The Mechanisms of Antitrypsin Deficiency an Their Role in the Pathogenesis of Pulmonary Emphysema Jack L i e b e r m a n * D e p a r t m e n t of R e s p i r a t o r y D i s e a s e s , City of Hope National M e d i c a l C e n t e r , D u a r t e , C M i f o r n i a 91010, U . S . A .
A l p h a l - a n t i t r y p s i n is one of the b o d y ' s d e f e n s e m e c h a n i s m s that p r o t e c t s a g a i n s t a d e g e n e r a t i v e d i s e a s e of the lungs we r e c o g n i z e as pulm o n a r y e m p h y s e m a [1]. A n t i t r y p s i n c o m p r i s e s a l m o s t the e n t i r e alpha 1globulin peak on s e r u m - p r o t e i n e l e e t r o p h o r e s i s . It is a g e n e r a l i z e d p r o t e a s e i n h i b i t o r c a p a b l e of inhibiting such e n z y m e s a s t r y p s i n , c h y m o t r y p s i n , e l a s t a s e , c e r t a i n b a c t e r i a l p r o t e a s e s , and the n e u t r a l l e u k o c y t i c p r o t e a s e s . The l e v e l o f i n h i b i t o r in the blood is d e t e r m i n e d by the m o l e c u l a r v a r i a n t s of the i n h i b i t o r i n h e r i t e d t h r o u g h two a u t o s o m a l codominant genes. L a u r e l l and E r i k s s o n of Sweden [2] w e r e the f i r s t to r e c o g n i z e an inherited deficiency of the alpha l-antitrypsin protein, and they observed that the severe and intermediate deficiencies resulted from the homozygous and heterozygous inheritance of an abnormal gene, respectively. These investigators suggested that the homozygeus state strongly predisposed to the development of pulmonary emphysema, but that the heterozygous state most likely did not. Evidence has since been accumulating to correct the role of the intermediate deficiency, since it is now felt that an intermediate deficiency also contributes to the development of pulmonary emphysema.
PATHOGENESIS OF
EMPHYSEMA
The mechanism whereby antitrypsin deficiency predisposes to pulmonary emphysema is believed to involve the proteolytic enzymes of leukocytes and macrophages. In the event of intrapulmonary inflammation or infection, the proteolytic enzymes of these cells function to digest inflammatory debris. In the event of enzyme leakage from the cells, as occurs during the act of phagocytosis or in the event of cellular death, the living tissues are protected from proteolytic attack by the presence of alphal-antitrypsin. The inhibitor is a low molecular weight protein
*Supported by G r a n t 14HL1339~ f r o m the N a t i o n a l I n s t i t u t e s of Health.
P n e u m o n o l o g i e 152, 7-14 (1975)
8
(54,000) capable of easily diffusing out of the vascular channels into the interstitial spaces. However, when antitrypsin is absent or deficient, the proteolytic enzymes from the phagocytes are free to attack and digest the alveolar walls resulting in the pathologic picture of pulmonary emphysema [31. The presence of leukocytes and their intrinsic proteases within the lung spaces can result from infection or inflammation within the lungs, but in addition, it has been recognized that leukocytes may be sequestered within the basilar portions of the lungs as a natural storage phenomenon [41. Thus, the mere presence of stored leukocytes within the basilar parts of the lungs may contribute to the development of basilar emphysema in patients who have a deficiency of alpha I - antit rypsin.
DETECTION
OF
AINTITRYPSIN
DEFICIENCY
A s e v e r e h o m o z y g o u s d e f i c i e n c y of a l p h a 1- a n t i t r y p s i n c a n b e d e t e c t e d by simple serum-protein electrophoresis on cellulose acetate. Total a b s e n c e of t h e a l p h a l - g l o b u l i n p e a k o r a s m a l l p l a t e a u in t h e a r e a w h e r e a s m a l l s h a r p p e a k s h o u l d o c c u r i s d i a g n o s t i c of t h e s e v e r e d e f i c i e n c y s t a t e ( F i g . 1), An i n t e r m e d i a t e d e f i c i e n c y a l s o r e s u l t s in a r e d u c t i o n i n
?
°
F i g . 1. S e r u m p r o t e i n e l e c t r o phoresis patterns on cellulose acetate membranes from indiv i d u a l s w i t h a) n o r m a l a l p h a 1a n t i t r y p s i n , b) i n t e r m e d i a t e d e f i c i e n c y , a n d c) s e v e r e d e f i c i e n c y of a n t i t r y p s i n
s i z e of t h e a l p h a l - g l o b u l i n p e a k , b u t c a r e f u l q u a n t i t a t i o n i s r e q u i r e d b e f o r e a m e a s u r e of t h e a l p h a l - g l o b u l i n c a n b e u s e d a s a ~ c r e e n i n g t e s t . No i n d i v i d u a l w i t h d e f i c i e n t l e v e l s of a l p h a l - a n t i t r y p s i n in t h e b l o o d h a s b e e n f o u n d in o u r l a b o r a t o r y to h a v e a n a l p h a l - g l o b u l i n p e a k g r e a t e r t h a n 0 . 2 g% [ 5 ] . Other methods for evaluating the atphal-antitrypsin c o n t e n t of t h e b l o o d i n v o l v e e i t h e r t h e q u a n t i t a t i v e m e a s u r e m e n t of t h e a l p h a l - a n t i trypsin protein or a measurement of t h e s e r u m - t r y p s i n inhibitory capacity (STIC). The measurement of a l p h a l - a n t i t r y p s i n protein by radial i m m u n o d i f f u s i o n r e q u i r e s a r e l i a b l e s t a n d a r d and s u c h s t a n d a r d s v a r y f r o m o n e s o u r c e t o a n o t h e r . We h a v e f o u n d t h e m e a s u r e m e n t of S T I C to b e m o r e r e l i a b l e f o r d e t e c t i n g a n i n t e r m e d i a t e d e f i c i e n c y and f o r s c r e e n i n g l a r g e n u m b e r s of p a t i e n t s ~6~.
