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Quantitation of Conjugated Dienes by Second-Derivative UV Spectroscopy
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Quantitation of Conjugated Dienes by Second-Derivative UV Spectroscopy F. P. Corongiu, S. Banni, and B. Lombardi
INTRODUCTION For familiarization w i t h t h e p r i n c i p l e s a n d p r a c t i c e o f derivative U V spect r o s c o p y , t h e r e a d e r is referred t o a f e w available s o u r c e s ( 1 - 4 ) . D e r i v a t i v e U V s p e c t r o s c o p y is particularly suited for analysis o f c o m p l e x m i x t u r e s in w h i c h b a n d o v e r l a p s o c c u r , o r s h o u l d e r s o n s l o p e s are p r e s e n t in c o n v e n t i o n a l absorpt i o n spectra. I n first-derivative s p e c t r o s c o p y t h e rate o f c h a n g e o f a b s o r b a n c e w i t h w a v e l e n g t h (dA/dk) is m e a s u r e d , resulting in a r e d u c t i o n o f t h e b a n d w i d t h o f i n d i v i d u a l c o m p o n e n t s and, therefore, in spectra o f greater r e s o l u t i o n ( 2 ) . I n s e c o n d - d e r i v a t i v e s p e c t r o s c o p y , a differential is m a d e o f t h e first2 2 d e r i v a t i v e s p e c t r u m (d A/d\ ), a t r e a t m e n t w h i c h is particularly effective in e x tracting distinct signals o u t o f t h e s h o u l d e r s o n absorption s l o p e s ( 2 ) . A s e x p l a i n e d e l s e w h e r e i n this v o l u m e ( 5 ) , t h e latter is e x a c t l y t h e situation a n d difficulty o n e is c o n f r o n t e d w i t h in t h e d e t e c t i o n a n d d e t e r m i n a t i o n o f c o n j u gated dienes by means of conventional U V spectroscopy. Figure 1 shows the conventional ( l o w e r c u r v e ) and second-derivative (upper c u r v e ) a b s o r p t i o n s p e c t r a o f a p r e p a r a t i o n o f partially p e r o x i d i z e d c o d liver oil. In t h e l o w e r c u r v e t h e c o n j u g a t e d d i e n e s h o u l d e r o n t h e e n d a b s o r p t i o n s l o p e o f t h e lipid is c l e a r l y visible b e t w e e n 2 5 0 and 2 2 0 n m . I n t h e u p p e r c u r v e , h o w ever, t w o distinct signals are p r e s e n t in t h e s a m e r e g i o n . I n t h e s e c o n d derivative spectral a s s i g n m e n t s are m a d e o n t h e basis o f m i n i m u m , rather than m a x i m u m , w a v e l e n g t h o f a b s o r p t i o n ( 2 ) . T h e signal w i t h a m i n i m u m at 2 3 3 n m ( a , Fig. 1 ) is d u e t o t h e p r e s e n c e o f fatty a c i d isomers w i t h trans, trans -conjugated d i e n e s , a n d that w i t h a m i n i m u m at 2 4 2 n m (b) is c a u s e d b y i s o m e r s w i t h eis, trans - c o n j u g a t e d d i e n e s (6—8). T h i s e x a m p l e also illustrates w e l l t h e greater r e s o l u t i o n afforded b y s e c o n d - d e r i v a t i v e U V s p e c t r o s c o p y . E a c h signal has a m a x i m u m (a\ b') as w e l l as a m i n i m u m , and t h e difference b e t w e e n the t w o (a'—a, b'—b) r e p r e s e n t s t h e signal h e i g h t (h, b'). G i v e n that in s e c o n d - d e r i v a t i v e s p e c t r o s c o p y t h e e n d a b s o r p t i o n o f lipids d o e s not interfere w i t h t h e c o n j u g a t e d d i e n e bands, t h e r e is n o l o n g e r a n e e d t o base t h e U V d e t e c t i o n a n d quantitation o f c o n j u g a t e d d i e n e s o n t h e s p e c t r u m difference b e t w e e n a p e r o x i d i z e d a n d a n o n p e r o x i d i z e d p r e p a r a t i o n o f t h e s a m e lipid ( 5 ) , greatly simplifying t h e M E T H O D S I N T O X I C O L O G Y , Volume I B Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Figure 1 Conventional (lower curve) and second-derivative (upper curve) UV absorption spectra of a sample of partially peroxidized cod liver oil. For an explanation see text. Abscissa, Wavelength ( λ ) in nanometers; ordinate, absorption units (A. U.).
