α1-Antitrypsin with unusual behaviour

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Clinica Chimica Acta, 72 (1976) 83-87 0 Elsevier/North-Holland Biomedical Press, Amsterdam -

Printed in The Netherlands

CGA 7980

cx1 -ANTITRYPSIN

WITH UNUSUAL

BEHAVIOUR

LUAN ENG LIE-INJO Hooper School

Foundation of Medicine,

and Department San Francisco,

of International Health, Calif. 94143 (U.S.A.)

University

of California

(Received April 8th, 1976)

Summary The unusual behaviour of an c~~antitrypsin, probably genetically determined, found in the serum of a Filipino in San Francisco is described. On starch gel electrophoresis the cr,-antitrypsin did not show any pattern or a very weak Pi MM type pattern. However, the protein level estimated by the immunodiffusion method and the trypsin inhibitory capacity was normal.

Introduction Deficiency of CY,-antitrypsin, the major protease inhibitor (Pi) in human serum has attracted much attention because of its association with pulmonary emphysema [l] and cirrhosis of the liver [2]. More than twenty different variants of ai-antitrypsin detectable by acid starch gel electrophoresis [3] have been reported. So far, deficiency of crl-antitrypsin is found to be associated with certain electrophoretic variants, the most severe deficiency resulting from a ZZ phenotype. Recently, complete deficiency was found to be associated with the absence of any pattern on starch gel electrophoresis [4]. In this preliminary communication the unusual behaviour of an al-antitrypsin not described before is reported. Observations

and methods

During a survey of (Y,-antitrypsin phenotypes (Pi types) by a slightly modified (as suggested by Dr. F. Kueppers, personal communication) Fagerhol’s acid starch gel electrophoretic method [3] in Filipinos in San Francisco, we noticed that the serum of one woman among more than two hundred persons examined, showed consistently no or a very weak pattern (Fig. 1) on acid starch gel electrophoresis, while her serum ol,-antitrypsin protein level and trypsin inhibitory activity were normal. If a shadow of a pattern was detectable, it resembled the MM phenotype. This weakness of pattern in comparison with normals was

Fig. 1. Acid starch gel electrophoresis [31 showing normal and deficient al-antitrypsin patterns. Slots 1, 3 and 5 al-antitrypsin patterns of three different normal controls. Slots 2 and 4 deficient patterns of Propositus, who had normal STIC and normal serum cvlantitrypsin protein level studied by radial immunodiffusion method. Fig. 2. Cross immunoelectrophoretie patterns of cYl-antitrypsin after acid starch gel electrophoresis. a, normal serum with MM phenotype: b, abnormal serum.

demonstra~d repeatedly by staining with amido black, ponceau red and nigrosine, as well as coomassie blue. The serum trypsin inhibitory capacity (STIC) using benzoylarginine-p-nitroanilide (BAPNA) as a substrate [ 51 showed normal activity, which on different occasions ranged between 1.44 and 1.77 mg trypsin inhibited per ml of serum (our normal mean was 1.40 f 0.23 mg trypsin inhibited per ml of serum). Also, the al-antitrypsin protein level was found to be normal by the radial immunodiffusion method of Mancini [6] using M Partigen plates from Behring Diagnostics, ranging between 205 and 290 mg percent (our normal mean was 238 + 54 mg percent). The phenomenon was seen with or without using filter paper for inserting the samples in the starch gel slots and therefore cannot be attributed to the influence of the filter paper. The failure to show a clear pattern on starch gel is also not due to an inability to bind protein stain because the immunodiffusion rings obtained in the immunodiffusion plates [6] could clearly be stained in normal controls as well as in the abnormal one. It was therefore thought that the a,-antitrypsin disappeared during the run in the gel. Indeed, when immunodiffusion was performed after acid starch gel electrophoresis was carried out (antigen-antibody crossed immunoelectrophoresis [ 7,8]), the resulting pattern produced by the abnormal serum was very small in comparison with that of normal controls. Two small peaks were seen corresponding to the two central, major peaks of the MM phenotype (Fig. 2). It was thought that the buffer used for preparing the gel might have an influence on the disappe~ance of the pattern during the run. When the abnormal serum was diluted 1 in 200 in the diluted acid gel buffer (21 g of citric acid and 23 g of tris(hydroxymethyl)aminometh~e dissolved in one liter of dis-

