Isotopes and Chemical Principles

9 Isotope Chemistry and Biology J O S E P H J.

KATZ,

R O B E R T A. U P H A U S , and H E N R Y L . C R E S P I

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Chemistry Division, Argonne National Laboratory, Argonne, Ill. 60439 M A R T I N I. B L A K E Department of Pharmacy, University of Illinois, College of Pharmacy, Chicago, Ill. 60612

I.

Introduction

The p a s t decade has seen a keen and growing intere s t in t h e application o f s t a b l e i s o t o p e s t o c h e m i c a l and biological problems ( 1 , 2 ) . There have been t h r e e main driving f o r c e s f o r this interest: (a) The applic a t i o n s o f magnetic resonance t e c h n i q u e s t o many complex c h e m i c a l and biological problems a r e greatly facilitated by j u d i c i o u s use o f s t a b l e i s o t o p e s ; ( b ) , t h e utility o f gas chromatographic-mass s p e c t r o m e t r i c t e c h n i q u e s i n m e t a b o l i c and e n v i r o n m e n t a l t r a c e r s t u d i e s is greatly enhanced by adjustment o f t h e isotopic c o m p o s i t i o n ; and, ( c ) , t h e availability of fully d e u t e r a t e d compounds and o r g a n e l l e s from fully d e u t e r a t e d m i c r o o r g a n i s m s (3) has made p o s s i b l e t h e investigation o f many r e f r a c t o r y problems o f biological interest. These, t o g e t h e r w i t h an e v e r i n c r e a s i n g availability o f C, N, O and d e p l e t e d C and N in h i g h isotopic p u r i t y have t r i g g e r e d a wide r e s p o n s e ( 4 ) . D e s p i t e t h e many uses t o which s t a b l e i s o t o p e s c a n be p u t , certain i n h e r e n t limitations still prevail. Deuterium as heavy water is available in large q u a n t i t i e s a t a relatively low p r i c e , but i n c o r p o r a t i o n o f deuterium i n t o living organisms can have s e v e r e t o x i c effects on h i g h e r p l a n t s and animals because o f p o s s i b l e l a r g e kinetic i s o t o p e e f f e c t s (5). The heavy i s o t o p e s o f c a r b o n , n i t r o g e n and oxygen a r e far less t o x i c t o living organisms t h a n is d e u t e r ium, b u t t h e quantities a v a i l a b l e a r e limited and t h e price tends t o be q u i t e h i g h . Hydrogen o f mass 2, d e u t e r i u m , was d i s c o v e r e d by Urey and his co-workers in 1932 (6). I n t h e decade f o l l o w i n g t h i s d i s c o v e r y t h e r e d e v e l o p e d a l a r g e body o f o f t e n c o n f u s i n g d a t a (7,8) c o n c e r n i n g t h e b i o l o g y o f heavy w a t e r . These e a r l y s t u d i e s were i n t e r p r e t e d t o i n d i c a t e t h a t d e u t e r i u m is toxic, and t h a t h i g h 13

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Rock; Isotopes and Chemical Principles ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

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κΑτζ ET AL.

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concentrations are fundamentally incompatible with c e l l u l a r growth and r e p r o d u c t i o n . However, i n I 9 6 0 , a u t o t r o p h i c f r e s h - w a t e r green and b l u e - g r e e n a l g a e were s u c c e s s f u l l y c u l t u r e d i n heavy w a t e r c o n t a i n i n g 9 9 . 7 atom p e r c e n t H. T h i s work was s h o r t l y f o l l o w e d by t h e c u l t i v a t i o n o f a wide v a r i e t y o f f u l l y d e u t e r a t e d h e t e r o t r o p h i c m i c r o o r g a n i s m s (3,8). These s u c c e s s e s l e d to a considerable e f f o r t to achieve f u l l deuteration of h i g h e r p l a n t s and mammals, b u t t o d a t e t h e s e more com p l e x systems have r e s i s t e d f u l l r e p l a c e m e n t o f ^H by H. T o x i c e f f e c t s i n mammals, f o r example, a r e appar e n t even a t 1 0 - 1 5 atom p e r c e n t H i n t i s s u e f l u i d s . The marked b i o l o g i c a l e f f e c t s o f d e u t e r i u m a r e n o t o b s e r v e d when C i s s u b s t i t u t e d f o r C , o r N f o r The s o u r c e o f t h i s d i f f e r e n t i a l e f f e c t i s l i k e l y the much l a r g e r k i n e t i c i s o t o p e e f f e c t a s s o c i a t e d w i t h d e u t e r i u m , as compared t o the heavy, s t a b l e i s o t o p e s o f c a r b o n and n i t r o g e n . The s u b s t i t u t i o n o f a heavy i s o t o p i c s p e c i e s i n a c h e m i c a l bond may change the r a t e o f any r e a c t i o n t h a t i n v o l v e s s c i s s i o n o f t h i s bond. In simple terms, the e f f e c t on the r e a c t i o n r a t e w i l l de pend on the mass r a t i o o f t h e i s o t o p i c atoms i n question. Thus, the mass r a t i o f o r the hydrogen i s o t o p e s , ^H/^H, i s 2, w h i l e the 1 3 c / l C r a t i o i s o n l y 1 . 0 8 , and t h a t of 1 5 N / N i s only 1 . 0 7 . Consequently, k i n e t i c isotope e f f e c t s perhaps an o r d e r o f magnitude l e s s would be e x p e c t e d f o r heavy carbon and n i t r o g e n t h a n f o r heavy hydrogen. The s u b s t i t u t i o n o f H for H also affects equi l i b r i u m c o n s t a n t s , p a r t i c u l a r l y the i o n i z a t i o n constants o f weak a c i d s and bases d i s s o l v e d i n D 0 (10_, 1 1 ) . The r a t e s o f a c i d - b a s e c a t a l y z e d r e a c t i o n s may be g r e a t l y d i f f e r e n t i n ! H 0 as compared t o H 0 (2,12). Deuter ium s u b s t i t u t i o n w i l l t e n d t o i n c r e a s e s l i g h t l y t h e s t r e n g t h o f hydrogen bonds, and d e u t e r i u m has a s i g n i f i c a n t l y s m a l l e r s t e r i c r e q u i r e m e n t t h a n does ^H. Thus, r a t e s o f c o n f o r m a t i o n a l i n t e r c h a n g e i n d e u t e r a t e d b i o p o l y m e r s can be markedly d i f f e r e n t from those o f normal i s o t o p i c c o m p o s i t i o n . I t i s t h e r e f o r e not at a l l s u r p r i s i n g t h a t the o v e r a l l b i o l o g i c a l e f f e c t o f deu t e r i u m can be e x c e e d i n g l y complex. The i s o t o p e s o f c a r b o n and n i t r o g e n may be e x p e c t e d t o have q u a l i t a t i v e l y s i m i l a r e f f e c t s , but the magnitude o f t h e e f f e c t s o f t h e s e i s o t o p e s a r e g e n e r a l l y s m a l l enough t o be w i t h i n the range o f the normal c e l l u l a r c o n t r o l mechanisms. 2

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II.