m e t h o d o l o g y . M o r e o v e r , s e c o n d - d e r i v a t i v e s p e c t r o s c o p y affords a m o r e direct a n d p r e c i s e m e a n s t o quantitate c o n j u g a t e d dienes, s i n c e t h e linear relationship b e t w e e n s a m p l e absorption ( p e a k h e i g h i ) and c o n c e n t r a t i o n , as g o v e r n e d b y t h e L a m b e r t - B e e r law, is unaffected b y differentiation ( 2 ) . T h e c o n j u g a t e d die n e c o n t e n t o f a single s a m p l e o f lipids c a n thus b e d e t e r m i n e d easily w i t h the aid o f a standard c u r v e p r e p a r e d w i t h a suitable reference c o m p o u n d (8—11).
CONSTRUCTION OF A STANDARD CURVE AND OPTIMIZATION OF INSTRUMENT SETTINGS Reagents: Reagent-grade c y c l o h e x a n e Standard: A s a m p l e o f as h i g h l y purified and c o m p l e t e l y p e r o x i d i z e d an unsaturated fatty a c i d as is available; w e h a v e b e e n u s i n g (9—11 ) ( ± )-9h y d r o p e r o x y o c t a d e c a d i e n o i c a c i d ( H P O D E ; purity > 9 8 % , O x f o r d Biomedical Research, Oxford, M I )
Procedure A s t o c k s o l u t i o n o f the standard s a m p l e is p r e p a r e d in c y c l o h e x a n e at a c o n c e n tration o f —100 M g / m l a n d p l a c e d in a small glass-stoppered flask. T h e flask is partially i m m e r s e d in l i q u i d N 2 t o r e p l a c e its air s p a c e w i t h N 2 , c l o s e d , sealed (Parafilm), a n d stored at - 8 0 ° C . If the s a m p l e is o b t a i n e d from a c o m m e r c i a l s o u r c e , it is transferred from its c o n t a i n e r into a c o n i c a l glass-stoppered flask, s u c h as C o r n i n g C a t . N o . 4 3 1 5 (Fisher C a t . N o . 10-060-5D, Pittsburgh, P A ) ,
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a l o n g w i t h r e p e a t e d w a s h e s o f t h e c o n t a i n e r w i t h t h e solvent in w h i c h the samp l e w a s d e l i v e r e d . T h e solvent, if o t h e r than c y c l o h e x a n e , is dried d o w n u n d e r v a c u u m , o r u n d e r a stream o f 0 2 - f r e e nitrogen, before p r e p a r i n g t h e s t o c k solution. T o p r e p a r e w o r k i n g solutions, t h e s t o c k s o l u t i o n is t h a w e d t o r o o m temperature, a n d t h e c o n c e n t r a t i o n o f t h e s a m p l e is c h e c k e d b y c o n v e n t i o n a l U V spect r o s c o p y u s i n g its coefficient o f e x t i n c t i o n at the w a v e l e n g t h o f m a x i m u m absorption, o r t h e e x t i n c t i o n coefficient and w a v e l e n g t h p r o v i d e d b y t h e supplier. W o r k i n g s o l u t i o n s are p r e p a r e d at c o n c e n t r a t i o n s ranging from 0.5 t o 1 5 - 2 0 μ-g/ml o f c y c l o h e x a n e , a n d t h e c o n c e n t r a t i o n s are c h e c k e d as d o n e w i t h t h e s t o c k s o l u t i o n . T r i p l i c a t e s o f e a c h s o l u t i o n are s c a n n e d b e t w e e n 3 0 0 and 2 2 0 n m w i t h an i n s t r u m e n t e q u i p p e d w i t h an e l e c t r o n i c unit for s e c o n d - d e r i v a t i v e s p e c t r o s c o p y , s u c h as are t h e P e r k i n - E l m e r ( N o r w a l k , C T ) , M o d e l L a m b d a 15 o r H i t a c h i ( N a p e r v i l l e , I L ) , M o d e l 5 5 7 s p e c t r o p h o t o m e t e r s . In u s i n g t h e s e instrum e n t s , five p a r a m e t e r s r e q u i r e s o m e p r e l i m i n a r y c o n s i d e r a t i o n and o p t i m i z a t i o n ( a - e ) : ( a ) t h e s c a n s p e e d ; ( b ) t h e bandpass ( s l i t ) ; ( c ) t h e r e s p o n s e t i m e ; ( d ) t h e Δ w a v e l e n g t h ; a n d ( e ) t h e o r d i n a t e s c a l e ( d e p e n d i n g o n the instrument sensitivity a n d h e i g h t o f