Determination of PseudoCholinesterase Activity by an Enzyme Coupling Method in the .... Pathway Enzyme Deficiencies: Pyruvate. Kinase and Cytochrome bc ...
Selected Topics in Clinical Enzymology Volume 2 Proceedings (selected) of the Fourth International Congress on Clinical Enzymology Washington, D. C , USA July 30 - August 2,1983
Editors Mario Werner • David M. Goldberg
W DE G
Walter de Gruyter • Berlin • New York 1984
CONTENTS
Introduction
List
b y M.
Werner
a n d D.M.
Goldberg
of Contributors
SECTION
I.
Improving
V
XIII
CHANGING METHODS
the Analytical
Sensitivity
of
E n z y m e A s s a y s b y R.C. R o c k S t a n d a r d i z a t i o n o f Enzyme A s s a y s B a s e d D e t e r m i n a t i o n s by R e f e r e n c e Methods b y D. L a u e
3 on 15
A U n i v e r s a l I n c l u s i v e Reference System f o r C l i n i c a l Enzymology. Proposal: The I n t e r n a t i o n a l C l i n i c a l Enzyme S c a l e b y G.y. B o w e r s , J r
23
Amylase Isoenzymes: Influence of Temperature a n d S t o r a g e o f S a m p l e s b y M. Z a n i n o t t o , P. R i z z o t t i , G* S a I m e r i , C\ C o c e o , D. Zanini, M. P l e b a n i a n d A . B u r l i n a
43
4 - N i t r o p h e n y l O l i g o s a c c h a r i d e s as S u b s t r a t e s f o r a Serum a-Amylase: S e l e c t i o n of O p t i m u m C h a i n L e n g t h by E.G. Hagele E. Rauscher E. S c h a l e h P. L e h m a n n > //. B u r k a n d A.W, Wahlefeld
51
Reliability Activity K. W u l f f
65
3
s
A
3
o f a New A s s a y f o r C h y m o t r y p s i n i n S t o o l by P. K a s p a r „ U. Neumann> a n d A.W, Wahlefeld
S e n s i t i v e Method f o r Assay o f L i p a s e A c t i v i t y b y C o u p l i n g w i t h ß-Oxidation E n z y m e s o f F a t t y A c i d s b y S. I m a m u r a a n d H. M i s a k i
73
VIII
Determination of PseudoCholinesterase Activity by an Enzyme C o u p l i n g M e t h o d i n t h e Serum of P a t i e n t s w i t h L i v e r Disease b y K . - J . Hsiao S . - J . W u j ¥. K a s a h a r a a n d W.-K. Ting
>
79
Measurement o f Serum C h o l i n e s t e r a s e A c t i v i t y U s i n g a C e n t r i f u g a l A n a l y z e r b y G.C\ Moses a n d A.R. Henderson
89
Enzymatic Determination of T o t a l B i l i r u b i n i n S e r u m b y A . K o s a k a , K. T s u d a , Y. M o r i s h i t a a n d K. N a k a n e
97
A s s a y o f Human M i t o c h o n d r i a l A s p a r t a t e A m i n o t r a n s f e r a s e by R o c k e t I m m u n o e l e c t r o p h o r e s i s a n d A u t o r a d i o g r a p h y b y 0. K o l d k j a e r
109
D e t e r m i n a t i o n o f C y t i d i n e Deaminase i n B l o o d by H i g h P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y b y T. R u s s o , A . C o l o n n a . j F. S a l v a t o r e a n d F. Cimino F l u o r i m e t r i c Assay of Angiotensin-I-Converting E n z y m e i n C e r e b r o s p i n a l F l u i d b y H. Schweisfurth a n d S. S c h i o b e r g - S c h i e g n i t z
SECTION I I .
DIAGNOSIS
S t r a t e g i e s i n the Enzymologic Diagnosis Myocardial I n f a r c t i o n b y A. B u r l i n a How
125
R e l i a b l e i s the Acute Myocardial
Laboratory Infarction
of 133
Diagnosis of b y /•/. W e r n e r
S t r a t e g i e s i n the Enzymological Diagnosis P a n c r e a t i c D i s e a s e b y D.M. Goldberg Pancreatic Cholesterol Ester Hydrolase: E s t i m a t i o n of I t s A c t i v i t y i n the Small B o w e l U s i n g t h e P a n c r e o l a u r y l - T e s t and Comparison w i t h P a n c r e a t i c Exocrine F u n c t i o n b y J . F r e i s e a n d F.W. Schmidt
14 7
of 171
199
Assay o f M i t o c h o n d r i a l Aspartate Aminotransf e r a s e (m-AST) b y I m m u n o p r e c i p i t a t i o n a n d Column C h r o m a t o g r a p h y i n S e r a o f P a t i e n t s w i t h L i v e r D i s e a s e b y F.W. S c h m i d t , E. S c h m i d t , S. O h l e n d o r f a n d R. R a u p a c h P e r s i s t e n t A c i d L i p a s e A c t i v i t y i n a Case o f Cholesterol Ester Storage b y A. L a g e r o n , I i B e u c l e r a n dJ.-P. Chagnon Nature and C l i n i c a l S i g n i f i c a n c e o f A l k a l i n e Phosphatase-Immunoglobulin Complexes b yA . Burlina, M* P l e b a n i , C. D e c h e c c h i , M.S. G r a z i a n i a n d P. R i z z o t t i B i o c h e m i c a l T a r g e t s and I n d i c a t o r s o f Drug A c t i o n b y G. W e b e r S t r a t e g i e s i n the Enzymologic Diagnosis o f M u s c u l a r a n d C o n n e c t i v e T i s s u e D i s e a s e by E. K a i s e r C a r b o x y p e p t i d a s e N: Spectrophotometric Assay a n d C l i n i c a l S i g n i f i c a n c e b y H. S c h w e i s f u r t h , J . H e i n r i c h a n d E. B r u g g e r
SECTION
I I I .
PATHOPHYSIOLOGY, THERAPEUTICS
PHARMACOLOGY
Enzymes i n I n f l a m m a t i o n : Aspects P a t h o p h y s i o l o g y b y R.W. D o r n e r
AND
of
Granulocyte Proteinases as Mediators o f Unspecific P r o t e o l y s i s i n Inflammation: A R e v i e w b y H. F r i t z , M. L o c h u m , K.-H. D u s w a l d , H. D i t t m e r , H. K o r t m a n n , 5. N e u m a n n a n d H, L a n g Enzymes i n C o l l a g e n b y R. E. P r i e s t
S y n t h e s i s and
Degradation
P r o t e o l y t i c Enzymes R e l a t e d t o B l o o d R e g u l a t i o n b y M. R o t h ,
Pressure
X
U r i n a r y Enzyme P a t t e r n i n M a l i g n a n t H y p e r t e n s i o n o f S a l t - L o a d e d D a h l R a t s by D. M a r u h n , G. Luckhaus, D. P a a r , B. G a r t h o f f a n d 3. K a z d a
353
E f f e c t o f Indomethacin on A c t i v e and I n a c t i v e R e n i n i n S o d i u m - R e p l e t e Man a t R e s t a n d Düring E x e r c i s e by P. L i e n e n , D. Groeseneken, R. F a g a r d , J . S t a e s s e n a n d A . A m e r y
359
The
Enzymes o f C a t e c h o l a m i n e M e t a b o l i s m i n Man: C a t e c h o l - O - M e t h y l t r a n s f e r a s e , Monoamine O x i d a s e a n d P h e n o l S u l f o t r a n s f e r a s e by R.M. Weinshilboum
3^3
E n z y m a t i c C o n v e r s i o n o f GABA a n d 2 - P y r r o l i d o n e : An A p p r o a c h t o t h e B i o c h e m i s t r y o f A n x i e t y by L . G a l z i g n a , A . B e r t a z z o n , A . B a r t h e z a n d G. Q u a d r o
399
Serum C r e a t i n e K i n a s e F o l l o w i n g T h r o m b o l y s i s i n A c u t e M y o c a r d i a l I n f a r c t i o n b y T. G. K w o n g , P.G. F i t z p a t r i c k a n d R.L. R o t h b a r d
4.1
C r e a t i n e Kinase Isoenzyme C o m p o s i t i o n i n S k e l e t a l Muscle B i o p s i e s Obtained from M a r a t h o n R u n n e r s by F . 5 . A p p l e , M.A. Rogers, W.M, S h e r m a n a n d J . L . I v y
4i9
U r i n e E n z y m e P a t t e r n s i n t h e R a t A f t e r Damage t o D i f f e r e n t P a r t s o f t h e N e p h r o n by D. M a r u k n , F. B o m h a r d a n d D. P a a r
4 29
U r i n a r y E n z y m e s a n d I s o e n z y m e s o f N-Acetyl-ßD-Glucosaminidase i n t h e Assessment o f N e p h r o t o x i c i t y by ,7. H a l m a n , R. G. P r i c e a n d J.S.L. Fowler
435
E f f e c t o f P a r a t h y r o i d Hormone a n d C a l c i u m on L i v e r a n d K i d n e y E n z y m e s by F. B o g i n a n d y. E a r o n ,
445
Enzymatic Removal o f L i p i d H y d r o p e r o x i d e s i n R a t L i v e r M i t o c h o n d r i a a n d M i c r o s o m e s by L . C a v a l l i n i , IL V a l e n z e a n d A . B i n d o l i
4 53
XI
E f f e c t o f Mesenteric Lymphatic O b s t r u c t i o n on S m a l l I n t e s t i n a l M u c o s a l E n z y m e s b y W. WeIlmann, K. T s a t c h e v , H. H u b e r t , K. S c h m i d , E. S c h m i d t a n d F.W. S c h m i d t
SECTION I V .