Deuterium

1930,

Through t h e p a s t 45 y e a r s , s i n c e i t s d i s c o v e r y i n t h e s t a b l e , r a r e hydrogen i s o t o p e o f mass two


ISOTOPES

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(deuterium) has been an i n c r e a s i n g l y u s e f u l t o o l i n c h e m i s t r y and b i o l o g y . The h i s t o r y o f d e u t e r i u m has shown a p r o g r e s s i o n from the s i m p l e t o the complex, from t r a c e r a p p l i c a t i o n s t o massive s u b s t i t u t i o n , from k i n e t i c i s o t o p e e f f e c t s i n simple m o l e c u l e s t o i s o t o p e e f f e c t s i n l i v i n g organisms, and from i t s use t o s i m p l i f y the p r o t o n magnetic resonance ( Hmr) spectrum o f e t h y l a l c o h o l t o the s i m p l i f i c a t i o n o f the iHmr s p e c t r a o f proteins. These most r e c e n t a p p l i c a t i o n s r e s u l t from the a b i l i t y t o grow a l g a e and o t h e r m i c r o o r g a n i s m s i n heavy water, H 0 , whose hydrogen c o n t e n t i s 99.8 atom p e r c e n t H and o n l y 0.2 atom p e r c e n t 2

2

2

Algae. A f t e r a succession of f a i l u r e s i n other l a b o r a t o r i e s , Chorney and co-workers (13) succeeded i n c u l t u r i n g two s p e c i e s o f f r e s h - w a t e r green a l g a e i n heavy water. T h i s work was q u i c k l y f o l l o w e d by the s u c c e s s f u l c u l t u r e o f a number o f o t h e r a l g a e i n heavy water. The e x t r a c t i o n o f o r g a n i c s u b s t r a t e s (14) from t h e s e H - a l g a e then made p o s s i b l e the c u l t i v a t i o n o f a number o f h e t e r o t r o p h i c b a c t e r i a and f u n g i i n f u l l y deut e r a t e d form. Many k i n d s o f a l g a e r e q u i r e d a l e n g t h y p e r i o d o f a d a p t a t i o n b e f o r e r o u t i n e c u l t u r e i n heavy water was p o s s i b l e . O f t e n a s m a l l n u t r i t i o n a l s u p p l e ment ( i n the form o f y e a s t e x t r a c t ) h e l p e d the a l g a e t o overcome the problems o f t o t a l k i n e t i c r e o r g a n i z a t i o n i n the new c u l t u r e medium. Near a n a e r o b i c c o n d i t i o n s were a l s o b e n e f i c i a l , as t h e r e were i n d i c a t i o n s t h a t r e s p i r a t i o n i n a d a p t i n g organisms was u n c o n t r o l l e d . A f t e r a d a p t a t i o n , however, t h e a l g a e grew i n a normal manner b u t a t a s l o w e r than normal r a t e . A k i n e t i c i s o t o p e e f f e c t ( k / k ) o f 3.5 was observed i n the l i g h t s a t u r a t e d growth r a t e o f s e v e r a l green and b l u e - g r e e n algae. However, because the l a r g e - s c a l e p r o d u c t i o n o f i s o t o p i c a l l y - a l t e r e d a l g a e u s u a l l y i n v o l v e s growth under l i g h t l i m i t i n g c o n d i t i o n s , t h i s l a r g e i s o t o p e e f f e c t i s n o t a major h a n d i c a p i n the p r o d u c t i o n o f l a r g e amounts o f f u l l y d e u t e r a t e d a l g a e . 2

H

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Deuterium Organisms i n E s r . The a v a i l a b i l i t y o f f u l l y d e u t e r a t e d a l g a e has p e r m i t t e d some s i g n i f i c a n t b i o l o g i c a l problems t o be a t t a c k e d i n new and e f f e c t i v e ways. Deuterium had been used as a t o o l t o a i d i n the s i m p l i f i c a t i o n and i n t e r p r e t a t i o n o f e l e c t r o n s p i n resonance (esr) and n u c l e a r magnetic resonance (nmr) s p e c t r a o f r e l a t i v e l y s i m p l e o r g a n i c m o l e c u l e s f o r many


9. κΑτζ

ET


AL.

Biology

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years.* In t h e s e c a s e s H was i n t r o d u c e d by c h e m i c a l synthesis. A s i m i l a r t e c h n i q u e can now be a p p l i e d t o some e x t r e m e l y complex b i o l o g i c a l m o l e c u l e s . One o f the more i m p o r t a n t r e s u l t s o b t a i n e d by s e l e c t i v e i n c o r p o r ation of and H has been the e l u c i d a t i o n o f t h e c h l o r o p h y l l f r e e r a d i c a l species t h a t i s i n v o l v e d i n the l i g h t c o n v e r s i o n a c t o f p h o t o s y n t h e s i s (15). E s r s t u d i e s on H - c h l o r o p h y l l s and H - o r g a n i s m s have shown t h a t the f r e e r a d i c a l s p e c i e s formed by the e x c i t i n g l i g h t i s comprised o f a p a i r o f c h l o r o p h y l l m o l e c u l e s arranged i n a s p e c i a l c o n f i g u r a t i o n which p e r m i t s them t o a c t as an energy t r a p . 2

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D e u t e r a t e d P r o t e i n s i n Nmr, Deuterated p r o t e i n s can a l s o s e r v e as t h e b a s i s f o r t h e s i m p l i f i c a t i o n o f the h i g h l y complex ^Hmr s p e c t r a o f t h e s e m o l e c u l e s . Three b a s i c experiments t o t h i s end have been d e s c r i b e d i n the l i t e r a t u r e (16). (1) An a u t o t r o p h i c organism growing i n H 0 can be i n d u c e d t o u t i l i z e an lH-amino a c i d ; t h e o r g a n i s m t h e n b i o s y n t h e s i z e s H - p r o t e i n con t a i n i n g !H-amino a c i d r e s i d u e s embedded i n i t ; ( 2 ) , an 1 H p r o s t h e t i c group can be bound t o a f u l l y d e u t e r a t e d a p o p r o t e i n , making i t p o s s i b l e t o d e t e c t e a s i l y the p r o s t h e t i c group by Hmr; and ( 3 ) , the s l o w l y exchange a b l e amide p r o t o n s can be o b s e r v e d i n o t h e r w i s e f u l l y d e u t e r a t e d p r o t e i n s by o b s e r v i n g t h e time dependence o f t h e iHmr spectrum when t h e p r o t e i n i s d i s s o l v e d i n Ή α We d e s c r i b e here an example o f a t y p e (3) experiment, i e . , t h e o b s e r v a t i o n by J-Hmr o f amide p r o t o n s i n a f u l l y deuterated protein. ^ The p r o t e i n resonance l i n e s o b t a i n e d i n an Hmr experiment a r e g e n e r a l l y q u i t e b r o a d . The l a r g e p r o t e i n m o l e c u l e s tumble s l o w l y i n s o l u t i o n , and many d i p o l e - d i p o l e magnetic i n t e r a c t i o n s and c h e m i c a l s h i f t a n i s o t r o p i e s a r e n o t averaged o u t . In g e n e r a l , amide p r o t o n resonance peaks i n m o l e c u l e s w i t h m o l e c u l a r w e i g h t s o f 10-20,000 d a l t o n s have l i n e w i d t h s o f the o r d e r o f 30-35 Hz. However, i n a d e u t e r a t e d p r o t e i n , the d i p o l e - d i p o l e i n t e r a c t i o n i s much d e c r e a s e d and most amide p r o t o n s have l i n e w i d t h s i n t h e range o f 15-18 Hz. These amide p r o t o n s appear 6 t o 11 ppm downf i e l d from t e t r a m e t h y l s i l a n e , and t h e p r o t o n resonances 2

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Η has a s p i n o f 1/2 and a magnetic moment o f 2.793 n u c l e a r magnetons. H has a s p i n o f 1 and a magnetic moment o f 0.857 n u c l e a r magnetons. Thus, when a deu t e r i u m atom i s s u b s t i t u t e d f o r a p r o t i u m atom, c o u p l i n g c o n s t a n t s a r e r e d u c e d by a f a c t o r o f 6.5 and t h e f r e quency a t w h i c h energy l e v e l t r a n s i t i o n s t a k e p l a c e i s a l s o reduced by a f a c t o r o f 6.5. 2


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t h a t o r i g i n a t e from t h e a r o m a t i c amino a c i d s i d e chains t h a t a r e n o r m a l l y p r e s e n t i n t h i s r e g i o n o f t h e Hmr spectrum a r e o f course absent i n t h e f u l l y d e u t e r a t e d protein. Thus, a c l e a r , w e l l - r e s o l v e d view o f t h e sLowl y exchangeable amide p r o t o n s can be had. These protons are p a r t o f t h e hydrogen-bonded s t r u c t u r e o f t h e h e l i x and p l e a t e d s h e e t p o r t i o n s o f p r o t e i n s and thus have considerable s t r u c t u r a l significance. F i g u r e 1 shows an example o f an ^Hitir study o f amide p r o t o n s i n a d e u t e r ated a l g a l f e r r e d o x i n . 1