Pi Phenotypes
ZZ
Fig. 2. Alpha l-antitrypsin phenotype patterns obtained by acid-starch electrophoresis and crossed i m m u n o - electrophoresis. The M M pattern is normal
10 One problem involved in detecting intermediate alpha I- antitrypsin deficiency relates to the fact that this protein is one of the acute-phase reactant proteins whose concentration increases during acute or chronic infection, cancer, post-operatively, during pregnancy, and with estrogen therapy. Thus, the levels in individuals with intermediate deficiency taking birth control pills can be elevated into the low normal range on both the quantitative and activity assays. We believe that anyone with an STIC value of I. 2 units or less while receiving estrogens should be suspected of having an intermediate deficiency of alphaFantitrypsin [7, 8 ] . M o s t of t h e p r o b l e m s i n v o l v e d w i t h d e t e c t i n g h e t e r o z y g o t e s h a v e b e e n c i r c u m v e n t e d b y F a g e r h o l of N o r w a y [9] who d i s c o v e r e d a m e t h o d for phenotyping the alphal-antitrypsin protein. This procedure involves acid- starch electrophoresis followed by crossed-immuno- electrophorests the alphal-antitrypsin p r o t e i n m o v e s t o w a r d t h e a n o d e and b r e a k s i n t o a p a t t e r n of t h r e e m a j o r and f i v e m i n o r b a n d s . T h e r e l a t i v e m o b i l i t y a n d p a t t e r n of t h e p r o t e i n b a n d s l e a d s t o t h e d e t e r m i n a t i o n of t h e p h e n o t y p e ( F i g . 2). T h e n o r m a l a n t i t r y p s i n p r o t e i n h a s a m e d i u m m o b i l i t y on a c i d - s t a r c h and h a s b e e n l a b e l e d M. V a r i a n t s w i t h f a s t e r m o b i l i t y on t h e s t a r c h h a v e l o w e r l e t t e r s in t h e a l p h a b e t and v a r i a n t s w i t h s l o w e r mobility have been assigned higher letters. The slowest moving protein and t h e o n e c o n t r i b u t i n g t h e m o s t to a n t i t r y p s i n d e f i c i e n c y i s t h e Z variant. The homozygous ZZ phenotype primarily accounts for the s e v e r e d e f i c i e n c i e s of a l p h a l - a n t i t r y p s i n . The MZ phenotype accounts f o r m o s t of t h e i n t e r m e d i a t e d e f i c i e n c i e s . A n o t h e r s l o w m o v i n g v a r i a n t , l a b e l e d S i s c o m m o n l y s e e n and i t s h o m o z y g o u s s t a t e (SS) c a n c a u s e a n i n t e r m e d i a t e d e f i c i e n c y s t a t e ; t h e MS h e t e r o z y g o u s p h e n o t y p e c a u s e s i n t e r m e d i a t e d e f i c i e n c y in o n l y 15 to 20% of s u c h c a s e s . O t h e r v a r i a n t s r a r e l y c a u s e i n t e r m e d i a t e d e f i c i e n c y e x c e p t in t h e e v e n t t h a t m i x t u r e s of t h e s e v a r i a n t s o c c u r s u c h a s a n SS, S Z , IS, o r F S , e t c . , p h e n o t y p e
[i0].