ENZYME
461
HETEROGENEITY
Monoclonal Antibodies: New T o o l s f o r B i o c h e m i c a l , Immunochemical and G e n e t i c A n a l y s e s o f E n z y m e s a n d I s o z y m e s b y S. V o r a and S.L. M o r r i s o n
469
Gene R e - A r r a n g e m e n t a n d M e s s e n g e r P r o c e s s i n g As C a u s e s o f Enzyme H e t e r o g e n e i t y b y J . - C . D r e y f u s a n d A. Kahn
505
P o s t - T r a n s l a t i o n a l l y M o d i f i e d Forms o f Enzymes o f D i a g n o s t i c I m p o r t a n c e by D.W. M o s s
517
Human L i v e r S o r b i t o l D e h y d r o g e n a s e : Evidence f o r Two F o r m s by D.A. N e a l o n a n d R. R e j
535
Studies o f Alanine Aminopeptidase, Dipeptidyl A m i n o p e p t i d a s e I a n d I I o f t h e Human S e m i n a l F l u i d a n d P r o s t a s o m e s by T. Vanha-Perttula
54 5
SECTION V.
VETERINARY
O v e r v i e w o f V e t e r i n a r y Enzyme Pathophysiology i n R u r a l a n d U r b a n P r a c t i c e b y S. N a t e l s o n
567
Animal Models o f Erythrocyte G l y c o l y t i c Pathway Enzyme D e f i c i e n c i e s : Pyruvate K i n a s e a n d C y t o c h r o m e b c R e d u c t a s e (NADHMethemoglobin Reductase) b y J . J . K a n e k o
583
XII
Animal Models o f E r y t h r o c y t e Pentose Phosphate and G l u t a t h i o n e D e t o x i f i c a t i o n Pathways b y J . E . S m i t h
597
N a t u r a l l y - O c c u r r i n g Models o f Inborn E r r o r s of Metabolism: D e f i n i t i o n of the Enzymatic D e f e c t s by P.F. J e z y k , M.E. H a s k i n s a n d D.F. Patterson
605
The U t i l i z a t i o n o f C l i n i c a l E n z y m o l o g y i n t h e P r a c t i c e o f V e t e r i n a r y M e d i c i n e b y J . W. H a r v e y
....
615
T r a n s f e r o f Gamma-Glutamyl T r a n s f e r a s e from M a t e r n a l C o l o s t r u m t o t h e Serum o f Newborn G o a t s , F o a l s a n d C a l v e s by J . P . B r a u n , D. T a i n t u r i e r , P. B e z i l l e a n d A.C. R i c o
627
The P r o t e c t i v e R o l e o f S e l e n i u m i n G e n t a m i c i n I n d u c e d R e n a l Damage i n t h e R a t b y E.G. 11 g a h a
635
Species D i f f e r e n c e s i n the I n V i t r o Response o f P l a s m a A n g i o t e n s i n - C o n v e r t i n g Enzyme t o P r e c u r s o r s and A c t i v e Forms o f C o n v e r t i n g E n z y m e I n h i b i t o r s by D.-S. Chen, U.R. B r u n n e r a n d B. W a e b e r
641
L e v o - C a r n i t i n e i n Serum, H e a r t , M u s c l e and Tumoral T i s s u e i n R a t s w i t h Y o s h i d a Tumor by E. D e L a M o r e n a , G.G. G o n z a l e z , L, S o l o r z a z o a n dL. C a n t e r o
647
Subject
653
Index
Index
of Contributors
665
304
10.
P r i e s t R.E.: In S e l e c t e d T o p i c s J.n C l i n i c a l E n z y m o l o g y , V o l . 2, D.M. G o l d b e r g , M. W e r n e r ( e d s ) , W a l t e r De G r u y t e r , B e r l i n , New Y o r k , 1 9 8 5 .
11.
H a r r i s E.D., D i B o n o D.R., 48, 2104-2113 ( 1 9 6 9 ) .
12.
A n d r i o p o u l o s N.A., M e s t e c k y J . , M i l l e r E . J . , B e n n e t t J.C.: C l i n I m m u n o l I m m u n o p a t h o l 6, 2 0 9 - 2 1 2 ( 1 9 7 6 ) .
13.
T r e n t h a m D.E., T o w n e s A . S . , 857-868 ( 1 9 7 7 ) .
14.
S t u a r t J.M., H u f f s t u t t e r E.H., T o w n e s A . S . , A r t h r i t i s Rheum 2 6 , 8 3 2 - 8 4 0 ( 1 9 8 3 ) .
15.
H o h n D.C., M e y e r s A . J . , G h e r i n i S.T., B e c k m a n A., M a r k i s o n R.E., C h u r g A.M.: Surg 88, 48-58 ( 1 9 8 0 ) .
16.
W e b s t e r R.O., L a r s e n G.L., 70, 1177-1183 (1982) .
17.
J a c o b s H.S., G o l d s t e i n I.M., S h a p i r o I . , C r a d d o c k P.R., H a m m e r s c h m i d t D.E., W e i s s m a n G.: A r c h I n t Med 1 4 1 , 134-136 (1981) .
18.
W e t s e l R.A., (1983) .
19.
W e i s s m a n G., Z u r i e r R.B., S p i e l e r J E x p Med 1 3 4 , 1 4 9 S - 1 6 5 S ( 1 9 7 1 ) .
P.J., Goldstein
20.
S m i t h G.P., P e t e r s T . J . : 304-308 (1982) .
Biophys
Kolb
W.P.:
K r a n e S.M.:
K a n g A.H. :
J Clin
J E x p Med
H e n s o n P.M.:
J E x p Med
Biochim
157,
Invest
Kang
J Clin
14 6, A.H.:
Invest
2029-2048
Acta
I.M.:
719,
G R A N U L O C Y T E P R O T E I N A S E S AS M E D I A T O R S I N INFLAMMATION: A REVIEW Hans F r i t z ,
M.
OF U N S P E C I F I C P R O T E O L Y S I S
Jochum
Department o f C l i n i c a l Chemistry and C l i n i c a l U n i v e r s i t y o f M u n i c h , M u n i c h , FRG.
K.-H.
Duswald,
H. D i t t m e r ,
Surgical Clinic s i t y of Munich,
S. N e u m a n n
Kortmann
C i t y and S u r g i c a l C l i n i c M u n i c h , FRG.
a n d H.
Biochemical
H.
