Clin. Biochem. 9, (3) 165-167 (1976)
Errors in Measuring Enzyme Activity By Reaction-Rate Methods A. R. HENDERSON Department of Clinical Biochemistry, University Hospital, University of Western 0ntarm, London, Ontario
CLBIA, 9, (3) 165-167 (1976)
Clin. Biochem.. Henderson, A. R.
Department of Clinical Biochemistry, University Hospital, U~liversity of Western O~tario, Lo~do~l, Ontario. ERRORS 1N M E A S U R I N G ENZYME A C T I V I T Y BY R E A C T I O N - R A T E METHODS 1. The two types of assay used in clinical enzymologsr - - reaction-rate and two-point assays are usually performed with different types of analyzers. A third type, the multi-point analyzer, has been introduced recently. 2. Reaction-rate. and two-point enzyme assays have sources of error inherent in their methodologies. There are also major errors due to the analytical system itself; these are considered systematically.
THE VELOCITY OF MOST ENZYME REACTIONS i~t vitro falls o f f w i t h time. The reasons f o r t h i s a r e many. F o r example, the r e a c t i o n p r o d u c t s m a y i n h i b i t the enzyme, t h e s u b s t r a t e c o n c e n t r a t i o n m a y fall to levels a t which t h e enzyme is u n s a t u r a t e d ; the r e v e r s e r e a c t i o n m a y become s i g n i f i c a n t ; or the enzyme, or its s u b s t r a t e ( s ) or c o f a c t o r ( s ) , may become i n a c t i v a t e d . These f a c t o r s a r e i m p o r t a n t , as the m e a s u r e m e n t of enzyme a c t i v i t y is t h e only p r a c t i c a b l e method of d e t e r m i n i n g active enzyme mass. The fallacies i n h e r e n t in t h i s a s s u m p t i o n a r e o b v i o u s ; however, since clinical b i o c h e m i s t r y , like medicine, is a p r a g m a t i c a c t i v i t y , we m u s t a d o p t v a r i o u s o p e r a t i o n a l d e f i n i t i o n s . T h u s it is i m p o r t a n t t h a t the c o n d i t i o n s u n d e r which an enzyme r e a c t i o n is m e a s u r e d a r e such t h a t only i n i t i a l velocities a r e obs e r v e d {i.e., d u r i n g the phase of z e r o - o r d e r k i n e t i c s ) , when the f a c t o r s m e n t i o n e d above a r e not o p e r a t i n g .
Methods of measurements T h e r e a r e two basic m e t h o d s f o r m e a s u r i n g enzyme a c t i v i t y d u r i n g its z e r o - o r d e r p h a s e : a) reaction-rate o r kinetic methods, in which t h e r e a c t i o n is c o n t i n u o u s ly m o n i t o r e d ( t h u s checking t h a t t h e i n i t l a l velocity is m a i n t a i n e d ) ; and b) fixed incubation or two-point assays, in which c u m u l a t i v e c h a n g e is m e a s u r e d . Usually, a d i f f e r e n t t y p e of e n z y m e - a n a l y z e r is used f o r each method. (a) R e a c t i o n - r a t e a n a l y z e r s include a n y t y p e of s p e c t r o p h o t o m e t e r t h a t can be used to follow an enzyme r e a c t i o n continuously, and ' d e d i c a t e d ' a n a l y z e r s such as the L K B r e a c t i o n - r a t e analyzer, t h a t p r o v i d e s a u t o m a t i c p r e l i m i n a r y i n c u b a t i o n and in-
i t i a t e s the enzyme r e a c t i o n s e q u e n t i a l l y for up to 100 samples, and the P y e - U n i c a m AC 60 system. (b) Nonm e c h a n i s e d s p e c t r o p h o t o m e t e r s can be used f o r f i x e d i n c u b a t i o n methods, b u t when n u m e r o u s enzyme a s s a y s a r e to be done these methods can be a u t o m a t e d (e.g., b y the Technicon continuous-flow s y s t e m ) . Between these e x t r e m e s a r e the i n c r e a s i n g l y numerous h y b r i d i n s t r u m e n t s v a r i o u s l y d e s c r i b e d as r e a c t i o n - r a t e or k i n e t i c enzyme-analyzers. B u t a r e t h e y ? F o r example, E l e c t