LIBRARY Michigan Stave University This is to certify that the thesis entitled CROSSED IMMUNOELECTROPHORESIS OF ALPHA-l-ANTITRYPSIN USING THIN LAYER ISOELECTROFOCUSING IN POLYACRYLAMIDE AS THE FIRST DIMENSION presented by David George Delforge has been accepted towards fulfillment of the requirements for M. S . degree in Clinical Laboratory Science (4 My r , I we, Major professor Lawrence D. Aronson, M.D. Date September 13, 1978 0-7 639 CROSSED IMMUNOELECTROPHORESIS OF ALPHA-l-ANTITRYPSIN USING THIN LAYER ISOELECTROFOCUSING IN POLYACRYLAMIDE AS THE FIRST DIMENSION BY David George Delforge A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1978 G/déé’c 7 ABSTRACT CROSSED IMMUNOELECTROPHORESIS OF ALPHA-l-ANTITRYPSIN USING THIN LAYER ISOELECTROFOCUSING IN POLYACRYLAMIDE AS THE FIRST DIMENSION By David George Delforge A two dimensional electrophoretic system, using high voltage thin layer isoelectrofocusing in polyacrylamide and crossed immunoelectrOphoresis in agarose, has been combined using a laying on technique. By laying on, all the associated problems due to electroendosmosis differences between the two media are circumvented. This system pro- vided increased resolution of the patterns associated with the polymorphic expression of alpha-l-antitrypsin (cl-AT). The system involved focusing 12.5 pg of al-AT from human serum on a 3.5-5.0 pH gradient across a 24 cm plate. Four millimeter wide polyacrylamide strips were next incised, inverted, and transferred to the surface of agarose just cathodal to agarose containing 3.0% (v/v) antibody to al-AT. Crossed immunoelectrophoresis was then carried out. The distribution of peaks was found to be similar to that seen using acid starch gel in the first dimension. A new zone just cathodal to M6 was observed. TO KATHY Your love and encouragement have made this all bearable ii ACKNOWLEDGEMENTS I wish to express my appreciation and gratitude to Dr. L. D. Aronson, my major professor, for his continuing support, counsel, and especially for the timely advice, both theoretical and practical, throughout the course of this study. I thank Martha Thomas, my academic advisor, especially for that smile and encouragement when the academic load seemed a bit too heavy to bear. My thanks also go to Dr. W. Leid for serving on my committee. Thanks also go to Dr. C. F. Pelzer for his technical advice at the beginning of this study. Thanks also go to the faculty and fellow students in the Clinical Laboratory Science program and Department of Pathology. I am grateful for the financial assistance of the Children's Leukemia Foundation of Michigan, 0RD 21195, which was available to me during the course of my graduate study. iii TABLE OF CONTENTS INTRODUCTION . Inhibitors in Human Plasma. Serine Proteases. Protease Mechanism. . Mechanism of Inhibition . . Mechanism of Inhibition by d1-AT. Biochemical Characterization. Genetics. Biochemical Abnormalities of Variants Physiological Function. Association with COPD . . Pathogenesis of d1-AT Deficiency. Intermediate Deficiency Controversy with COPD Hepatic Disease . Neonatal Respiratory Distress Syndrome (RDS). Rheumatoid Arthritis. . . . . . . . Pancreatic Fibrosis Celiac Disease. Asthma. Sex Chromosome Mosaicism. or Trisomy 21. Periodontal Disease LITERATURE REVIEW. OBJECTIVES MATERIALS AND METHODS. Human Serum . Phenotyping . Crossed ImmunoelectrOphoresis (CIEP). pH Determination. . . . . Joule Heating RESULTS. TLIEF Across the Length . . Crossed ImmunoelectrOphoresis (CIEP). DISCUSSION . SUMMARY AND CONCLUSIONS. iv Page LITERATURE CITED . . . . . . . . . . . . . . . . . . 74 VITA . . . . . . . . . . . . . . . . . . . . . . . . 87 Table LIST OF TABLES Protease inhibitors in human serum. al-AT and STIC levels in sera of various phenotypes. Pathlength of al-AT versus time of focusing Least squares analysis of pH gradient formation . Distribution of al-AT among zones in dif- ferent homozygous Pi phenotypes Comparison of the Pi-M distribution between acid starch gel and TLIEF vi Page 42 49 51 59 62 LIST OF FIGURES Figure Page 1 Analytical TLIEF of al-AT at pH 3.5-5.0 . . . 4O 2 Joule heating of TLIEF gels . . . . . . . . . 48 3 Voltage and amperage during 12 hours of focusing across the length. . . . . . . . . . 50 4 pH gradient formation during 12 hours of focusing across the length. . . . . . . . . . 52 5 CIEP of undiluted serum phenotyped as Pi-M. . 54 6 CIEP of serum phenotyped as Pi- M diluted to 0.5 mg/ml.. . . . . . . . . . . . . . SS 7 CIEP of a homozygous phenotype Pi-M . . . . . S7 8 CIEP of heterozygous phenotypes Pi-MZ and Pi-MS . . . . . . . . . . . . . . . . . . . . S8 9 CIEP of acute and chronic forms of the Pi-Z phenotype . . . . . . . . . . . . . . . . . . 60 vii INTRODUCTION Alpha-l-antitrypsin (cl-AT), quantitatively the most important inhibitor of proteolytic enzymes in human serum, is a protein which demonstrates a high degree of poly- morphism. The first indications of chemical heterogeneity 102 were given by Laurell and Eriksson in 1963. Since the development of an acid starch gel electrOphoretic pheno- typing technique in 196539 52,99 and aided by crossed immuno- electrOphoresis into agarose containing antibody specific to al-AT, by 1974 twenty-three different alleles had been described.27 With the advent of the study of al-AT with thin layer isoelectrofocusing (TLIEF) in polyacrylamide gels,4’6’7’92 the resolution has been greatly enhanced. Due to the increased resolution, the number of alleles described has 160 increased to 25. In addition, subtypes of alleles have 49’57’86’93 It seems reasonable that now been described. the increased resolution offered by TLIEF should be main- tained through a crossed immunoelectrophoresis step. Attempts have been reported combining the two techniques.7’107 They have not proven satisfactory because the resolution offered by TLIEF has not been preserved. The major problem is that they involved the molding in of the polyacrylamide, 2 which has an extremely low cathodal electroendosmosis (EEO), into agarose, which has measurably significant EEO. Such a technique will be important in defining the precise role of a deficiency of this protein with various pathological states. Inhibitors in Human Plasma Alpha-l-antitrypsin is a glyc0protein in human sera that inhibits several serine proteases. Normal concentra- tion in human sera is about 220-380 mg/dl. Under various physiological conditions including inflammation and preg- nancy its level increases considerably. 67 Heimburger lists six different protease inhibitors well characterized in human sera (Table 1). Table 1. Protease inhibitors in human serum - _.-_——_-..__——_———.._-.—_—_-__——.‘———n_ —- _———-__._ ——_—————————.—___‘___—p.—.__—— - ——-———.—.—_.—-——-———_—_— —_ _— ___..__- ‘_——————_—-__-_-——_ _—-_-_-——_—-—_—— ——o-————————.