t h e signals o b t a i n e d ) . W e h a v e b e e n u s i n g the f o l l o w i n g ( a - e ) : ( a ) 6 0 n m / m i n w i t h b o t h o f t h e a b o v e instruments; ( b ) 1 n m , w i t h b o t h o f the a b o v e instruments; ( c ) 2 s e c ( P e r k i n - E l m e r ) and s l o w setting ( H i t a c h i ) ; ( d ) 4 n m w i t h b o t h instruments; a n d ( e ) ordinates v a r y i n g from 0.05 t o 0.40 abs o r p t i o n units. S e e t h e w o r k o f Situnayake et al ( 8 ) for use o f a S h i m a d z u d o u b l e - b e a m s c a n n i n g s p e c t r o p h o t o m e t e r , if that is the instrument available. O n c e o p t i m i z e d , t h e s e p a r a m e t e r s are u s e d t h r o u g h o u t , e x c e p t for setting the instrum e n t baseline, in w h i c h c a s e t h e s c a n s p e e d is l o w e r e d ; for this p u r p o s e w e h a v e b e e n u s i n g s p e e d s o f 7.5 n m / m i n ( P e r k i n - E l m e r ) and 15 n m / m i n ( H i t a c h i ) . T h e b a s e l i n e is set b y s c a n n i n g c y c l o h e x a n e in b o t h c u v e t t e s . F i g u r e 2 s h o w s t y p i c a l c o n v e n t i o n a l ( l o w e r c u r v e ) and s e c o n d - d e r i v a t i v e ( u p p e r c u r v e ) s p e c t r a o f 2 μ, g o f H P O D E p e r 1 m l o f c y c l o h e x a n e . N o t e in the l o w e r c u r v e t h e l a c k o f major e n d a b s o r p t i o n a n d the s h o u l d e r d u e t o c o n j u g a t e d d i e n e s . I n t h e u p p e r c u r v e t w o distinct signals are again present, w i t h m i n i m a at 2 3 3 a n d 2 4 2 n m . After taking t h e s e c o n d - d e r i v a t i v e spectra, t h e h e i g h t s o f t h e t w o signals ( £ , h', Fig. 1 ) o b t a i n e d w i t h e a c h w o r k i n g s o l u t i o n are m e a s u r e d a n d s u m m e d . A p l o t is t h e n m a d e o f t h e s u m versus c o n c e n t r a t i o n , t o c o n s t r u c t a standard r e f e r e n c e c u r v e as s h o w n in Fig. 3. T h e c u r v e is u s e d rep e a t e d l y , as l o n g as i n s t r u m e n t settings 1—4 are not c h a n g e d .
ANALYSIS OF BIOLOGICAL LIPID SAMPLES T h e r e a g e n t s a n d p r o c e d u r e s d e s c r i b e d in this v o l u m e ( 5 ) for t h e preparation o f tissue, c e l l , o r s u b c e l l u l a r fraction s a m p l e s a n d the e x t r a c t i o n , purification, a n d q u a n t i t a t i o n o f their total lipids are fully a d e q u a t e also in t h e p r e s e n t case. It is
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H
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I I I I I I I 300 250
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Figure 2 Conventional (lower curve) and second-derivative (upper curve) UV absorption spectra of H P O D E . Note in the lower curve the lack of major end absorption and the shoulder due to conjugated dienes. In the upper curve two distinct signals are seen, with minima at 233 and 242 nm, due to the presence of trans, trans- and cis, trans -conjugated dienes, respectively. Abscissa, Wavelength ( λ ) in nanometers; ordinate, absorption units (A. U.). w o r t h y o f n o t e , h o w e v e r , that w h i l e c o n j u g a t e d d i e n e analyses l i m i t e d t o total lipids are satisfactory in studies d e a l i n g w i t h p r o o x i d a n t agents, s u c h as C C U , t h e y are n o t w h e n assessing w h e t h e r an agent is a p r o o x i d a n t . I n this c a s e initial analyses s h o u l d b e m a d e o n p h o s p h o l i p i d ( P L ) preparations, o r initial p o s i t i v e results s h o u l d b e validated b y analyses o f PL, s i n c e it is w e l l established that t h e target o f b i o l o g i c a l l i p o p e r o x i d a t i v e p r o c e s s e s are p o