r o - N u c l e o n i c s Gemsaec f a s t parallel a n a l y z e r does not c o n t i n u o u s l y m o n i t o r enzyme r e a c t i o n s : it t a k e s one r e a d i n g e v e r y 130 msec f o r a t o t a l of e i g h t r e a d i n g s , then pauses f o r 10, 20, o r even 30 sec or so b e f o r e t a k i n g a n o t h e r set of r e a d i n g s . Thus the r e a c t i o n is followed for a b o u t 1040"msec, d u r i n g which e i g h t points a r e collected to produce one ' m e a n d a t a point', and a set of these points is used to d e t e r m i n e enzyme a c t i v i t y . S i m i l a r l y , the A b b o t t A B A - 1 0 0 b i c h r o m a t i c a n a l y z e r t a k e s t h r e e r e a d i n g s at 15- or 30-sec i n t e r v a l s , the Gilford-300 a u t o m a t i c enzyme a n a l y z e r t a k e s r e a d i n g s at 10-sec intervals, and t h e Technicon S M A C s y s t e m uses some t h r e e - p o i n t analysis. Clearly, these h y b r i d a n a l y t i c a l a p p r o a c h e s do not provide t r u e m e a s u r e m e n t s of reaction rates, and the s y s t e m s p r o b a b l y should be r e f e r r e d to as m u l t i point enzyme-analyzers. K i n e t i c m e t h o d s of enzyme a s s a y a r e g e n e r a l l y reg a r d e d by most clinical e n z y m o l o g i s t s as the method of choice '~" ~'. However, t h e a p p a r a t u s may be b o t h expensive and complex, and t h e r e f o r e , most b i o c h e m i s t s use a blend o f k i n e t i c methods and t w o - p o i n t a s s a y syst e m s f o r the wide v a r i e t y of r o u t i n e enzyme analyses. W h e r e a s t h e two p o i n t a s s a y s y s t e m s have been v o c i f e r o u s l y criticised, the i n n a t e s u p e r i o r i t y of t h e kinetic method has tended to overshadow some of its defects '~). Thus, G o l d b e r g et a/'') have d r a w n a t t e n t i o n to t h e e f f e c t s of lag and of a c c e l e r a t e d and b l a n k r e a c t i o n s when d e t e r m i n i n g enzyme a c t i v i t i e s , p r o b lems i n h e r e n t in a m e c h a n i s e d s y s t e m w i t h a fixed t i m e cycle f o r r e a c t i o n i n i t i a t i o n and a b s o r b a n c e recording. Clearly, the k i n e t i c enzyme a n a l y z e r should be able to m o n i t o r its own p e r f o r m a n c e , a l t e r i n g t h e cycle t i m i n g to t a k e account of the a c t i v i t i e s b e i n g assayed. The p r i n c i p l e s of t w o - p o i n t enzyme a s s a y s have been well reviewed'SL One of the f u n d a m e n t a l objections to t h i s t y p e of a s s a y is t h a t it is impossible to know when d e v i a t i o n f r o m z e r o - o r d e r k i n e t i c s occ u r s : the only ( p r o s p e c t i v e ) method f o r checking t h i s is by t e s t i n g w i t h a wide v a r i e t y of s e r a and a s s u m i n g t h a t l i n e a r i t y will continue w i t h i n t h e e s t a b l i s h e d l i m i t a t i o n s . O t h e r o b j e c t i o n s a r e t h a t t h e y m u s t be c a l i b r a t e d by some o t h e r a s s a y systems, and t h a t t h e t e m p e r a t u r e at which an enzyme r e a c t i o n is c a r r i e d
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o u t is o f t e n in d o u b t w i t h e i t h e r a m e c h a n i s e d c o n t i n u ous-flow or d i s c r e t e - s a m p l e a s s a y system. D e s p i t e these objections, a g r e a t m a n y clinical b i o c h e m i s t r y l a b o r a t o r i e s use m e c h a n i s e d t w o - p o i n t a s s a y m e t h o d s w i t h a r e a s o n a b l e d e g r e e of success, and it m u s t be a c k n o w l e d g e d t h a t t h e i n s t r u m e n t s r e q u i r e d a r e cons i d e r a b l y c h e a p e r t h a n those f o r c o m p a r a b l e k i n e t i c methods.