Biochemistry,
Grosshadern,
Univer-
Lang
Research
Department,
E. M e r c k ,
Darmstadt,
FRG.
Abstract In
severe
cells, somal
inflammatory
proteinases
Such enzymes, gents
as w e l l as n o r m a l l y
produced
constituents first
during
used
connective
tor
(o^PI)
lial
polymorphonuclear
determined inal
Whereas crease with
tissue
lyso-
the circulation.
intracellular enhance
tissue
release
oxidizing
the
a-
inflammatory
s t r u c t u r e s , membrane
Using
elastase
Proteinase
inhibi-
and i s e l i m i n a t e d f i n a l l y
levels
immunosorbent assay,
o f E-a^PI
f o l l o w i n g major
m u l t i p l e trauma and p a n c r e a t o g e n i c
the operative
competes
as
complexes by t h e r e t i c u l o - e n d o t h e -
an e n z y m e - l i n k e d
t h e plasma
(E) a s a m a r k e r o f
i n c l u d i n g ct-^-proteinase
2
surgery,
and i n t o
The l i b e r a t e d
and a - m a c r o g l o b u l i n ,
enzyme-inhibitor
System.
and
and s o l u b l e p r o t e i n s by p r o t e o l y s i s o r o x i d a t i o n .
susceptible Substrates,
inactive
blood
granulocytes,
phagocytosis,
such r e l e a s e r e a c t i o n s . with
various
extracellularly
response by degrading
We
response,
i n c l u d i n g polymorphonuclear
we
abdom-
shock,
t r a u m a was f o l l o w e d b y up t o 3 - f o l d i n -
o f the E-a^PI, postoperative
a 10- t o 2 0 - f o l d i n c r e a s e .
septicemia
The i n c r e a s e
Selected Toplcs in Clinical Enzymology, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York-Printed in Germany
was
associated
o f E-a,PI and a
306
concomitant bin
decrease
I I I ,clotting
related. a PI
factor
Multiple
seems t o c o r r e l a t e the r e l a t i o n s h i p by
o f E-a-^PI c o i n c i d e
accident.
increase
The r e l e a s e d of injury,
o f plasma
transfusions. with
as antithrom-
a substantial
the severity
peaks
such
and e ^ - m a c r o g l o b u l i n , were c o r -
t o consumption
concomitant
factors,
causes
after with
cated
thrombin
XIII
trauma
u p t o 14 h o u r s
1
o f plasma
elastase
but assessing
factors
In acute
a massive
i s compli-
pancreatitis,
consumption
I I I and c ^ - m a c r o g l o b u l i n d u r i n g
o f E-
of
anti-
shock.
Introduction
Severe
injury
including ting,
or infection
the activation
fibrinolysis,
triggers
o f (a) s u c h
(b) c e l l u l a r
Systems, e s p e c i a l l y
and
lymphocytes,
but also
The
humoral
flammatory may
cells,
activate
stress
are often
phagocytes,
multiple
from
from
Similarly,
between
clot-
cells
cells.
of the i n these
cells
the repair
inflammation i n t h e i r
relationships
as
cascades,
mast
potent stimulators
Systems.
response,
Systems
hormone p r o d u c i n g
and c o n v e r s e l y e f f e c t o r s
some h u m o r a l
mechanisms r e s u l t i n g tablish
humoral
complement and k a l l i k r e i n - k i n i n
and
factors
an i n f l a m m a t o r y
aggregate es-
the various parts of
the organism ( 1 ) .
Lysosomal
We
proteinases
focus on t h e p o t e n t i a l
teinases cytes
contain lytic
released
such
pathological
extracellularly
as polymorphonuclear
many
lysosomes
potential
with
role
of lysosomal
during inflammation.
oxidizing
triggered,
serve
agents
Phago-
g r a n u l o c y t e s and macrophages
a powerful h y d r o l y t i c
(2). Normally, the lysosomal
along with
pro-
produced
two m a i n p u r p o s e s
and p r o t e o -
enzymes,
when p h a g o c y t o s i s i s (3) :
(a)
intracellular
307
protein
catabolism including
extracellular
endogenous
of
phagocytized
In
summary, l y s o s o m a l
physiological but
under
tors out
up
proteinases normally
function inside
thec e l l
t h ec e l l
during
normal
occurs
(3,19).
phagocytosis,
phagocytosis, structural
Only
higher
s m a l l amounts amounts escape
element
such
with
complement l y s i s ,
somal o r p h a g o l y s o s o m a l
L i b e r a t i o n
i sunable
fac-
leak during t o take
as a piece o f v a s c u l a r
Disintegration
b y endogenous o r exogenous e n d o t o x i n s ,
combination
their
i nt h e phagolysosomes,
when t h e p h a g o c y t e
p l a s m a membrane o r c a r t i l a g e . caused
fulfill
c o n d i t i o n s , major escape o f lysosomal
from
a larger
o f i n t r a - and
a n d (b) t h e d e g r a d a t i o n
v i r u s e s and b a c t e r i a .
certain
frustrated
the degradation
substances,
o f phagocytes possibly i n
r e l e a s e s most o r a l l l y s o -
constituents (Figure1 ) •
and
E f f e c t s
of
° Proteases v ^ v ^
° Hydro]ases
Lysosomal
Factors
Endotoxins (endogenous & exogenous)
>v \
° Hyeloperoxidase V
(^^)
m
K.) J \
Antigen-antibody complexes
H
2°2
X ^ ^ s P ^ ^
toxic peptides: blood pressure
Complement
edema, heart toxic, clotting inh.
degradation/inactivation: meibranes, connective tissue, plasma factors
activ. of system-specific proteases: thrombin, plasmin, C1-esterase, kallikrein
F i g u r e 1. L i b e r a t i o n a n d e f f e c t s o f l y s o s o m a l f a c t o r s d u r i n g inflammation. Degradation products present i nt h e phagolysosomes ( e . # . t o x i c p e p t i d e s ) a r e l i b e r a t e d d u r i n g c e l l Stimulation or disintegration. Released lysosomal o r phagolysosonal c o n s t i t u e n t s degrade o r i n a c t i v a t e n a t i v e s t r u c t u r a l and humoral f a c t o r s . System-specific proteinases activate the blood System cascades. I n t h i s way, a c t i v a t e d p r o t e i n a s e s nay a l s o d e g r a d e h u m o r a l f a c t o r s , a n d t h e r e f o r e , t o x i c P o l y p e p t i d e s may b e g e n e r a t e d a s w e l l . f
308
Released
extracellularly,
inflammation teolysis erate
i n two m a j o r ways
a c t i v a t e s proenzymes,
biologically
potent
atoxins.
The f o r m e d
tion
specific
with
s p e c i f i c
blood
soluble tural
(Figure 2 ) :
peptides
such
inhibitor
Systems h e r e ) ;
elements
such
as k i n i n s
and
participating
s u m p t i o n may p r o d u c e
Degradation
for the
i n the clotting, cascades
toxic
which
peptides
inhibit
Granulocyfes
Macrophages
I
I
L i b e r a t i o n
(call-
inactivates
I I I , or digests
induced
anaphyl-
interac-
(b) u n s p e c i f i c p r o t e o l y s i s
(4-8) .
products
a n d may g e n -
p r o t e i n s , as i s true
as antithrombin
enhance
(a) s e l e c t i v e p r o -
cofactors or both,
complement and k a l l i k r e i n - k i n i n
factors
radation
p r o t e i n a s e s may
p r o t e i n a s e s a r e e l i m i n a t e d by
consumption o f factors
fibrinolysis, ed
lysosomal
struc-
u n s p e c i f i c
con-
as f i b r i n - f i b r i n o g e n
deg-
clotting (9).