Deuterated N u c l e i c A c i d s . Deuterated algae are a s o u r c e o f d e u t e r a t e d s u b s t r a t e s f o r t h e growth o f bac t e r i a , y e a s t and molds (14). D e u t e r a t e d b a c t e r i a have been found u s e f u l as a s o u r c e o f d e u t e r a t e d n u c l e i c a c i d s f o r u s e i n u l t r a c e n t r i f u g a l d e n s i t y g r a d i e n t an a l y s i s o f t h e i n t e r a c t i o n s o f homologous DNA m o l e c u l e s . F u l l y d e u t e r a t e d DNA ( a l l C- H bonds) has a bouyant d e n s i t y 0.04 g/cm g r e a t e r than normal (normal DNA s have bouyant d e n s i t i e s i n t h e range o f 1.70-1.71 g/cm ) . T h i s t e c h n i q u e has been e x p l o i t e d r e c e n t l y i n t h e study o f t h e i n t e r a c t i o n o f b a c t e r i a l and b a c t e r i o p h a g e n u c l e i c acids (17). 2

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Deuterated M e t a b o l i t e s . A number o f s t u d i e s on the p r o d u c t i o n o f e x t r a c e l l u l a r m e t a b o l i t e s by d e u t e r a t e d organisms have been completed i n r e c e n t y e a r s . These s t u d i e s have i n v o l v e d t h e p r o d u c t i o n and c h a r a c t e r i z a t i o n o f a l k a l o i d s (18), a n t i b i o t i c s (19), and t h e v i t a min r i b o f l a v i n produced by organisms grown i n 99.8 atom p e r c e n t H 0 (20_) . The use o f v a r i o u s *Η ( n a t u r a l abundance) s u b s t r a t e s i n c o n j u n c t i o n w i t h iHmr a n a l y s i s o f t h e p r o d u c t has made i t p o s s i b l e t o o b t a i n a q u a n t i t a t i v e assessment o f t h e e x t e n t o f s o l v e n t p a r t i c i p a t i o n i n t h e b i o s y n t h e s i s o f these n a t u r a l p r o d u c t s . As might be e x p e c t e d , heavy water s t r o n g l y i n h i b i t s t h e production o f the e x t r a c e l l u l a r m a t e r i a l i n a l l o f the e x p e r i m e n t s . More r e c e n t l y , however, i t has been ob s e r v e d t h a t r i b o f l a v i n p r o d u c t i o n by t h e fungus Eremothecium a s h b y i i i s s t i m u l a t e d by heavy water (Table 1 ) . E v i d e n t l y , t h e r e i s an i n v e r s e i s o t o p e e f f e c t on t h i s p a r t i c u l a r m e t a b o l i c pathway. 2

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H i g h e r P l a n t s . The e f f e c t s o f d e u t e r i u m on h i g h e r p l a n t s have a t t r a c t e d p a r t i c u l a r a t t e n t i o n . The e a r l y l i t e r a t u r e has been reviewed by Morowitz and Brown Π ) and more r e c e n t l y by Flaumenhaft e t a l . (Β). The f i r s t d e t a i l e d study o f t h e e f f e c t s o f e x t e n s i v e s u b s t i t u t i o n o f hydrogen by d e u t e r i u m on t h e growth and development o f h i g h e r p l a n t s was d e s c r i b e d by B l a k e e t a l . (21) who


9. ΚΑτζ E T A L .

Isotope Chemistry and Biology

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Ferredoxin Oxidized Full Back Exchange

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ppm Figure 1. Proton magnetic resonance spectra at 220 MHz (Varian HR 220 spectrometer in the fast fourier transform pulse mode) of fully deuterated algal ferre doxin. About 26 slowly exchangeable protons (Spectrum I) are observable after back exchange in H 0 buffer at pll 9. After 3 hrs at 60°C in Ή Ό buffer, pH 7.2, 11 of these protons have completely out exchanged and 15 have been quite resistant to exchange (Spectrum II). These data indicate that this ferredoxin molecule contains two segments of helix (or pleated-sheet) secondary structure. 1

2


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Table I S t i m u l a t i o n o f R i b o f l a v i n P r o d u c t i o n by E. a s h b y i i S u b c u l t u r e d from ^-E O i n t o V a r i o u s L e v e l s o f ^H 0 n

o

Atom P e r c e n t Deuterium i n N u t r i e n t Water

Day 0

0 25 50 99

0 0 0 0

Riboflavin Production (mg/liter) Day Day Day 22 15 8 2.7 3.5 8.5 15.8

4.6 5.4 13.0 23.5

5.6 7.3 15.0 25.1

used peppermint (Mentha p i p e r i t a L.) as the s t u d y p l a n t . Above the 20 p e r c e n t D 0 l e v e l t h e r e was a p r o g r e s s i v e i n h i b i t o r y e f f e c t on growth w i t h i n c r e a s e i n the D0 c o n t e n t o f the n u t r i e n t as r e f l e c t e d i n e l o n g a t i o n growth o f the a x i a l s h o o t . V e r y pronounced r e p r e s s i v e e f f e c t s were o b s e r v e d a t the 50 p e r c e n t l e v e l , and growth e s s e n t i a l l y s t o p p e d i n 70 p e r c e n t D 0. In a subsequent r e p o r t the h i s t o l o g i c e f f e c t s o f d e u t e r a t i o n on peppermint were r e p o r t e d by Crane e t a l . (22). The major e f f e c t o f d e u t e r i u m on the growth o f peppermint appeared t o be i n h i b i t i o n o f c e l l d i v i s i o n . In the deut e r a t e d p l a n t s parenchyma c e l l s were e n l a r g e d , w h i l e t h e r e appeared t o be a r e d u c t i o n i n the v a s c u l a r t i s s u e . In g e n e r a l , t h e e f f e c t s o f d e u t e r a t i o n were more appare n t i n a c t i v e l y growing t i s s u e s t h a n i n t i s s u e exposed t o D 0 i n the n u t r i e n t a f t e r d i f f e r e n t i a t i o n had occurred. The e f f e c t s o f c e r t a i n growth r e g u l a t o r s on peppermint grown a t d i f f e r e n t l e v e l s o f D 0 i n the nut r i e n t s o l u t i o n were r e p o r t e d by B l a k e e t a l . (23). The i n h i b i t o r y e f f e c t s o f d e u t e r i u m on the c e l l u l a r l e v e l were not r e v e r s e d by g i b b e r e l l i c a c i d , naphthal e n e - a c e t i c a c i d or i n d o l e a c e t i c a c i d . In some i n s t a n c e s the i n h i b i t o r y e f f e c t s on growth were even g r e a t e r than t h a t a t t r i b u t a b l e t o the D 0 i n the nutrient. I t i s i n t e r e s t i n g to note t h a t maleic hydraz i d e , u s u a l l y c o n s i d e r e d t o be a p l a n t growth i n h i b i t o r , a c t u a l l y s t i m u l a t e d the growth o f peppermint c u l t u r e d i n d e u t e r a t e d media. Perhaps the most i n t e n s i v e l y s t u d i e d h i g h e r p l a n t i s Lemna p e r p u s i l l a (duckweed), which was s u b j e c t e d t o e x t e n s i v e d e u t e r i u m r e p l a c e m e n t by Cope e t a l . (24). A l a r g e number o f growth f a c t o r s , i n d i v i d u a l l y and i n c o m b i n a t i o n , were i n c l u d e d i n the n u t r i e n t s o l u t i o n and t h e i r e f f e c t on the growth o f duckweed a t h i g h l e v e l s o f d e u t e r a t i o n was o b s e r v e d . A t d e u t e r i u m l e v e l s i n 2