Errors inherent in the analytical s}~tem A r e c e n t r e v i e w c~' of t h i s p r o b l e m s t r e s s e d the imp o r t a n c e of t h i s a s p e c t in clinical enzymology. E n z y m e a c t i v i t y is calculated f r o m t h e c h a n g e in a b s o r b a n c e w i t h t i m e b y the following f o r m u l a : E n z y m e a c t i v i t y ( u n i t s / l i t r e ) = l / L " TC " 1 / 1 0 - s e " [ ( V s + VR)/Vs] " 1/~St" A A • 1000, w h e r e L = p a t h l e n g t h of cuvette, TC = t e m p e r a t u r e c o r r e c t i o n f a c t o r , e = m o l a r a b s o r p t i v i t y , Vs = s a m p l e volume, V,~ = r e a g e n t volume, A t = t i m e interval, and /xA = abs o r b a n c e ch.ange. E r r o r in m e a s u r e m e n t of A A can be c l a s s i f i e d as s p e c t r o p h o t o m e t r i c i n a c c u r a c y and n o n - l i n e a r i t y , s t r a y e n e r g y , effect of band width, w a v e l e n g t h inaccuracy ~nd imprecision, and s p e c t r o p h o t o m e t r i c noise. Maclin et al '~ considered the m a x i m a l possible e r r o r in each component of the system, and it is an i n s t r u c t i v e exercise to e x a m i n e the sources of e r r o r in each of these components. P a t h - l e n g t h o f ~uvettes. R a n d (quoted by Maclin et al ~2) showed t h a t n o m i n a l and a c t u a l p a t h - l e n g t h s of commercial cuvettes d i f f e r by as much as ± 5%, a r a t h e r unexpected source of e r r o r . A corollary is t h e s t a t e of cleanliness of the cuvette faces. Ideally, cuvettes should be c a r e f u l l y matched and should a l w a y s be placed w i t h t h e same face t o w a r d s the l i g h t source - - conditions n o t usually p r a c t i s e d . T h e cleaning and s t o r a g e of cuvettes has g e n e r a t e d a g r e a t deal of l i t e r a t u r e , b u t d e s p i t e this a wide v a r i e t y of cleaning methods is common w i t h i n a l a b o r a t o r y . Temperatu~'e-correction factor. Maclin et al"-' used the f a c t o r s produced by the B o e h r i n g e r M a n n h e i m Corp o r a t i o n to calculate the e f f e c t of t e m p e r a t u r e v a r i a tion on enzyme a c t i v i t y . I n o u r l a b o r a t o r y , however, we have f o u n d t h a t t e m p e r a t u r e v a r i a t i o n is d e p e n d e n t upon a s s a y conditions (i.e., t h e k i t b e i n g used) ; t h e r e fore, c o r r e c t i o n f a c t o r s should be used w i t h caution. Even the species of enzyme b e i n g m e a s u r e d can a f f e c t the t e m p e r a t u r e c h a r a c t e r i s t i c s , as has been dem o n s t r a t e d f o r s e r u m a m y l a s e and m a c r o - a m y l a s e . Nonetheless, an a w a r e n e s s of the i m p o r t a n c e of temp e r a t u r e control, and knowledge of the l i m i t s of dev i a t i o n f r o m a set t e m p e r a t u r e , a r e n e c e s s a r y to an a p p r e c i a t i o n of possible e r r o r s a r i s i n g f r o m t h i s source. M o O r absorbance. This m a y a l t e r w i t h a s s a y t e m p e r a t u r e . F o r example, t h e m o l a r a b s o r b a n c e of N A D H a t 340 n m c h a n g e s by a p p r o x i m a t e l y 4 % , and a t 366 n m by 14%, when the a s s a y t e m p e r a t u r e is r a i s e d f r o m 15 ° to 35 °3. A n o t h e r p r o b l e m 6oncerns t h e a c t u a l value of c to be used on a p a r t i c u l a r i n s t r u m e n t ' 4 ' ' : t h i s is v e r y i m p o r t a n t when several s p e c t r o p h o t o m e t e r s a r e used f o r t h e same enzyme a s s a y (e.g., f o r r o u t i n e and 'stat' analyses).
V o l u m e m e a s u r e m e n t . The +-- 3 % e r r o r s t h a t Maclin et aI '2) r e c o r d e d in t h e e x a m p l e t h e y w e r e c o n s i d e r i n g a p p r o x i m a t e t h e w i t h i n - b a t c h c o e f f i c i e n t of v a r i a t i o n of m a n y enzyme assays. Such d a t a a r e usually o b t a i n e d u n d e r n e a r ideal conditions, however, and m u c h g r e a t e r e r r o r s in p i p e t t i n g a r e common in r o u t i n e practice. The q u a l i t y - c o n t r o l checks in each a s s a y b a t c h can be (and often are!) more carefully pipetted than the clinical samples, a n d d i f f e r e n c e s in s p e c i m e n h a n d l i n g m a y go u n d e t e c t e d if a b l i n d - d u p l i c a t e check is n o t made, daily, w i t h a selected p a t i e n t sample. Thus, -----3 % m u s t be t a k e n as t h e lower l i m i t of e r r o r of t h e s y s t e m in r o u t i n e use. A b s o r b a n c e clwmge. A c c u r a c y and l i n e a r i t y of t h e s p e c t r o p h o t o m e t r i c r e s