid extract were separated by thin layer chromatography. Fifty micro- liters of extract were spotted on Whatman 00-41 (0.3 mm. layers) cellulose powder and developed unidimensionally in ethyl acetate: pyridine: water (2:122). The plates were dried and rechromatographed in the same direction with the same solvent system. The reducing sugars were detected by carefully immersing the plates in a solution of silver nitrate in acetone. The solution was prepared by dissolv- ing 0.122 grams of silver nitrate in 1 m1. of water and 13 adding the solution to 200 m1. of acetone. The fine preci- pitate which formed was redissolved with a minimum amount of water (Sherwood and Jermyn, 1965). The plates were re— moved from the bath, dried and sprayed with 0.5N NaOh in ethanol. The reducing sugars were indicated by dark brown spots on a light brown background. The amounts of the sugars were determined by a spot area technique. Known quantities of sugars were spotted, dipped and sprayed. The Spots were traced on mm.2 graph paper, and their areas deter— mined. The areas were plotted on semilog paper (areas along the linear axis, weights along the log axis) (Randerath, 1965). Linearity was observed to hold from one to forty micrograms of glucose. Serum Protein Analyses Serum albumins, alpha—globulins, beta—globulins, and gamma-globulins were measured by the phosphate turbidimetric method using standard phosphate solutions prepared by Hycel, Inc. (Moran, 1963). Plasma protein analysis involved immunoelectrophoresis, acrylamide disc electrophoresis, and Cohn fractionation followed by disc electrophoresis. Immunoelectrophoresis was performed by the micro method of Scheidegger (1955). Prepared antibodies were obtained from Hyland Laboratories (Hyland Laboratories, 4501 Colorado Blvd., Los Angeles, Calif.). The electrophoretic medium consisted of two grams of Difco agar noble, fifty milliliters of borate buffer 14 (pH 8.2), fifty milliliters of distilled water, and ten milligrams of sodium azide. The electrode vessel buffer was prepared with 3.092 grams boric acid, 1 m1. 8N NaOH, and-3 ml. 1N H01. The pH of this solution was 8.4. The electrode buffer was diluted 75:25 with distilled water to give the gel buffer, pH 8.2. The agar mixture was heated to 100 degrees on a hot plate until dissolved. Three milliliters of solution were applied to each micrOSCOpe slide and allowed to solidify. Antigen wells and the slit for the antiserum were prepared with a cork device con— sisting of two parallel double edged razor blades and two glass capillary tubes. Figure 1.--Slot and Well Cutter for Immunoelectro- phoresis. 15 The antigen wells are 1 mm. in diameter and situated 3 mm. from the antiserum trough. The dimensions of the antiserum trough are 1.0 mm. x 45 mm. The antigen-well plugs were removed by suction with a Pasteur pipette connected to an aspirator. One lambda of plasma was introduced to each antigen well such that patient and control were run on opposite sides of the antiserum trough. The wells were sealed with-cooled liquid agar. A potential gradient of 45 volts was applied to each slide for 45 minutes. The ,. electrophoresis apparatus is shown in Figure 2. Buffer Chambers Water Bath Cover //I / Slide Supportw AV H20 9% 7/// Figure 2.