Endothelial cells
Mast cells
I
l
P r o t e i
of
n
a s e s
l Selective
proteolysis
Unspec ific
proteolysis
• proenzyme/cofactor activation
•
inactivation by degradation
» biol. active peptides released
•
toxic peptides produced
Specific consumption reaction
Unspecific consumption reaction
E* I
tEI)
AT III
Q c t i v e
i 2 Ü 5 ! ! * AT m
inactive
F i g u r e 2. R e a c t i o n pathways caused by l y s o s o m a l p r o t e i n a s e s i f l i b e r a t e d from v a r i o u s body c e l l s . E = enzyme; I = i n h i bitor; [ E I ] = e n z y m e - i n h i b i t o r c o m p l e x ; AT I I I = a n t i t h r o m b i n III.
Clinical
organ
multiple
injury
These organs blasts, all, the
failure
precipitated
affects
are rieh
primarily
polymorphonuclear during
lungs,
liver
i n endothelial cells,
and macrophages, which
lungs
by severe
inflammation or and k i d n e y s .
mast c e l l s ,
c o n t a i n many l y s o s o m e s .
granulocytes
the inflammation.
may a c c u m u l a t e
fibroAbove
rapidly i n
Hence, t h e r e l a t i o n s h i p s
309
between tent
t h e sequence o f organ
Among k n o w n l y s o s o m a l neutrophil thepsin
partate
proteinases,
polymorphonuclear
i n fully
active
proteinases.
cluding
Both
form,
such
They
like
inhibitors
thiol
feature
cathepsin
(4,11),
(8),
under
p o t e n t i a l o f lysosomes
physiological conditions
of
and c a i n the and a s -
of both
elas-
G i s non-specificity
as w e l l
(6) a n d c o l l a g e n
tive
of over
elastase
are stored
the acid
The most s t r i k i n g
as e l a s t i n
con-
proteinases
enzymes degrade numerous h u m o r a l
Proteinase
elements,
thesis
the neutral
granulocytes,
and t h e c h y m o t r y p s i n - l i k e
(3,10).
and the lysosomal
consideration.
G are of special interest.
lysosomes
tase
failures
o f d i f f e r e n t Organs m e r i t s
as
type
(Figure
i s demonstrated
1 g of neutral proteinases
factors,
i n -
structural I I Iand IV
3).
The
diges-
by t h e d a i l y s y n -
i n man.
Substrates of PMN Granulocyte Neutral Proteinases P r o t e i n a s e * Elastase
B i o l o g i c a l
S u b s t r a t e s
• elastin, collagen III & IV, proteoglycans, FN
+
* clotting & fibrinolysis factors * canplement factors & Immunoglobulins * Proteinase inhibitors (AT 111 ot PI, C1 INA; ITI) 9
2
* transport proteins (transferrin, prealbumin) Cathepsin
G
• collagen II & I, proteoglycans, fibronectin * clotting & complement factors
Collagenase * >1 g daily turnover
* collagen I & II & III +
fibronectin ( R E S
function)
F i g u r e 3. N a t u r a l Substrates o f n e u t r a l p r o t e i n a s e s from p o l y m o r p h o n u c l e a r (PMN) g r a n u l o c y t e s . AT I I I = a n t i t h r o m b i n I I I . ; ot2PI = c t 2 - p l a s m i n i n h i b i t o r ; C l I N A = C l - i n a c t i v a t o r ; ITI = i n t e r - a - t r y p s i n inhibitor.
310
Plasma P r o t e i n a s e
Functional are
kept
coated
inhibitors
aspects.
Within
cell,
lysosomal
by
their
localization
under c o n t r o l f i r s t
o r g a n e l l e s , and
cytosol
(12).
s e c o n d by
Lysosomal
potent
antagonists,
(13).
a -Macroglobulin as
as
the
(a M) 2
w e l l as
proteinases.
thiol
The
high
aspartate
the
called
concentration
fluid
and
rapidly fold
responding
inhibitor
of
chymase.
The
are
clearly
represent albumin
acute
plasma
interactions enzymes
of
Hageman
of
also
in
major
of
in
interstitial
inflammation,
(a^AC) , up
is a
G and
a l l other
to
6-
mast
cell
inhibitors
inhibitor
the
proteins other
plasma
a
potent
Proteinase
(AT
cascade.
existence
and
kallikrein
checked
by
complement last and
and
inhibitors i n Figure
III) regulates
This
kallikrein,
factor
be
sketched
fibrinolysis,
2
of
the
reactant, reaching to
Proteinase
are
may
classical
The
ulation
the
60%
between
III
(a PI)
plasma
occurs
concentrations
i n plasma
antithrombin
ulation
phase
Still,
enzyme a c t i v a t i o n
the
me-
normally
proteins than
Immunoglobulins.
The
both
and
a^-Proteinase
c^-antitrypsin),
neutrophil cathepsin
approximately
and
face
serine
c^M
a-^-Antichymotrypsin
i n response
lower.
get
hibitor
but
the
proteins
neutrophil elastase, i s present
i n blood,
lysosomal
of
v a s c u l a r bed.
mucous s e c r e t i o n s .
concentration
weight
in
cell
(cysteine), molecular
(c^PI, formerly
high
the
inhibitor inhibits
i t s function to
lysosomal
i n membrane
effectively
inhibitor
of
proteinases
inhibitors
escaping
Proteinase
restricts
antagonist
Proteinase
proteinases
such
2
proteinases, tallo
the
three
and
4.
main
clotting,
pathway and
the
Hageman f a c t o r
or
tar-
inhibitors: a -plasmin
in-
2
Cl-inactivator
function results
their
Excessive
(Cl
INA)
intrinsic from
the
coag-
inhibition
28,000
dalton
fragment.
i n plasma of or
plasmin
enzyme c a s c a d e s
complexes between suggests
an
in certain
a -macroglobulin 2
involvement
i n the
reg-
pathological conditions.
311
However,
t h e predominant r o l e
unspecific proteinases thiol ase
o f c^M s e e m s t o b e p r e v e n t i o n o f
p r o t e o l y s i s by i n h i b i t i n g
a l l types
of
lysosomal
including neutrophil elastase, cathepsin
proteinases
cathepsin
cathepsin
D and m e t a l l o
G, t h e
B, H, L , t h e a s p a r t a t e P r o t e i n enzymes
such as c o l l a g e n a s e .
Toxins (exogenous, endogenous) or
a-M
noxae
-i Cell
S t i m u l a t i o n / disintegration n
PI
thromboplastins
a i
Plasminogen activators
ot M
—K> Proteinase
_^
^.
2
release
lysosomal elostase(s), cathepsin G*/B, H, L/D
A
o^AC*
Selective activation
Proteinase
Unspecific degradation
inhibitors
inactivation
AT III
• blood system factors
or PI
• Proteinase inhibitors
of
blood Systems
•clotting
>SFMK «
• fibrinolysis > FDP • kallikrein
«-
2
• immunoglobulins
> Kinins J
• complement > C3a/5a
platelets, mast
4
(0|)
C1 INA
E+l — El
c e l l s , phagocytes
• tissue etc. proteins
phagocytes etc. lymphocytes
RES
F i g u r e 4. A c t i v a t i o n and c o n s u m p t i o n r e a c t i o n s c a u s e d b y proteinases r e l e a s e d d u r i n g c e l l Stimulation or d i s i n t e g r a tion: S y s t e m - s p e c i f i c p r o t e i n a s e s { e . g . , t h r o m b o k i n a s e s and Plasminogen a c t i v a t o r s ) t r i g g e r a c t i v a t i o n o f b l o o d Systems, whereby b i o l o g i c a l l y h i g h l y a c t i v e P o l y p e p t i d e s a r e formed, e.g., SFMK ( s o l u b l e f i b r i n monomer c o m p l e x e s ) , FDP ( f i b r i n / ogen d e g r a d a t i o n p r o d u c t s ) , k i n i n s , and a n a p h y l a t o x i n s (C 3 a and C 5 a ; l e f t p a r t ) . Unspecific degradation or inactivat i o n o f p l a s m a and t i s s u e f a c t o r s i s c a u s e d by lysosomal p r o t e i n a s e s and/or o x i d i z i n g agents ( r i g h t p a r t ) . In both cases, l i b e r a t e d P o l y p e p t i d e s may s t i m u l a t e s u i t a b l e c e l l u l a r S y s tems. F i n a l l y , complex formation o f a c t i v a t e d o r l i b e r a t e d e n z y m e s (E) o c c u r s w i t h t h e P r o t e i n a s e i n h i b i t o r s ( I ) , e . g . , a^PI ( a ^ - p r o t e i n a s e i n h i b i t o r ) , a M ( c ^ - m a c r o g l o b u l i n ) , a^AC ( a ^ - a n t i c h y m o t r y p s i n ) , AT I I I ( a n t i t h r o m b i n I I I ) , ^ P I ( 2 ~ p l a s m i n i n h i b i t o r ) , a n d C l INA ( C l - i n a c t i v a t o r ) . The enzymei n h i b i t o r complexes [EI]a r e e l i m i n a t e d by phagocytes o f the r e t i c u l o - e n d o t h e l i a l s y s t e m (RES; c e n t r a l p a r t ) . 2
a
312
is
also
which
the
can
i n s by
proteolysis,
f a c t o r s and
Peptides. an
Despite
acute
neutrophil trypsin
inhibitor
of
a c t i v a t e enzyme c a s c a d e s
selective
various
not
most potent
structural this
phase
broad
reactant
e l a s t a s e as
and
bacterial
chymotrypsin,
or
pancreatic trypsins
i n blood
can
and
elements while inhibitory
i n man.