—- —— Normals (mg/d1) dl-Antitrypsin al-AT 180-280 al-Antichymotrypsin al-X 30-60 Inter-a-trypsin inhibitor IaI 20‘70 Antithrombin III AT III 22-39 Cl-Inactivator Cl INA 15-35 az-Macroglobulin dz-M 150-350 males 175-420 females With the exception of al-X, which is specific for chymo- trypsin, these inhibitors are capable of inactivating several 3 proteases. Alpha-l-antitrypsin and aZ-M are present in highest concentration. Both have a wide inhibitory capacity against serine proteases including trypsin, chymo- trypsin, plasmin, kallikrein, elastase, and thrombin. Probably the most pathophysiologically important are -AT 0‘1 and derivatives of IdI, because they have the specific ability to neutralize the elastolytic proteases of leuko- cytes. These enzymes are mediators of inflammation; they attach basal membranes and are probably responsible for the destruction characterizing pulmonary emphysema. Serine Proteases The serine proteases are a class of proteolytic enzymes characterized by the presence of a uniquely reactive serine residue. Two families have been well studied, trypsin and subtilisn. The trypsin family includes trypsin, chymotrypsin, elastase, collagenase, thrombin, plasmin, and the proteo- lytic enzymes of the complement system. The serine proteases specifically bind the tetrahedral transition state-like complex characteristic of acyl transfer reactions. All of the known covalent inhibitors of the serine proteases form tetrahedral adducts with similar geometry. The binding template is made up of a number of elements 90 acting together: (1) an antiparallel B-binding site for the acylat- ing polypeptide chain of the substrate (2) specific side chain binding sites dependent on the enzyme 4 (3) a less specific leaving group site (4) a site for hydrogen bonding to the tetrahedral oxyanion (S) the reactive serine for covalent binding to the substrate's carbonyl carbon atom. The charge relay system, consisting of a histidine imidazole nitrogen hydrogen bonded to an aspartic acid carboxylate oxygen, can also be regarded as aiding the other imidazole nitrogen at the active site in binding a proton. In the reaction sequence, this facilitates the process of proton transfer between nucleophiles, i.e., between the serine and the leaving group of the substrate. Protease Mechanism The hydrolysis of the peptide bond is initiated when the hydroxyl of the action site serine attacks the carbonyl of the substrate. Simultaneously, the proton of the hydroxyl is transferred to the nearby histidine and the serine hydroxyl oxygen forms a covalent bond with the carbonyl carbon. The substrate's carbonyl oxygen assumes at least an incipient negative charge and the carbonyl bond angles change from a planar sp2 to a sp3 tetrahedral configuration. This tetra- hedral adduct is stabilized by both the covalent bonding to the serine oxygen and by hydrogen bonding between the carbonyl oxygen and other proton donor group on the enzyme. The pro— ton delivered to the histidine is transferred to the peptidyl -NH- of the leaving group of the substrate. Another proton is "recoiled" from the other side of the histidine, S transferring the proton to the aspartate carboxylate, the third catalytically important amino acid at the active site. The peptide bond is broken after the nitrogen in the substrate receives the proton from the histidine. The leaving group is then free to break away, taking a proton from the enzyme. The acyl part of the substrate has now formed an acyl-enzyme intermediate. The carbonyl is now reverted to a planar configuration, hydrogen bonding to the carbonyl oxygen is broken, but the covalent bond to the serine oxygen remains intact. Subsequent deacylation of the acyl-enzyme is the reverse of the acylation process with the leaving group replaced by water or other nucleophile. The hydroxyl group of water forms another tetrahedral intermediate with the acyl enzyme. The proton is transferred to the serine with breaking of the covalent bond between substrate and enzyme. The serine protease molecule has now resumed its original state and is ready to hydrolyze another peptide bond. Mechanism of Inhibition Hixson and Laskowski7O presented evidence that trypsin binds to soybean trypsin inhibitor (STI) in a manner similar to the acylation of the enzyme with its substrate. Trypsin associates with either virgin (Arg-64-I1e bond intact) or with modified (Arg-64-Ile bond cleaved) STI to form a stable complex. Both the carboxyl end of the Arg and the amino end of the Ile are necessary for the inhibitory 6 function. In the modified inhibitor the resultant two chains are held together by a disulfide bridge. The mechanism entails the establishment of an equilibrium between native inhibitor, enzyme substratecomplex, and the modified inhibitor with the peptide bond hydrolyzed. ‘L* ————>¢_—T + I* T+ I‘__—;.-‘L<____?C\_ where T is trypsin, I and 1* are virgin and modified inhibitors, respectively, and L and L* are loose, nonco- valent, Michaelis-Menton type complexes between trypsin and virgin and modified inhibitors, respectively. The 5 values of Km and Km* are of the order of 10- M and inde- pendent of pH above 4.5. Since dissociation of trypsin 5 inhibitor complexes is less than 10- M at pH above 4.5, the stable complex (C) predominates above pH 4.5. In an earlier paper, Ozawa and Laskowski132 argue that the reactive center need not necessarily require an arginine in the reactive site. Since trypsin is also spe- cific for cleavage adjacent to lysine, such bonds should not be ruled out of consideration. Lysyl substitutions do not affect the trypsin inhibiting activity of STI and of chicken ovomucoid. However, lysyl substitution destroys the trypsin inhibiting activity of pancreatic trypsin inhibitor, lima bean trypsin inhibitor and turkey ovomucoid. X-ray crystallography by Ruhlmann et al.143 using bovine trypsin and bovine pancreatic trypsin inhibitor, showed the complex to be a tetrahedral adduct with a covalent bond 7 between the carbonyl carbon of Lys-lSl of the inhibitor and the gamma oxygen of the active site serine of the enzyme. Mechanism of Inhibition by dl-AT Few studies have investigated the inhibition of trypsin by dl-AT. Studies at low pH, which were used to study other antiproteases, cannot be done because dl-AT is inac- 59 tivated at low pH. Under dispute is which of the two amino acids is critical in the active site of al-AT. 22 Cohen treated the inhibitor with phenylglyoxal hydrate under conditions that modified the arginine residues; the result was blocking of the inhibitory action of al-AT on trypsin. However, investigations by Busby and Can16 suggest that the reagent also modifies lysyl residues on the a 68 l-AT. Heimburger et al. modified lysyl residues with maleic anhydride to give a polymaleoyl derivative; the result was a loss of inhibition of trypsin. 