Statement on methods for detecting alpha 1 -antitrypsin abnormalities.

Notes Statement on Methods for Detecting Alpha,-antitrypsin Abnormalities* Alpha,-antitrypsin (a,-at), the principal alpha1- globulin of human plasma, inhibits a number of proteolytic enzymes including trypsin, chymotrypsin, elastase, plasmin, thrombin, and leucocytic and bacterial proteases. Genetic heterogeneity for a,-at has been demonstrated and the term " Pi system" (for proteinase inhibitor) is recommended for refer- ences to the phenotypes of this protein. The system appears to be controlled by a number of codominant alleles at a single locus. Although 21 phenotypes have been identified to date, in general population surveys more than 900% of sera are of the MM phenotype and most of the remaining sera are of either the MZ or the MS phenotypes. Abnormalities of the Pi system are associated with chronic obstructive lung disease and infantile cir- rhosis of the liver. The basis for this association is uncertain, but it appears to be related to the quantity of a,-at. The phenotype ZZ is most clearly related to disease and is associated with the lowest levels of a,-at. However, it is possible that the pathogenic mechanisms are related to the phenotype per se or some other disturbance linked to phenotype. Currently, there are three ways of evaluating serum a,-at: (1) genetic typing, (2) quantification of the serum a,-at concentration, and (3) measurement of serum inhibitory capacity against trypsin or pancreatic elastase. The determination of pheno- type requires the use of acid-starch-gel electropho- resis (Fagerhol, 1968) and antigen-antibody crossed electrophoresis (Laurell, 1965; Kueppers, 1969). Serum a,-at is measured by immunodiffusion (Mancini et al., 1965). Serum inhibitory capacity is measured, usually with spectrophotometric tech- niquLes (Hcmer et al., 1963), by determining the effectiveness of serum in blocking the action of trypsin or pancreatic elastase on a suitable sub- strate. The inhibitory methods also measure the activity of the inter-alpha trypsin inhibitor. Unfortunately, these three ways of evaluating serum al-at are not entirely interchangeable. A sim- ple relationship appears to exist between the quan- tity of a1-at and the proteinase inhibitory capacity of * This statement was drafted by the signatories listed on page 836. the serum. This suggests that inactive forms of a1-at do not occur, although rare phenotypes may have different spectra of action against different pro- teinases. The relationship between phenotype and either a,-at concentration or proteinase inhibitory capacity is less predictable, since a phenotype may be expressed in a range of protein concentrations and similar concentrations may be associated with different phenotypes. The only exception to these generalizations is the ZZ phenotype, which can be reliably and easily detected by quantitative techniques. No other phe- notype has been detected with such low protein levels or inhibitory capacities. The likelihood of a ZZ homozygote developing chronic obstructive lung disease is considerably increased. However, the exact risk cannot be estimated from available data, since most homozygotes have been found by screening groups of patients. There are a number of phenotypes associated with an " intermediate deficiency". The two most common in a general population are the MZ and MS heterozygotes. These phenotypes may pre- dispose to respiratory disease in populations exposed to certain environmental factors such as cigarette smoke. Using quantitative methods, phenotypes giving an intermediate deficiency can be distinguished from the severe deficiency of the ZZ homozygotes, but cannot be distinguished from one another. Further, as shown in the table below (Kueppers, 1971), overlap exists between the level of protein in this intermediate range and that in the normal range. Phenotype Alpha -antitrypsin Concentration(mg/JOO Ml) (± S.D.) MM 212 (32) MS 167 (37) MZ 120 (46) SS 80, 112 (2 patients only) ZZ 25 (6) Similar results are seen with the inhibitory me- thods. The alpha,-antitrypsin level in MZ and MS heterozygotes may rise into the normal range under the influence of stimuli such as surgery, infection, pregnancy, or oestrogen administration. The magni- 2 775A 835 836 NOTES tude of the response of this acute phase reactant protein appears to be related to the phenotype. In ZZ homozygotes the response is slight and the level rarely rises into the intermediate range. Anti- trypsin levels in the normal range can be seen in a number of phenotypes, but in a general population the overwhelming majority of such individuals will be MM homozygotes. Despite this overlap between the MM and other phenotypes, it is probable that limits of antitrypsin and inhibitory capacity can be determined for pre- dicting, with a high degree of accuracy, whether a given serum is or is not an MM phenotype. Such criteria apply most effectively in individuals who are not suffering from an infection, not receiving oestrogens, and are not otherwise stimulated. Sera that fall near the limits would have to be phenotyped, since a reliable prediction could not be made other- wise. Selection of the proper method for studying serum a,-at depends upon the purpose of the study. It is simpler and cheaper to measure a,-at concentra- tion or serum inhibitory capacity than to do pheno- typing. Moreover, these quantitative techniques can be carried out more quickly and can be automated. Phenotyping is required for definitive genetic assess- ment or to survey patients for a possible association between different phenotypes and disease. On the other hand, quantification of serum a1-at or serum proteinase inhibitory capacity is probably adequate for identifying persons who may be susceptible to a,-at-deficiency-related disease, since disease seems most directly related to protein quantity. Of the various quantitative methods now available, none appears to have an inherent advantage. The comparison of results obtained in different laboratories is hampered by non-standard assay methods, by variations in the activity of commer- cially available crystalline trypsin, and by changes in a,-at in diluted serum or samples stored under cer- tain conditions. The development of a reference laboratory, or standardized procedures, nmight cir- cumvent these problems. For example, a calibrated soybean trypsin inhibitor standard is being evaluated, as is the use of a molar titration of trypsin (Chase & Shaw, 1967). Each laboratory should validate its assay method with adequate abnormal and control sera. REFERENCES Chase, T. & Shaw, E. (1967) Biocheln. biophYls. Res. Communi., 29, 508 Fagerhol, M. K. (1968) Ser. Haemnatol., 1, 153 Homer, G. M., Katchman, B. J. & Zipf, R. E. (1963) Clin. Chem., 9, 428 Kueppers, F. (1969) Biochem. Genet., 3, 283 Kueppers, F. (1971) Humnangenetik, 11, 177 Laurell, C.-B. (1965) Analyt. Biochemn., 10, 358 Mancini, M., Carbonara, A. 0. & Heremans, J. F. (1965) Immunochem., 2. 234 J. Adamson M. Laskowski A. L. Baker C.-B. Laurell M. K. Fagerhol J. Liebermann G. A. Falk I. Mandl E. F. Freier R. W. Moskowitz H. H. Fudenberg R. L. Myerowitz H. Gans J. A. Pierce L. Greene R. M. Senior F. Kueppers R. Talamo