ROLE
OF
INTERMEDIATE
ANTITRYPSIN
DEFICIENCY
In 1967 I b e c a m e s u s p i c i o u s of t h e p o s s i b i l i t y t h a t a n i n t e r m e d i a t e d e f i c i e n c y , a s w e l l a s a s e v e r e d e f i c i e n c y of a l p h a l - a n t i t r y p s i n , may pred i s p o s e to p u l m o n a r y e m p h y s e m a [6]. In a p r e l i m i n a r y s u r v e y of p a tients discharged from the Long Beach Veterans Administration Hospit a l a n d t h e C i t y of H o p e M e d i c a l C e n t e r in C a l i f o r n i a , 18%0 of 126 p a tients with emphysema were found to have an intermediate deficiency, a s m e a s u r e d b y t h e S T I C , and 8% h a d a s e v e r e o r a p p a r e n t l y h o m o zygous deficiency. This compared to the 4.7% intermediate deficiency and 0 . 4 % w i t h h o m o z y g o u s d e f i c i e n c y f o u n d in 1200 c o n t r o l s u b j e c t s . T h u s , 26% of o u r t o t a l e m p h y s e m a p o p u l a t i o n h a d e i t h e r h o m o z y g o u s
11 or heterozygous alphal-antitrypsin deficiency. The percentage rose to approximately 50% when the older individuals were eliminated and only those cases 50 years of age or younger were considered. It is believed that alphal-antitrypsin deficiency causes pulmonary emphysema at a younger age, a more severe disease, and an earlier death than occurs in individuals with emphysema from other etiologies. Eighty-three percent of individuals in this survey with an intermediate deficiency state had a Z variant in their phenotype. The other few cases with intermediate deficiency had either an SS, MS, or an IM phenotype. Thus, it appears as if the Z variant is primarily responsible for most significant deficiency states of alphal-antitrypsin. Certain differences are apparent between individuals with emphysema who have a severe deficiency of antitrypsin as compared to those with an intermediate deficiency. First of all, the individuals with a homozygous deficiency are younger averaging 45 _+ 8 years as compared to a mean age of 57 _+ P years for the heterozygotes. Secondly, there is a difference in the amount of cigarette smoking that the homozygotes have done as compared to the heterozygotes. Homozygotes had a mean smoking history of 24 pack-years as compared to 43 pack-years of cigarette smoking for the heterozygotes. The type of lung disease and the symptoms associated with the onset of their disease did not differ significantly between the two types of patients in that cough or insidious onset of dyspnea occurred in equal percentages in both groups. When asymptomatic individuals with the intermediate deficiency state were tested in the pulmonary function laboratory, certain significant differences appeared in comparison to other asymptomatic individuals with normal antitrypsin levels [Ii]. First of all, obstructive abnormalities, as demonstrated by reduced FEVI, were seen more frequently in individuals with intermediate antitrypsin deficiency than in controls, but only in smokers. Other abnormalities, on the other hand, relating to p u l m o n a r y perfusion and diffusion capacity, were also seen with greater frequency in heterozygotes than in the controls, but these abnormalities w e r e not necessarily related to a history of cigacette smoking. Thus, it is believed that heterozygotes w h o were a s y m p tomatic m a y s h o w abnormalities of p u l m o n a r y perfusion or diffusion irrespective of whether or not they s m o k e cigarettes, but obstructive changes most likely leading eventually to symptomatic lung disease are related to a history of cigarette smoking. Another interesting feature related to the detection of an intermediate deficiency of alphal-antitrypsin is the apparent ethnic and national distribution of the deficiency gene. Prevalence rates ranging f r o m 5 to 12% for the heterozygote state w e r e observed primarily in those individuals derived f r o m Northern E u r o p e a n countries such as Ireland, England, G e r m a n y , France, and Scandinavia. Relatively few individuals with the deficiency were found in Italians, those of Jewish heritage, Blacks, M e x i c a n - A m e r i c a n s , or A m e r i c a n Indians. T h e s e studies of necessity w e r e all p e r f o r m e d on A m e r i c a n s and their national origin w a s determined by taking their histories.
12 MECHANISMS
FOR
ANTITRYPSIN
DEFICIENCY
One way to evaluate alphal-antitrypsin deficiency is to study the dynamic variation in antitrypsin response to an appropriate stimulus. We administered 3 mg of diethylstilbestrol daily for 14 days to measure the increase in STIC that occurs in individuals with different antitrypsin phenotypes [81. Individuals with the homozygous ZZ phenotype were totally incapable of responding to stilbestrol whereas those with the MZ heterozygous phenotype had approximately half the normal response capability. Other heterozygous phenotypes had a slightly reduced response to stilbestrol but did not differ significantly from normal. This included MS, MP, and FS phenotypes. The SS phenotype was studied in two instances and mimicked the response seen with the MZ heterozygotes. A few individuals with an apparent MM phenotype also had intermediate deficiency levels of alphal-antitrypsin and half the response capability to stilbestrol. We postulated that these individuals had inherited an inactive or null gene for alpha 1- antitrypsin as part of their phenotype [101. T a l a m o of Boston [12] su-bsequently reported an individual with essentially no antitrypsin in his blood whatsoever w h o appeared to be a homozygote for the null or inactive gene. Thus, inheritance of a null gene m a y be one of the causes for antitrypsin deficiency, although this is probably rare. The m a j o r cause of antitrypsin deficiency is associated with the Z variant and