——Immunoelectrophoresis Apparatus 16 The electrode and inner buffer chambers were connected by Whatman 3mm filter paper bridges. Buffer solution was conveyed to the microscope slides by Whatman 3mm filter paper bridges by an agar bridge of the same consistency as that on the slides. Following the electrophoresis the antiserum trough plug was carefully removed with a razor blade. Five one—hundredths milliliter of antiserum was applied uniformly along the groove with a 100 lambda pipette. The optimal concentration of antiserum was deter- mined by trial and error. The slides were placed on a level surface in a moist chamber (glass dishes containing a vial of water and covered with cellophane), and placed in an incubator set at 18C 0. The precipitin lines were fully developed after 36 hours. The slides were washed for 36 hours in three changes of 0.8% NaCl and dried under filter paper. The dried slides were fixed in 2% acetic acid for five minutes, stained in 0.5% Buffalo Black 10B (Amido- schwarz 10B) innwthanol-glacialacetic acid 9:1 for five to ten minutes, and washed three times in MeOHzHOAc (9:1) for fifteen minutes each (Scheidegger, 1955). The resultant immunoelectropherograms are stable indefinitely. Total human antiserum and specific human protein antiserums were used and are listed below: Anti—Human'Serum Anti-Human Ceruloplasmin Anti—Human Alpha-2—Haptoglobin Anti—Human Beta Lipoprotein Anti—Human Beta lC/Beta 1A Globulin Anti—Human Gamma G l— J \] Anti—Human Immunoglobulins Anti-Human Transferrin Anti—Human Alpha-2—Globu1in Anti-Human Gamma M Anti—Human Gamma A Anti-Human Orosomucoid The Cohn alcohol fraCtionation technique (method 10) was modified to separate serum, leukocyte, and erythrocyte proteins (Cohn gt_al., 1950). The same modification was used for each series of proteins in order to permit possible comparisons between them. Ten milliliters of a solution consisting of 250 m1. of 95% ethanol and 2.5 m1. of sodium acetate (0.4 x 10-6g. per ml.) per liter were added to 2.5 ml. of serum at 00 C. The solution was added gradually over a period of one minute. The reaction mixture was ad- justed to pH 5.8-5.9 with 0.1 N H01. (In this step and all succeeding steps, 0.5 ml. of reaction mixture was diluted to 2.5 ml. with 0.02M NaCl. The pH of the diluted mixture was adjusted to the desired pH. The number of drops of 0.1N H01 or 0.1N NaOH required to adjust the pH to the de— sired range was then multiplied by a factor which was deter- mined by trial and error, and the requisite number of drops added to the master tubes.) The suspension was stirred fifteen minutes at -50 C. and centrifuged at 4000 r.p.m. for thirty minutes at -50 C. The pellet contained fractions I, II, and III; and the supernatant contained fractions IV, V, and VI. All manipulations from this point on were carried out at —50 C. One milliliter of a solution con— taining 100 ml. 95% ethanol and 27.4 g. zinc acetate 18 dihydrate per liter was added to the decanted supernatants. The protein suspensions were allowed to stand for thirty minutes, centrifuged thirty minutes, and the supernatants decanted. The supernatants contained Fraction VI. The volumes were measured and 1 ml. was withdrawn.for a biuret determination. Seventeen and one—half milliliters of a solution composed of 160 ml. 95% ethanol, 2.6 g. barium acetate, 20 m1. 1M sodium acetate, and 7.28 ml. 1M acetic acid per liter were added to the precipitates; the pH was adjusted to 5.5—5.6; and the suspension was stirred for one hour. The tubes were centrifuged for thirty minutes and the supernatants (Fraction V) decanted. The volumes were measured, and 1 m1. withdrawn for prOtein determi— nations. Two and one—half milliliters of a solution con- sisting of 160 ml. 95% ethanol, 0.10 g. zinc acetate dihydrate, and 50.0 ml. 1M sodium acetate per liter were added to the pellets, and the pH was adjusted to 6.1-6.2. The suspensions were stirred for one hour, centrifuged thirty minutes, and the supernatants (Fractions IV—6+7) decanted. The volumes were measured and 1 m1. withdrawn for biuret determinations. Precipitate IV—l was resuspended in 2 ml. of 0.15M sodium chloride, and the proteins were brought into solution with 10 drops of 0.5M sodium citrate. The volumes were measured, and 1 m1. withdrawn for protein determinations. Precipitates I+II+IIIwere stirred into a paste and resuspended in 5 ml. of