22 Cohen suggested the proteases might combine with al-AT, similar to other substrates; however, one of the intermediates was stable. Subsequent investigations have proposed two somewhat opposing hypotheses. Moroi and Yamaski126 concluded that the al-AT-trypsin complex was an acyl intermediate of trypsin through a new carboxyl terminal residue, probably an arginine or lysine and the y-oxygen of Ser-183 of the enzyme. Johnson and Travis87 found that by releasing the complex with the nucleophile benzamide hydrochloride, the released fragment of al-AT had an amino 8 terminal threonine and a carboxy terminal Ser-Lys dipep- tide similar to that found in native purified inhibitor. They concluded that trypsin cleaves al-AT at a threonine near the amino terminal end to activate the inhibitor and then becomes bound by an unknown mechanism to a carboxyl group at a different site of the al-AT. Cohen et al.23 proposed a hypothesis explaining data from both investi- gations. They found that a Lys-Thr bond is cleaved during base-catalyzed disruption of the complex and that a new carboxyl terminal lysyl residue is formed. Cohen et a1.24 18OH labeled base. The new 18 disrupted the complex using carboxyl terminal lysyl residue became labeled with O. This conforms to the known distribution of oxygens which occurs during the base-catalyzed hydrolysis of acyl esters, which would occur if the al-AT-trypsin complex were an acyl ester or if an acyl intermediate formed during the base-catalyzed hydrolysis of a tetrahedral adduct. They then hypothesized that trypsin binds to a Lys-Thr bond near the amino terminal end of the al—AT. The bond is probably analogous to that usually formed between trypsin and its substrates. Since there are no intrachain disulfide bonds at the amino terminal end in al-AT, a tetrahedral complex is most likely. Biochemical Characterization Alpha-l-antitrypsin is a glycoprotein with a carbo- hydrate moiety of 12%, consisting of galactose, mannose, acetylhexosamine, N-acetylneuraminic acid, and fucose. The 9 amino acid composition does not show any unusual features except for relatively high concentrations of aSpartate (9.8%), glutamate (12.9%), and leucine (9.9%). The protein contains two moles of cysteine per mole, which would give 94 Reported molecular weights range from 45,000 to 54,000 daltons. Glaser,58 using rise to one disulfide bridge. ultracentrifugation, reported a molecular weight of 52,100 18 reported the complete daltons. Recently, Chan et al. amino acid sequence of a CNBr-fragment of 109 amino acid residues. Genetics 102 . . . on routine exam1nat10n of Laurell and Eriksson, serum electrophoretic patterns, noted some with a low alpha-one band. Further examination revealed low levels of al-AT. Laurell100 noted slower electrophoretic mobility of the alpha-one fraction in individuals with low levels. Acid starch gel electrophoresis, thin layer isoelectro- focusing (TLIEF), and crossed immunoelectrophoresis (all to be discussed in detail in the literature review) have all been used to study the genetics of al-AT. Most obser- vations are consistent with the interpretation that low values are caused by homozygosity for a "deficiency” gene, intermediate values by heterozygosity, and normal values by homozygosity for the "normal" gene. Hence, the normal Pi-M phenotype will usually have a normal serum level of tflie protein, the extreme deficiency Pi-Z phenotype will 10 have levels of about 10-15% of normal, and the heterozygous Pi-MZ phenotype will have intermediate levels of al-AT. Fagerhol and Gedde-Dahl4O proposed a genetic model with multiple autosomal co-dominant alleles at one locus. The locus is designated Pi for protease inhibitor. Indi- vidual alleles are designated PiM, PiZ , etc. An example of a heterozygous genotype would be PiM/Piz, with a result- ing phenotype Pi-MZ. Pi-M is the most common allele with a frequency of about 0.9. Other alleles are designated by letters of the alphabet, the position in the alphabet giving an approximation of the electrophoretic mobility of a particular variant. To date, the following alleles have been reported: B, C, D, E, E F, G, I, L, M, M 86 L, MB, N, 55 2’ P, S, V, W, W X, Y, Y2 and Z. Gedde-Dahl et a1. 2, reported linkage between the Gm and the Pi loci. Rare cases of a null allele have been reported.117’148 Within the limit of sensitivity, no dl-AT could be detected in these two patients. As determined by the individual families, these findings were thought to be incompatible with co-dominant inheritance. These individuals were desig- nated as null alleles (Pi-) and relatives were assigned genotypes consistent with this allele. The genotype PiM/Pi' would give a Pi-M- and would have al-AT levels about one- half of what would be expected for the normal Pi-M. Martin et 31.116 described a patient with an dl-AT level of S ug/ml. They were able to detect this extreme low amount by rising radiolabeled specific antibody. Using these same zintibodies for crossed immunoelectrophoresis, they were ll able to demonstrate a pattern identical to that of a normal Pi-M. The existence of this null allele is therefore controversial. Recent work indicates that the M allele may be further divided into a number of subtypes. The designation is subtypes because the major allele products show just a slightly altered mobility, usually discernible only on TLIEF using narrow pH gradients. Johnson86 reported MLamb and M subtypes with acid starch gel electrophoresis. Baldwin Kueppers93 reported a heterozygous subtype Pi-MM Frants 49 66 I' and Harada et al. all reported Mle sub- 57 and Eriksson types. Genz et al. further divided the subtypes into the homozygous types Ma, Mb, and Mc and the heterozygotes into Mab, Mac, and Mbc. There is considerable overlap in the different author terminologies of these subtypes. A standard nomenclature has yet to be determined by workers * in this field. Biochemical Abnormalities of Variants The interest is in variants that are associated with a lower than normal concentration of al-AT, specifically the Z, S, and null alleles. The biochemical differences for all the different alleles have not been elucidated. 161 Yoshida et al. determined two amino acid substitutions in.the 2 versus the M protein. Glutamate was substituted * At the time of this writing (July 1978), a con- fkarence is being held in Paris, France, to decide on a nomenclature . 12 for by a lysine and another glutamate substituted for by a glutamine. The sialic acid content was also decreased by 26%, accounting for the slower electrophoretic mobility. None of the usual carbohydrate linking amino acids, aspargine, threonine, or serine, was substituted. Therefore, the low sialic acid content may be due to a conformational change introduced by the other two amino acid substitutions. Yoshida et a1.160 determined the difference of the S pro- tein to be one amino acid substitution of a glutamate by a valine. The lower amount of sialic acid on the Z protein is significant in elucidating the pathophysiology of diseases associated with a deficiency of serum al-AT. The liver is 144 found periodic the only known site of synthesis. Sharp acid-Schiff (PAS) positive inclusion bodies in the peri- portal areas of hepatocytes in Pi-Z patients with severe deficiency. Electron micrographs indicated the material to be located in the rough endOplasmic reticulum. Using fluorescent antibody against al-AT, he demonstrated this material to be antigenically related to serum dl-AT. Eriksson and Larsson34 purified and partially characterized material from PAS inclusion bodies from liver of Pi-Z patients. They noted no immunological differences between serum and this hepatic al—AT using three different immuno- llogical techniques. The material had a marked tendency to aéggregate, had no sialic acid residues, and had the same HKDlecular weight on SDS electrOphoresis. These results Snggest the material to be an asialo form of ol—AT. 13 Possible mechanisms for the low serum dl-AT in indi- viduals with the Pi~Z phenotype or other variants are: 1) less secretion, 2) decreased rate of production, and 3) increased rate of removal. The accumulation of the asialolated al-AT in the rough endoplasmic reticulum suggests the first mechanism. Sialic acid is generally accepted as important for the transport of glycoproteins from the cell. Bell and Carrell14 postulated that the incomplete addition of sialic acid causes the protein to accumulate intracellu- larly with only small amounts escaping by passive diffusion. Kuhlenschmidt et al.97 reported a deficiency of the Golgi membrane sialyltransferase. Such a deficiency could prevent sialylation. As mentioned, the low sialic acid content may be due to a conformational change introduced by the two amino acid substitutions. Reduction in the rate of synthesis is frequently associated with variant proteins and enzymes; thus, the second alternative should not be ruled out. 124 Morell et al. demonstrated asialo proteins to be rapidly eliminated from the circulation. Pricer and Ashwell137 demonstrated that asialo proteins bound preferentially to the membrane fractions of liver cells. Thus, low serum con- centration and the accumulation of the Z protein in hepato- cytes could be explained on this basis. 