involves a defect in either synthesis or release of the antitrypsin protein f r o m its site of synthesis within the liver. Sharp [13] w a s the first to observe the presence of unusual hepatocellular globules in individuals with the Z variant. These globules can be seen under the electron microscope to be c o m p o s e d of a rough endoplasmic r e t i c u h m dilated by the presence of a granular storage material. These globules have an affinity for binding fluorescein- or peroxidase-labeled antibody against alpha I- antitrypsin, and thus appear to contain large amounts of alpha l-antitrypsin despite the shortage of the protein within the blood. It is therefore postulated that the deficiency associated with the Z variant results f r o m inability of the protein to leave the liver cell. Another cause of antitrypsin deficiency relates to the protein's increased lability and spontaneous degradation within the blood. This observation was m a d e in our laboratory w h e n we observed that certain bloods contained a second m i n o r antigen for alphal-antitrypsin El4]. T h e antigen w a s seen in m o s t eases of h o m o z y g o u s or heterozygous individuals with Z, S, I or P variants, and in relatively few individuals with the normal M M phenotype. Since the antigen could be produced in normal blood by degradation of antitrypsin by heat or low pH, it w a s eoneluded that it w a s associated with an inactive, degraded f o r m of alphal-antitrypsin. Subsequent heat lability tests on bloods of vacious phenotypes confirmed that the abnormal antitrypsin variants did have increased heat lability and were more rapidly inactivated at 55°C [15]. Three reasons for alphal-antitrypsin deficiency are: i) A defect in the release of the Z variant from its site of synthesis in the liver. (This is the major cause of antitrypsin deficiency. ) 2) Inheritance of a
13
null or inactive gene for antitrypsin production. 3) Increased antitrypsin variants resulting in in vivo degradation,
NEED
FOR
ANTITRYPSIN
SCREENING
lability of
PROGRAMS
S c r e e n i n g p r o g r a m s for d e t e c t i n g i n t e r m e d i a t e alpha 1 - a n t i t r y p s i n defic i e n c y a r e i n d i c a t e d at t h i s t i m e b e c a u s e s y m p t o m a t i c p u l m o n a r y e m p h y s e m a need not o c c u r in t h o s e i n d i v i d u a l s with the d e f i c i e n c y if t h e y do not s m o k e c i g a r e t t e s or w o r k in i n d u s t r y w h e r e t h e y a r e e x p o s e d to u n u s u a l f u m e s and d u s t s [ 1 6 ]. Thus, d e t e c t i o n of the d e f i c i e n t s u b j e c t s at a r e l a t i v e l y e a r l y age and c o u n s e l l i n g t h e m a g a i n s t c i g a r e t t e s m o k i n g , etc., could v e r y well p r e v e n t t h e i r d e v e l o p i n g this s e r i o u s lung d i s e a s e . The t e s t i n g of e m p h y s e m a p a t i e n t s and then the f a m i l y m e m b e r s of d e f i c i e n t p a t i e n t s would be m o s t r e w a r d i n g , since l a r g e r n u m b e r s of d e f i c i e n t relatives could be discovered and counselled. Tests for alphal-antitrypsin deficiency, should eventually become an important part of preemployment examinations in industry so that susceptible individuals can be kept out of those work areas where the risk would be greatest. A cure or treatment of alphal-antitrypsin deficiency does not currently exist, although work is in progress to devise a means for L'eplenishing deficient levels of serum alphal-antitrypsin or to perform normal liver transplants for taking over this job. These treatments, of course, lie in the future but suggest that alphal-antitrypsin deficiency may be one of the correctable genetic defects that plague mankind.
REFERENCES
I. Eriksson, S. : Studies in alphal-antitrypsin deficiency. Acta Med. Scand. 177, (Suppl. 432)1 (1965) 2. Laurell, C. -B. and Eriksson, S. : Electrophoretic alphal-globulin pattern of s e r u m alphal-antitrypsin deficiency. Scand. J. Clin. Lab. Invest. 15, 132 (1963) 3. bieberma---n, J. and Gawad, M. A. : Inhibitors and activators of leukocytic proteases in purulent sputum. Digestion of h u m a n lung and inhibition by alphal-antitrypsin. J. Lab. & Clin. Med. 7__77,713 (1971) 4. Bierman, H.R., Kelly, K.H., and Cordes, F.h.: The sequestration and visceral circulation of leukocytes in man. Ann. N. Y. Acid. Sci. 59, 850 (1955) 5. Lieberman, J. , Mittman, C., and Schneider, A. S, : Screening for homozygous and heterozygous alpha l-antitrypsin deficiency. Protein electrophoresis on cellulose acetate membranes. J. Amer, Med. Ass. 210, 2055 (19~9) 6. Lieberman, J. : Heterozygous and homogous alphal-antitrypsin deficiency in patients with pulmonary emphysema. New Engl. J. Med, 281, 279 (1969) 7. Lieberman, J. , Mittman, C., and Kent, J. R. : Sreening for heterozygous alpha l-antitrypsin deficiency. III. A provocative test with Diethylstilbestrol and effect of oral contraceptives. J. Amer. Med. Ass. 217, 1198 (1971)
14
8. L i e b e r m a n , J. and M i t t m a n , C. : D y n a m i c r e s p o n s e of a l p h a l - a n t i t r y p s i n v a r i a n t s to d i e t h y l s t i l b e s t r o l . A m e r . J. H u m a n G e n e t i c s 25, 610 (1974) 9. F a g e r h o l , M. K. : The P i - S y s t e m . G e n e t i c v a r i a n t s of s e r u m alpha 1a n t i t r y p s i n . S e r i e s , H a e m a t o l . 1, 153 (1968) I0. Lieberman, J., Gaidulis, L., Garoutte, B., and IYlittman, C. : Identification and characteristics of the common alphal-antitrypsin phenotypes. Chest 62, 557 (1972) ii. Mittman, C., Lieberman, J., Marasso, F. , and Miranda, A. : Smoking and chronic obstructive lung disease in alphal-antitrypsin deficiency. Chest 60, 214 (1971) 12. Talamo, R. C., Langley, C. E., Reed, C. E., and Makino, S. : Alpha lantitrypsin deficiency: A variant with no detectable alphal-antitrypsin. Science 181, 70 (1973) 13. Sharp, II. A. : Alphal-antitrypsin deficiency. Hosp. Pract. 6, 83 (1971) 14. Lieberman, J. : A new "double ring" screening test for alp~al-antitrypsin variants. Amer. Rev. Resp. Dis. 108, 248 (1973) 15. Lieberman, J. : Heat labilily of alphal-antitrypsin variants. Chest 64, 579 (1973) 16. Kueppers, F. and Black, L. F. : Alphal-antitrypsin and its deficiency. Amer. Rev. Resp. Dis. ii0, 176 (1974) Dr. J. L i e b e r m a n Dept. of R e s p i r a t o r y D i s e a s e s City of Hope National Medical Center Duarte, Calif. 91010, USA