w e l l as
release
unspecifically
releasing toxic
specificity,
c^PI
kin-
degrade
strongly
c^M
is
inhibits
p a n c r e a t i c e l a s t a s e , chymo-
elastases.
o^AC
neutrophil cathepsin
inhibits
G and
pancreatic
mast c e l l
chymase
(13) .
Inhibition toxic
of
Stimulation as
proteinases
peptides, by
such
Chemotaxis
In essence,
plasma
plexes
their
with
reversibly bind
still
cleave
However, cleared
the by
proteolysis
inactivated same i s t r u e and
activated
(13,17) .
Proteinase t a r g e t s by
by
c^M
inhibitors which
striking
such
anaphylatoxin-
about
this
formed
system by
are
lysosomal
a,PI
and
(14,15).
rapidly
of
factors. by
unspecific
lysosomal is
rapidly
by
The
Cl-inactivator
(11) ,
is proteolytically
Proteinase
the
i t can
(16) .
potential
inhibitor
o x i d a t i o n of
as
complex
neutrophil elastase.
inhibitor
thiol
com-
isi r -
rule,
10,000 d a l t o n s
I I I (4), f o r example,
amounts of
lysosomal
of
to
resulting
complexes
f r o m m a c r o p h a g e s , and
site
or
as
Systems,
activity
pathological effect
o^-plasmin
Moreover,
enzyme-reactive
the
inhibiting
c^-proteinase
the
enzyme
catalytic
inhibitors
Proteinase
catalytic
for
or
much
form equimolar
exception and
below
Antithrombin by
vasoactive
cellular
aggregation
i s an
Proteinase
is a
of
granulocytes.
reticulo-endothelial
the
similarly,
metallo
platelet
Polypeptides
proteinases.
formation
a n a p h y l a t o x i n s , as
products o f
enzyme-inhibitor
the
of
or
enzyme m o l e c u l e s ,
Consumption of Reduction
of
blocked.
two
prevents
kinins
proteolytic
thrombin-triggered
induced
can
as
cathepsin
a bacterial
methionine
g r e a t l y reduces
the
B,
ina
elastase
residue
i n the
affinity
to
313
neutrophil droxyl
elastase
r a d i c a l s and hydrogen
amounts
i n the phagolysosomes
tein
breakdown,
with
lysosomal
Thus, ase with and
and these enzymes
agents
peroxide,
proteins
proteinases,
a r e produced
i n three
pro-
along
t o t h e same S t i m u l i ( 3 ) .
consumption
ways:
of Protein-
(a) c o m p l e x
formation
(b) i n a c t i v a t i o n b y p r o t e o l y t i c denaturation.
elastase
degradation, The
special i n t e r e s t i n connection
e f f e c t s on O ^ P I on n e u t r o p h i l
hy-
i n large
intracellular
s u b s t a n c e s may b e r e l e a s e d
i n response
(c) i n a c t i v a t i o n by o x i d a t i v e deserves
as Superoxide,
to facilitate
i n j u r y o r i n f e c t i o n can induce
inhibitor
mechanism tive
(18). Oxidizing
last
with
binding
oxida-
(Figure
5) .
Oxidation and (Met residuels) in o^PI
H 0 2
a
i
p,
2
Proteolysis oxidized to Met sulfoxide)
& Myetoperoxidase or 0\
a.PI 1
native
n
PHN Elastase or pancreas
a
i
p ,
Qf 1 PI oxjd
native-E|
elastin
oxid
Elastase
- PHNE
•
pancreas
Elastase
high affinity Substrate
PMN E
digestion
F i g u r e 5. A f f i n i t y o f native and o x i d i z e d a ^ - p r o t e i n a s e i n h i b i t o r ( c ^ P I ) t o p o l y m o r p h o n u c l e a r (PMN) e l a s t a s e ( E ) a n d p a n c r e a t i c e l a s t a s e (E) , r e s p e c t i v e l y . T h e o x i d i z e d a]_PI r e a c t s much more s l o w l y ( a p p r o x i m a t e l y 2 0 0 0 t i m e s ) w i t h PMN e l a s t a s e than native i * In addition, the elastase complex with o x i d i z e d c^PI i s d i s s o c i a t e d by Substrates with h i g h a f f i n i t y t o PMN e l a s t a s e , t h u s a g a i n l i b e r a t i n g t h e a c t i v e enzyme. O x i d i z e d c ^ P I does n o t r e a c t w i t h pancreatic elastase. P
I
314
Clinical
studies
Assay o f l i b e r a t e d n e u t r o p h i l elastase
i s found i n blood
tase-a^-proteinase
lin,
elastase
b u t t h e E-a^M c o m p l e x
from t h e c i r c u l a t i o n Consequently, treme not
apply
(E-a^PI) complex. may b e b o u n d
2
body
fluids,
studies
ontibodies to elostose l
o
b
- E J
(20).
by an e n z y m e - 1 i n k e d
+
elastase-
eliminated
(t ~ 1 0 ' v s , 1 h). -1 l
r e q u i r e m e n t may
such as s y n o v i a l
2
— ^
small
i n plasma r e q u i r e s ex-
(16), but this
E - a M c o m p l e x e s may c l e a r s l o w l y clinical
neutrophil
t o a^-macroglobu-
than t h e E-a,PI complex l
sensitivity
i n other
A
i s much more r a p i d l y
assay o f E-a M complex
analytical
Liberated
p r i m a r i l y i n t h e form o f the e l a s -
inhibitor
amount o f n e u t r o p h i l
elastase.
fluid,
where
We a s s a y E-a-^PI i n
immunoassay
ab-E/E-OtiPl
(Figure 6 ) .
-habtoOcrPI,
! P r o t e i n a s e inhibitor
labelled w i t h
complex
alk. P h o s p h a t a s e
lE-aiPl]
l a b - 0 ^ PH-API]
•
ab-E/E-OciPI/ab-(XiPI (-AP)
p-nitrophenol
~*
(00^05)
p-nitrophenylPhosphate
F i g u r e 6. R e a c t i o n scheme o f t h e s o l i d p h a s e e n z y m e - l i n k e d immunoassay used f o r d e t e c t i n g t h e complex o f p o l y m o r p h o n u c l e a r (PMN) e l a s t a s e ( E ) w i t h a . - p r o t e i n a s e i n h i b i t o r (a-^PI) in b i o l o g i c a l samples. AP = a l R a l i n e P h o s p h a t a s e .