a solution containing 150 ml. 95% 19 ethanol, 2.0 ml. 1M sodium acetate, 1.40 ml. 1M acetic acid, and 45.0 g. glycine per liter. (This solution will be referred to as solution 7.) The pH was adjusted to 5.5. The reaction mixtures were stirred for fifteen minutes, centrifuged thirty minutes, and the supernatants decanted. The volumes were measured, and 1 ml. withdrawn for biuret determinations. This fraction was designated fraction II. Precipitate I+III was stirred into a paste, and 10 ml. of a solution composed of 160 ml. 95% ethanol, 45 g. glycine, 2.5 ml. of solution 7, 3.2 m1. of 0.5M disodium phosphate, and 2.4 ml. of monosodium phosphate per liter added. The pH was adjusted to 6 8-6.9. The suspension was stirred for one hour, centrifuged forty-five minutes, and the super- natants (fraction Ill-0) decanted. The volumes were mea- sured, and 1 ml. withdrawn for protein determinations. The precipitates were stirred into a paste and treated with 2.5 m1. of a solution consisting of 160 ml. 95% ethanol, 1.2 ml. 1M citric acid, and 120 ml. 1M sodium citrate per liter. The protein suspensions were adjusted to pH 7 l-7.2, stirred for one hour, centrifuged thirty minutes, and the supernatants (fraction III—1,2) decanted. Their volumes were measured, and 1 m1. withdrawn for biuret determinations. Precipitate I+III-3 was redissolved in 2 ml. of 0.02M sodium citrate. The volumes were measured, and 1 ml. withdrawn for protein determinations. 20 The solutions in the preceding paragraphs were pre- pared fresh for each set of fractionations. The biuret determinations were performed as follows. One milliliter of the fraction was diluted to 2 ml. with 0.8% sodium chloride. Two milliliters of the dilution were added to 5 m1. of biuret working reagent. The stock biuret solution contained 11.25 g. of sodium potassium tartrate, 100 ml. 0.2N sodium hydroxide, 3.75 g. cOpper sulfate pentahydrate, and 1.25 g. potassium iodide per 250 ml. The working solution was derived from the stock reagent by diluting it one to five with 0.2N sodium hydrox— ide (Weichselbaum, 1965). The reaction mixtures were placed in a water bath at 37° C. for ten minutes, cooled five min— utes, and read in a spectrophotometer at 552.5 millimicrons. A standard curve was constructed using aliquots from a standard serum. The serum was standardized by microkjeldahl total nitrogen and non-protein nitrogen techniques (Wong, 1923; Folin, 1919). The average of four determinations on a pooled serum sample was used for the standard value. Total sera and the serum fractions were subjected to disc electrophoresis in polyacrylamide gels. The gel column included a 7% gel, 5% gel, stacking gel, and sample gel (Fig. 3). The Gel compositions were adapted from Davis (1962). The gel compositions are presented in Table 10 21 Sample Gel (1.00m) ——)- Stacking Gel (0.5cm) -—fi> "" 5% Gel (1.5cm) __€> 7% Gel (6.5cm) -—e» I.D. 0.5cm Figure 3.——Polyacrylamide Gel Columns The tubes were made from flint glass tubing and placed vertically in rubber caps (available from Canalco). The gel solutions were introduced sequentially using a 10 cc. disposable syringe with a 21 gauge needle. The 7% gel was layered first followed by a 2 mm. layer of distilled water added drop by drop down the side of the glass tube with the same syringe. The gels (in caps) were placed upright in a test tube rack, put in the dark, and allowed to polymerize for thirty minutes. The water layer was removed by invert— ing the tube and gently blotting the water on paper toweling. The 5% gel was then layered on the 7% gel and the above 22 TABLE 1.--Stock and working solutions for disc acrylamide gels. pH 8.3 I. Stock Solutions A. IN H01 48 ml. B. 1N HCl approx. 