A l p h a l - a n t i t r y p s i n is one of the b o d y ' s d e f e n s e m e c h a n i s m s that p r o t e c t s a g a i n s t a d e g e n e r a t i v e d i s e a s e of the lungs we r e c o g n i z e as pulm o n a r y e m p h y s e m a [1]. A n t i t r y p s i n c o m p r i s e s a l m o s t the e n t i r e alpha 1globulin peak on s e r u m - p r o t e i n e l e e t r o p h o r e s i s . It is a g e n e r a l i z e d p r o t e a s e i n h i b i t o r c a p a b l e of inhibiting such e n z y m e s a s t r y p s i n , c h y m o t r y p s i n , e l a s t a s e , c e r t a i n b a c t e r i a l p r o t e a s e s , and the n e u t r a l l e u k o c y t i c p r o t e a s e s . The l e v e l o f i n h i b i t o r in the blood is d e t e r m i n e d by the m o l e c u l a r v a r i a n t s of the i n h i b i t o r i n h e r i t e d t h r o u g h two a u t o s o m a l codominant genes. L a u r e l l and E r i k s s o n of Sweden [2] w e r e the f i r s t to r e c o g n i z e an inherited deficiency of the alpha l-antitrypsin protein, and they observed that the severe and intermediate deficiencies resulted from the homozygous and heterozygous inheritance of an abnormal gene, respectively. These investigators suggested that the homozygeus state strongly predisposed to the development of pulmonary emphysema, but that the heterozygous state most likely did not. Evidence has since been accumulating to correct the role of the intermediate deficiency, since it is now felt that an intermediate deficiency also contributes to the development of pulmonary emphysema.
PATHOGENESIS OF
EMPHYSEMA
The mechanism whereby antitrypsin deficiency predisposes to pulmonary emphysema is believed to involve the proteolytic enzymes of leukocytes and macrophages. In the event of intrapulmonary inflammation or infection, the proteolytic enzymes of these cells function to digest inflammatory debris. In the event of enzyme leakage from the cells, as occurs during the act of phagocytosis or in the event of cellular death, the living tissues are protected from proteolytic attack by the presence of alphal-antitrypsin. The inhibitor is a low molecular weight protein
*Supported by G r a n t 14HL1339~ f r o m the N a t i o n a l I n s t i t u t e s of Health.
P n e u m o n o l o g i e 152, 7-14 (1975)
8
(54,000) capable of easily diffusing out of the vascular channels into the interstitial spaces. However, when antitrypsin is absent or deficient, the proteolytic enzymes from the phagocytes are free to attack and digest the alveolar walls resulting in the pathologic picture of pulmonary emphysema [31. The presence of leukocytes and their intrinsic proteases within the lung spaces can result from infection or inflammation within the lungs, but in addition, it has been recognized that leukocytes may be sequestered within the basilar portions of the lungs as a natural storage phenomenon [41. Thus, the mere presence of stored leukocytes within the basilar parts of the lungs may contribute to the development of basilar emphysema in patients who have a deficiency of alpha I - antit rypsin.
DETECTION
OF
AINTITRYPSIN
DEFICIENCY
A s e v e r e h o m o z y g o u s d e f i c i e n c y of a l p h a 1- a n t i t r y p s i n c a n b e d e t e c t e d by simple serum-protein electrophoresis on cellulose acetate. Total a b s e n c e of t h e a l p h a l - g l o b u l i n p e a k o r a s m a l l p l a t e a u in t h e a r e a w h e r e a s m a l l s h a r p p e a k s h o u l d o c c u r i s d i a g n o s t i c of t h e s e v e r e d e f i c i e n c y s t a t e ( F i g . 1), An i n t e r m e d i a t e d e f i c i e n c y a l s o r e s u l t s in a r e d u c t i o n i n
?
°
F i g . 1. S e r u m p r o t e i n e l e c t r o phoresis patterns on cellulose acetate membranes from indiv i d u a l s w i t h a) n o r m a l a l p h a 1a n t i t r y p s i n , b) i n t e r m e d i a t e d e f i c i e n c y , a n d c) s e v e r e d e f i c i e n c y of a n t i t r y p s i n
s i z e of t h e a l p h a l - g l o b u l i n p e a k , b u t c a r e f u l q u a n t i t a t i o n i s r e q u i r e d b e f o r e a m e a s u r e of t h e a l p h a l - g l o b u l i n c a n b e u s e d a s a ~ c r e e n i n g t e s t . No i n d i v i d u a l w i t h d e f i c i e n t l e v e l s of a l p h a l - a n t i t r y p s i n in t h e b l o o d h a s b e e n f o u n d in o u r l a b o r a t o r y to h a v e a n a l p h a l - g l o b u l i n p e a k g r e a t e r t h a n 0 . 2 g% [ 5 ] . Other methods for evaluating the atphal-antitrypsin c o n t e n t of t h e b l o o d i n v o l v e e i t h e r t h e q u a n t i t a t i v e m e a s u r e m e n t of t h e a l p h a l - a n t i trypsin protein or a measurement of t h e s e r u m - t r y p s i n inhibitory capacity (STIC). The measurement of a l p h a l - a n t i t r y p s i n protein by radial i m m u n o d i f f u s i o n r e q u i r e s a r e l i a b l e s t a n d a r d and s u c h s t a n d a r d s v a r y f r o m o n e s o u r c e t o a n o t h e r . We h a v e f o u n d t h e m e a s u r e m e n t of S T I C to b e m o r e r e l i a b l e f o r d e t e c t i n g a n i n t e r m e d i a t e d e f i c i e n c y and f o r s c r e e n i n g l a r g e n u m b e r s of p a t i e n t s ~6~.