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the n u t r i e n t between 50 and 63 p e r c e n t numerous abnor m a l i t i e s were produced, b u t t h e s e were l a r g e l y e l i m i n a t e d by t h e a d d i t i o n o f k i n e t i n . None o f t h e o t h e r ad d i t i v e s produced a b e n e f i c i a l r e s p o n s e . Even k i n e t i n f a i l e d t o evoke a p r o t e c t i v e e f f e c t when t h e D 0 l e v e l exceeded 63 p e r c e n t . The i n c l u s i o n o f Η-glucose i n the n u t r i e n t s o l u t i o n (50 atom p e r c e n t D 0) r a i s e d t h e f i x e d d e u t e r i u m i n t h e p l a n t DNA t o 75 p e r c e n t . I n b e l l a d o n n a p l a n t s d e u t e r a t i o n had a d r a s t i c e f f e c t on f l o w e r development (25). The number o f c a l y x l o b e s , c o r o l l a l o b e s , and stamens, w h i l e i n v a r i a b l y 5 i n c o n t r o l f l o w e r s , i n c r e a s e d t o as many as 9 o r 10 i n p l a n t s grown i n 70 p e r c e n t D 0 medium. Abnormally shaped b e r r i e s formed i n p l a n t s grown i n heavy water. The e x t e n t o f m a l f o r m a t i o n depended on t h e D 0 c o n t e n t o f t h e medium and how l a t e i n t h e l i f e c y c l e o f t h e p l a n t t h a t t h e b e r r y formed. The shapes ranged from pear-shaped t o dumbbell-shaped t o c y l i n d r i c a l . The misshapened b e r r i e s r e s u l t e d from t h e t e n a c i t y w i t h which t h e c o r o l l a remained a t t a c h e d t o t h e r i p e n i n g b e r r y i n d e u t e r a t e d p l a n t s . As t h e b e r r y e n l a r g e d , a c o n s t r i c t i o n d e v e l o p e d where t h e c o r o l l a was a t t a c h e d t o t h e b e r r y . The s i z e and number o f seeds were s e v e r e l y reduced i n d e u t e r a t e d b e r r i e s w i t h o n l y a few r u d i m e n t a r y seeds a p p a r e n t i n t h e 70 p e r c e n t b e r r i e s . A s i m i l a r study w i t h A r a b i d o p s i s t h a l i a n a gave much the same r e s u l t s ( 2 6 ) . A replacement c u l t u r e t e c h n i q u e was d e v e l o p e d by Crane e t a l . (27) t o study t h e e f f e c t o f d e u t e r a t i o n on a l k a l o i d production i n Atropa belladonna. P l a n t s were grown t o m a t u r i t y i n an aqueous (H^O) medium and were then t r a n s p l a n t e d t o media c o n t a i n i n g 50, 60, 75 and 99.7 p e r c e n t D 0. The p l a n t s i n 99.7 p e r c e n t D 0 show ed t h e d r a s t i c e f f e c t s o f d e u t e r a t i o n almost immediate l y and a l l p l a n t s d i e d i n s e v e r a l days. Plants trans p l a n t e d i n t o 75 p e r c e n t D 0 s u r v i v e d about 3 weeks, and t h e 50 and 60 p e r c e n t D 0 p l a n t s w i t h s t o o d t h e s t r e s s e s imposed by deuterium. These p l a n t s were h a r v e s t e d a f t e r a growth p e r i o d o f 7.5 months. Alkaloid p r o d u c t i o n was reduced t o from o n e - t h i r d t o o n e - t e n t h t h a t o f t h e c o n t r o l p l a n t s . The a b s o l u t e amount o f a l k a l o i d formed and t h e t o t a l amount o f p l a n t m a t e r i a l produced were t o o s m a l l t o p e r m i t i s o l a t i o n o f a l k a l o i d . I t appeared from t h i s study t h a t a l k a l o i d p r o d u c t i o n was c o m p l e t e l y i n h i b i t e d upon t r a n s f e r o f t h e p l a n t s from normal growth i n H-O t o t h e d e u t e r a t e d medium. G e r m i n a t i o n o f seeds has been the o b j e c t o f study. Crumley and Meyer (28) o b s e r v e d a d e l a y i n t h e i n i t i a t i o n of germination i n four species of p l a n t s , the e x t e n t o f which i n c r e a s e d w i t h t h e d e u t e r i u m 2

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c o n c e n t r a t i o n o f the s o l v e n t . However, t h e number o f seeds which f i n a l l y germinated was o n l y s l i g h t l y lower f o r pure D 0 t h a n f o r w a t e r . More r e c e n t l y , S i e g e l e t a l . (29) s t u d i e d the g e r m i n a t i o n o f 11 s p e c i e s o f seeds i n h i g h c o n c e n t r a t i o n s o f D 0, and o b s e r v e d o n l y r y e seeds were c a p a b l e o f g e r m i n a t i o n a t h i g h D 0 concentrations. B l a k e e t a l . (30) found a s i m p l e r e l a t i o n s h i p between the s i z e o f the seed and the g e r m i n a t i o n c a p a c i t y i n high deuterium c o n c e n t r a t i o n s . Larger seeds a r e a p p a r e n t l y more s u c c e s s f u l i n g e r m i n a t i n g because they c o n t a i n l a r g e r h y d r o g e n - c o n t a i n i n g f o o d r e s e r v e s . M e t a b o l i c d i f f i c u l t i e s a s s o c i a t e d w i t h deut e r i u m i n c o r p o r a t i o n a r e t h u s d e l a y e d u n t i l the hydrogen r e s e r v e s o f the seed a r e exhausted. I f the b a s i s f o r the o b s e r v e d e f f e c t s i s c o r r e c t , then the s p e c i e s s p e c i f i c d e u t e r i u m e f f e c t s s h o u l d be s e p a r a b l e from the e f f e c t o f h y d r o g e n - c o n t a i n i n g f o o d r e s e r v e s by growing t h e embryo i n t h e absence o f the h y d r o g e n - c o n t a i n i n g endosperm. Removal o f the seed s t r u c t u r e s from the embryos reduces t o a minimum the a v a i l a b i l i t y o f ^H. Crane e t a l . (31) s t u d i e d the e f f e c t o f d e u t e r i u m r e placement on t h e e l o n g a t i o n o f e x c i s e d embryos o f seve r a l s p e c i e s o f seeds and noted t h a t a l l embryos s u f f e r e d growth i n h i b i t i o n i n D 0. Added s u c r o s e m i t i g a t e d t h e r e p r e s s i v e e f f e c t s o f d e u t e r i u m on growth. S i e g e l and G a l s t o n (32) demonstrated t h a t b i o s y n t h e s i s does take p l a c e when w i n t e r r y e seeds a r e germinated i n 99.7 p e r c e n t D 0. F i v e i s o p e r o x i d a s e s i s o l a t e d from seeds germinated i n D 0 were shown t o be d e u t e r a t e d , and i t was c o n c l u d e d t h a t they were b i o s y n t h e s i z e d d u r i n g the g e r m i n a t i o n p r o c e s s . In g e n e r a l , h i g h e r p l a n t s show more complex D 0 e f f e c t s than do m i c r o o r g a n i s m s . T h i s i s t o be e x p e c t e d as t h e i r s t r u c t u r e i s more h i g h l y o r g a n i z e d . The r e sponse t o D 0 i n h i g h e r p l a n t s i s a graded one w i t h 60 t o 70 p e r c e n t the maximum l e v e l t o l e r a t e d i n the nut r i e n t medium. A p r i m a r y response t o d e u t e r a t i o n app e a r s t o be s u p p r e s s i o n o f the p r o d u c t i o n o f i m p o r t a n t metabolites including a l k a l o i d s , a n t i b i o t i c s , proteins, c a r b o h y d r a t e s , e t c . Some b e n e f i c i a l e f f e c t s i n h e l p i n g h i g h e r p l a n t s adopt t o d e u t e r i u m have been noted w i t h the p l a n t growth s t i m u l a n t k i n e t i n and w i t h the growth i n h i b i t o r maleic hydrazide. The complex n a t u r e o f h i g h e r p l a n t s makes d i f f i c u l t a s i m p l e e x p l a n a t i o n o f t h e o b s e r v e d e f f e c t s o f growth r e g u l a t o r s i n H 0, and f u r t h e r s y s t e m a t i c s t u d i e s w i l l have t o be undertaken i n o r d e r t o b e t t e r u n d e r s t a n d the phenomena t a k i n g place. The replacement t e c h n i q u e used s u c c e s s f u l l y f o r c u l t u r i n g c e r t a i n f u n g i and molds was u n f o r t u n a t e l y not found t o be a p p l i c a b l e f o r h i g h e r p l a n t s . 2

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B i o l o g i c a l E f f e c t s o f Carbon-13