14 Physiological Function The precise physiological function of al-AT is not known. Nor is it known why there is an increase in certain physiological and pathological conditions. Alpha-l-anti- trypsin is an acute phase reactant protein. Many of the conditions in which the serum trypsin inhibitory capacity (STIC) is raised involve inflammation. Increases have been noted in gout, rheumatoid arthritis, diabetes melli- tus, gastric disease, cirrhosis, hepatitis, jaundice, acute pancreatitis, renal disease, cardiac disease, cancer, myocardial infarction, and with bacterial infection.30 53 Ganrot and Bjerre noted doubling of base levels of dl-AT as a normal feature of pregnancy. This increase was cor- related with levels of estrogen hormones when Laurel et 103 al observed that estrogen-progestin oral contraceptives elevated levels of al-AT. Lieberman and Mittman110 con- firmed this elevation but noted that patients homozygous for the 2 protein did not show any increase of STIC. Heterozygous patients doubled their intermediate STIC levels, suggesting that the single PiM 91 gene alone responds to the hormone. Kueppers noted similar increases after intravenous injections of typhoid vaccine. Recent work by Arora et al.10 suggests that al-AT plays an immunoregulatory role in the suppression of antigen dependent B+cell response without affecting adherent or T-cells. Work is presently in progress on this campus to determine the mode of action of dl-AT on cellular proteases. (I) t: 15 Association with COPD Eriksson32 reported the association of a marked reduc- tion in serum al-AT with pulmonary emphysema. The disease was unusual in that the first symptoms were noted at an early age with the onset usually occurring below 40 years of age in 60% of his population and 50 years of age in 90%. In his group of 33 patients, 23 had definite evidence of chronic obstructive pulmonary disease (COPD). The patients did not have frequent episodes of recurrent pul- monary infection. The incidence of primary emphysema was about 50%. The most noticeable abnormality was a symmetric decrease in the peripheral vasculature which was most prominent in the lower lung fields. There is general agreement that the homozygous deficiency state lends itself to a significantly higher risk of developing COPD.65 Compared to other types of emphysema, the basal predilec- tion is the most striking feature. This basal predilection is important for two reasons. It represents an important diagnostic tool and it fits a theory of pathogenesis. Pathogenesis of al-AT Deficiengy The major factor in COPD in patients with a severe al-AT deficiency seems to be a loss of elastic recoil. 15 I31ack et al. reported the loss of elastic recoil to be Iwesponsible for the decreased expiratory flow rates. In tflese patients the mechanical properties of the lung were SiJnilar to papain-induced emphysema. The ratio of collagen 144 to elastin in normal individuals is 3.4:1. In patients 16 with al-AT deficiency and advanced emphysema, this ratio increases to 4.9:1. These data indicate a decreased pul- monary elastin in emphysema. Human leukocyte proteases can degrade elastin,8O 105 75 all 79 collagen, basement membrane and arterial wall, constituents of pulmonary tissue. Janoff and Scherer demonstrated human polymorphonuclear leukocytes (PMNs) to contain large amounts of elastase. MacrOphages also 81,158 have a lysosomal elastase-like esterase. Lieberman and Gawad109 isolated two proteases from purulent sputum, a "labile" protease and a "stable" protease, which they believed to be a leukocyte elastase. They demonstrated proteolysis of lung tissue with this purulent sputum. Both proteases were shown to be inhibited by serum a —AT. 1 The correlation between the degree of inhibition of the leukoproteases and the serum trypsin inhibitory capacity indicated that individuals with an inherited deficiency of al-AT are also deficient in leukoprotease inhibitors. Janoff77 demonstrated human PMN elastase to be inhibited 130 demonstrated that two by al-AT. Ohlsson and Olsson neutral granulocytic collagenases are also inhibited by al-AT. The general working hypothesis revolves around a I>rotease—protease inhibitor imbalance in emphysema. This h)7pothesis rests with the assumption that proteolytic erlzyme activity may exceed proteolytic inhibitory activity irl the lung. The hypothesis is supported by experimental 17 models of emphysema which can be produced by intratracheal instillation or aerosolization of proteolytic enzymes. Papain has been used experimentally to produce emphysema 60,134 118 instilled human PMN in hamsters. Mass et al. homogenates to produce emphysematous lesions in dogs. Elastic fiber destruction was noted with these emphysema models. In view of the ability of the enzyme to degrade elastic fibers and basement membrane at neutral pH, elastase was considered by Janoff76 as a possible mediator of lung damage in emphysema. Janoff et 31.82 instilled purified PMN elastase into dog lung. They found that elastase, acting alone, was capable of rapidly inducing emphysematous lesions. They demonstrated with immunohistochemical techniques that the elastase was localized in close proximity to lung elastic fibers. Ultrastructural immunohistochemical techniques demonstrated that the elastase penetrated into intercellular regions of the connective tissue interstitium and attached to interstitial elastic fibers. These studies were done in vitro and in viva. Other PMN proteolytic enzymes cannot be excluded. The generalized destruction of alveolar elements suggests that alveolar glycoproteins and basement membrane may also be susceptible. Wilson et al.159 demonstrated that sequestered granulo- cytes damaged the capillary membrane of the lung. The mechanism probably involved microsomal proteases because degranulation was usually present. Discontinuity of the alveolar capillary membrane is considered to be the earliest 18 156 assumed morphological sign of emphysema. Welch et al. this process led to an accelerated develOpment of COPD in al-AT deficiency. Heinmann and Fishman69 demonstrated filtration of granulocytes in pulmonary capillaries to be a continuous normal process. The greater perfusion of and, hence, sequestration of granulocytes in the lower lobe compared with the upper could explain the greater extent of tissue destruction seen in the lower zones in al-AT deficiency. The protease-protease inhibitor imbalance theory of emphysema suggests that proteolysis of lung connective tissue can result either from a deficiency of a protease inhibitor