p o n s e can v a r y by --< 1% a n d 3 % r e s p e c t i v e l y '~). The e f f e c t s of s t r a y e n e r g y ( r a d i a tion of w a v e l e n g t h s o t h e r t h a n t h a t p a s s i n g t h r o u g h the sample) depend on t h e i n i t i a l a b s o r b a n c e of t h e t e s t solution, b u t h i g h s t a r t i n g a b s o r b a n c e s a r e quite common in clinical enzyme assays. I t has been calculated'-" t h a t a t y p i c a l ( 1 % ) s t r a y e n e r g y can r e s u l t in - - 4% e r r o r in t h e enzyme assay. B a n d - w i d t h also can a f f e c t the a c c u r a c y o f t h e m e a s u r e d a b s o r b a n c e . This can be c o r r e c t e d for by a l t e r i n g the value of m o l a r a b s o r b a n c e used in c a l c u l a t i n g enzyme a c t i v i t y . W h e n several i n s t r u m e n t s a r e in use in a l a b o r a t o r y , t h i s value should be d e t e r m i n e d f o r each i n s t r u m e n t , t h e r e by c o r r e c t i n g f o r e f f e c t s of t h e i r d i f f e r e n t bandw i d t h s . Maclin et al '2' c o n s i d e r t h e e f f e c t of w a v e l e n g t h i n a c c u r a c y s i m i l a r to t h a t of b a n d - w i d t h . Noise is a m a j o r source o f e r r o r : t h e s a m e w o r k e r s f o u n d t h a t t h i s c o n t r i b u t e d an e r r o r of n e a r l y 14% to t h e calculation of enzyme a c t i v i t y . T a k i n g into account all e r r o r s due to i n s t r u m e n t a tion, Maclin et al concluded t h a t the g r o s s e r r o r could r a n g e between + 35% to - 4 0 % in m e a s u r e m e n t s of enzyme a c t i v i t y . I n p r a c t i c e t h e e r r o r s a r e usually c o n s i d e r a b l y less, b u t these a u t h o r s ' conclusion t h a t t h e r e is c o n s i d e r a b l e room f o r i m p r o v e m e n t seems more t h a n j u s t i f i e d .
Instrument quality control A l t h o u g h some e r r o r s due to the s p e c t r o p h o t o m e t e r a p p e a r to be beyond the u s e r ' s control, he can t a k e c e r t a i n m e a s u r e s to o b t a i n o p t i m a l p e r f o r m a n c e . F o r all s p e c t r o p h o t o m e t e r s , c o n s t a n t m a i n t e n a n c e is essent i a l and the u s e r m u s t be p r e p a r e d to check p e r f o r m a n c e r e g u l a r l y . R a n d ' s r e p o r t ~ , w h i c h is an invaluable source of r e f e r e n c e on s p e c t r o p h o t o m e t r i c s t a n d a r d s , s u g g e s t s t h a t t h e s e checks include w a v e l e n g t h and p h o t o m e t r i c accuracy, and m e a s u r e m e n t of s t r a y l i g h t and p h o t o m e t r i c l i n e a r i t y to e n s u r e t h a t the i n s t r u m e n t is p e r f o r m i n g w i t h i n t h e m a n u f a c t u r e r ' s specifications. F i n a l l y , it is n e c e s s a r y to p o i n t out t h a t a n a l y t i c a l balances a p p e a r to receive even less a t t e n t i o n . To be r e a l l y effective, a n y c o n t r o l p r o g r a m of a n a l y t i c a l p r o c e d u r e s in a clinical l a b o r a t o r y (and t h a t in i t s e l f is a r a r e occurrence) m u s t include s p e c t r o p h o t o m e t e r s a n d a n a l y t i c a l balances. Q u a l i t y control m a y seem a long w a y f r o m c o n s i d e r a t i o n of enzyme assays, b u t a s s a y e r r o r m u s t include all p r o c e d u r e s and i n s t r u m e n t s d i r e c t l y o r i n d i r e c t l y involved in p r o d u c i n g a
CLINICAL ENZY.MOLOGY ASSAY value for enzyme activity. Only when all these aspects are fully controlled can we be c e r t a i n that enzyme assays are a n a l y t i c a l l y satisfactory. REFERENCES 1. Goldberg, D. M., Ellis, G. and Wilcock, A. R. (1971). Ann. Clin. Biochvm. 8, 189-194. 2. Maclin, E., Rohlfing, D. and Ansour, M. (1973). Clin. Chant. 19, 832-837. 3. Malcolm, A. D. B. (197~). Anal. Biochenz. 55, 278-281.
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4. Morell, S. A. and Doumas, B. T. (1973). Clin. Chem. 19, 783, 785. 5. Moss, D. W. (1972). Clin. Chem. 18, 1449-1454. 6. Rand, R. N. (1969). Clin. Chem. 15, 839-863. 7. Schales, O. (1973). Clin. Chem. 19, 434 435. " 8. Schwartz, M. K. (1972). Technicon Q. 4, 12-19. 9. Wilkinson, J. H. (1971). In Enzym6 assays in ~wdicine, ed. by McGowan, G. K. and Walters, G. J. CHn. Pathol. Suppl. 24, 14-21. #t
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Errors in Measuring Enzyme Activity By Reaction-Rate Methods A. R. HENDERSON Department of Clinical Biochemistry, University Hospital, University of Western 0ntarm, London, Ontario
CLBIA, 9, (3) 165-167 (1976)
Clin. Biochem.. Henderson, A. R.