48 ml. TRIS* 36.6 g. TRIS 5.98 g. TEMEDX 0.23 ml. TEMED 0.46 ml. H90 to 100 m1. H20 to 100 ml. ’ pH 8.9 Adjust pH to 6.7 with 1N HCl. 0. AcrylamideX 28.0 g. D. Acrylamide 10.0 g. BISXo 0.735 g. BIS 2.5 g. H20 to 100 ml. H2O to 100 m1. E. Riboflavin A mg. F. Sucrose 40 g. H20 to 100 m1. H30 to 100 m1. G. Acrylamide 10.0 g. BIS 0.368 g. H20 to 50 ml. 11. ‘Worklng Solutions Sample and 7% Gel 5% Gel Stacking Gels 1 part A 1 part A 1 part B 2 parts C 2 parts G 2 parts D 1 part H20 1 part H20 1 part E 4 parts 0.14% 4 parts A. P. 4 parts A. P. Ammonium Persulfate (A. P.) III~ salts: TRIS 6.0 g. The buffer was diluted 1 Glycine 28.8 g to 10 before use H20 to 1 liter pH 8.3 * Sigma 7—9, Sigma Chemical Company, St. Louis, Mo. 'TEMED: N,N,N',N'-Tetramethy1ethylenedlamine BIS: N,N'—Methy1enebisacry1amlde XAvailable from C“na1 Industrial Corp. (Canalco), Rockville, Md. 23 procedure repeated. After the water was removed, the stack— ing gel was introduced to the tube, overlayed with water, and the gels allowed to polymerize in fluorescent light. This was accomplished by standing the tubes approximately three inches from a desk type fluorescent lamp. Polymer- ization time was approximately fifteen minutes. The water layer was removed, and the sample layered on the stacking gel with a ten lambda micropipet. Ten ml. of serum, 10 ml. of serum fraction IV—l, or 30 ml. of the other serum fractions were applied to the column. The sample gel was gently squirted against the side of the tube such that the sample was mixed and trapped within it. This gel was over- layed with water and allowed to polymerize in fluorescent light. Polymerization time was approximately thirty min- utes. The length of the sample gel had to be increased proportionally with increased sample volume, approximately 0.5 mm. per ten lambdas of volume. Completion of polymer- ization was signified by the appearance of a distinct line at the gel—water interface. This was accompanied by the disappearance of the yellow color and appearance of a slight cloudiness in the riboflavin—catalyzed gels. The tubes were removed from the base caps and in- serted, sample gels uppermost, into silicone stoppers (Canalco) such that they protruded slightly above the stopper. The balance of the tubes was filled with diluted buffer. The electrophoresis apparatus was constructed from 24 pine, glass tubing, rubber bands, platinum wire (upper electrodes), a pyrex baking dish, copper wire, one-holed rubber stoppers, and galvanized clothesline (lower electrode). Top View 00000 00000 If... Bottom 1 Electrode ‘ll L.- J . r r .. W \h i,ss \ u . L, j v Figure u.-—Disc Acrylamide Electrophoresis Apparatus. The silicone stoppers were inserted into the bottoms of the buffer tubes, and diluted buffer poured down the glass rod and the buffer tube walls to a point one-inch from the top. This buffer contained a few drops of 0.5% aqueous Bromphenol Blue as a traCKing dye. Diluted buffer was poured into the baking dish, and the electrophoresis 25 circuit completed. The tracking dye, which marks the buffer front, was allowed to approach a point 1 cm. from the lower end of the arylamide tube. The gels were gently removed from the tubes with a 10 cc. syringe equipped With a 21 gauge needle and filled with buffer. The needle was slowly inserted between the gel and the tube wall while applying a gradual pressure on the syringe plunger. The tube was then slowly turned (pressed tightly against the needle), and a steady gentle pressure maintained on the plunger. This procedure intro— duced a film of buffer between the gel and the tube. The tube was inverted, and the technique repeated. The gel then fell free into a container filled with buffer. The gels were stained individually in test tubes with 0.5% Buffalo Black in 7% acetic acid for one hour or longer. Destaining was accomplished electrophoretically in a bath of 7% acetic acid. The gels were preserved in stoppered test