l y u n s a t u r a t e d fatty a c i d s p r e s e n t in c e l l u l a r m e m b r a n e P L ( 1 2 ) . A t least o n e c a s e has b e e n r e p o r t e d in the r e c e n t literature in w h i c h t h e p r e s e n c e o f c o n j u g a t e d d i e n e s in tissue total lipids, b u t n o t PL, w a s s h o w n t o result from assimilation o f stable fatty a c i d isom e r s w i t h c o n j u g a t e d d i e n e s o f dietary origins, rather than from a c e l l u l a r l i p o p e r o x i d a t i v e p r o c e s s (9—11). Isolation a n d purification o f PL, o r subclasses thereof, c a n b e satisfactorily a c h i e v e d b y several established c h r o m a t o g r a p h i c p r o c e d u r e s ( 9 - 1 1 ) . H o w e v e r , t o o b t a i n sufficient a m o u n t s o f P L for analysis, larger ( t w o t o t h r e e t i m e s ) initial s a m p l e s o f tissue, cells, o r c e l l u l a r subfractions s h o u l d b e used. O n c e isolated, t h e lipids are r e d i s s o l v e d in c y c l o h e x a n e at a c o n c e n t r a t i o n o f —250 / x g / m l [ 1 3 ; s e e also the c h a p t e r b y G l e n d e a n d R e c k n a g e l ( 5 ) for a des c r i p t i o n o f a c o l o r i m e t r i c d e t e r m i n a t i o n o f lipids], a n d their s e c o n d - d e r i v a t i v e
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μ§/πι1 Figure 3 Standard curve prepared by plotting micrograms of H P O D E per milliliter of cyclohexane (abscissa) and the height sum (h + h') (ordinate, arbitrary units) of the signals at 233 and 242 nm obtained in second-derivative UV spectroscopy. U V a b s o r p t i o n s p e c t r u m is taken, u s i n g i n s t r u m e n t settings as i n d i c a t e d for t h e c o n s t r u c t i o n o f a standard c u r v e . T h e h e i g h t s o f t h e signals w i t h m i n i m a at 2 3 3 a n d 2 4 2 n m are m e a s u r e d a n d s u m m e d , a n d t h e c o n c e n t r a t i o n o f c o n j u g a t e d d i e n e s in t h e d e t e r m i n e d w i t h r e f e r e n c e t o t h e standard c u r v e . T h e results are e x p r e s s e d as m i c r o g r a m s o f c o n j u g a t e d d i e n e e q u i v a l e n t s p e r m i l l i g r a m of lipid ( 9 - 1 1 ) .
REFERENCES 1. A. Schmitt, "Applied UV Spectroscopy. Derivative Spectroscopy. An Introduction with a Practical Example," N o . 1 E, pp. 3-10. Bodenseewerk Perkin-Elmer, Uberlingen, Germany. 2. D . Botten, T. Honkawa, and S. Tohyama, Perkin-Elmer Appl. Data Bull. X - 7 7 , 1 (1977). 3. J . E. Cahill, Perkin-Elmer Appl. Data Bull. A D S - 1 1 5 , 1 (1979). 4. J . E. Cahill, and F. G . Padera, Perkin-Elmer Appl. Data Bull. A D S - 1 2 2 , 1 (1980). 5. E. A. Glende, Jr., and R. O . Recknagel, this volume, C h . 33. 6. F. P. Corongiu and A. Milia, Chem.-Biol. Interact. 44, 289 (1983). 7. F. P. Corongiu, G . Poli, M. U. Dianzani, K. H. Cheeseman, and T. F. Slater, Chem.-Biol. Interact. 59, 1 4 7 ( 1 9 8 6 ) .
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8. R. D. Situnayake, B. J . Crump, Α. V. Zevulka, M. Davis, B. McConkey, and D. I. Thurnham, Ann. Clin. Biochem. 27, 258 (1990). 9. S. Banni, R. E. Basford, F. P. Corongiu, and B. Lombardi, Adv. Biosci. 76, 187 (1989). 10. S. Banni, R. W. Evans, M. G . Salgo, F. P. Corongiu, and B. Lombardi, Carcinogenesis 1 1 , 2047 (1990). 11. S. Banni, M. G . Salgo, R. W. Evans, F. P. Corongiu, and B. Lombardi, Carcinogenesis 1 1 , 2053 (1990). 12. R O . Recknagel, E. A. Glende, Jr., and R. S. Britton, in "Hepatotoxicology" (R. G Meeks, S. D. Harrison, and R. J . Bull, eds.), p. 401. C R C Press, Boca Raton, Florida, (1991). 13. S. P. Chiang, C . F. Gessert, and O . H. Lowry, in "Research Report 56-113". Air University School of Aviation Medicine, U. S. Air Force, Texas, 1957.