Department of Clinical Biochemistry, University Hospital, U~liversity of Western O~tario, Lo~do~l, Ontario. ERRORS 1N M E A S U R I N G ENZYME A C T I V I T Y BY R E A C T I O N - R A T E METHODS 1. The two types of assay used in clinical enzymologsr - - reaction-rate and two-point assays are usually performed with different types of analyzers. A third type, the multi-point analyzer, has been introduced recently. 2. Reaction-rate. and two-point enzyme assays have sources of error inherent in their methodologies. There are also major errors due to the analytical system itself; these are considered systematically.
THE VELOCITY OF MOST ENZYME REACTIONS i~t vitro falls o f f w i t h time. The reasons f o r t h i s a r e many. F o r example, the r e a c t i o n p r o d u c t s m a y i n h i b i t the enzyme, t h e s u b s t r a t e c o n c e n t r a t i o n m a y fall to levels a t which t h e enzyme is u n s a t u r a t e d ; the r e v e r s e r e a c t i o n m a y become s i g n i f i c a n t ; or the enzyme, or its s u b s t r a t e ( s ) or c o f a c t o r ( s ) , may become i n a c t i v a t e d . These f a c t o r s a r e i m p o r t a n t , as the m e a s u r e m e n t of enzyme a c t i v i t y is t h e only p r a c t i c a b l e method of d e t e r m i n i n g active enzyme mass. The fallacies i n h e r e n t in t h i s a s s u m p t i o n a r e o b v i o u s ; however, since clinical b i o c h e m i s t r y , like medicine, is a p r a g m a t i c a c t i v i t y , we m u s t a d o p t v a r i o u s o p e r a t i o n a l d e f i n i t i o n s . T h u s it is i m p o r t a n t t h a t the c o n d i t i o n s u n d e r which an enzyme r e a c t i o n is m e a s u r e d a r e such t h a t only i n i t i a l velocities a r e obs e r v e d {i.e., d u r i n g the phase of z e r o - o r d e r k i n e t i c s ) , when the f a c t o r s m e n t i o n e d above a r e not o p e r a t i n g .
Methods of measurements T h e r e a r e two basic m e t h o d s f o r m e a s u r i n g enzyme a c t i v i t y d u r i n g its z e r o - o r d e r p h a s e : a) reaction-rate o r kinetic methods, in which t h e r e a c t i o n is c o n t i n u o u s ly m o n i t o r e d ( t h u s checking t h a t t h e i n i t l a l velocity is m a i n t a i n e d ) ; and b) fixed incubation or two-point assays, in which c u m u l a t i v e c h a n g e is m e a s u r e d . Usually, a d i f f e r e n t t y p e of e n z y m e - a n a l y z e r is used f o r each method. (a) R e a c t i o n - r a t e a n a l y z e r s include a n y t y p e of s p e c t r o p h o t o m e t e r t h a t can be used to follow an enzyme r e a c t i o n continuously, and ' d e d i c a t e d ' a n a l y z e r s such as the L K B r e a c t i o n - r a t e analyzer, t h a t p r o v i d e s a u t o m a t i c p r e l i m i n a r y i n c u b a t i o n and in-
i t i a t e s the enzyme r e a c t i o n s e q u e n t i a l l y for up to 100 samples, and the P y e - U n i c a m AC 60 system. (b) Nonm e c h a n i s e d s p e c t r o p h o t o m e t e r s can be used f o r f i x e d i n c u b a t i o n methods, b u t when n u m e r o u s enzyme a s s a y s a r e to be done these methods can be a u t o m a t e d (e.g., b y the Technicon continuous-flow s y s t e m ) . Between these e x t r e m e s a r e the i n c r e a s i n g l y numerous h y b r i d i n s t r u m e n t s v a r i o u s l y d e s c r i b e d as r e a c t i o n - r a t e or k i n e t i c enzyme-analyzers. B u t a r e t h e y ? F o r example, E l e c t r o - N u c l e o n i c s Gemsaec f a s t parallel a n a l y z e r does not c o n t i n u o u s l y m o n i t o r enzyme r e a c t i o n s : it t a k e s one r e a d i n g e v e r y 130 msec f o r a t o t a l of e i g h t r e a d i n g s , then pauses f o r 10, 20, o r even 30 sec or so b e f o r e t a k i n g a n o t h e r set of r e a d i n g s . Thus the r e a c t i o n is followed for a b o u t 1040"msec, d u r i n g which e i g h t points a r e collected to produce one ' m e a n d a t a point', and a set of these points is used to d e t e r m i n e enzyme a c t i v i t y . S i m i l a r l y , the A b b o t t A B A - 1 0 0 b i c h r o m a t i c a n a l y z e r t a k e s t h r e e r e a d i n g s at 15- or 30-sec i n t e r v a l s , the Gilford-300 a u t o m a t i c enzyme a n a l y z e r t a k e s r e a d i n g s at 10-sec intervals, and t h e Technicon S M A C s y s t e m uses some t h r e e - p o i n t analysis. Clearly, these h y b r i d a n a l y t i c a l a p p r o a c h e s do not provide t r u e m e a s u r e m e n t s of reaction rates, and the s y s t e m s p r o b a b l y should be r e f e r r e d to as m u l t i point enzyme-analyzers. K i n e t i c m e t h o d s of enzyme a s s a y a r e g e n e r a l l y reg a r d e d by most clinical e n z y m o l o g i s t s as the method of choice '~" ~'. However, t h e a p p a r a t u s may be b o t h expensive and complex, and t h e r e f o r e , most b i o c h e m i s t s use a blend o f k i n e t i c methods and t w o - p o i n t a s s a y syst e m s f o r the wide v a r i e t y of r o u t i n e enzyme analyses. W h e r e a s t h e two p o i n t a s s a y s y s t e m s have been v o c i f e r o u s l y criticised, the i n n a t e s u p e r i o r i t y of t h e kinetic method has tended to overshadow some of its defects '~). Thus, G o l d b e r g et a/'') have d r a w n a t t e n t i o n to t h e e f f e c t s of lag and of a c c e l e r a t e d and b l a n k r e a c t i o n s when d e t e r m i n i n g enzyme a c t i v i t i e s , p r o b lems i n h e r e n t in a m e c h a n i s e d s y s t e m w i t h a fixed t i m e cycle f o r r e a c t i o n i n i t i a t i o n and a b s o r b a n c e recording. Clearly, the k i n e t i c enzyme a n a l y z e r should be able to m o n i t o r its own p e r f o r m a n c e , a l t e r i n g t h e cycle t i m i n g to t a k e account of the a c t i v i t i e s b e i n g assayed. The p r i n c i p l e s of t w o - p o i n t enzyme a s s a y s have been well reviewed'SL One of the f u n d a m e n t a l objections to t h i s t y p e of a s s a y is t h a t it is impossible to know when d e v i a t i o n f r o m z e r o - o r d e r k i n e t i c s occ u r s : the only ( p r o s p e c t i v e ) method f o r checking t h i s is by t e s t i n g w i t h a wide v a r i e t y of s e r a and a s s u m i n g t h a t l i n e a r i t y will continue w i t h i n t h e e s t a b l i s h e d l i m i t a t i o n s . O t h e r o b j e c t i o n s a r e t h a t t h e y m u s t be c a l i b r a t e d by some o t h e r a s s a y systems, and t h a t t h e t e m p e r a t u r e at which an enzyme r e a c t i o n is c a r r i e d
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o u t is o f t e n in d o u b t w i t h e i t h e r a m e c h a n i s e d c o n t i n u ous-flow or d i s c r e t e - s a m p l e a s s a y system. D e s p i t e these objections, a g r e a t m a n y clinical b i o c h e m i s t r y l a b o r a t o r i e s use m e c h a n i s e d t w o - p o i n t a s s a y m e t h o d s w i t h a r e a s o n a b l e d e g r e e of success, and it m u s t be a c k n o w l e d g e d t h a t t h e i n s t r u m e n t s r e q u i r e d a r e cons i d e r a b l y c h e a p e r t h a n those f o r c o m p a r a b l e k i n e t i c methods.