or from a protease overload. The above cited models of a protease overload are examples of the latter. Familial emphysema associated with al—AT deficiency is an important form of the former. Intermediate Deficiency Controversy with COPD The prevalence of homozygotes Pi-Z in the general popu- lation is only about 0.1 to 0.2%; hence, homozygosity will account for only a very small proportion of patients with COPD. Of greater importance to the etiology and patho- genesis of COPD is the potential significance of the heterozygous state. Frequency estimates of the heterozygous state are estimated between 5 and 14% of the general popu- 1ation.65’121 There is considerable controversy regarding the clinical irnportance of intermediate levels of al-AT. In analogy, the 19 assumption could be made that the heterozygote usually has some dysfunction that is only fully deve10ped in the 108 112 deficient homozygote. Lieberman and Lieberman et al., using the STIC assay, identified heterozygotes in 17 and 18%, respectively, in COPD populations. This frequency was higher than the 4.7% found in control populations. He concluded that heterozygosity predisposes to COPD. In contrast, Larson et al.,98 also using the STIC assay, reported 8.0% heterozygotes in a chest clinic population and 15.2% in their control p0pulation. These data refute the hypothesis of intermediate deficiency as a cause of COPD. Kueppers et al.,95 using antigen-antibody crossed immunoelectrophoresis, reported 25.5% heterozygotes in a COPD population and 11.7% in their control group. The higher percentages reflect the increased sensitivity offered by crossed immunoelectrophoresis. About half of the patients classified as heterozygous are missed if only STIC levels are assayed. The more recent studies to evaluate the susceptibility of the MZ are basically of four types: (1) physiological, radionuclide, and pathological exam of symptomatic Pi-MZ's compared with Pi-M subjects (2) the distribution of Pi phenotypes in patients with COPD and in a control group (3) pulmonary function of asymptomatic Pi-MZ's, usually found as relatives of patients with COPD, compared with Pi-M controls (4) pulmonary function and/or symptoms in a large p0pulation. 20 Kanner et al.88 conducted a study of the first type. They concluded that heterozygosity was an important factor in the development of COPD. The remarkable result from their study was that, by using regional 133 Xe ventilation, perfusion scans and statistical analysis, they could classify Pi-M, Pi-MZ and Pi-Z into three separate pul- monary dysfunction groups. This indicated that lung disease in the heterozygote was different than that found in the deficient patient. In the second type of study Cox et al.29 concluded that there was an increased risk of COPD in the Pi-MZ but not in the Pi-MS. They pointed out the extreme importance of accurate phenotyping pro- cedures when assessing the relative risk of the hetero- 35 . . . u51ng a consecutlve series of zygote. Eriksson et al., autopsies, concluded that heterozygosity predisposed to both liver and lung disease but did not influence the survival rate. It should be noted that phenotyping could not be done in this study. Deficiencies were inferred by the presence of PAS positive granules in the liver. Using the third type of study, Mittman et al.122 noted the presence of lung parenchymal disease was seen more fre- quently in the relatives with intermediate STIC levels than in relatives with a normal STIC. However, a high percentage of the relatives were smokers. These investigators were the first to postulate the role of external irritants as having a synergistic role in the develOpment of COPD in the heterozygote. Aronson et al.,9 using a selected population of young asymptomatic heterozygotes who were not smokers, 21 gave preliminary evidence that all asymptomatic Pi-MZ's have evidence of small airway disease. Studies of the 135,152 fourth type in large unselected pOpulations revealed no evidence that the Pi-MZ phenotype puts the person at an increased risk for the development of COPD. The differences noted in the last two types of studies suggest that studies done on Pi-MZ groups made up of rela- tives of patients with COPD may be detecting other familial factors that, in conjunction with a moderate degree of al-AT deficiency, could lead to COPD. Galston et al.51 studied the relationship between a familial variation of leukocyte lysosomal protease and al-AT. What they found was that normal elastase-like esterase and leukoprotease activities appeared to be associated with an unfavorable clinical course in patients with intermediate or low STIC. Low activity was associated with a favorable course in those patients with intermediate or low STIC's. Taylor 149 clouded this conclusion by electrOphoresing and Kueppers granulocytic lysosomal extracts at pH 8.6. Three mobili- ties of elastase were noted--fast, intermediate, and slow. The esterase activity was lowest in the slow electrophoretic category. Patients with COPD had a four-fold prevalence of this slower mobility elastase. These observations are the opposite of what would be expected if the only deter- minant in the proteolysis theory were an increase in protease concentration. The authors suggested a potential nuechanism to explain this dilemma. The existence of struc- tnrrally and/or functionally altered elastase that is 22 incompletely inhibited by al-AT, or an abnormal elastase and abnormal al-AT may contribute to an abnormal interac- tion between protease and protease inhibitor. Results from both these studies imply that further investigations are necessary . Hepatic Disease The first hint of a relationship between childhood liver disease and a deficiency of al-AT was noted by Sharp et al.145 When surveying serum protein electrophoretic patterns, the first serum with a low alpha-globulin band was that of a child with hepatic cirrhosis. This child and a brother were shown to be al-AT deficient. Ten unrelated infants of the Pi-Z phenotype were reported who had hepatitis or cirrhosis of the liver. Other causes of hepatic cirrhosis were excluded. The disease starts with hepatosplenomegaly, elevated bilirubin, alkaline phospha- tase and transaminases. During the following six months, bilirubin returns to normal but alkaline phosphatase, transaminases, cholesterol and triglycerides remain moder- ately elevated. Histologic findings are cholestasis and periportal fibrosis. Only about 10-20% of Pi-Z infants develop the disease.1 All with the disease show signs of a slowly progressive cirrhosis even though cholestasis diminishes. Heterozygous infants apparently have no clinical manifestation of the disease. In the adult, cirrhosis is a well established hepatic manifestation of 61-AT deficiency. It does not differ from 23 cirrhosis from other causes. Cirrhosis may be seen 12,25 associated with emphysema or may be the only manifes- tation of a deficiency.”’74 In both children and adults, the major abnormality is a deposit of an amorphous substance in the parenchymal 144 was the first to describe these liver cell. Sharp deposits. They were best seen in the cytoplasm of a variable number of hepatocytes, after saliva or diastase digestion as PAS positive globules. The material was identified as similar to al-AT by fluorescent tagged anti- al-AT. This accumulated material is an