tubes containing 7% acetic acid. The protein bands were quantitated on a Densicord scanning photodensitometer (Photovolt, N. Y., N. Y.) using white light, The curves were automatically integrated, and the protein content calculated from the percentages (area under peak A/total area) and the total prOtein in the fraction (biuret deter- mination), Leukocyte and Erythrocyte Proteins This procedure was repeated on erythrocyte and leukocyte extracts. Ten lambdas of erythrocyte fraction .1 VI or thirty lambdas o: the remaining erythrocyte fractions were introduced to the gel columns. Forty lambdas of leukocyte extract were employed in the electrophoresis. Leukocytes were isolated from 10 ml. of heparinized blood by allowing the blood to stand at room temperature for approximately one hour. The erythrocytes sedimented out leaving the leukocytes suspended in the plasma. The leukocytes were cultured three days in a medium consisting -aboratories, Detroit, Michigan) to L— -| of l0 ml. TC-l99 (Difco which 0.1 cc. of Penicillin/loo ml. (300,000 units/cc.) and 0.025 cc. of Streptomycin/lOO ml. (1 g./2 5 cc.) had been added, 2.5 ml. of serum, and four or five drops of Baoto- phytohemagglutinin (P: (the latter may be obtained from Difco). The leukocytes were spun down in a clinical centrifuge washed in 0.85% sodium chloride six times, suspended in 0.5 ml. of saline and sonicated fifteen seconds at the maximum setting. The extracts were electrophoresed in the pH 8.3 buffer and gel system mentioned previously, and at pH 4.5 according to Reisfeld egflal. (1962). The compositions of the stock and working solutions for the latter system are presented in Table 2. The arrangement of the gels is identical to that in Figure 3. TABLE 2.—-Solutions for 27 disc Acrylamide electrophoresis pH 4-5. I° II. A. lN KOH Glatia Acid TEMED H20 to BIS H20 to Ribofl H90 to Sucros H20 to Working 8 Stock Solutions IL AGE +4.1!”; tHOAc) PH “.3 Acrylamide avin e olutions 7% l part A 2 parts F 1 part H2 A parts suffers Gel O 0.28% A.P. Beta Alanine HOAc H20 to pH 4.5 —- *‘tm--m .v— .m- _v . *A.P, 48 ml. B. lN KOH 48 ml. HOAc 2.87 ml. 17.2 ml. TEMED 0.46 ml. “.0 ml. H?0 to 100 ml. 100 ml. ‘ pH 6.8 l0 g. D. Acrylamide 10 g. 2.5 g BIS 0.373%. lOO ml H20 to 50 mi. A-0 mg. F. Acrylamide 30 g. l00 ml. BIS 0.8 g. H20 to 100 ml. 90 g. l00 ml. Sample and 5% Gel Stacking Gels 1 part A 1 part B 2 parts 0 2 parts 0 l part H.0 1 part E * A parts .28% A.P. A parts G 3l.2 g. The buffer was diluted 8.0 ml- one to ten with distilled l000 ml. water before use '1'. uzzmbfiwtflmvw_ . m“ viz-If— Ammonium Persglfate. 28 The erythrocytes were washed six times in 0.85% saline. The supernatant following the final centrifu— gation was decanted, and the cells sonicated at the maxi— mum setting for fifteen seconds. The erythrocyte extracts were electrophoresed in the pH 8.3 buffer and gel system. Serum Enzyme Activities Activities of the serum enzymes associated with glutamic acid were measured by colorimetric procedures. Serum glutamic-oxalacetic transaminase and serum glutamic- pyruvic transaminase were assayed by the methods of Reitman and Frankel (1957). The procedures were modified by halv— ing the quantities of serum and reagents they used. A serum blank was prepared for each sample by omitting the incubation period and adding 2,A—dinitrophenylhydrazine to 0.1 ml. of serum and 0.5 ml. 0 85% sodium chloride immediately. The reagent blank was modified to include 0.6 ml. of distilled water, 0.5 ml. of 2,A-dinitrophenylhydrazine, and 5 ml. of 0.AN sodium hydroxide. In both cases, standard curves were prepared as suggested to give results comparable to those obtained by the ultraviolet method (Karmen e:flal., 1955). All tubes were read at 505 millimicron 0: Serum glutaminase was determined by a method modified from the L—asparaginase technique of Mashburn and Wriston and the L-glutaminase procedure of Moister tMashburn et al., 1963; Meister, 1955). The substrate contained 0 08M L—glutamine in 0 05M Tris buffer, pH 8.6. Six-tenths 29 TABLE 3.