Errors inherent in the analytical s}~tem A r e c e n t r e v i e w c~' of t h i s p r o b l e m s t r e s s e d the imp o r t a n c e of t h i s a s p e c t in clinical enzymology. E n z y m e a c t i v i t y is calculated f r o m t h e c h a n g e in a b s o r b a n c e w i t h t i m e b y the following f o r m u l a : E n z y m e a c t i v i t y ( u n i t s / l i t r e ) = l / L " TC " 1 / 1 0 - s e " [ ( V s + VR)/Vs] " 1/~St" A A • 1000, w h e r e L = p a t h l e n g t h of cuvette, TC = t e m p e r a t u r e c o r r e c t i o n f a c t o r , e = m o l a r a b s o r p t i v i t y , Vs = s a m p l e volume, V,~ = r e a g e n t volume, A t = t i m e interval, and /xA = abs o r b a n c e ch.ange. E r r o r in m e a s u r e m e n t of A A can be c l a s s i f i e d as s p e c t r o p h o t o m e t r i c i n a c c u r a c y and n o n - l i n e a r i t y , s t r a y e n e r g y , effect of band width, w a v e l e n g t h inaccuracy ~nd imprecision, and s p e c t r o p h o t o m e t r i c noise. Maclin et al '~ considered the m a x i m a l possible e r r o r in each component of the system, and it is an i n s t r u c t i v e exercise to e x a m i n e the sources of e r r o r in each of these components. P a t h - l e n g t h o f ~uvettes. R a n d (quoted by Maclin et al ~2) showed t h a t n o m i n a l and a c t u a l p a t h - l e n g t h s of commercial cuvettes d i f f e r by as much as ± 5%, a r a t h e r unexpected source of e r r o r . A corollary is t h e s t a t e of cleanliness of the cuvette faces. Ideally, cuvettes should be c a r e f u l l y matched and should a l w a y s be placed w i t h t h e same face t o w a r d s the l i g h t source - - conditions n o t usually p r a c t i s e d . T h e cleaning and s t o r a g e of cuvettes has g e n e r a t e d a g r e a t deal of l i t e r a t u r e , b u t d e s p i t e this a wide v a r i e t y of cleaning methods is common w i t h i n a l a b o r a t o r y . Temperatu~'e-correction factor. Maclin et al"-' used the f a c t o r s produced by the B o e h r i n g e r M a n n h e i m Corp o r a t i o n to calculate the e f f e c t of t e m p e r a t u r e v a r i a tion on enzyme a c t i v i t y . I n o u r l a b o r a t o r y , however, we have f o u n d t h a t t e m p e r a t u r e v a r i a t i o n is d e p e n d e n t upon a s s a y conditions (i.e., t h e k i t b e i n g used) ; t h e r e fore, c o r r e c t i o n f a c t o r s should be used w i t h caution. Even the species of enzyme b e i n g m e a s u r e d can a f f e c t the t e m p e r a t u r e c h a r a c t e r i s t i c s , as has been dem o n s t r a t e d f o r s e r u m a m y l a s e and m a c r o - a m y l a s e . Nonetheless, an a w a r e n e s s of the i m p o r t a n c e of temp e r a t u r e control, and knowledge of the l i m i t s of dev i a t i o n f r o m a set t e m p e r a t u r e , a r e n e c e s s a r y to an a p p r e c i a t i o n of possible e r r o r s a r i s i n g f r o m t h i s source. M o O r absorbance. This m a y a l t e r w i t h a s s a y t e m p e r a t u r e . F o r example, t h e m o l a r a b s o r b a n c e of N A D H a t 340 n m c h a n g e s by a p p r o x i m a t e l y 4 % , and a t 366 n m by 14%, when the a s s a y t e m p e r a t u r e is r a i s e d f r o m 15 ° to 35 °3. A n o t h e r p r o b l e m 6oncerns t h e a c t u a l value of c to be used on a p a r t i c u l a r i n s t r u m e n t ' 4 ' ' : t h i s is v e r y i m p o r t a n t when several s p e c t r o p h o t o m e t e r s a r e used f o r t h e same enzyme a s s a y (e.g., f o r r o u t i n e and 'stat' analyses).
V o l u m e m e a s u r e m e n t . The +-- 3 % e r r o r s t h a t Maclin et aI '2) r e c o r d e d in t h e e x a m p l e t h e y w e r e c o n s i d e r i n g a p p r o x i m a t e t h e w i t h i n - b a t c h c o e f f i c i e n t of v a r i a t i o n of m a n y enzyme assays. Such d a t a a r e usually o b t a i n e d u n d e r n e a r ideal conditions, however, and m u c h g r e a t e r e r r o r s in p i p e t t i n g a r e common in r o u t i n e practice. The q u a l i t y - c o n t r o l checks in each a s s a y b a t c h can be (and often are!) more carefully pipetted than the clinical samples, a n d d i f f e r e n c e s in s p e c i m e n h a n d l i n g m a y go u n d e t e c t e d if a b l i n d - d u p l i c a t e check is n o t made, daily, w i t h a selected p a t i e n t sample. Thus, -----3 % m u s t be t a k e n as t h e lower l i m i t of e r r o r of t h e s y s t e m in r o u t i n e