Quantitation of Conjugated Dienes by Second-Derivative UV Spectroscopy F. P. Corongiu, S. Banni, and B. Lombardi
INTRODUCTION For familiarization w i t h t h e p r i n c i p l e s a n d p r a c t i c e o f derivative U V spect r o s c o p y , t h e r e a d e r is referred t o a f e w available s o u r c e s ( 1 - 4 ) . D e r i v a t i v e U V s p e c t r o s c o p y is particularly suited for analysis o f c o m p l e x m i x t u r e s in w h i c h b a n d o v e r l a p s o c c u r , o r s h o u l d e r s o n s l o p e s are p r e s e n t in c o n v e n t i o n a l absorpt i o n spectra. I n first-derivative s p e c t r o s c o p y t h e rate o f c h a n g e o f a b s o r b a n c e w i t h w a v e l e n g t h (dA/dk) is m e a s u r e d , resulting in a r e d u c t i o n o f t h e b a n d w i d t h o f i n d i v i d u a l c o m p o n e n t s and, therefore, in spectra o f greater r e s o l u t i o n ( 2 ) . I n s e c o n d - d e r i v a t i v e s p e c t r o s c o p y , a differential is m a d e o f t h e first2 2 d e r i v a t i v e s p e c t r u m (d A/d\ ), a t r e a t m e n t w h i c h is particularly effective in e x tracting distinct signals o u t o f t h e s h o u l d e r s o n absorption s l o p e s ( 2 ) . A s e x p l a i n e d e l s e w h e r e i n this v o l u m e ( 5 ) , t h e latter is e x a c t l y t h e situation a n d difficulty o n e is c o n f r o n t e d w i t h in t h e d e t e c t i o n a n d d e t e r m i n a t i o n o f c o n j u gated dienes by means of conventional U V spectroscopy. Figure 1 shows the conventional ( l o w e r c u r v e ) and second-derivative (upper c u r v e ) a b s o r p t i o n s p e c t r a o f a p r e p a r a t i o n o f partially p e r o x i d i z e d c o d liver oil. In t h e l o w e r c u r v e t h e c o n j u g a t e d d i e n e s h o u l d e r o n t h e e n d a b s o r p t i o n s l o p e o f t h e lipid is c l e a r l y visible b e t w e e n 2 5 0 and 2 2 0 n m . I n t h e u p p e r c u r v e , h o w ever, t w o distinct signals are p r e s e n t in t h e s a m e r e g i o n . I n t h e s e c o n d derivative spectral a s s i g n m e n t s are m a d e o n t h e basis o f m i n i m u m , rather than m a x i m u m , w a v e l e n g t h o f a b s o r p t i o n ( 2 ) . T h e signal w i t h a m i n i m u m at 2 3 3 n m ( a , Fig. 1 ) is d u e t o t h e p r e s e n c e o f fatty a c i d isomers w i t h trans, trans -conjugated d i e n e s , a n d that w i t h a m i n i m u m at 2 4 2 n m (b) is c a u s e d b y i s o m e r s w i t h eis, trans - c o n j u g a t e d d i e n e s (6—8). T h i s e x a m p l e also illustrates w e l l t h e greater r e s o l u t i o n afforded b y s e c o n d - d e r i v a t i v e U V s p e c t r o s c o p y . E a c h signal has a m a x i m u m (a\ b') as w e l l as a m i n i m u m , and t h e difference b e t w e e n the t w o (a'—a, b'—b) r e p r e s e n t s t h e signal h e i g h t (h, b'). G i v e n that in s e c o n d - d e r i v a t i v e s p e c t r o s c o p y t h e e n d a b s o r p t i o n o f lipids d o e s not interfere w i t h t h e c o n j u g a t e d d i e n e bands, t h e r e is n o l o n g e r a n e e d t o base t h e U V d e t e c t i o n a n d quantitation o f c o n j u g a t e d d i e n e s o n t h e s p e c t r u m difference b e t w e e n a p e r o x i d i z e d a n d a n o n p e r o x i d i z e d p r e p a r a t i o n o f t h e s a m e lipid ( 5 ) , greatly simplifying t h e M E T H O D S I N T O X I C O L O G Y , Volume I B Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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2.0 π
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λ [nm]
Figure 1 Conventional (lower curve) and second-derivative (upper curve) UV absorption spectra of a sample of partially peroxidized cod liver oil. For an explanation see text. Abscissa, Wavelength ( λ ) in nanometers; ordinate, absorption units (A. U.).
m e t h o d o l o g y . M o r e o v e r , s e c o n d - d e r i v a t i v e s p e c t r o s c o p y affords a m o r e direct a n d p r e c i s e m e a n s t o quantitate c o n j u g a t e d dienes, s i n c e t h e linear relationship b e t w e e n s a m p l e absorption ( p e a k h e i g h i ) and c o n c e n t r a t i o n , as g o v e r n e d b y t h e L a m b e r t - B e e r law, is unaffected b y differentiation ( 2 ) . T h e c o n j u g a t e d die n e c o n t e n t o f a single s a m p l e o f lipids c a n thus b e d e t e r m i n e d easily w i t h the aid o f a standard c u r v e p r e p a r e d w i t h a suitable reference c o m p o u n d (8—11).