-—Solutions for deriving SGOT standard curve. m1. Standard m1. Water Unit Equivalent 0.0 2.2 0 0.1 2.1 20 0.2 2.0 55 0.3 1.9 95 0.A 1.8 148 0.5 1.7 216 1 ml. of 2,A—dinitrophenylhydrazine and 10 m1. of 0.4N sodium hydroxide were added to each tube as outlined in the above method. milliliter of serum was added to 1.7 m1. of substrate and incubated at 37C 0. for thirty minutes. One—tenth milli- liter of 1.5M trichloracetic acid (TCA) was added to stop the reaction, and the mixture was centrifuged for five minutes. Serum blanks were prepared by adding TCA before the enzyme to 1.7 m1. of substrate and centrifuging. Five— tenths milliliter of the supernatant was combined with 7.0 m1. of water and 1.0 ml. of Nessler's Reagent. A reagent blank was prepared by adding l.0 ml. Nessler's Reagent to 8.7 ml. of water. After ten minutes, the color was read at 480 millimicrons. An ammonium sulfate solution contain— ing 1A micrograms of nitrogen per 0.5 ml. was used as a standard. Activity was expressed as: micrograms N liberated/mg. serum protein/min -i... . 1A _i _.- U) C“) Leukocyte Enzyme Analyses Peripheral leukocytes from 10 cco of blood were iso- lated, and their extracts prepared as outlined about (Leukocyte and Erythrocyte Proteins)o Leukocyte L—glutamic dehydrogenase activity was measured by the procedure of Strecker {1955)0 The NADP-specific enzyme was tested by substituting 001 mlc of NADP solution (3 micromoles/mlo water) for the NAD specified by Streckero In both the NAB—specific analysis and the NADP—specific analysis, 003 mln of leukocyte extract was substituted for the 001 ml° of purified enzymeo RESULTS Biochemical Studies Silver's report of an elevation in serum phospholipids was not upheld in this sample (Table A). The patient mean was actually lower than the control mean, although the difference was not significant. It is interesting that one of the controls had a value exceeding that quoted in Silver's paper (12.8 mg/100 ml.) (Silver, 196“). The data contained in Table A fail to show deviations by serum amino acid nitrogen, glucose, and cholesterol from control ranges. Control 3 and patient 3, both of whom had glucose levels below 50 mg.% are now deceased. Ptacek et_al. reported a low gamma globulin level in his patients (Ptacek et_al., 1963). The phosphate buffer fractionation failed to demonstrate significant differences between patient and control gamma globulin concentrations (Table 5). Other evidence tends to support Ptacek's finding and will be reported below. Serum albumins, alpha globulins, and beta globulins fell within the control ranges. Thin layer chromatography displayed fifteen amino acids. Positive identification was hampered somewhat by trailing caused by the excess acid, however, gradual heating 31 :32 TABLE U.--General clinical tests. Phospholipids A. A. Nitrogen Glucose Cholesterol (mg%) (mg%) (mg%) (mg%) Control 1 9.7 7.0 . 76.0 192 2 10.3 9.1 52.7 165 3 13.7 7.3 A3.7 200 A 9 7 8.9 72.1 170‘ 5 9.7 9.7 62.6 250 BDL 1 8 u 5.8 70.6 208 2 8 5 A.A 5A.5 2AA 3 0 u 8.5 u7.0 220 A 11.8 10.0 71.5 180 5 9.2 8.2 83.8 20M Accepted Range 2-1-7 5—83 50—90b 150—250a YC i scc 10.6 i 0 8 8.9 i 0.5 61.9 i 6.0 195 i 15 I? i St, 9.2 i 0 6 7.u i 1 0 65.5 i 13.1 211 i 10 F (Crit. Reg.)c 0.10u, 9.60M (0.05,u,u) Fd 1.919 0.277 1.093 2.095 t (c. B.) -2.306, 2.306 (0.0558) t 1.u58 0.881 -0.u62 -0.870 aThese values were taken from Hawk's Physiological Chemistry, lUth ed., B. L. Oser, ed., New York: McGraw-Hill, 1965, pp. 977—979. DJ. E. Middleton and w. J. Griffiths, Brit. Med. J., 2 1525, 1957. CThe pairs of numbers represent the upper and lower bounds, respectively, of the critical region. dTest on homogeneity of variances. .pcmopoa Empw CH commomaxo mum 0090p 20 005005H 33 000.0- 000.0- 000.01 000.0 000.0 0 Ammmodv 000.0 .000.0. .0 .0 o 000.H . 