use. A b s o r b a n c e clwmge. A c c u r a c y and l i n e a r i t y of t h e s p e c t r o p h o t o m e t r i c r e s p o n s e can v a r y by --< 1% a n d 3 % r e s p e c t i v e l y '~). The e f f e c t s of s t r a y e n e r g y ( r a d i a tion of w a v e l e n g t h s o t h e r t h a n t h a t p a s s i n g t h r o u g h the sample) depend on t h e i n i t i a l a b s o r b a n c e of t h e t e s t solution, b u t h i g h s t a r t i n g a b s o r b a n c e s a r e quite common in clinical enzyme assays. I t has been calculated'-" t h a t a t y p i c a l ( 1 % ) s t r a y e n e r g y can r e s u l t in - - 4% e r r o r in t h e enzyme assay. B a n d - w i d t h also can a f f e c t the a c c u r a c y o f t h e m e a s u r e d a b s o r b a n c e . This can be c o r r e c t e d for by a l t e r i n g the value of m o l a r a b s o r b a n c e used in c a l c u l a t i n g enzyme a c t i v i t y . W h e n several i n s t r u m e n t s a r e in use in a l a b o r a t o r y , t h i s value should be d e t e r m i n e d f o r each i n s t r u m e n t , t h e r e by c o r r e c t i n g f o r e f f e c t s of t h e i r d i f f e r e n t bandw i d t h s . Maclin et al '2' c o n s i d e r t h e e f f e c t of w a v e l e n g t h i n a c c u r a c y s i m i l a r to t h a t of b a n d - w i d t h . Noise is a m a j o r source o f e r r o r : t h e s a m e w o r k e r s f o u n d t h a t t h i s c o n t r i b u t e d an e r r o r of n e a r l y 14% to t h e calculation of enzyme a c t i v i t y . T a k i n g into account all e r r o r s due to i n s t r u m e n t a tion, Maclin et al concluded t h a t the g r o s s e r r o r could r a n g e between + 35% to - 4 0 % in m e a s u r e m e n t s of enzyme a c t i v i t y . I n p r a c t i c e t h e e r r o r s a r e usually c o n s i d e r a b l y less, b u t these a u t h o r s ' conclusion t h a t t h e r e is c o n s i d e r a b l e room f o r i m p r o v e m e n t seems more t h a n j u s t i f i e d .
Instrument quality control A l t h o u g h some e r r o r s due to the s p e c t r o p h o t o m e t e r a p p e a r to be beyond the u s e r ' s control, he can t a k e c e r t a i n m e a s u r e s to o b t a i n o p t i m a l p e r f o r m a n c e . F o r all s p e c t r o p h o t o m e t e r s , c o n s t a n t m a i n t e n a n c e is essent i a l and the u s e r m u s t be p r e p a r e d to check p e r f o r m a n c e r e g u l a r l y . R a n d ' s r e p o r t ~ , w h i c h is an invaluable source of r e f e r e n c e on s p e c t r o p h o t o m e t r i c s t a n d a r d s , s u g g e s t s t h a t t h e s e checks include w a v e l e n g t h and p h o t o m e t r i c accuracy, and m e a s u r e m e n t of s t r a y l i g h t and p h o t o m e t r i c l i n e a r i t y to e n s u r e t h a t the i n s t r u m e n t is p e r f o r m i n g w i t h i n t h e m a n u f a c t u r e r ' s specifications. F i n a l l y , it is n e c e s s a r y to p o i n t out t h a t a n a l y t i c a l balances a p p e a r to receive even less a t t e n t i o n . To be r e a l l y effective, a n y c o n t r o l p r o g r a m of a n a l y t i c a l p r o c e d u r e s in a clinical l a b o r a t o r y (and t h a t in i t s e l f is a r a r e occurrence) m u s t include s p e c t r o p h o t o m e t e r s a n d a n a l y t i c a l balances. Q u a l i t y control m a y seem a long w a y f r o m c o n s i d e r a t i o n of enzyme assays, b u t a s s a y e r r o r m u s t include all p r o c e d u r e s and i n s t r u m e n t s d i r e c t l y o r i n d i r e c t l y involved in p r o d u c i n g a
CLINICAL ENZY.MOLOGY ASSAY value for enzyme activity. Only when all these aspects are fully controlled can we be c e r t a i n that enzyme assays are a n a l y t i c a l l y satisfactory. REFERENCES 1. Goldberg, D. M., Ellis, G. and Wilcock, A. R. (1971). Ann. Clin. Biochvm. 8, 189-194. 2. Maclin, E., Rohlfing, D. and Ansour, M. (1973). Clin. Chant. 19, 832-837. 3. Malcolm, A. D. B. (197~). Anal. Biochenz. 55, 278-281.
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4. Morell, S. A. and Doumas, B. T. (1973). Clin. Chem. 19, 783, 785. 5. Moss, D. W. (1972). Clin. Chem. 18, 1449-1454. 6. Rand, R. N. (1969). Clin. Chem. 15, 839-863. 7. Schales, O. (1973). Clin. Chem. 19, 434 435. " 8. Schwartz, M. K. (1972). Technicon Q. 4, 12-19. 9. Wilkinson, J. H. (1971). In Enzym6 assays in ~wdicine, ed. by McGowan, G. K. and Walters, G. J. CHn. Pathol. Suppl. 24, 14-21. #t
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