asialated form of al-AT, and possible mechanisms for its accumulation have already been discussed in this manuscript. Electron microscopy located this material in the lumen of the rough endoplasmic reticulum, which was markedly dilated. No globules were observed in the Golgi apparatus. The number of globules varied between hepatocytes. Ishak et al.74 111 demonstrated the globule-containing and Lieberman et al. cells to be randomly distributed through the lobule, but more abundant in periportal areas and around hepatic veins. A periportal distribution is evident in livers with portal fibrosis or cirrhosis, the globule-containing cells being adjacent to collagen septa. With fibrosis or cirrhosis, necrosis was also observed. Aagenaes et al.2 noted the globules only in the parenchymal cells, not in the Kupffer cells. They also estimated that between 20-60% of the hepmtocytes contained the globules and that the percentage twas higher in the homozygous deficient than the 24 heterozygous patient. In adult patients, the specific lesions have been identified in the heterozygous Pi-FZ,45’46 17,111,133 111 Pi-SZ and Pi-MZ as well as the Pi-Z phenotype. The association between cirrhosis and a heterozygous deficiency of al-AT may be only a fortuitous one,48’125 or there may be a definite correlation as argued by Palmer et al.133 150 and Triger et al. As with the early studies of the heterozygote and COPD, the discrepancies arise because definitive phenotyping procedures were not used or were not available. The synergistic role of alcohol and other xenobiotics cannot be ruled out. There is no obvious relationship between the accumu- lation of al-AT in the hepatocyte and hepatic damage. It is not known why a decrease in serum a -AT and/or its 1 accumulation in the liver may lead to hepatocellular necrosis, fibrosis, or cirrhosis. The hypothesis of a protease-protease inhibitor imbalance has not successfully 54 been applied to the liver. Gans suggested leukocyte or Kupffer cell proteases could be responsible for the damage when serum concentrations are low. Lieberman et 31.111 proposed that increased vulnerability results from the globules of al-AT in the hepatocytes. Neonatal Respiratory Distress Syndrome (RDS) 36 Evans et al. observed that 12 of 14 infants with R08 had low STIC and elastase inhibitory capacity. They noted that levels returned to normal when the infant improved. Mathis et a1.119 observed that in a series of 25 34 Pi-M infants half had significantly lower serum levels of al-AT. The severity of RDS was inversely proportional to the serum levels of al-AT. With improvement, levels returned to normal. The lung hyaline membranes stained with fluorescein-labeled antibody to a -AT. The initial 1 low levels can probably be explained by absorption to the membrane. Levels return to normal with disappearance of the membrane. Rheumatoid Arthritis 28 Cox and Huber reported a significantly higher inci- dence in adults of heterozygotes having classical or definite rheumatoid arthritis. Children with juvenile rheumatoid arthritis had no difference in their Pi pheno- types. Since elastase and other proteolytic enzymes, released from leukocytes in the synovial fluid, are known to attack cartilage,78 inadequate amounts of the major proteolytic inhibitor could be hypothesized as having a role in intensifying the cartilage destruction. Pancreatic Fibrosis 50 Freeman et al. presented a case report of pancreatic fibrosis in a Pi-Z woman who also had emphysema. A search for other causes of the fibrosis yielded negative results. They then speculated on whether al-AT plays a role in pro- tecting the pancreas. C)‘ 7—1“ I]? m 26 Celiac Disease 151 Walker-Smith and Andrews, on examination of 13 children with untreated celiac disease, found one child with severe deficiency of 01 -AT and four with intermediate 1 levels. In another group of 15 children on a gluten free diet with the same disease, five had intermediate levels 61 reported a patient with of al-AT. Greenwald et a1. severe deficiency having emphysema, cirrhosis and intes- tinal mucosal atrophy. Asthma Katz et a1.89 screened a population of children with severe atopic bronchial asthma. They found the al-AT levels and phenotypes to be similar to a control group. However, one difference was noted. The children with steroid-dependent severe asthma had a higher incidence of heterozygosity for the 2 protein than did the non-steroid dependent and the control groups. Sex Chromosome Mosaicism or Trisomy 21 Aarskog and Fagerhol3 suggested a causal link between sex chromosomal mosaicism and severe or heterozygous variants of al-AT. Kueppers et al.96 found a significant increase of heterozygotes in a small pOpulation with sex 47 also suggested chromosome mosaicism. Fineman et al. heterozygosity as an etiological factor in trisomy 21. The question of a possible role of al-AT in cell division has not been investigated. 27 Periodontal Disease Allen and Spicer5 reported an apparent interaction of Pi variants with the ABO blood system. The Pi-M2 with an A or B blood type is more susceptible to perio- dontal disease, while Pi-M2 with an 0 blood type is more refractory toward the disease than the normal Pi-M. LITERATURE REVIEW Various electrophoretic procedures over the last 15 years have played an integral role in the study of al-AT, especially in elucidating the genetics of the protein. Laurell and Eriksson,102 after screening 1500 paper gel electrophoretic patterns from diseased patients, noted five patients with the d1 band missing. Agar gel electro- phoresis showed no band sharpening of the a zone. The l l lipoprotein and orosomucoid levels were normal. Three a of their five patients were noted to have widespread pul- monary lesions. The sister of one of the patients had the same lung disease. In addition, one patient had a history of rheumatoid arthritis. The suggestion of a link with a deficiency of the al zones and the pulmonary lesions prompted Eriksson32 to further investigate one of the families. The pr0positus had dyspnea at age 34 which steadily progressed. At age 36, the diagnosis of emphysema was made. Agar gel electro- phoresis revealed an absence of the 61 zone in both this patient and a sister who was also diagnosed as having emphysema. A brother, who had died earlier of complications of emphysema, was presumed to have had an absence of the al zone. The electrOphoretic distribution of the 28 29 antitryptic activity, determined by using DL-arginine p-nitroanilide hydrochloride as a substrate, was determined after separation in agar. Alpha-l-antitrypsin levels were determined by subtracting the aZ-macroglobulin activity from the total antitryptic activity. Eriksson found three levels of 6 -AT in this family: 1 normal, 60% and 10% of normal. Combined with the agar gel electrophoresis information, he concluded the deficiency and emphysema correlation to be familial. The deficient woman married a normal man and had four children with 60% of al-AT. These data indicated a recessive mode of inheritance. Laurell99 introduced and gave experimental details of crossed immunoelectrOphoresis. The technique involves separation of protein in one direction followed by immuno- precipitation against antibody at right angles to the first direction. In this first demonstration, Laurell used agarose gel electrOphoresis of whole human sera at pH 