CONSTRUCTION OF A STANDARD CURVE AND OPTIMIZATION OF INSTRUMENT SETTINGS Reagents: Reagent-grade c y c l o h e x a n e Standard: A s a m p l e o f as h i g h l y purified and c o m p l e t e l y p e r o x i d i z e d an unsaturated fatty a c i d as is available; w e h a v e b e e n u s i n g (9—11 ) ( ± )-9h y d r o p e r o x y o c t a d e c a d i e n o i c a c i d ( H P O D E ; purity > 9 8 % , O x f o r d Biomedical Research, Oxford, M I )
Procedure A s t o c k s o l u t i o n o f the standard s a m p l e is p r e p a r e d in c y c l o h e x a n e at a c o n c e n tration o f —100 M g / m l a n d p l a c e d in a small glass-stoppered flask. T h e flask is partially i m m e r s e d in l i q u i d N 2 t o r e p l a c e its air s p a c e w i t h N 2 , c l o s e d , sealed (Parafilm), a n d stored at - 8 0 ° C . If the s a m p l e is o b t a i n e d from a c o m m e r c i a l s o u r c e , it is transferred from its c o n t a i n e r into a c o n i c a l glass-stoppered flask, s u c h as C o r n i n g C a t . N o . 4 3 1 5 (Fisher C a t . N o . 10-060-5D, Pittsburgh, P A ) ,
35.
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a l o n g w i t h r e p e a t e d w a s h e s o f t h e c o n t a i n e r w i t h t h e solvent in w h i c h the samp l e w a s d e l i v e r e d . T h e solvent, if o t h e r than c y c l o h e x a n e , is dried d o w n u n d e r v a c u u m , o r u n d e r a stream o f 0 2 - f r e e nitrogen, before p r e p a r i n g t h e s t o c k solution. T o p r e p a r e w o r k i n g solutions, t h e s t o c k s o l u t i o n is t h a w e d t o r o o m temperature, a n d t h e c o n c e n t r a t i o n o f t h e s a m p l e is c h e c k e d b y c o n v e n t i o n a l U V spect r o s c o p y u s i n g its coefficient o f e x t i n c t i o n at the w a v e l e n g t h o f m a x i m u m absorption, o r t h e e x t i n c t i o n coefficient and w a v e l e n g t h p r o v i d e d b y t h e supplier. W o r k i n g s o l u t i o n s are p r e p a r e d at c o n c e n t r a t i o n s ranging from 0.5 t o 1 5 - 2 0 μ-g/ml o f c y c l o h e x a n e , a n d t h e c o n c e n t r a t i o n s are c h e c k e d as d o n e w i t h t h e s t o c k s o l u t i o n . T r i p l i c a t e s o f e a c h s o l u t i o n are s c a n n e d b e t w e e n 3 0 0 and 2 2 0 n m w i t h an i n s t r u m e n t e q u i p p e d w i t h an e l e c t r o n i c unit for s e c o n d - d e r i v a t i v e s p e c t r o s c o p y , s u c h as are t h e P e r k i n - E l m e r ( N o r w a l k , C T ) , M o d e l L a m b d a 15 o r H i t a c h i ( N a p e r v i l l e , I L ) , M o d e l 5 5 7 s p e c t r o p h o t o m e t e r s . In u s i n g t h e s e instrum e n t s , five p a r a m e t e r s r e q u i r e s o m e p r e l i m i n a r y c o n s i d e r a t i o n and o p t i m i z a t i o n ( a - e ) : ( a ) t h e s c a n s p e e d ; ( b ) t h e bandpass ( s l i t ) ; ( c ) t h e r e s p o n s e t i m e ; ( d ) t h e Δ w a v e l e n g t h ; a n d ( e ) t h e o r d i n a t e s c a l e ( d e p e n d i n g o n the instrument sensitivity a n d h e i g h t o f t h e signals o b t a i n e d ) . W e h a v e b e e n u s i n g the f o l l o w i n g ( a - e ) : ( a ) 6 0 n m / m i n w i t h b o t h o f t h e a b o v e instruments; ( b ) 1 n m , w i t h b o t h o f the a b o v e instruments; ( c ) 2 s e c ( P e r k i n - E l m e r ) and s l o w setting ( H i t a c h i ) ; ( d ) 4 n m w i t h b o t h instruments; a n d ( e ) ordinates v a r y i n g from 0.05 t o 0.40 abs o r p t i o n units. S