13 C u l t u r e Systems f o r the Growth o f C-Qrganisms. The c u l t u r e o f m i c r o o r g a n i s m s i n systems r e q u i r i n g o n l y the s u b s t i t u t i o n o f d e u t e r i u m p r e s e n t s no complex p r o b lems i n a p p a r a t u s d e s i g n . U s u a l l y , the i s o t o p e i s p r e s e n t e d i n the form of H 0, and o n l y the p r e v e n t i o n o f exchange w i t h i s o t o p i c a l l y normal water i n the ambient atmosphere i s r e q u i r e d . U t i l i z a t i o n of 13 presents somewhat more complex problems, because the i s o t o p e i n i t s most e c o n o m i c a l form i s a v a i l a b l e as 1 3 c o . Of c o u r s e , c a r b o n d i o x i d e i s the optimum s u b s t r a t e f o r t h e p r o d u c t i o n o f p h o t o s y n t h e t i c organisms, b u t i t does p r e s e n t problems i n m a n i p u l a t i o n and c o n s e r v a t i o n . The c u r r e n t p r i c e o f t h i s i s o t o p e makes mandatory a com p l e t e l y s e a l e d growth system, n o t , i n t h i s c a s e p r i m a r i l y t o p r e v e n t i s o t o p i c a t t e n u a t i o n w i t h the e x t e r n a l c a r b o n d i o x i d e , b u t t o p r e v e n t l o s s o f the r a r e , ex pensive m a t e r i a l . Equipment f o r t h e c u l t u r e o f p h o t o s y n t h e t i c micro organisms i n the l i q u i d phase on 13C0 p r e s e n t s no l a r g e problem i n d e s i g n o r c o n s t r u c t i o n . A l l t h a t i s r e quired, i n a d d i t i o n to a s u i t a b l e , t i g h t container f o r t h e a l g a l c u l t u r e , i s a s u f f i c i e n t l y l a r g e volume t o c o n t a i n t h e gas phase a t a r e a s o n a b l e c a r b o n d i o x i d e l e v e l and a c i r c u l a t i n g pump t o c a r r y the gas m i x t u r e o v e r the c u l t u r e . L i g h t i n g and o t h e r arrangements may be a r r a n g e d as i n the c u l t u r e o f t h e same organisms i n 2H 0. A u s e f u l c o n c e n t r a t i o n of carbon d i o x i d e i s around 20%, w i t h n i t r o g e n as a c a r r i e r gas. C u l t u r e of s a p r o p h y t i c microorganisms w i t h C c o m p o s i t i o n s i s o f t e n f a c i l i t a t e d by d i r e c t use o f sub s t r a t e s d e r i v e d from 13c a l g a e . Alternatively, specific m e t a b o l i t e s o r p r e c u r s o r s p r e p a r e d by o r g a n i c s y n t h e t i c methods may be added t o the s u b s t r a t e s o f whatever yeasts, f u n g i , b a c t e r i a , e t c . are d e s i r e d . The t e c h n i q u e s u s e f u l i n many such growth e x p e r i m e n t s have been d e s c r i b e d (33). Growth o f t e r r e s t r i a l h i g h e r p l a n t s from C0 presents s e v e r a l complications i n apparatus design. The system must be c a p a b l e o f s u s t a i n i n g normal p l a n t s growth f o r l o n g p e r i o d s i n a c o m p l e t e l y s e a l e d con dition. O b v i o u s l y , p r o v i s i o n must be made t o a s s u r e i n o r g a n i c n u t r i e n t s u p p l i e s , adequate c a r b o n d i o x i d e l e v e l s , c o n t r o l o f temperature, h u m i d i t y , s o i l m o i s t u r e and l i g h t i n t e n s i t y . Such systems have been c o n s t r u c t e d and o p e r a t e d t o produce t o b a c c o and o t h e r h i g h e r p l a n t s a t l e v e l s o f o v e r 90% e n r i c h e d 1 C, and a r e d e s c r i b e d i n d e t a i l elsewhere (34J. I t i s t o be emphasized t h a t t h e s e growth chambers d i f f e r i n one i m p o r t a n t a s p e c t 2

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from most p r e v i o u s l y d e s c r i b e d " p h y t o t r o n s " o r " b i o t r o n s " (35). Whereas most s p e c i a l growth chambers i n use a r e by n e c e s s i t y n o t s e a l e d and a r e c l o s e l y l i n k e d t o the o u t s i d e environment t o p e r m i t easy c o n t r o l o f tempera t u r e and h u m i d i t y , and i n many c a s e s f o r a c o n t i n u o u s s u p p l y o f carbon d i o x i d e , the growth chambers d i s c u s s e d h e r e were d e s i g n e d w i t h the same p h i l o s o p h y used where manipulation of radioactive or other t o x i c materials must be employed. Our growth chambers thus a r e provided w i t h g l o v e p o r t s f o r i n t e r n a l m a n i p u l a t i o n s , hermetically s e a l e d bulkheads, e t c . The p r i n c i p a l s t r u c t u r a l f e a t u r e s a r e as f o l l o w s : c e i l i n g and w a l l s a r e 1/4" thick methacrylate p l a s t i c ; f l o o r and s t r u c t u r a l elements were aluminum. A p p r o x i mate dimensions a r e : 1 χ 2 χ 3 M. A h e a t exchanger s u p p l i e d by e x t e r n a l c h i l l e d water p r o v i d e d temperature and h u m i d i t y c o n t r o l . Sensors b u r i e d i n the v e r m i c u L i t e or sand s u p p o r t medium a l l o w e d m o n i t o r i n g o f s o i l mois t u r e c o n t e n t . Carbon d i o x i d e was p r o v i d e d from e x t e r n a l c y l i n d e r s , and n u t r i e n t was s u p p l i e d t o each p l a n t through a s e a l e d b u l k h e a d . Alternatively, solid i n o r g a n i c n u t r i e n t was p r o v i d e d from a p e l l e t b u r i e d i n the s a n d / v e r m i c u l i t e s o i l medium, and the condensed water o f t r a n s p i r a t i o n r e c y c l e d and d i s t r i b u t e d t o each p l a n t by means o f an a u t o m a t i c d i s p e n s e r c o n t r o l l e d by a p r e s e t t i m e r . T h i s system was p r o b a b l y most s a t i s f a c t o r y from the v i e w p o i n t o f e l i m i n a t i n g p e r i o d i c a d d i t i o n s o f e x t e r n a l l i q u i d n u t r i e n t and subsequent w i t h d r a w a l o f t h e condensed water o f t r a n s p i r a t i o n . Such an automated system i s c a p a b l e of smooth f u n c t i o n i n g f o r l o n g p e r i o d s o f time w i t h o u t a t t e n d a n c e . A view o f t h e s e systems i s shown i n F i g u r e 2; t h e c r o p i s t o b a c c o , e n r i c h e d t o a l e v e l o f > 90% C. 3

13 . . . . . B i o l o g i c a l E f f e c t s of C Substitution i n Living Organisms. The consequences o f s u b s t i t u t i o n o f 1 C f o r i ^ C i n b i o l o g i c a l systems a r e o f i n t e r e s t as a problem i n i s o t o p e b i o l o g y , as w e l l as c a r r y i n g e n t i r e l y p r a g matic i m p l i c a t i o n s . Numerous s u g g e s t i o n s have been made (36) i n v o l v i n g p o s s i b l e uses o f 13c compounds i n c l i n i c a l d i a g n o s i s and o t h e r m e d i c a l a p p l i c a t i o n s . It i s t h e r e f o r e o f paramount importance t o determine the e x t e n t o f any p o s s i b l e d e l e t e r i o u s e f f e c t s o f 13c sub s t i t u t i o n i n l i v i n g organisms. G i v e n the s m a l l d i f f e r e n c e i n the masses o f C and C , r e l a t i v e t o t h o s e o f hydrogen and d e u t e r i u m , i t would be e x p e c t e d t h a t the k i n e t i c i s o t o p e e f f e c t s and t h e r e f o r e t h e d i s r u p t i o n o f m e t a b o l i c f u n c t i o n when t h e s e i s o t o p e s are i n t e r c h a n g e d , would be much l e s s J