000.0 000.0 000.0 000.0 m A0.:m00.00 000.0 .00H.0 .0 .0 0 0H.0H00.0 00.0H00.0 0H.0H0H.H 00.0H00.0 00.0M0H.0 000 H 0M 00.0Hm0.0 00.0H00.0 00.0HNH.H 00.0H00.0 00.0H0H.s 000 H 0m mm.H 00.0 00.0 00.0 00.0 , 0 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 00.0 00.0 0 00.0 He.0 00.0 00.0 00.0 0 00.0 00.0 00.H 00.: 00.0 a 400 mmé mm.o mmé ma.m mm.w 0 0.0.0 mm.o Hmé om.: 00;. z 2.0 00.0 :mé 00.: mmé m 00.0 00.0 00.0 00.: 00.0 m 00.0 00.0 00.0 00.0 00.0 H Homoeoo mEEmw mpmm MQQH< wcflESQH< Hmpoe mcafisooao .oocpoe coapmpfidflompa Hoozm ”mam>oa aflopopq Edpomll.m mqm mo omSMomo com: pwmp :2: hoopflczuccm: * .mpocpo on» no o xMoQ oo ocoomopmoo 0p mpmodom s xmom .moampococa pocpo 0:» mo 3 smog mpwEonpamm m ocm .m.: mxmoo mo saw one .mpocpo on oaowmeEoo ohm OH one .©.w.m.m.a mxmmm .opommmoumuoc canoaonomm 3.m 3.0 ©.o 03 m.owm.m m.H w.m m.m a.3 m 3oo.o- m.oflm.m 3.3H3.m m.m 3.3 m.e m.s 9.0 s.m 3 mmm.o+ 3.3Ho.mH m.mum.om a.om m.ms a.03 m.63 0.0m - m.ma a m30.3n a.m3Ho.mm 3.mHm.s© 3.mm $.3m a.©m s.ae 3.m®3 “.mm m.em m.m3 n.003 m.ma 3.003 e 3mm. I m.mmws.mom m.mmflo.ss3 m.m3m s.som m.mom 0.0mm m.mm3 u.ma m.3m3 m.mm3 3.3mm m.oam m eao.o+ 3.mmflw.oa3 m.omflm.sa3 m.mam w.smm m.mam m.omm 0.3m3 m.om3 3 33a m.smm 0.6mm 3.3m3 ©.m33 3 mmo.o+ 3.sHHm.mm3. o.m3wm.033 n.33a m.sam a.mm3 m.mma 3.3a m.nm3 H.0e _.3©3 m.maa m.003 o.om3 m "memwa a.mws.33 5.0Hm.m ®.a 3.33 m.s 3.3 3.3 m.3 3.3 3.04 m.mm 5.3 3.33 m s3o.m- m.ou3.m s.owm.3 a.m m.3 m.o m.3 v.0 m.m 3.3 s.o s.0 3 p mmmflaw ommmow s10 one muc 3-0 am.“ mum 3-0 31amm mnaam muaam Huqam 366m .mCOflthpcoocoo sampona opzoomcszMIl.HH mqmm mm.o 0.: **.m .m wom.o msH.o m.ma mo.H m.: *.m .w mmm.o mmm.o o.m: sm.o :.H .w .m mmo.o mmm.o m.ma mm.o o.H .U .2 mmm.o :om.o o.m: mm.o s.: **.o .2 :ma.o mmm.o 0.5: mm.o m.: .w .m omfi.o mmm.o m.sm ms.o w.m *.w .m Aemv Aewv A.ws Ampficov Aemsv Aawev =x= um \.QHE\mpHcDV Boom cm< mH cflmposa use .mwumaz .eowm .eaom oapmpsaw opmxumgafim .eflom shampsamuu.ma mamas 64 are listed in Table 17. Table 16 contains additional data for glutamic acid, alpha-keto glutaric acid, and serum glutamic oxalacetic transaminase (SGOT). The parental controls were selected from parents, twenty to forty years of age, of patients being committed to Lapeer State Home. The parents had no known biochemical disorders. The Scheffé analysis demonstrated significant differences be- tween patient and control, patient and family, and family and control means for glutamic acid. A similar pattern was observed for alpha-keto glutaric acid. SGOT levels differed significantly between patient and control, patient and family, but not family and control means. The patients appear to have low levels of gamma globulin; however, the differences between their levels and those of either the controls or their relatives were not statistically signifi- cant. Both patient and family means for Protein "X" were significantly higher than that of the controls, but they did not differ from each other. Although the family mean for leukocyte NAD-linked glutamic dehydrogenase appears to be lower than that of the controls, the difference is not statistically significant. L. B., the twin sister of K. B. lacked the NAD- glutamic dehydrogenase and had high levels of glutamic acid and alpha-keto glutarate, but her SGOT activity was normal. 65 TABLE l6.--Serum glutamate, serum alpha—keto glutarate, and SGOT. GA AKG SGOT (ms%) (ms%) , (Units) A. Patient Controls 1 0.40 0.89 2 1.05 0.40 3 1.85 0.57 4 0.70 0.96 5 1.00 0.23 Refer to 6 1.00 0.10 Table 7 7 0.95 0.38 8 1.00 0.60 9 1.80 0.23 10 1.85 0.22 11 1.80 0.34 12 1.00 0.27 EA 1 SEA 1.20 i 0.14 0.43 i 0.08 25.4 i 3.5 B. Parental Controls 1 1.30 0.85 31.8 2 1.70 0.68 17.5 3 1.95 0.48 23.1 4 1.45 0.66 16.5 5 1.02 0.40 25.0 6 1.09 0.57 27.5 7 0.90 0.79 40.0 8 0.45 0.51 27.0 9 0.67 0.71 40.0 10 1.12 1.07 25.3 IE 1 SEE 1.16 i 0.14 0.67 i 0.06 27.4 i 2.5 t 0.196 —7.717** -0.424 **Significant at the 0.01 level. 