Pi Phenotypes
ZZ
Fig. 2. Alpha l-antitrypsin phenotype patterns obtained by acid-starch electrophoresis and crossed i m m u n o - electrophoresis. The M M pattern is normal
10 One problem involved in detecting intermediate alpha I- antitrypsin deficiency relates to the fact that this protein is one of the acute-phase reactant proteins whose concentration increases during acute or chronic infection, cancer, post-operatively, during pregnancy, and with estrogen therapy. Thus, the levels in individuals with intermediate deficiency taking birth control pills can be elevated into the low normal range on both the quantitative and activity assays. We believe that anyone with an STIC value of I. 2 units or less while receiving estrogens should be suspected of having an intermediate deficiency of alphaFantitrypsin [7, 8 ] . M o s t of t h e p r o b l e m s i n v o l v e d w i t h d e t e c t i n g h e t e r o z y g o t e s h a v e b e e n c i r c u m v e n t e d b y F a g e r h o l of N o r w a y [9] who d i s c o v e r e d a m e t h o d for phenotyping the alphal-antitrypsin protein. This procedure involves acid- starch electrophoresis followed by crossed-immuno- electrophorests the alphal-antitrypsin p r o t e i n m o v e s t o w a r d t h e a n o d e and b r e a k s i n t o a p a t t e r n of t h r e e m a j o r and f i v e m i n o r b a n d s . T h e r e l a t i v e m o b i l i t y a n d p a t t e r n of t h e p r o t e i n b a n d s l e a d s t o t h e d e t e r m i n a t i o n of t h e p h e n o t y p e ( F i g . 2). T h e n o r m a l a n t i t r y p s i n p r o t e i n h a s a m e d i u m m o b i l i t y on a c i d - s t a r c h and h a s b e e n l a b e l e d M. V a r i a n t s w i t h f a s t e r m o b i l i t y on t h e s t a r c h h a v e l o w e r l e t t e r s in t h e a l p h a b e t and v a r i a n t s w i t h s l o w e r mobility have been assigned higher letters. The slowest moving protein and t h e o n e c o n t r i b u t i n g t h e m o s t to a n t i t r y p s i n d e f i c i e n c y i s t h e Z variant. The homozygous ZZ phenotype primarily accounts for the s e v e r e d e f i c i e n c i e s of a l p h a l - a n t i t r y p s i n . The MZ phenotype accounts f o r m o s t of t h e i n t e r m e d i a t e d e f i c i e n c i e s . A n o t h e r s l o w m o v i n g v a r i a n t , l a b e l e d S i s c o m m o n l y s e e n and i t s h o m o z y g o u s s t a t e (SS) c a n c a u s e a n i n t e r m e d i a t e d e f i c i e n c y s t a t e ; t h e MS h e t e r o z y g o u s p h e n o t y p e c a u s e s i n t e r m e d i a t e d e f i c i e n c y in o n l y 15 to 20% of s u c h c a s e s . O t h e r v a r i a n t s r a r e l y c a u s e i n t e r m e d i a t e d e f i c i e n c y e x c e p t in t h e e v e n t t h a t m i x t u r e s of t h e s e v a r i a n t s o c c u r s u c h a s a n SS, S Z , IS, o r F S , e t c . , p h e n o t y p e
[i0].
ROLE
OF
INTERMEDIATE
ANTITRYPSIN
DEFICIENCY
In 1967 I b e c a m e s u s p i c i o u s of t h e p o s s i b i l i t y t h a t a n i n t e r m e d i a t e d e f i c i e n c y , a s w e l l a s a s e v e r e d e f i c i e n c y of a l p h a l - a n t i t r y p s i n , may pred i s p o s e to p u l m o n a r y e m p h y s e m a [6]. In a p r e l i m i n a r y s u r v e y of p a tients discharged from the Long Beach Veterans Administration Hospit a l a n d t h e C i t y of H o p e M e d i c a l C e n t e r in C a l i f o r n i a , 18%0 of 126 p a tients with emphysema were found to have an intermediate deficiency, a s m e a s u r e d b y t h e S T I C , and 8% h a d a s e v e r e o r a p p a r e n t l y h o m o zygous deficiency. This compared to the 4.7% intermediate deficiency and 0 . 4 % w i t h h o m o z y g o u s d e f i c i e n c y f o u n d in 1200 c o n t r o l s u b j e c t s . T h u s , 26% of o u r t o t a l e m p h y s e m a p o p u l a t i o n h a d e i t h e r h o m o z y g o u s
11 or heterozygous alphal-antitrypsin deficiency. The percentage rose to approximately 50% when the older individuals were eliminated and only those cases 50 years of age or younger were considered. It is believed that alphal-antitrypsin deficiency causes pulmonary emphysema at a younger age, a more severe disease, and an earlier death than occurs in individuals with emphysema from other etiologies. Eighty-three percent of individuals in this survey with an intermediate deficiency state had a Z variant in their phenotype. The other few cases with intermediate deficiency had either an SS, MS, or an IM phenotype. Thus, it appears as if the Z variant is primarily responsible for most significant deficiency states of alphal-antitrypsin. Certain differences are apparent between individuals with emphysema who have a severe deficiency of antitrypsin as compared to those with an intermediate deficiency. First of all, the individuals with a homozygous deficiency are younger averaging 45 _+ 8 years as compared to a mean age of 57 _+ P years for the heterozygotes. Secondly, there is a difference in the amount of cigarette smoking that the