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pronounced. T h i s e x p e c t a t i o n tends t o be b o r n out by e x p e r i m e n t a l f i n d i n g s . In t h e case o f u n i c e l l u l a r , p h o t o s y n t h e s i z i n g organisms, o n l y r e l a t i v e l y s m a l l d i f f e r e n c e s a r e seen i n growth r a t e , s i z e d i s t r i b u t i o n o r g r o s s morphology (37), when 13c i s s u b s t i t u t e d f o r 12c a t l e v e l s above 90% enrichement. D i s t u r b a n c e o f normal b i o l o g i c a l f u n c t i o n by -*-C s u b s t i t u t i o n i s somewhat more e a s i l y demonstrated when t h i s i s o t o p e i s s u b s t i t u t e d i n c o n j u n c t i o n w i t h d e u t e r i u m and o t h e r s t a b l e isotopes. .^ The e f f e c t o f a h i g h l e v e l o f C s u b s t i t u t i o n on complex organisms, p l a n t s o r a n i m a l s , i s ambiguous a t present. Experiments aimed a t growth o f l ^ C t o b a c c o and o t h e r f l o w e r i n g p l a n t s a t l e v e l s o f i s o t o p i c en richment around 90% p r o v i d e some e v i d e n c e . Compared t o c o n t r o l s , the i s o t o p i c a l l y s u b s t i t u t e d p l a n t s showed s l i g h t l y r e t a r d e d f l o w e r i n g , the p r o d u c t i o n o f fewer f l o w e r s , a b s c i s s i n g more f r e q u e n t l y , and s e e m i n g l y ab normal p o l l e n . Any a b n o r m a l i t y i n t h e r e p r o d u c t i v e c y c l e i s p a r t i c u l a r l y s u g g e s t i v e , i n l i g h t o f the ex p e r i e n c e w i t h d e u t e r i u m s u b s t i t u t i o n and i t s marked e f f e c t s on r e p r o d u c t i o n . E x a m i n a t i o n o f 13c p o l l e n by the t e c h n i q u e o f s c a n n i n g e l e c t r o n m i c r o s c r o p y r e v e a l e d a h i g h degree o f m o r p h o l o g i c a l a b n o r m a l i t y . Subsequent experiments w i t h morning g l o r y tended t o s u p p o r t t h e s e observations. I n v e s t i g a t i o n s o f v a r i o u s 13c p o l l e n s are s t i l l i n p r o g r e s s and any c o n c l u s i o n s a t t h i s time must be r e g a r d e d as t e n t a t i v e . The s u b s t i t u t i o n o f C at high levels i n highly e v o l v e d a n i m a l s evokes q u e s t i o n s o f g r e a t i n t e r e s t , o f b o t h p r a c t i c a l and t h e o r e t i c a l c h a r a c t e r . Conclusive answers are not, as y e t , e v i d e n t . The growth o f 13c tobacco provided p e r i p h e r a l evidence. In the c o u r s e o f c u l t i v a t i o n o f 13c t o b a c c o an a d v e n t i t i o u s i n f e s t a t i o n o f w h i t e f l i e s took p l a c e i n one o f the growth chambers, which had been i s o l a t e d from the e x t e r n a l environment f o r s e v e r a l weeks. S u p e r f i c i a l e x a m i n a t i o n by a t i m e o f - f l i g h t mass s p e c t r o m e t e r o f the waxes e l a b o r a t e d by the wings o f t h e s e i n s e c t s i n d i c a t e d a h i g h l e v e l o f 13c s u b s t i t u t i o n . The mass spectrum peaks are shown i n F i g u r e 3. I t was e s t i m a t e d t h a t the i n s e c t has somewhat more than 50% 13c i n i t s i s o t o p i c c a r b o n make up. T h i s u n s c h e d u l e d event p r o b a b l y produced the f i r s t highly enriched l c insect. Whether the d i s p a r i t y w i t h the s u b s t r a t e was due t o complex m e t a b o l i c f r a c t i o n a t i o n p a t t e r n s o r whether performed embryonic t i s s u e made a s i g n i f i c a n t c o n t r i b u t i o n cannot now be d e c i d e d . An o b v i o u s l e s s o n t o be drawn would suggest the ease o f p r o d u c i n g 13c p a r a s i t i c a n i m a l s , g i v e n a s u i t a b l e 13c plant substrate. 3

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Long term experiments i n v o l v i n g 13c s u b s t i t u t i o n i n mice have been attempted, w i t h i n c o n c l u s i v e r e s u l t s on t h e p o s s i b l e d e l e t e r i o u s e f f e c t s o f the i s o t o p e (38). These a n i m a l s were f e d on a d i e t o f performed l c mater i a l s d e r i v e d from l a b o r a t o r y s y n t h e s i s , t o g e t h e r w i t h added v i t a m i n s , e t c . o f i s o t o p i c a l l y normal c o n t e n t . A t p r e s e n t a l o n g term s t u d y i s under way t o determine p o s s i b l e b i o l o g i c a l e f f e c t s o f 13c i n f e t a l mice tissue (16). 3

13 A p p l i c a t i o n s of C S u b s t i t u t i o n . The w i d e s p r e a d use o f h i g h l y e n r i c h e d J-^C f o r use i n i n c o r p o r a t i o n i n compounds and organisms of s p e c i a l i n t e r e s t has o n l y begun, but a l r e a d y i t i s e v i d e n t t h a t 13c w i l l have extensive f u t u r e uses. L i m i t a t i o n s a r e imposed o n l y by the i n v e s t i g a t o r ' s s p e c i f i c i n t e r e s t s and the ease o f s y n t h e s i z i n g the m a t e r i a l s d e s i r e d , whether by the b i o s y n t h e s i s i n a s p e c i f i c organism o r by l a b o r a t o r y organic synthesis. I t i s c l e a r , from a l r e a d y r e p o r t e d a p p l i c a t i o n s i n e s r (39) and nmr (40), t h a t the use o f 13c may w e l l become a r o u t i n e t e c h n i q u e i n c e r t a i n k i n d s o f magnetic resonance s t u d i e s . The s i g n i f i c a n t p r o p e r t y o f l c h e r e i s the non-zero n u c l e a r s p i n , b u t o t h e r a p p l i c a t i o n s depend upon the i s o t o p i c mass d i f ference i t s e l f . S e p a r a t i o n o f c o n s t i t u e n t s by d e n s i t y g r a d i e n t u l t r a c e n t r i f u g a t i o n , as f i r s t used i n the c a s e o f 15N (£1) i s an o b v i o u s p o s s i b i l i t y . Another a p p l i c a t i o n , a l r e a d y w i d e l y e x p l o i t e d i s the use o f 13c in mass s p e c t r o m e t r y , e i t h e r w i t h h i g h l y e n r i c h e d systems or a t n a t u r a l abundance l e v e l s . The p r o p o s e d a p p l i c a t i o n s i n c l i n i c a l m e d i c i n e , w i t h mass s p e c t r o m e t r y used i n c o n j u n c t i o n w i t h c h r o m a t o g r a p h i c t e c h n i q u e s , has a l r e a d y been mentioned (42^) . 3

IV.

Nitrogen-15

Nitrogen-14 n u c l e i have a q u a d r u p o l e moment, which means t h a t nmr s i g n a l s from N a r e b r o a d and d i f f i c u l t to detect. An a d d i t i o n a l d i s a d v a n t a g e i s the f a c t t h a t c o u p l i n g between N and o t h e r n u c l e i i s o f t e n u n o b s e r v a b l e due t o the v e r y s h o r t r e l a x a t i o n time o f the N n u c l e u s . Thus, 1 4 N - H c o u p l i n g i s not ob s e r v e d i n the p e p t i d e bond. I t i s t h e r e f o r e advanta geous t o p e r f o r m magnetic resonance s t u d i e s w i t h b i o l o g i c a l m a t e r i a l s s u b s t i t u t e d w i t h 1%, which has a s p i n o f 1/2 and no q u a d r u p o l e moment. D e u t e r a t e d p r o t e i n c o n t a i n i n g N a l l o w s d i s c r i m i n a t i o n between hydrogen bonds i n v o l v i n g amide groups o r h y d r o x y l groups. 1 4