The
a s s a y i s p e r f o r m e d b y (a) i n c u b a t i n g
complex
produced i n v i t r o )
Standards { i . e . ,
the
o r unknowns f o r 1 h i n p o l y s t y r e n e
315
tubes (c)
coated
with
sheep
antibodies
i n c u b a t i n g t h e tubes
tase
with
phatase (d)
a n d (e) d e t e r m i n a t i o n
Phosphatase
activity
specimen.
ly
i n this
of
the complex
ma
s p e c i m e n s was e x c e l l e n t .
pooled from 26
with
as the
and found t o
o f the elas-
reacted
linear-
1 t o 80 p 1 .
and added
Within-run
p l a s m a w a s 4 t o 8%, b e t w e e n - r u n
Recovery
to different
plas-
V a r i a t i o n (CV) w i t h V a r i a t i o n (CV) r a n g e d
ränge i n c i t r a t e d
p l a s m a w a s 98 +
u g / 1 (mean + s . d . , n = 4 3 ) .
release
prospective
repetitively
induced
study, i n over
lowed by e i t h e r
by major
30 c a s e s
recovery
temperature
> 38.5° C; l e u k o c y t e s
tients, to
< 100,000/mm
Plasma
value;
fourteen
septicemia
without
of infection
3
survived
blood
criteria
positive
estimated
drops
of sepsis:
culture;
body
> 30% below t h e
culture.
( g r o u p B) w h i l e
(group C ) ; eleven
surgery f o l -
> 1 5 , 0 0 0 o r < 5,000/mm ;
or platelet
positive
controls
Of t h e s e
sixteen
pa-
succumbed
( g r o u p A)
recovered
complications.
levels
o f E-c^PI a r e below
viduals or preoperative 3-fold increase
the
with
In
o r by s e p t i c e m i a ( 2 1 ) .
met a c c e p t e d
site
preoperative
and s e p t i c e m i a .
o f major abdominal
unequivocal
platelets
surgery
plasma E-a^PI complexes were 120 c a s e s
uncomplicated
Among t h e l a t t e r ,
a
alka-
w a s 0.25 n g o f c o m p l e x e d Normal plasma
i n v i t r o
The n o r m a l
inhibitor,
i n t h e sample.
sample volumes from
produced
3 t o 8%.
Elastase a
assay
complex
Phos-
fixed
p-nitrophenylphosphate
o f the assay
i n the test
phase
the concentration
inhibitor
detection limit
of solid
a t 4 0 5 nm i s m e a s u r e d
correlated with
tase-a^-proteinase
elastase
with
The a b s o r b a n c e
linearily
The
alkaline
l a b e l e d r a b b i t - a n t i b o d i e s t o ot-^-proteinase
Substrate. be
(b) w a s h i n g ,
complex o f e l a s -
c^-proteinase inhibitor for 1 h with
washing,
line
to elastase,
containing the fixed
preoperative
patients.
(Figure
7).
mean v a l u e
100 ng/ml
i n healthy
The O p e r a t i o n
causes
R e t r o s p e c t i v e l y , we n o t e d
o f g r o u p C was a l r e a d y
indiup t o that
elevated,
316
(n.r.
E - ^ P l
ng/ml
«60-110)
( m e a n t S.E.M.)
1000
p< 0.0025 p< 0.0005 p< 0.0005
n.s. p < 0.012 p< 0.0125
F i g u r e 7. M e a n p l a s m a l e v e l s o f e l a s t a s e - a ^ - p r o t e i n a s e i n h i b i t o r complex (E-a,PI) i n g r o u p s o f p a t i e n t s s u b j e c t e d t o maj o r abdominal surgery: Group A p a t i e n t s (n = 11) b e i n g w i t h o u t p o s t o p e r a t i v e i n f e c t i o n ; Group B p a t i e n t s (n = 14) s u r v i v i n g p o s t o p e r a t i v e s e p t i c e m i a ; G r o u p C p a t i e n t s (n = 16) d y i n g due t o s e p t i c e m i a . T h e E-a-.PI l e v e l s a r e g i v e n a s m e a n v a l u e s (+ SEM) f o r t h e d a y b e f o r e O p e r a t i o n , t h e d a y a f t e r Operation, as w e l l as f o r t h e p o s t o p e r a t i v e phase before s e p s i s , a t onset o f s e p s i s , and during septicemia. Last determ i n a t i o n s were done o n d a y o f d i s c h a r g e (D) f o r G r o u p A , o n d a y o f r e c o v e r y (R) f o r G r o u p B , a n d b e f o r e d e a t h (D) f o r G r o u p C. n r = n o r m a l r ä n g e ; n s = n o t s i g n i f i c a n t ; p = s i g n i ficance.
possibly
due t o i n c r e a s e d
ed
before
es
may h a v e c a u s e d
tion.'
Operation.
ation. levels
elevated
individual
recovery
increased peaks
as high
decrease
i nboth
high
until
focus-
maintained
clinically,
after
Oper-
E-a^PI
g r o u p s B a n d C, w i t h
a s 2,500 n g / m l .
E-c^PI remained
infect-
f o l l o w i n g Opera-
B and C
f o r s e v e r a l days
was d i a g n o s e d
manyfold
was a c c o m p a n i e d
ränge.
mean
i n6 patients
of theinfection
g r o u p A, g r o u p s
E-ct-^PI l e v e l s
When s e p t i c e m i a were
septicemia,
mal
theslight
Contrasting with
moderately
concentrations
S u r g i c a l removal
With
death
persisting
(group C ) , w h i l e
by a decrease o f E-a^PI
t o t h e nor-
317
Elastase Other with
release
indicators E-a-^PI.
important related ing
and plasma
Concentrations
inhibitor
t o E-a-^PI
the course
factor
of inflammation
consumption
were
of antithrombin
of the c l o t t i n g
(Figure
assayed
8) .
of septicemia,
i n septicemia. in parallel
I I I , t h e most
c a s c a d e , were i n v e r s e l y
Particularly antithrombin
at onset
and
I I I reached
dur-
clini-
F i g u r e 8. Mean p l a s m a l e v e l s o f t h e i n h i b i t o r y a c t i v i t y o f a n t i t h r o m b i n I I I (AT I I I ) i n g r o u p s o f p a t i e n t s s u b j e c t e d t o major abdominal surgery. F o r d e t a i l s , s e e l e g e n d t o F i g u r e 7. n v = n o r m a l v a l u e ; % d . NW = p e r c e n t o f n o r m a l v a l u e (reference plasma = 100%).
cally
critical
lopathy ly and
concentrations
or disseminated
lowered
Severe subunit
the risk
enzyme
were observed factor
XIII
consumption of o^-macroglobulin of factor
XIII
of
hypercoagu-
intravascular coagulation.
concentrations
for coagulation
posing
(which
i s easily
for
Similar-
c^-macroglobulin
(Figure 9).
along
with
the
susceptible to
carrier degra-
318
dation subunit dences ing
by l y s o s o m a l (which
and t h e e n z y m a t i c a l l y
t h e i m b a l a n c e among v a r i o u s
septicemia.
suggest
elastase)
i s p r i m a r i l y consumed d u r i n g
The c o n c o m i t a n t
the causal
circulation
role
and there
blood
consuming
evi-
enzyme c a s c a d e s
e l e v a t i o n o f E-a^PI
of lysosomal
active
clotting)
proteinases
Proteinase
released
inhibitor
dur-
levels into
proteins.
Plasma levels, elevated (f) or decreased (I): (M) highly signif.
(n) normal
M) signif.
Parameter
Sepsis Prefinal Survival c
H
H
n
Antithrombin m
a
it
II
n
Factor XDJ
a
ü
II
n
cx -Macroglobulin
a
Ii
Ii
n
c
Ii
II
n-l
C - reactive protein
c
it
ff
n
oi - Proteinase inhibitor
a
n-j
n-|
n-|
oc -P(asnin inhibitor
a
n
n
n
-Antichymotrypsin
c
1
n
n
a
n-|
n
n-|
c
n
n
n
E-
PI
complex
2
1
2
C1 Inactivator
activity assay
concentration assay
F i g u r e 9. C o r r e l a t i o n s b e t w e e n mean p l a s m a l e v e l s o f e l a s t a s e - a - P r o t e i n a s e i n h i b i t o r complex (E-a,PI) and o t h e r p l a s ma f a c t o r s i n p a t i e n t s s u f f e r i n g f r o m s e p t i c e m i a a f t e r m a j o r abdominal surgery. S i g n i f i c a n t l y elevated plasma l e v e l s o f E-a,PI and t h e u n s p e c i f i c acute phase r e a c t a n t C - r e a c t i v e prot e i n are i n v e r s e l y correlated to a high s t a t i s t i c a l l y signific a n t c o n s u m p t i o n o f a n t i t h r o m b i n I I I , f a c t o r X I I I a n d c^-mac r o g l o b u l i n d u r i n g s e p s i s and p r e f i n a l ; a l l p r o t e i n l e v e l s n o r m a l i z e d i n p a t i e n t s who s u r v i v e d t h e i n f e c t i o n . The o t h e r given P r o t e i n a s e i n h i b i t o r s , which a r e a l s o acute phase react a n t s , showed n o r m a l o r o n l y s l i g h t l y i n c r e a s e d p l a s m a l e v e l s .