e e t h e w o r k o f Situnayake et al ( 8 ) for use o f a S h i m a d z u d o u b l e - b e a m s c a n n i n g s p e c t r o p h o t o m e t e r , if that is the instrument available. O n c e o p t i m i z e d , t h e s e p a r a m e t e r s are u s e d t h r o u g h o u t , e x c e p t for setting the instrum e n t baseline, in w h i c h c a s e t h e s c a n s p e e d is l o w e r e d ; for this p u r p o s e w e h a v e b e e n u s i n g s p e e d s o f 7.5 n m / m i n ( P e r k i n - E l m e r ) and 15 n m / m i n ( H i t a c h i ) . T h e b a s e l i n e is set b y s c a n n i n g c y c l o h e x a n e in b o t h c u v e t t e s . F i g u r e 2 s h o w s t y p i c a l c o n v e n t i o n a l ( l o w e r c u r v e ) and s e c o n d - d e r i v a t i v e ( u p p e r c u r v e ) s p e c t r a o f 2 μ, g o f H P O D E p e r 1 m l o f c y c l o h e x a n e . N o t e in the l o w e r c u r v e t h e l a c k o f major e n d a b s o r p t i o n a n d the s h o u l d e r d u e t o c o n j u g a t e d d i e n e s . I n t h e u p p e r c u r v e t w o distinct signals are again present, w i t h m i n i m a at 2 3 3 a n d 2 4 2 n m . After taking t h e s e c o n d - d e r i v a t i v e spectra, t h e h e i g h t s o f t h e t w o signals ( £ , h', Fig. 1 ) o b t a i n e d w i t h e a c h w o r k i n g s o l u t i o n are m e a s u r e d a n d s u m m e d . A p l o t is t h e n m a d e o f t h e s u m versus c o n c e n t r a t i o n , t o c o n s t r u c t a standard r e f e r e n c e c u r v e as s h o w n in Fig. 3. T h e c u r v e is u s e d rep e a t e d l y , as l o n g as i n s t r u m e n t settings 1—4 are not c h a n g e d .
ANALYSIS OF BIOLOGICAL LIPID SAMPLES T h e r e a g e n t s a n d p r o c e d u r e s d e s c r i b e d in this v o l u m e ( 5 ) for t h e preparation o f tissue, c e l l , o r s u b c e l l u l a r fraction s a m p l e s a n d the e x t r a c t i o n , purification, a n d q u a n t i t a t i o n o f their total lipids are fully a d e q u a t e also in t h e p r e s e n t case. It is
418
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Lipid Peroxidation
H
<
0.5
Π
I
220
I I I I I I I 300 250
λ [nm]
Figure 2 Conventional (lower curve) and second-derivative (upper curve) UV absorption spectra of H P O D E . Note in the lower curve the lack of major end absorption and the shoulder due to conjugated dienes. In the upper curve two distinct signals are seen, with minima at 233 and 242 nm, due to the presence of trans, trans- and cis, trans -conjugated dienes, respectively. Abscissa, Wavelength ( λ ) in nanometers; ordinate, absorption units (A. U.). w o r t h y o f n o t e , h o w e v e r , that w h i l e c o n j u g a t e d d i e n e analyses l i m i t e d t o total lipids are satisfactory in studies d e a l i n g w i t h p r o o x i d a n t agents, s u c h as C C U , t h e y are n o t w h e n assessing w h e t h e r an agent is a p r o o x i d a n t . I n this c a s e initial analyses s h o u l d b e m a d e o n p h o s p h o l i p i d ( P L ) preparations, o r initial p o s i t i v e results s h o u l d b e validated b y analyses o f PL, s i n c e it is w e l l established that t h e target o f b i o l o g i c a l l i p o p e r o x i d a t i v e p r o c e s s e s are p o l y u n s a t u r a t e d fatty a c i d s p r e s e n t in c e l l u l a r m e m b r a n e P L ( 1 2 ) . A t least o n e c a s e has b e e n r e p o r t e d in the r e c e n t literature in w h i c h t h e p r e s e n c e o f c o n j u g a t e d d i e n e s in tissue