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Recent work by Boxer e t a l . (4_3) has l e d t o the assignment o f f o u r 3* N nmr t r a n s i t i o n s i n c h l o r o p h y l l a and i t s magnesium-free d e r i v a t i v e p h e o p h y t i n . An analy s i s o f the c h e m i c a l s h i f t changes i n d u c e d by complexa t i o n w i t h magnesium r e v e a l e d t h a t the magnesium atom s e l e c t i v e l y p e r t u r b s low l y i n g ηττ* s t a t e s . The charge d e n s i t y i n the f o u r p y r r o l e r i n g s becomes more n e a r l y e q u i v a l e n t so t h a t the energy d i f f e r e n c e s among lowl y i n g π π * s t a t e s are decreased. 5

V. B i o l o g i c a l E f f e c t s of M u l t i p l e Isotope S u b s t i tution Taken t o g e t h e r , compounds o f the elements hydro gen, oxygen, n i t r o g e n and carbon c o n s t i t u t e o v e r 99% o f the mass o f l i v i n g p r o t o p l a s m . A l l o f t h e s e elements p o s s e s s s t a b l e , r a r e heavy i s o t o p e s . Although k i n e t i c i s o t o p e e f f e c t s i n i s o t o p e s o f elements o t h e r t h a n hydrogen might be e x p e c t e d t o have g r e a t l y l e s s e n e d k i n e t i c i s o t o p e e f f e c t s , and t h e r e f o r e d i m i n i s h e d b i o l o g i c a l e f f e c t s , i t i s o f i n t e r e s t t o c o n s i d e r the r e s u l t s o f s u b s t i t u t i o n o f more than one element by i t s heavy i s o t o p e . The o v e r a l l p r e s e n t c o s t s o f H , " C , 1% and 18Q p r o h i b i t growth o f organisms on any b u t t h e s m a l l e s t s c a l e , i f a l l o f t h e s e heavy i s o t o p e s are t o be i n c o r p o r a t e d s i m u l t a n e o u s l y . There have been o n l y two e x t e n s i v e i n v e s t i g a t i o n s t o d a t e o f organisms i n c o r p o r a t i n g deuterium plus other s t a b l e isotopes at h i g h enrichment. The b a s i s f o r a l l such s t u d i e s must be a t o t a l l y deuterium-adapted organism, t o which the o t h e r i s o t o p e s o f i n t e r e s t may be added i n the c o u r s e o f c u l t u r e . An o b v i o u s c o m b i n a t i o n i s p r e s e n t e d by d e u t e r i u m and 13c; t h i s was c a r r i e d o u t by Flaumenhaft e t a l . a t e n r i c h ment l e v e l s o f 99% + D and 95% C (44). C . vulgaris was grown i n a l l combinations o f 1H,~~2*H, l ^ C and l ^ C . The v a r i o u s i s o t o p i c a l l y a l t e r e d c e l l s were compared on the b a s i s o f s i z e and shape, g r o s s morphology, and s t r u c t u r a l changes i n s u b c e l l u l a r o r g a n e l l e s , as i n d i c a t e d by c y t o l o g i c a l s t a i n i n g t e c h n i q u e s . The i n c o r p o r a t i o n o f had no o b v i o u s e f f e c t on the growth c y c l e o f the a l g a e . The c - H a l g a e grew a t about the same r a t e as * C - H c u l t u r e s , as the 1 3 c - I H grew a t the r a t e seen i n normal 1 C - ! H c u l t u r e s . An e x a m i n a t i o n o f c e l l s i z e d i s t r i b u t i o n , however, r e v e a l e d t h a t i n the change o f i s o t o p i c c o m p o s i t i o n from H C t o H - C , a marked change t a k e s p l a c e , as seen i n F i g u r e 4. E v i d e n t l y , the s u b s t i t u t i o n o f in a d e u t e r i u m adapted c e l l r e s u l t s i n a tendency t o undo some o f the d i s r u p t i v e e f f e c t s o f deuterium, a t l e a s t 2

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as r e g a r d s such a g r o s s and s t a t i s t i c a l parameter as c e l l size distribution. S t u d i e s on s u b c e l l u l a r morphology and c y t o l o g y tended t o b e a r o u t t h e f i n d i n g s on s i z e d i s t r i b u t i o n . The p a r t i a l r e v e r s a l o f e f f e c t s a t t r i b u t a b l e t o d e u t e r ium s u b s t i t u t i o n were b o r e o u t by i n v e s t i g a t i o n o f t h e following features: (1) N u c l e a r s i z e . Nuclei of C - H c e l l s were l a r g e r t h a n t h o s e o f 1 3 C - 1 H c e l l s , but s m a l l e r than those o f the C - H cells. (2) Nudear shape. The n u c l e a r s i z e range seen i n 1 3 C - 1 H c e l l s i s about t h a t seen i n 1 2 C - 1 H c e l l s ? C - H c e l l s have l a r g e r n u c l e i and a r e l e s s s p h e r o i d a l . (3) Nucleic a c i d c o n t e n t . DNA d i s t r i b u t i o n was d e t e r m i n e d by c y t o chemical s t a i n i n g . The s t a i n i n g f o r DNA and RNA o f 13 H c e l l n u c l e i was found t o be i n t e r m e d i a t e between t h o s e o f t h e 1 C - 1 H c e l l s and t h o s e o f 1 C - H . As r e gards s t a i n i n g o f c y t o p l a s m i c DNA, a g a i n , t h e c h a r a c t e r of C - H c y t o p l a s m was somewhere between t h a t seen i n 1 3 C - 1 H c y t o p l a s m and t h a t o f 1 C - H . The c o n c l u s i o n s i n r e g a r d t o the RNA was somewhat a t v a r i a n c e t o t h e above p a t t e r n . 1 3 C - 2 H c e l l s produced RNA t a k i n g t h e form o f t h r e a d - l i k e a c c r e t i o n s . These s t r u c t u r e s were never seen i n c e l l s made up o f 1 C , whether i n combin a t i o n w i t h hydrogen o r d e u t e r i u m . (4) P r o t e i n and amino acid content. Staining of c e l l u l a r p r o t e i n material d i s c l o s e d about t h e same p a t t e r n s o f i n t e n s i t y and o c c u r r e n c e as was seen f o r the n u c l e i c a c i d s . The s t a i n i n g f o r f r e e amino a c i d s i n d i c a t e d , a g a i n , t h a t c e l l s w i t h t h e 1 C - H c o m p o s i t i o n s t a i n e d most h e a v i l y . Other f e a t u r e s r e v e a l e d by c y t o l o g i c a l s t u d y i n c l u d e d i n d i c a t i o n s t h a t t h e RNA c o n t e n t may be more n e a r l y normal i n C - H c e l l s than i n t h e p r e v i o u s l y much s t u d i e d 1 C - H c e l l s . T h i s o b s e r v a t i o n must be q u a l i f i e d , as t h e d i s t o r t i o n o f c y t o p l a s m i c RNA was so g r e a t i n c c o n t a i n i n g c e l l s t h a t comparisons were difficult. The most o b v i o u s changes b r o u g h t about by 13c s u b s t i t u t i o n , as seen by phase c o n t r a s t m i c r o s c o p y , was i n the t h i c k n e s s o f c e l l w a l l s . C e l l w a l l s o f both t y p e s o f 13c-grown c e l l s were much t h i c k e r than e v e r seen i n C c e l l s , t h e 1 3 c - H showing up as b e i n g even t h i c k e r than those of 13C-1H c e l l s . A l l these observations i n d i c a t e that p r e d i c t i o n o f l i k e l y changes when two heavy i s o t o p e s a r e s u b s t i t u t e d i n an o r g a n i s m i s n o t p o s s i b l e . Simple assump t i o n s o f l i n e a r a d d i t i v e k i n e t i c i s o t o p e e f f e c t s can n o t a c c o u n t f o r t h e v a r i a t i o n s seen i n t h i s s t u d y . The second c o n c l u s i o n , t h a t s u b s t i t u t i o n o f C for 1 C in d e u t e r a t e d organisms t e n d s t o d e c r e a s e t h e magnitude o f the a b n o r m a l i t y produced a f t e r a d a p t a t i o n t o a i 3