8.6 as the first dimension. A strip of agarose was then cut along the axis of migration and set into a trough of equal dimension in agarose containing antibody to whole human sera. In this technique, current is then passed through the antibody containing agarose. The antigens (different proteins in the sera) are forced into the antibody contain- ing agarose. They migrate until the zone of equivalence is reached, where they precipitate as antigen-antibody complexes. Hence, the size of an antigen peak is 30 pr0portional to the antigen concentration and inversely proportional to the amount of antibody in the agarose. The crossed immunoelectrOphoresis technique was used by Axelsson and Laurell11 to give the first indication of the electrophoretic polymorphism of al-AT. Using agarose gel electrophoresis as the first dimension followed by crossed immunoelectrOphoresis in the second against an antibody specific for al-AT, they came up with three zone patterns,i.e., normal, slow and double. This distribu- tion of zones and the presence of normal and deficient STIC led the authors to conclude that the phenotypes could be explained by different combinations of three allelo- morphic, autosomal genes for a -AT. 1 Independent of these observations, Fagerhol and Braend,39 using a horizontal discontinuous acid starch gel electrophoretic procedure at pH 4.95, revealed a pat- tern of three or four "prealbumin" bands of varying strength. These patterns were found in five different combinations. The "prealbumins" for the majority of the patterns appeared as a three band distribution of "one weak in front of two relatively heavy bands." This pattern was designated MM for medium mobility. A slower moving pattern of three bands was designated as SS for slow mobility. Four zone patterns of lower staining intensity which seemed to be a combination of the MM and either SS or a faster mobility were designated as MS, FS, and PM. They did not observe an FF pattern. The authors proposed a genetic theory of three codominant alleles which they 31 called PrF, PrM and PrS. The MM pattern was found in 96% of 390 randomly selected donors. Fagerhol and Laurell41 then joined forces, exchanged sera and determined that Fagerhol's "prealbumin" and Laurell's al-AT were identical. By using discontinuous acid starch gel in the first dimension and crossed immuno- electrOphoresis, they described six zones attributed to al-AT. The original three band pattern was identified as bands 2, 3 and 4. One more anodal band and two more cathodal bands were demonstrated by the sensitive immuno- precipitation step. All the homozygous patterns except the ZZ had the same six zone pattern. Only three zones were seen with the 22, probably because of the low serum concentration associated with the 22. Heterozygote pat- terns fit the pattern of one allele product superimposed on another, with the major zones showing about half the staining intensity. The terminology of Pr (prealbumin) was replaced by Pi (protease inhibitor) in this paper. Pi was chosen for al-AT because it is the major protease inhibitor in human serum and trypsin is only one protease that it will inhibit. In his review article, Fagerhol37 recognized that the Pi system consisted of at least seven codominant alleles. The seven known alleles at that time were PiF, PiI, PiM, PiS, Piv, Pix, and P12. At that time there were no known excep- tions to the codominant mode of inheritance. Fagerhol38 modified the acid starch gel electrophoresis procedure and was able, in addition to the six zones, to 32 demonstrate two more zones in the homozygous phenotype after crossed immunoelectrOphoresis. The two new zones were labeled 3 and 5 and were close to and in front of the two major zones now labeled zones 4 and 6. The rela- tive protein content of each zone, 1 through 8, was (in percent): 2.5, 14.4, 5.3, 40.9, 3.7, 33.6, 2.8 and 2.4. The three major zones 2, 4 and 6 accounted for 89% of the total al-AT in each of the homozygous phenotypes. The exception was again the 22, where zones 2 and 4 con- tained relatively more protein and zone 6 relatively less. These quantitative results are questionable because the height of each peak was the only measured parameter. No attempt was made to estimate the area under each peak. Also, the heights were compared with a standard curve obtained by using "rocket" immunoelectrOphoresis101 to quantitate the al—AT. The crossed immunoelectrophoresis is a modification of "rocket" immunoelectrOphoresis. With "rocket" immunoelectrOphoresis the area under the peak is not critical to quantitation of the peaks. As would be predicted by earlier experimentation, the distribution of zones from each allele was maintained in the heterozygous sera. Laurell and Persson104 developed what they considered to be a simpler method of Pi phenotyping. The system con- sisted of agarose gel electrophoresis at pH 5.15 followed by crossed immunoelectrophoresis. To reduce the EEO of the agarose and to sharpen the zones of the al region, linear acrylamide was incorporated into the agarose. The 33 system mandated that an internal reference solution be used. They used a carbamylated immunoglobulin L chain of the lambda type. Also, because the stain was poor in the first dimension, it was imperative that a crossed immunoelectrOphoresis be performed with every sample. The homozygous phenotype was resolved into only five zones in this system. With acid starch gel electrophoresis and crossed immunoelectrOphoresis techniques, 23 alleles had been described.27 The isoelectric point range of al-AT is about 4.2-to 4.7; albumin is about pH 4.9. Also, the heterogeneity of al-AT is only seen at acid pH. Thus, thin layer iso- electric focusing106 should have the capability of separat- ing the Pi phenotypes. Three separate reports utilizing acid pH isoelectrofocusing on thin layer polyacrylamide 4,7,92 The gels were published at about the same time. experimental protocols were similar in all three papers, and each utilized a pH gradient approximately 3.5 to 5.0. Densitometry by Allen et al.4 showed a distribution of peaks similar to that obtained by acid starch gel, followed by crossed immunoelectrOphoresis.38 The high resolution obtained by TLIEF and the sensitivity of Coomassie Blue dye allowed the majority of phenotypes to be ascertained simply by this one dimensional procedure. An explanation for the microheterogeneity had not been offered up to this point. Because the heterogeneity was similar with TLIEF, the microheterogeneity was attributed 34 to charge alone. Mega and Yoshida123 separated the com- ponents by preparative starch gel electrophoresis and DEAE-ion exchange. The anodal components contained more sialic acid per molecule than the cathodal components. The molecular size of each component was identical. The increased resolution with TLIEF is expected since proteins are maximally resolved when focusing is completed. In contrast, with the acid starch gel procedure, proteins will diffuse with time once they begin to unstack follow- ing passage of the moving boundary. Within the time span of about two years the number of recognized alleles increased to 25 and described pheno- types to about 40.136 Since the protein is inherited as an autosomal codominant allele, the combinations of possible phenotypes including the homozygous phenotypes is 325. As an aid in documenting new possible phenotypic expressions, some authors attempted to develop a crossed immunoelectro- focusing technique from the polyacrylamide gel. Arnaud et al.6 first used the technique to document that the area around the zones was indeed al-AT. The resolution was poor, as even the two major zones could not be resolved. 