85

tilled deionized water adjusted to pH 4.6 with HCI and again diluted 15 times as used for preparing the gel), instead of in the Tris buffer pH 7.6, as done in the standard assay method [ 5) and incubated at 37°C for 2 h before the assay was carried out, a great difference in the influence upon normal and abnormal serum was observed. While it decreased the trypsin inhibitory activity of normal sera slightly or moderately, a considerable drop of activity,was observed in the abnormal serum. The cuI-antitrypsin in the abnormal serum was probably sensitive to the very dilute acid buffer as used for preparing the gel, leading to the disappearance of the 01,-antitrypsin during the prolonged electrophoretic run. The abnormality was shown to be consistent, because the serum of freshly drawn blood of the woman with the unusual ff~-antit~psin obtained 13 months after the first examination, while she was in a healthy condition, showed the same characteristic behaviour. Both parents of the carrier of this unusual cr,-antitrypsin were examined. The at-antitrypsin pattern of both parents on acid starch gel electrophoresis was easily detectable as MM phenotype. Incubation of their sera in 1 in 200 dilution in dilute acid gel buffer prior to carrying out the assay gave a higher percentage drop in activity than the average drop seen in normal controls. However, it was not as great as seen in the daughter and overlapped with findings in normal controls (Table I). TABLE I PERCENT DECREASE OF SERUM TRYPSIN INHIBITORY al-ANTITRYPSIN AND OF NORMAL CONTROLS AFTER ACID BUFFER AT 37OC

CAPACITY @TIC) OF AN UNUSUAL 2 h INCUBATION IN DILUTE CITRIC

The assay carried out with 1 in 200 serum dilution. Activity expressed in mg trypsin inhibited per ml

serum.

Propositus Propositus (13 months later) Father of propositus Mother of propositus Normal controls 1 2 3 4 5 6 7 3 9 10 11 12 13 14

Standard STIC assay

STIC assay after 2-h incubation in dilute acid buffer at 37’C

% Decrease

1.60 1.44 1.72 1.77 1.60 1.64

0.92 0.80 0.84 0.86 1.22 1.22

74.0 80.0 104.8 105.8 31.1 34.4

1.38 1.14 0.88 1.25 1.26 1.30 1.78 1.66 1.76 1.36 1.64 1.44 1.52 1.25

1.27 1.08 0.80 1.02 1.14 1.28 1.70 1.56 1.76 1.14 1.60 1.08 1.32 1.04

8.7 5.6 10.0 22.5 10.5 1.6 4.7 6.4 0.0 19.3 2.5 33.3 15.2 20.1 Average decrease in normals

11.5

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Discussion It is tempting to postulate that the daughter in this Filipino family with the unusual serum ~~-antit~psin described here had the homozygous condition for a genetically determined abnormality and expressed the abnormality in a greater degree than the parents, who had only the heterozygous condition. Although the findings in the parents overlap with findings in normal controls, such findings are not unusual in heterozygous states of enzyme abno~~ities. However, more cases have to be discovered and family studies carried out to elucidate the mode of inheritance more clearly. Also, further biochemical studies may shed more light on the nature of the instability and unusual behaviour of this ~~-~ntit~psin. The possibility that a factor in the water used for diluting the buffer (despite that distilled deionized water was used), had a more profound influence on the variant than on normals and was responsible for the unusual behaviour of the a,-dntitrypsin cannot be excluded. Genetic variants of the Pi system have so far been designated by letters of the alphabet according to their electrophoretic mobility and the absence of it by the designation Pi-. Soon the number of letters of the alphabet will be used up for this purpose or become confusing due to different centers giving the same letter to different cwlantitrypsin variants, as,has been the case with the abnormal hemoglobins and glucoseS-phosphate dehydrogenase systems some years ago. It would not be surprising to find a great number of cu,-antitrypsin variants in the future if more elaborate kinetic studies are carried out. If the abnormal behaviour of the al-antitrypsin described in this paper is indeed due to a genetically determined variant, it should be designated aI-antitrypsin M San Francisco since it has the same electrophoretic mobility as the M type, yet behaves differently. Despite the great amount of work carried out in the field of cul-antitrypsin, its precise physiological functions and the way it affects the function of certain organs are still not clear. The abnormal behaviour of the al-antitrypsin reported in the present paper may be of clinical significance, since its function under certain environmental conditions in the body may be reduced, despite the fact that its activity assayed according to standard methods in vitro is normal.

This work was supported by Research grant HL-10486 and Research grant AI-10051 to the University of California In~rnation~ Center for Medical Research (UC ICMR), both from the NIH US Public Health Service. I am grateful to Dr. E.A. Zarate and his staff from 4804 Mission Clinic, San Francisco for collecting blood samples from Filipinos and to Mr. Alejandro R. Herrera for technical assistence. References 1 2 3 4

Erikson, S. (1965) Acta Med. &and. 177 Suppl. 432 Berg, N.O. and Eriksson, S. (1972) N. En&J. Med. 287.1264 Fagerhol. M.K. (1968) Ser. Haematol. 1, 153 Talamo, R.C., Langley. C.E., Reed, C.E. and Makino, S. (1973)

Science 181. 70

87 5 6 7 6

Briscoe. W.A.. Kueppers, F., Davis, A.L. and Beam, A.G. (1966) Am. Rev. Resp. Dis. 94, 529 Mancini. G., Carbonara, A.O. and Heremans, J.F. (1965) Immunochemistry 2,235 Laurel& C.B. (1965) Anal. Biochem. 10,358 Kueppers, F. (1969) Biochem. Gene& 3,283