66 .CHHSQOHu mEEmw .mocwHHm> Ho mHmzamcm zmzloco OHHpoemedlco: momHHmznamxmsnm 3 "mm .oocmHHm> Ho mHmmamcm mmzloco H mm .m can < Ho came oopstmz on» ma m+flw mHOHHo pmmocmpm H mm mAwHCmHmQ 6cm mwcHHnHmv mucmema mo6H Ho.o we» 66 pemoaeficwamo .H6>6H No.0 esp pm enmefloaemame .He>ea mo.o an» em pcmoaeacmamm o e e m+ew I em 0 O 0 .mwmlmM o o o o 0 AM I mm - ememeem emmm.ma. emom.m emoa.s mm eoama.mm emmsm.mm . emmmm.em _ mm m.: H m.ea omo.o H sma.o mmo.o H omm.o m.m H :«mm oa.o H ms.o mm.o H mm.m mmm H mm no.0 H no.0 woa.o H smH.o mHo.o H maa.o s.w H m.ws ma.o H mm.m mw.o H mo.m mmm H em m.m H e.sm oo.o H No.0 ea.o H 0H.H mmm H mm s.m H w.mm eao.o H meo.o omo.o H ewm.o m.m H e.mm wo.o H me.o :H.o H om.H «mm H «N impasse game game Ampaeev. games. games QUIQ¢Z :N: 00 BOUW UM< <0 cameoaa : .mcmme mHHEmH cam .pcmHHMQ .Hoppzoo Ho comHHwQEooll.>H mqmmom czocxcs xmm .mstp msommNHQ mmeHHmomHz mom ocmnosm 3. 3.3 3 28...... @— Pedigree of M. B “TED >H HHH HH 86 Pedigree of T. G. .anHn pm COHuwaswcmem choc HMOHHHQE: .vmmmmomo mum .mSHcoE m pm mHsossch .pmmwmooo H .mHmonozma m>HmmoHdop OHCMS z .zaco mooswswoaam HHonmnd Ho mHmmn on» Go pmcHEHcpoo szmosz .wcfizp HmoapcmoH nflu mom ocmnoam . . assists. 2 HHH HH H 87 S. Pedigree of D. pcmcmmpm MHcoESmQQ Hapoa .pmmmoooa .m mwm um «quESmca Ho ooHQ .mamggmoosng .m Ho own map pm poHU chSOO mHm .CSOquz comp .UmpHdpom hem quoosm /NAH . u >H HHH 88 Pedigree of L. Bu. .pmpmop mcmepcw pooao ma nWMV Ho pso ma HOH pcwvaoocoo .mcHsp mzomhmocoz .czocxcs comp .Qphmp pcmHQH Ho owMHHHwomHz a hex .ostOHw / .. >H HHH HH 0 D H m < 89 Figure 15.--Tota1 Protein Profiles of BDL Family. Figure l6.-—Fraction III—1,2. APPENDIX II 90 The pH 8.3e1ectr0phoretic system revealed one band that had a mobility intermediate between those of albumin and transferrin. The pH 4.5 system displayed three bands. There were no differences between patients and controls. The leukocytes did not proliferate sufficiently to yield adequate protein for these analyses. pH 8.3 pH 4.5 III: — - Front -- - Figure 17.--Electr0phoresis of Leukocyte Extracts. Two patients who presented several traits of the Brachmann-De Lange Syndrome were tested for enzyme 91 92 activity, amino and keto acid levels, and protein concen- trations. Patient M who showed a greater resemblance to the BDL patients that patient G did not have an elevated SGOT activity but did tend to show a tendency to follow the remaining BDL pattern. It-is further evident from this data that Peak 37 in Fraction III-1,2 is not connected to the syndrome. TABLE II-1.-—Enzyme, protein, amino and keto acid levels in two fringe cases. SGOT GA AKG GG P."X" (Units) (ms%) (ms%) (3%) (5%) M 25.0 3.1 1.68 0.100 0.620 G 19.0 0.8 0.85 0.140 0.332 A. J.* 79.2 2.02 0.168 0.352 “Patient retest The urinary amino acids were quantitated by thin layer chromatography and elution for A. J. Earlier paper chromatograms which were not quantitated exhibited a visible decrease in intensity of the spots of the patients in comparison to those of the controls. 93 TABLE II-2.--Urina1ysis of creatinine and total amino acids. mg. AA/mg. mgAA/24 hr. mg. Creatine/24 hr. Creatinine A. J. 99.7 190.0 0.52 This value borders the lower range quoted by Carver (0.46—0.95) for patients three to ten years old. Since the thin layer technique gives values which are slightly higher than those obtained by column chromatography, this value is probably a high estimate. lCarver, M., and Paska, R., Ion—exchange chromato- graphy of urinary amino acids. 1. Normal Children, Clin. Chim. Acta, 6:721, 1961.