319
Remarkably,
a^-proteinase
antichymotrypsin, changed ure
at
9).
the
In
contrast
inhibitors
their
synthesis
be
can
tion get
of
tions,
the
the
Stimulus
phase
occur.
C-reactive specific
concentrations
infection
Assay
and
than
methods
ment.
Jointly
immunological inactive allows
and
10).
We
onset
using
a
assay
of
the the
this
i s the
did both 9),
rapidly
eliminated
assay
probably
trial,
formation
B
or and
inhibitors
phase
no
fur-
allow
patients at onset p a t i e n t s i n the
severity
the
disof course
E - a ^ P I more
of
to
plasma
and
d i d not
that
a
reactant
Operation,
C
tar-
condi-
time f o r
showed h i g h e s t
C
with
inflammatory
enough
T h u s , CRP
can
consump-
given
first
acute
after
(assaying
speci-
postoperative
assay,
principles
the
Only
a
for
similar
a -M-proteinase 2
an
together)
functional ct2-macroresults
complexes
are
circulation.
acute
cases of
produce
and
inhibitors
i s desired,
choice.
com-
inhibitor,
complex
actual consumption of Information
special
active
enzyme-inhibitor
method o f
and
deserve
Inhibition
assay
because
from
shock, nine
inhibitors
functional
and
Unless
Pancreatogenic
an
i t
f o r the
Under the the
and
increased
Thus,
Proteinase
believe
and
for Proteinase
(Figure
second
days
septicemia.
both
inhibitor
inhibition globulin
plasma
(CRP),
concentration
11).
considerably
allowing
activity,
three
reactants,
compensates
patients,
(Fig-
o^-macroglobulinr
phase
complex
a^-
CRP.
calculation
(Figure
be
by
between group B
(Figure
reflects
infection
to
septicemia
in infection.
trauma presented
of
of
inhibitor,
measurably
I I I and
inactivation.
between group A
septicema
fically
proteins
protein
during
to
organism
inhibitor
up
increase
crimination the
the
response
without
of
expected
other
operative
course
acute
response or
inhibitory by
c^-plasmin were not
antithrombin
i n non-infected
füll a c u t e
ther
be
that
enzymes, o r
the
known t o be
postoperative
hypothesized
during
to
are
inhibitor,
Cl-inactivator
onset or
these
during
and
inflammation
acute
pancreatitis
in general. were
In
studied.
a
320
E-MP1
" 1
5
+ CRP
10
15
20
30
[d]
Operation
F i g u r e 10. Comparison of the plasma l e v e l s of acute phase r e a c t a n t C - r e a c t i v e p r o t e i n (CRP) a n d e l a s t a s e - a - ^ - P r o t e i n a s e i n h i b i t o r complex (E-a,PI) o f t h r e e p a t i e n t s s u b j e c t e d t o major abdominal surgery: Patient A being without postoperative i n fection; Patient B surviving postoperative septicemia; Pat i e n t C d y i n g due t o s e p t i c e m i a . L a s t d e t e r m i n a t i o n was d o n e o n d a y o f d i s c h a r g e f o r P a t i e n t A, o n d a y o f r e c o v e r y f o r P a t i e n t B a n d b e f o r e d e a t h f o r P a t i e n t C.
321
Proteinase Inhibitors as Acute Phase Reactants (o^PI • o^AT, oc PI, C1 INA, o^AC) 2
Concentration (immunol. assay)
Inhibitor level
enzyme - inhibitor complex formation
Antienzymatic activity resulting Inflannarion (hours or doys)
F i g u r e 11. Reasons f o r t h e d i s c r e p a n c y between measureable p r o t e i n c o n c e n t r a t i o n and a n t i - e n z y m a t i c a c t i v i t y o f plasma P r o t e i n a s e i n h i b i t o r s r e s p o n d i n g as acute phase r e a c t a n t s during inflammation. Consumption by complex f o r m a t i o n and o t h e r i n a c t i v a t i o n p r o c e s s e s may b e c o m p e n s a t e d b y i n c r e a s e d p r o d u c t i o n , r e s u l t i n g i n an i n h i b i t o r y a c t i v i t y c l o s e t o t h e normal ränge. See legend o f F i g u r e 4 f o r a b b r e v i a t i o n s .
In
these
cularly plasma and
patients,
i n t h e shock
antithrombin
teinase
phase
biochemical
responds
shock,
Faculty in
accord
elsewhere).
enzyme
with
cascades.
the following
inflammation,
such
the liberated
n e u t r o p h i l e l a s t a s ec o r -
as s e p t i c e m i a and
o f the inflammatory
(1984),
of the University
detail
data
elas-
consumption o f Pro-
on t h e b l o o d
condition (for clinical
( 1 9 8 2 ) a n d H. K o r t m a n n
o^-macroglobulin
the release of neutrophil by concomitant
and c l i n i c a l
to the severity
the c l i n i c a l
correlated with
and e f f e c t s
In acute
pancreatogenic
parti-
( F i g u r e 1 2 ) , As i n s e p t i c e m i a ,
I I I , i . e . ,
accompanied
inhibitors
hypothesis:
as
E - a ^ P I was h i g h l y e l e v a t e d ,
E - a ^ P I was i n v e r s e l y
t a s e was a g a i n
Our
plasma
data,
Habilitation
o f Munich;
response
they
s e e H.
theses,
will
as w e l l Duswald Medical
be p u b l i s h e d
Imbalance o f the p h y s i o l o g i c a l
equili-
322
2200
Eiastase-fX/j PI Complex
1800 I
1400
c 1000 ] 600200100 80 %
a2-Macroglobulin
604020 1001 T 80
%
60
k
Antithrombin III
.
4020I
A
I
I
I
012 24
I
I
l
J/' I »
t
48 72 96h D
F i g u r e 12. M e a n p l a s m a l e v e l s o f t h e E-a-^PI c o m p l e x a s w e l l a s o f AT I I a n d a^M i n 9 p a t i e n t s s u f f e r m g f r o m p a n c r e a t o genic shock I n t h e mean v a l u e o f t h e l o w e r E - a , P I c u r v e , a p a t i e n t i s e x c l u d e d w h i c h showed 6 0 - f o l d i n c r e a s e i n E - a ^ P I pancreacomplex i n the shock phase. A = diagno.sis o f a c u t e t i t i s ; 0 = p a n c r e a t o g e n i c s h o c k pha s e f o l l o w e d b y r e c o v e r y ( a b s c i s s a In h o u r s ) ; D = d i s c h a r g e T h e l e v e l s o f o^M a n d AT (100%, c f . l e f t o r III are r e l ated t o pool plasma as Standard d i n a t e s ) . E- a ^ P I l e v e l s a r e g i v e n i n n g / m l .