homozygotes have done as compared to the heterozygotes. Homozygotes had a mean smoking history of 24 pack-years as compared to 43 pack-years of cigarette smoking for the heterozygotes. The type of lung disease and the symptoms associated with the onset of their disease did not differ significantly between the two types of patients in that cough or insidious onset of dyspnea occurred in equal percentages in both groups. When asymptomatic individuals with the intermediate deficiency state were tested in the pulmonary function laboratory, certain significant differences appeared in comparison to other asymptomatic individuals with normal antitrypsin levels [Ii]. First of all, obstructive abnormalities, as demonstrated by reduced FEVI, were seen more frequently in individuals with intermediate antitrypsin deficiency than in controls, but only in smokers. Other abnormalities, on the other hand, relating to p u l m o n a r y perfusion and diffusion capacity, were also seen with greater frequency in heterozygotes than in the controls, but these abnormalities w e r e not necessarily related to a history of cigacette smoking. Thus, it is believed that heterozygotes w h o were a s y m p tomatic m a y s h o w abnormalities of p u l m o n a r y perfusion or diffusion irrespective of whether or not they s m o k e cigarettes, but obstructive changes most likely leading eventually to symptomatic lung disease are related to a history of cigarette smoking. Another interesting feature related to the detection of an intermediate deficiency of alphal-antitrypsin is the apparent ethnic and national distribution of the deficiency gene. Prevalence rates ranging f r o m 5 to 12% for the heterozygote state w e r e observed primarily in those individuals derived f r o m Northern E u r o p e a n countries such as Ireland, England, G e r m a n y , France, and Scandinavia. Relatively few individuals with the deficiency were found in Italians, those of Jewish heritage, Blacks, M e x i c a n - A m e r i c a n s , or A m e r i c a n Indians. T h e s e studies of necessity w e r e all p e r f o r m e d on A m e r i c a n s and their national origin w a s determined by taking their histories.
12 MECHANISMS
FOR
ANTITRYPSIN
DEFICIENCY
One way to evaluate alphal-antitrypsin deficiency is to study the dynamic variation in antitrypsin response to an appropriate stimulus. We administered 3 mg of diethylstilbestrol daily for 14 days to measure the increase in STIC that occurs in individuals with different antitrypsin phenotypes [81. Individuals with the homozygous ZZ phenotype were totally incapable of responding to stilbestrol whereas those with the MZ heterozygous phenotype had approximately half the normal response capability. Other heterozygous phenotypes had a slightly reduced response to stilbestrol but did not differ significantly from normal. This included MS, MP, and FS phenotypes. The SS phenotype was studied in two instances and mimicked the response seen with the MZ heterozygotes. A few individuals with an apparent MM phenotype also had intermediate deficiency levels of alphal-antitrypsin and half the response capability to stilbestrol. We postulated that these individuals had inherited an inactive or null gene for alpha 1- antitrypsin as part of their phenotype [101. T a l a m o of Boston [12] su-bsequently reported an individual with essentially no antitrypsin in his blood whatsoever w h o appeared to be a homozygote for the null or inactive gene. Thus, inheritance of a null gene m a y be one of the causes for antitrypsin deficiency, although this is probably rare. The m a j o r cause of antitrypsin deficiency is associated with the Z variant and involves a defect in either synthesis or release of the antitrypsin protein f r o m its site of synthesis within the liver. Sharp [13] w a s the first to observe the presence of unusual hepatocellular globules in individuals with the Z variant. These globules can be seen under the electron microscope to be c o m p o s e d of a rough endoplasmic r e t i c u h m dilated by the presence of a granular storage material. These globules have an affinity for binding fluorescein- or peroxidase-labeled antibody against alpha I- antitrypsin, and thus appear to contain large amounts of alpha l-antitrypsin despite the shortage of the protein within the blood. It is therefore postulated that the deficiency associated with the Z variant results f r o m inability of the protein to leave the liver cell. Another cause of antitrypsin deficiency relates to the protein's increased lability and spontaneous degradation within the blood. This observation was m a d e in our laboratory w h e n we observed that certain bloods contained a second m i n o r antigen for alphal-antitrypsin El4]. T h e antigen w a s seen in m o s t eases of h o m o z y g o u s or heterozygous individuals with Z, S, I or P variants, and in relatively few individuals with the normal M M phenotype. Since the antigen could be produced in normal blood by degradation of antitrypsin by heat or low pH, it w a s eoneluded that it w a s associated with an inactive, degraded f o r m of alphal-antitrypsin. Subsequent heat lability tests on bloods of vacious phenotypes confirmed that the abnormal antitrypsin variants did have increased heat lability and were more rapidly inactivated at 55°C [15]. Three reasons for alphal-antitrypsin deficiency are: i) A defect in the release of the Z variant from its site of synthesis in the liver. (This is the major cause of antitrypsin deficiency. ) 2) Inheritance of a