total lipids, b u t n o t PL, w a s s h o w n t o result from assimilation o f stable fatty a c i d isom e r s w i t h c o n j u g a t e d d i e n e s o f dietary origins, rather than from a c e l l u l a r l i p o p e r o x i d a t i v e p r o c e s s (9—11). Isolation a n d purification o f PL, o r subclasses thereof, c a n b e satisfactorily a c h i e v e d b y several established c h r o m a t o g r a p h i c p r o c e d u r e s ( 9 - 1 1 ) . H o w e v e r , t o o b t a i n sufficient a m o u n t s o f P L for analysis, larger ( t w o t o t h r e e t i m e s ) initial s a m p l e s o f tissue, cells, o r c e l l u l a r subfractions s h o u l d b e used. O n c e isolated, t h e lipids are r e d i s s o l v e d in c y c l o h e x a n e at a c o n c e n t r a t i o n o f —250 / x g / m l [ 1 3 ; s e e also the c h a p t e r b y G l e n d e a n d R e c k n a g e l ( 5 ) for a des c r i p t i o n o f a c o l o r i m e t r i c d e t e r m i n a t i o n o f lipids], a n d their s e c o n d - d e r i v a t i v e
35. Spectroscopy of Lipid-Conjugated Pienes
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μ§/πι1 Figure 3 Standard curve prepared by plotting micrograms of H P O D E per milliliter of cyclohexane (abscissa) and the height sum (h + h') (ordinate, arbitrary units) of the signals at 233 and 242 nm obtained in second-derivative UV spectroscopy. U V a b s o r p t i o n s p e c t r u m is taken, u s i n g i n s t r u m e n t settings as i n d i c a t e d for t h e c o n s t r u c t i o n o f a standard c u r v e . T h e h e i g h t s o f t h e signals w i t h m i n i m a at 2 3 3 a n d 2 4 2 n m are m e a s u r e d a n d s u m m e d , a n d t h e c o n c e n t r a t i o n o f c o n j u g a t e d d i e n e s in t h e d e t e r m i n e d w i t h r e f e r e n c e t o t h e standard c u r v e . T h e results are e x p r e s s e d as m i c r o g r a m s o f c o n j u g a t e d d i e n e e q u i v a l e n t s p e r m i l l i g r a m of lipid ( 9 - 1 1 ) .
REFERENCES 1. A. Schmitt, "Applied UV Spectroscopy. Derivative Spectroscopy. An Introduction with a Practical Example," N o . 1 E, pp. 3-10. Bodenseewerk Perkin-Elmer, Uberlingen, Germany. 2. D . Botten, T. Honkawa, and S. Tohyama, Perkin-Elmer Appl. Data Bull. X - 7 7 , 1 (1977). 3. J . E. Cahill, Perkin-Elmer Appl. Data Bull. A D S - 1 1 5 , 1 (1979). 4. J . E. Cahill, and F. G . Padera, Perkin-Elmer Appl. Data Bull. A D S - 1 2 2 , 1 (1980). 5. E. A. Glende, Jr., and R. O . Recknagel, this volume, C h . 33. 6. F. P. Corongiu and A. Milia, Chem.-Biol. Interact. 44, 289 (1983). 7. F. P. Corongiu, G . Poli, M. U. Dianzani, K. H. Cheeseman, and T. F. Slater, Chem.-Biol. Interact. 59, 1 4 7 ( 1 9 8 6 ) .
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8. R. D. Situnayake, B. J . Crump, Α. V. Zevulka, M. Davis, B. McConkey, and D. I. Thurnham, Ann. Clin. Biochem. 27, 258 (1990). 9. S. Banni, R. E. Basford, F. P. Corongiu, and B. Lombardi, Adv. Biosci. 76, 187 (1989). 10. S. Banni, R. W. Evans, M. G . Salgo, F. P. Corongiu, and B. Lombardi, Carcinogenesis 1 1 , 2047 (1990). 11. S. Banni, M. G . Salgo, R. W. Evans, F. P. Corongiu, and B. Lombardi, Carcinogenesis 1 1 , 2053 (1990). 12. R O . Recknagel, E. A. Glende, Jr., and R. S. Britton, in "Hepatotoxicology" (R. G Meeks, S. D. Harrison, and R. J . Bull, eds.), p. 401. C R C Press, Boca Raton, Florida, (1991). 13. S. P. Chiang, C . F. Gessert, and O . H. Lowry, in "Research Report 56-113". Air University School of Aviation Medicine, U. S. Air Force, Texas, 1957.