323
brium be
a
Our
between major
view
reason
the
Multiple of
the
of
by
potent
I I I and
trauma plasma
of
blood
E - a ^ P I was
To
quantify
an
injury scale
13,
plasma
jured
patients
tients while
with the
levels but
not
more
20-fold
plasmin
and
these
the
findings,
as
blood
release
of
by
a^Pl fused ther
explain plasma
of
the
factors
blood as
normal. mean
increases
showed
peak
E-a^PI
groups,
other
a
^ ^ / l (tjy
i n v e s t i g a t i o n s have elastase
2
due a
bout
of to
to
the 1 h)
several
lysosomal
III
are
with
release
and
the
stor-
is
adminthat
endogenous
faster clearance of
of
Fur-
relationship
consumption
of
E-
trans-
days n o r m a l l y ) .
show w h e t h e r a
stable
continuous
E-a^PI
that
in
other
quite a
for
blood
our
observed
than
could
Plasminogen,
hand,
alter
slow
normal
h a n d , we
with
appreciable
significantly
so in-
10-fold
d e t a i l e d i n v e s t i g a t i o n showed
not
Fig-
Pa-
antithrombin
was
in
in
injury
Substitution of
other
neutrophils
loss
moderately
liberated
and
pat-
multiple
shown
degree of
i n question the
although
shown),
factors
and
inconsistency
On
with
and
Prothrombin,
Extensive
this
from
Thus,
from c i r c u l a t i o n
tween n e u t r o p h i l
On
c^-macroglobulin
did
interesting
l e v e l s towards
c o r r e l a t i o n between
( r e s u l t s not
An
a l l three
E-c^PI
recovery.
transfusion,
plasma
In
the
consumption
14).
endotoxemia.
levels 5-fold showed
dimin-
including
by
the
patients
early
inhibitors
in patients
may
mechanisms.
i n which
after accident,
norm. of
preservation.
exogenous E-a^Pl level
the
during
elastase
(Figure
istered
the
induced
injuries
trauma p a t i e n t s .
c a s e s may
during
hours
inhibitors
factors
developed,
injured
decrease
inhibitor,
multiple
14
severe
demonstrate a
was
mean E - a ^ P I
above
occurred
elastase
age
had
XIII
correlated with
to
most h e a v i l y
continuous
values
8
plasma
tissue destruction
E-a^Pl
From
Proteinase
observed
injuries.
expressed,
(22),
transfusions.
accidents, ure
their
pathological
studies
various
factor
and
and
underlying animal
exogenous
consumption
antithrombin
tern
for
i s supported
application ished
endogenous p r o t e i n a s e s
be-
blood
324
[ngAnl]
2500-
E
-
oc
1
PI
2000 (Sit SEM)
1500-
1
1000
500 ^
^
*
1
^
*
I
(n = 12
100 ~4
12
20 I 28 34 40 461 52 1 2
S
V % 8 8 3
J 100 [h] After Accident 4 [d]
F i g u r e 13. Mean p l a s m a l e v e l s o f t h e e l a s t a s e - o ^ - p r o t e i n a s e i n h i b i t o r c o m p l e x ( E - a . . P I ) i n 27 p a t i e n t s a f t e r m u l t i p l e traumas. On t h e b a s i s o f a h o s p i t a l i n t e r n a l s c a l e ( H I S ) , r e f l e c t i n g the s e v e r i t y o f i n j u r y , p a t i e n t s were a l l i e d t o 3 groups: I (n = 1 2 ) , m o d e r a t e l y wounded ( H I S : 6 . 3 + 0 . 6 ) ; I I (n = 1 1 ) , s e v e r e l y i n j u r e d ( H I S : 1 0 . 0 + 1 . 0 ) ; I I I ( n = 4 ) , m o s t h e a v i l y i n j u r e d ( H I S : 15.3 + 1 . 0 ) . The n o r m a l ränge o f E - a ^ P I (60 - 1 1 0 n g / m l ) i s a l s o i n d i c a t e d .
325
Plasma Levels of E-oc PI 1
oc M, AT III and FII 2
in Stored Blood [%] 120-
1
U
1
21
28
35
[d]
Days of Storage
F i g u r e 14. Mean p l a s m a l e v e l s o f e l a s t a s e - a - p r o t e i n a s e i n h i b i t o r c o m p l e x (E-a, P I ) , a ^ - m a c r o g l o b u l i n (a M), antithrombin I I I (AT I I I ) a n d P r o t h r o m b i n (F I I ) i n s t o r e d b l o o d s a m p l e s (n = 1 1 ) a s a f u n c t i o n o f s t o r a g e t i m e ( i n d a y s ) . The l e v e l s o f a M , AT I I I a n d F I I a r e r e l a t e d t o p o o l p l a s m a a s Stand a r d (100%, c f . l e f t Ordinate). E-a^^PI l e v e l s a r e g i v e n i n ng/ml ( o f . r i g h t o r d i n a t e ) . ?
2
326
components as
well
i s measureable only
as lymph
inflamed
area
i n patients
Nevertheless, tients
i n e f f l u e n t s and lavage
and venous b l o o d
the high
samples
receiving large
E-c^PI
i n d i c a t e the strong
levels
from blood
or
transfusions.
i n severely
inflammatory
fluids
t h e wounded
injured pa-
response of the o r -
ganism.
Other of
clinical
plasma
(23)
studies.
neutrophil
elastase
and i n r h e u m a t o i d
bined
with
clinical
PMN
The p o s s i b l e was
arthritis
Information,
diagnostic
studied (20,23) plasma
significance
further (Figure
i n gestosis 15).
E-a-^PI s e e m s t o
Comallow
Elastase as a Marker of the Inflammatory Response
Range of application
Meaning of elevated E -oc1 PI levels
Postoperative infections
Diagnosis, course, prognosis (sepsis)
Multiple trauma
Severity, course, complications (sepsis)
Pregnancy: gestosis
Early diagnosis of ARDS
Rheumatoid arthritis Pleural effusion
Differentiation
Hemodialysis
Suitability / effect of dialyzer membranes
Myelocytic leukemia
Refined Classification: normal / defective
inflammatory / non-inflammatory inflammatory / malignant
enzyme equipment of leukemic cells
F i g u r e 15. P o l y m o r p h o n u c l e a r (PMN) e l a s t a s e a s a m a r k e r o f the inflammatory response under various p a t h o l o g i c a l c o n d i tions. See t e x t f o r l i t e r a t u r e references.
early and tory cal
diagnosis o f a d u l t
differentiation State. events
The l a t t e r leading
respiratory
distress
o f an i n f l a m m a t o r y seems
t o be t r u e
to pleural effusions
a^PI
i s a s e n s i t i v e marker
eign
surfaces
from a also
Syndrome
for pathologi-
(23).
f o r the stimulatory
on n e u t r o p h i l s
during
(ARDS)
non-inflamma-
hemodialysis
Further,
E-
e f f e c t o ff o r (23,24).
327
Finally,
investigations
leukemic
cells
on t h e l y s o s o m a l
and t h e r e l e a s e o f e l a s t a s e i n m y e l o c y t i c l e u -
kemia
patients
tions
represents y e t another
to
enzyme make-up o f
under
inflammation
certain
clinical
and t h e r a p e u t i c c o n d i -
application
not directly
related
(21,25).
Acknowledgements
Supported
by t h e Deutsche Forschungsgemeinschaft,
schungsbereich the FRG.
51 ( B / 3 0 ) a n d 0 2 0 7
Bundesministerium We
thank
(LP 8 ) , Munich,
fuer Forschung
SonderforFRG, a n d by
und T e c h n o l o g i e ,
M r s . U. H o f a n d M r s . C. S e i d l
Bonn,
f o r technical
assistance.
References
1.
Solomkin America
J . S . , Simmons R.L.: 63, 225-243 (1983).
Surg
Clinics
2.
D i n g l e J.T. ( e d ) : Lysosomes: A L a b o r a t o r y Handbook, E l s e v i e r N o r t h H o l l a n d , A m s t e r d a m , 1 9 7 7 , 323 p p .
3.
K l e b a n o f f S . J . , C l a r k R.A. ( e d s ) : The N e u t r o p h i l : F u n c t i o n and C l i n i c a l D i s o r d e r s , E l s e v i e r North H o l l a n d , A m s t e r d a m , 1 9 7 8 , 810 p p .
4.
J o c h u m M., L a n d e r S., H e i m b u r g e r N., F r i t z P h y s i o l Chem 362, 103-112 ( 1 9 8 1 ) .
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