107 7 Lebas and Arnaud et al. used the crossed immuno- electrofocusing technique to study the genetic polymorphism of the protein. The resolution was poor in that the resolving power offered by the TLIEF was lost in the immuno- precipitation step. 35 The problem with these attempts is that they involve molding in of charge free polyacrylamide which has vir- tually no EEO into agarose which has significantly measurable EEO. Briefly, two phenomena occur when this is attempted. A buffer flow will emerge on the anodal side of the polyacrylamide strip and the cathodal side of the strip will dry, breaking the electrical current. The buffer flow will cause loss of resolution, and the breakage of electrical current will distort the field strength in the agarose. Numerous investigators have attempted to reduce the EEO of the agarose. Reports include charge modification 63,64 115 of the agarose, treatment with alkali treatment, 138 an anion-exchange resin, purification and the addition of a nonionic water soluble polymer,84 purification and augmentation of ViSCOSity,8S,155 146 and mixing of polyacrylamide into the agarose. In addition, methods have also been described using technically tedious and time consuming modification of the crossed immunoelectrophoresis. Loft114 used a procedure for crossed-line immunoelectrophoresis that involved washing the polyacrylamide with barbital buffer, using low voltage for 35 hours, removing the poly- acrylamide strip, and finally filling in the gap with 62 agarose. Groc and Jendry described a special chamber for performing the crossed immunoelectrOphoresis. Another method of solving the problem was offered by 147 Soderholm and Smyth. They introduced a simple technique of laying on the polyacrylamide for crossed 36 immunoelectrofocusing. With this method, proteins on the surface of the acrylamide are transported by diffusion and electrOphoresis into the agarose gel. The field strength in the agarose is little affected by surface application of the acrylamide gel. 8 to mold One last attempt was made by Arnaud et al. the polyacrylamide into agarose which had also been mixed with polyacrylamide to reduce the EEO. The polyacrylamide strip was laid on a glass plate, agarose with anti-al-AT was poured up to the strip, and then the whole plate was overlaid with the agarose-polyacrylamide solution. This was a technically demanding technique that mandated all procedures to be run at 4 C in order to further reduce problems from EEO. Again, the resolution was not preserved through the second dimension. OBJECTIVES The objectives of this research were to: 1. Develop a method that would combine crossed immunoelectrophoresis with TLIEF and not lose resolution. Use this method to study the microheterogeneity of alpha-l—antitrypsin. Offer the method so that it could be used to study the microheterogeneity of other proteins. 37 MATERIALS AND METHODS Human Serum The majority of human sera received in this labora- tory are from relatives of patients presented at the Chest Clinic at Ingham Medical Center in Lansing, Michigan. We are currently screening relatives of these patients, search- ing for young asymptomatic, non-smoking individuals who are Pi-MZ. Whole blood is collected by venipuncture, allowed to clot, and serum taken off after centrifugation. Serum was stored at -20 C until analysis was performed. Occasionally, sera obtained from other clinical labora- tories are sent to our laboratory for phenotyping. Phenotyping Alpha-l-antitrypsin phenotypes are determined by culling information from three independent procedures. A. Thin Layer Isoelectrofocusing (TLIEF) Thin layer isoelectrofocusing was accomplished by modifications of the methods of Allen et al.4 and Kueppers.92 Acrylamide, N,N' methylene bisacrylamide, ammonium per- sulfate (Ames Co., Elkhart, IN) and sucrose (Fisher Scientific Co., Fair Lawn, NJ) were used to make the gel. Ampholines pH 3.5-5.0 (LKB Products Inc., Bromma, Sweden) were increased 38 39 to 2.4% (w/v) and l M glycine (Sigma Chemical Co., St. Louis, MO) was used for the cathode strip. The percent of crosslinking (C)71 was increased to 5%. N,N,N',N'- tetramethylethylene diamine (TEMED) was not used since the ampholytes themselves contain tertiary amino groups to catalyze the polymerization. The gel was prefocused for 30 minutes at a constant voltage of 500 to eliminate the persulfate. Focusing was accomplished with an LKB 2117 Multiphor Basic Unit and LKB 2130 D.C. Power Supply (LKB Products Inc., Bromma, Sweden) at 4 C with cooling supplied at 10 L/min with a Lauda K-Z/RD (Brinkman Instru- ments Inc., Westbury, NY) temperature regulator. Maximum power and amperage were set at 30 watts and 30 milliamperes, respectively. Maximum voltage was set at 850. With this system, dl-AT will migrate anodal to the majority of other serum proteins into a series of distinct reproducible zones. Major zones with TLIEF at pH 3.5-5.0 are generally sufficient to determine the phenotype. Figure 1 demonstrates some of the phenotypic expressions of al-AT. Pi-M will migrate as two major zones, M4 and M6. Minor zones M7 and M8 are noted cathodal to M6; M2 is located anodal to M4. Since the protein is inherited as an autosomal codominant allele, one would expect the heterozygote to show allele products from both loci. The Pi-MZ shows the allele products of both the M and the Z loci. With the Pi-MZ, the major zone Z4 is located just cathodal to M7 and 26 just cathodal to M8. Because of the low concentration donated from the Z loci, these are the only 2 zones generally noted. 40 Anode M‘— M _ blrfi M7/'. M8 (knhodo MMZZ Ms_ S MZMZ M Figure 1. Analytical TLIEF of al-AT at pH 3.5-5.0. The of : lhe Pi-‘s CU { ii 41 The Pi-Z is obvious by its cathodal mobility and absence of zones in the region where one sees the major M zones. The major zones 24 and 26 are located as noted with the Pi-MZ. The Pi-MS has as its main feature the major S6 zone between the minor M zones M7 and M8. The S4 zone is buried under the M6 zone. The Pi-S has the same distri- bution as a Pi-M, but the whole banding pattern is shifted cathodally. B. Alpha-l-antitrypsin The radial immunodiffusion (RID) method of Fahey and McKelvey42 was used to quantitate the al-AT. Quantiplate (Kallestad, Chaska, MN) immunodiffusion plates were used. Five ul of serum and standards were pipetted into the wells. At 18 hours, the precipitin rings were measured with a PEAK (Meloy Laboratories Inc., Springfield, VA) magnifying comparator capable of measuring to one-tenth millimeter. The log of the concentration was plotted against the diameter of the precipitin rings and a best line fit determined. Unknowns were extrapolated from the graph. Normals, determined by the manufacturer, were given as 220-380 mg/dl. C. Serum trypsin inhibitory capacity (STIC) Total STIC was measured by a modification of the method 72 <9f Homer et al. The 3X crystallized trypsin (Worthington Biochemicals, Freehold, NJ) was standardized by the method 19 (If Chase and Shaw. The assay was performed by incubating