13
null or inactive gene for antitrypsin production. 3) Increased antitrypsin variants resulting in in vivo degradation,
NEED
FOR
ANTITRYPSIN
SCREENING
lability of
PROGRAMS
S c r e e n i n g p r o g r a m s for d e t e c t i n g i n t e r m e d i a t e alpha 1 - a n t i t r y p s i n defic i e n c y a r e i n d i c a t e d at t h i s t i m e b e c a u s e s y m p t o m a t i c p u l m o n a r y e m p h y s e m a need not o c c u r in t h o s e i n d i v i d u a l s with the d e f i c i e n c y if t h e y do not s m o k e c i g a r e t t e s or w o r k in i n d u s t r y w h e r e t h e y a r e e x p o s e d to u n u s u a l f u m e s and d u s t s [ 1 6 ]. Thus, d e t e c t i o n of the d e f i c i e n t s u b j e c t s at a r e l a t i v e l y e a r l y age and c o u n s e l l i n g t h e m a g a i n s t c i g a r e t t e s m o k i n g , etc., could v e r y well p r e v e n t t h e i r d e v e l o p i n g this s e r i o u s lung d i s e a s e . The t e s t i n g of e m p h y s e m a p a t i e n t s and then the f a m i l y m e m b e r s of d e f i c i e n t p a t i e n t s would be m o s t r e w a r d i n g , since l a r g e r n u m b e r s of d e f i c i e n t relatives could be discovered and counselled. Tests for alphal-antitrypsin deficiency, should eventually become an important part of preemployment examinations in industry so that susceptible individuals can be kept out of those work areas where the risk would be greatest. A cure or treatment of alphal-antitrypsin deficiency does not currently exist, although work is in progress to devise a means for L'eplenishing deficient levels of serum alphal-antitrypsin or to perform normal liver transplants for taking over this job. These treatments, of course, lie in the future but suggest that alphal-antitrypsin deficiency may be one of the correctable genetic defects that plague mankind.
REFERENCES
I. Eriksson, S. : Studies in alphal-antitrypsin deficiency. Acta Med. Scand. 177, (Suppl. 432)1 (1965) 2. Laurell, C. -B. and Eriksson, S. : Electrophoretic alphal-globulin pattern of s e r u m alphal-antitrypsin deficiency. Scand. J. Clin. Lab. Invest. 15, 132 (1963) 3. bieberma---n, J. and Gawad, M. A. : Inhibitors and activators of leukocytic proteases in purulent sputum. Digestion of h u m a n lung and inhibition by alphal-antitrypsin. J. Lab. & Clin. Med. 7__77,713 (1971) 4. Bierman, H.R., Kelly, K.H., and Cordes, F.h.: The sequestration and visceral circulation of leukocytes in man. Ann. N. Y. Acid. Sci. 59, 850 (1955) 5. Lieberman, J. , Mittman, C., and Schneider, A. S, : Screening for homozygous and heterozygous alpha l-antitrypsin deficiency. Protein electrophoresis on cellulose acetate membranes. J. Amer, Med. Ass. 210, 2055 (19~9) 6. Lieberman, J. : Heterozygous and homogous alphal-antitrypsin deficiency in patients with pulmonary emphysema. New Engl. J. Med, 281, 279 (1969) 7. Lieberman, J. , Mittman, C., and Kent, J. R. : Sreening for heterozygous alpha l-antitrypsin deficiency. III. A provocative test with Diethylstilbestrol and effect of oral contraceptives. J. Amer. Med. Ass. 217, 1198 (1971)
14
8. L i e b e r m a n , J. and M i t t m a n , C. : D y n a m i c r e s p o n s e of a l p h a l - a n t i t r y p s i n v a r i a n t s to d i e t h y l s t i l b e s t r o l . A m e r . J. H u m a n G e n e t i c s 25, 610 (1974) 9. F a g e r h o l , M. K. : The P i - S y s t e m . G e n e t i c v a r i a n t s of s e r u m alpha 1a n t i t r y p s i n . S e r i e s , H a e m a t o l . 1, 153 (1968) I0. Lieberman, J., Gaidulis, L., Garoutte, B., and IYlittman, C. : Identification and characteristics of the common alphal-antitrypsin phenotypes. Chest 62, 557 (1972) ii. Mittman, C., Lieberman, J., Marasso, F. , and Miranda, A. : Smoking and chronic obstructive lung disease in alphal-antitrypsin deficiency. Chest 60, 214 (1971) 12. Talamo, R. C., Langley, C. E., Reed, C. E., and Makino, S. : Alpha lantitrypsin deficiency: A variant with no detectable alphal-antitrypsin. Science 181, 70 (1973) 13. Sharp, II. A. : Alphal-antitrypsin deficiency. Hosp. Pract. 6, 83 (1971) 14. Lieberman, J. : A new "double ring" screening test for alp~al-antitrypsin variants. Amer. Rev. Resp. Dis. 108, 248 (1973) 15. Lieberman, J. : Heat labilily of alphal-antitrypsin variants. Chest 64, 579 (1973) 16. Kueppers, F. and Black, L. F. : Alphal-antitrypsin and its deficiency. Amer. Rev. Resp. Dis. ii0, 176 (1974) Dr. J. L i e b e r m a n Dept. of R e s p i r a t o r y D i s e a s e s City of Hope National Medical Center Duarte, Calif. 91010, USA