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d e u t e r a t e d m e l i e u i s q u i t e unexpected and cannot be explained at present. The o t h e r s t u d y d e a l i n g w i t h m u l t i p l e i s o t o p i c s u b s t i t u t i o n i n c l u d e d a l l f o u r heavy i s o t o p e s o f hydrogen, carbon, oxygen and n i t r o g e n . Uphaus e t a l . (45) grew c u l t u r e s o f deuterium-adapted C. v u l g a r i s i n v o l umes o f 1-2 ml. The r e s u l t s o f t h i s study a r e o f cons i d e r a b l e i n t e r e s t , when t a k e n as a p r e l i m i n a r y e f f o r t , b u t t h e experiments s u f f e r e d from two s h o r t c o m i n g s . One o f t h e s e a r o s e from t h e use o f o n l y p a r t i a l l y enriched (55% m o l e - p e r c e n t 13c), which made any conc l u s i o n s r e g a r d i n g the p o s s i b l e e f f e c t s o f C substit u t i o n ambiguous. The o t h e r weakness o f t h e study was t h a t t h e g r e a t expense o f t h e i s o t o p e s made i t i m p o s s i b l e t o f o l l o w p a t t e r n s o f change i n the organism, i f each i s o t o p e were added s t e p w i s e i n sequence. The i s o t o p i c enrichments o f t h e m a t e r i a l s used were: H , 99%, C , 55%, N , 98%, 0, 97%. The r e s u l t s o f e x t e n s i v e i s o t o p i c s u b s t i t u t i o n o f a l l f o u r major elements i n t h e biomass r e s u l t e d i n d r a s t i c changes, t h e p r o c e s s e s o r c e l l u l a r m e t a b o l i c d i s r u p t i o n and growth a b n o r m a l i t y p r o g r e s s i n g f a r beyond t h a t seen d u r i n g d e u t e r i u m a d a p t a t i o n . The s i z e d i s t r i b u t i o n s of the i s o t o p i c s p e c i e s studied are given i n F i g u r e 5. V i s u a l o b s e r v a t i o n s by l i g h t m i c r o s c o p y gave the i m p r e s s i o n o f even g r e a t e r h e t e r o g e n e i t y t h a n t h a t i m p l i e d by t h e c u r v e s as determined w i t h a c e l l counter. I t i s p o s s i b l e t h a t the "monster" o r g i a n t c e l l s seen a f t e r complete i s o t o p i c s u b s t i t u t i o n may be p a r t o f the a d a p t i v e p r o c e s s and l i f e c y c l e o f t h e s e cells. C e l l d i v i s i o n may n o t always o c c u r . A l a r g e q u a n t i t y o f c e l l u l a r d e b r i s was p r e s e n t i n mature c u l t u r e s , much more i n mass t h a n c o u l d be accounted f o r by t o t a l d i s i n t e g r a t i o n o f the o r i g i n a l innocum, which was about 1% o f t h e f i n a l c e l l mass. C e l l s grown i n 1 H - 1 8 O - 1 3 C - 1 N media appeared t o resemble more c l o s e l y i s o t p p i c a l l y normal c e l l s than d i d those grown i n H - 0 - C - N media, b u t many o f t h e i r c h a r a c t e r i s t i c s were i n t e r m e d i a t e between the extremes. Cytological studies, using appropriate stains f o r v a r i o u s c e l l u l a r components, produced the f o l l o w i n g observations: 2 _16Q.13 .15 c e l l s : A g r e a t e r amount o f n u c l e i c a c i d was produced by t h e s e c e l l s than any o t h e r type s t u d i e d . L a r g e amounts o f DNA appeared i n b o t h the n u c l e u s and the p e r i p h e r y o f c h l o r o p l a s t s . By cont r a s t , the RNA c o n t e n t o f t h e s e c e l l s was t h e l o w e s t found f o r any system. N u c l e i o f t h e s e c e l l s were greatl y e n l a r g e d and had a u n i f o r m d i s t r i b u t i o n o f DNA, suggesting e i t h e r polyploidy or nuclear degeneration. The f a s t - g r e e n s t a i n used t o v i s u a l i z e p r o t e i n s appears t o I

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r

I I I 14 !6 18 2 0 SIZE, MICRONS

I 22

i 24

I 26

I 28

L_ 30

Size distributions of various Chlorella cells after multiple isotopic substitution


ISOTOPES

202

AND CHEMICAL

PRINCIPLES

be d e e p l y and u n i f o r m l y d i s t r i b u t e d throughout the cell. S t a i n i n g f o r carbohydrate d i s t r i b u t i o n revealed t h a t t h e s e c e l l s c o n t a i n e d much more such m a t e r i a l than e i t h e r o f the I H o r H c o n t r o l s o r the H - 1 8 o c e l l s . 2

2

°0- c°N C e l l s . These c e l l t y p e s showed t h e g r e a t e s t v a r i a b i l i t y i n s t a i n i n g o f components and the l e a s t l o c a l i z a t i o n o f a p a r t i c u l a r component b e i n g stained. Young c e l l s w i t h t h i s i s o t o p i c c o m p o s i t i o n tended t o resemble o r d i n a r y H - 1 0 - 1 C - 1 N c e l l s , w i t h round and p e r i p h e r a l l y l o c a t e d n u c l e i which tended w i t h a g i n g t o become m u l t i l o b a t e ; m u l t i p l e n u c l e i were commonly found. C h l o r o p l a s t s o f t h e s e c e l l s appeared t o produce more DNA than t h o s e o f o t h e r t y p e s . Protein s t a i n i n g i n d i c a t e d a l a r g e amount o f t h i s m a t e r i a l , p r o b a b l y l a r g e r than i n o t h e r i s o t o p i c t y p e s ; t h i s was a l s o true of carbohydrate content. These c e l l s had the most h e a v i l y s t a i n e d and t h e t h i c k e s t w a l l s o f any c e l l type. J

1

6

2

4

H - 0 - C - N Cells. The t r e n d s i n s t a i n i n g i n t h i s c e l l t y p e i n d i c a t e d t h a t i t s h o u l d be p l a c e d i n t e r m e d i a t e between c e l l s o f normal i s o t o p i c c o n t e n t and those of H - 1 0 - C - 1 % , t e n d i n g t o resemble the l a t t e r more than t h e former. N u c l e i appeared t o c o n t a i n more RNA than d i d the I H - I ^ O c o n t r o l . The most s i g n i f i c a n t d i f f e r e n c e between 1^0 and l ^ o - c o n t a i n i n g c e l l s appeared i n t h e n a t u r e o f t h e c a r b o h y d r a t e d i s t r i b u t i o n and content. Those c o n t a i n i n g the heavy oxygen i s o t o p e showed l e s s c l e a r s t a i n i n g o f the c e l l w a l l s . Perhaps the most i m p o r t a n t g e n e r a l i z a t i o n t o app e a r from t h i s study was t h a t i t i s q u i t e o b v i o u s t h a t d e v i a t i o n s from normalcy appear more and more as heavy i s o t o p e s a r e s u b s t i t u t e d i n t o t h e organism. The t e n dency t o form g i a n t c e l l s , some w i t h a volume s e v e r a l o r d e r s o f magnitude g r e a t e r than normal c e l l s , and a suggestion of increasing s u b c e l l u l a r disorganization, i s q u i t e common. The e x a c t c o n t r i b u t i o n o f each heavy i s o t o p e cannot be a s s e s s e d a t p r e s e n t and must await f u r t h e r s t u d i e s on o t h e r organisms and l a r g e s i z e d cultures. The a p p l i c a t i o n s o f m u l t i p l y s u b s t i t u t e d organisms i s a t p r e s e n t l i m i t e d by the h i g h c o s t o f the heavy i s o t o p e s , e s p e c i a l l y 1 0. The even r a r e r 0 may have g r e a t f u t u r e p o t e n t i a l , because o f i t s non-zero n u c l e a r spin. A s i d e from a r e a s o f o b v i o u s a p p l i c a t i o n , such as mass s p e c t r o m e t r y , t h e r e appears t o be g r e a t p o t e n t i a l f o r t h e study o f b i o l o g i c a l problems by means o f m u l t i p l e resonance t e c h n i q u e s as endor ( e l e c t r o n - n u c l e a r doab l e resonance), with the use of multiply substituted organisms. 1 8

1

2

1 3

6

1 5

1 2

8

i 7


9.

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