Observations on the periodide method for the estimation of choline

432

SHORT COMMUNICATIONS

VOL. 20 (Z956)

Observations on the periodide method for the estimation of choline A c c o r d i n g to BEST AND LUCAS~, who h a v e reviewed t h e m e t h o d s available for t h e q u a n t i t a t i v e d e t e r m i n a t i o n of choline, t h e m o s t sensitive microchemical m e t h o d d e p e n d s u p o n precipitation of choline as choline periodide. APPLETON, LA DU, LEVY, STEELE AND BROOm 2 h a v e recently described a procedure, b a s e d on this principle, for t h e d e t e r m i n a t i o n of choline in p l a s m a , b y which 0 . 0 5 - 0 . 5 / , m o l e c a n be accurately e s t i m a t e d . I n this m e t h o d t h e precipitate of choline periodide is dissolved in e t h y l e n e dichloride a n d t h e optical d e n s i t y of t h e solution c o m p a r e d at 365 m # with t h a t of k n o w n a m o u n t s of choline similarly treated. At this w a v e l e n g t h choline periodide s h o w s a s t r o n g absorption band, while interference b y traces of iodine c o n t a m i n a t i n g t h e precipitate is minimal. Possible losses of choline periodide entailed in w a s h i n g t h e precipitate as a necessary p r e l i m i n a r y to t h e chemical e s t i m a t i o n of t h e iodine p r e s e n t in it are t h u s avoided, a n d t h e m e t h o d h a s t h e a d v a n t a g e s of speed a n d simplicity. APPLETON el al. ~ p o i n t o u t h o w e v e r t h a t their procedure, as described for p l a s m a , c a n n o t be applied directly to t h e d e t e r m i n a t i o n of choline in tissue extracts. Nevertheless HAVAISHI A~D KORNSERG3 h a v e a d a p t e d a similar t e c h n i q u e to t h e e s t i m a t i o n of choline released from lecithin b y t h e action of a bacterial e n z y m e . T h e p r e s e n t c o m m u n i c a t i o n deals with a brief i n v e s t i g a t i o n into t h e m e t h o d , w i t h a view to its use for t h e m e a s u r e m e n t of choline released e n z y m i c a l l y from choline-containing p h o s p h o lipids b y tissue preparations. I n p r e l i m i n a r y e x p e r i m e n t s it was f o u n d t h a t precipitation as choline periodide g a v e satisf a c t o r y recovery of choline from a q u e o u s 5 % trichloroacetic acid solutions. F u r t h e r m o r e , chloroform (Hopkin a n d Williams, Analar) was f o u n d to be a s a t i s f a c t o r y s o l v e n t for t h e precipitate, a n d h a s t h e a d v a n t a g e over ethylene dichloride t h a t it c a n easily be o b t a i n e d pure. I n t h e techn i q u e finally adopted, 0. 5 ml s a m p l e s of 5 % (w/v) trichloroacetic acid c o n t a i n i n g suitable a m o u n t s of choline were placed in a finely p o i n t e d centrifuge t u b e a n d 0.2 ml of K I 3 reagent, consisting of 15.7 g of iodine (B.D.H., Analar) a n d 2o g p o t a s s i u m iodide (B.D.H., Analar) m a d e u p to ioo ml in distilled water, was added. After stirring, cooling in ice, centrifuging a n d r e m o v i n g t h e s u p e r n a t a n t fluid as described b y APPLETON et al. z, t h e precipitate was dissolved in Io m l chloroform a n d t h e optical d e n s i t y read in a n ultraviolet S p e c t r o p h o t o m e t e r a t 365 m # . Stable readings were o b t a i n e d over a period of several hours. E x p e r i m e n t s showed t h a t t h e optical densities of such solutions do n o t obey Beer's Law. Molar extinction coefficients ( ~ coefficients of variation) calculated from t h e optical densities obtained for s t a n d a r d s of Io, 20 a n d 4 ° g g of choline chloride in 16 replicate d e t e r m i n a t i o n s were 1.8. zo 4 ( ± 4.4 %), 2.3' IO4 ( ~ 1.8 %), a n d 2.6. Io 4 ( ± 1.3 %) respectively. The optical d e n s i t y of b l a n k s a v e r a g e d only 0.007. T h e s e v a l u e s agree well with t h e d a t a given b y APPLETON et al. 2 a n d b y HAYAZSrll AND KORNBERG 3. W i t h regard to t h e specificity of t h e m e t h o d , it was f o u n d t h a t t h e acetyl, propionyl, b u t y r y l , acetyl-fl-methyl a n d benzoyl esters of choline, a n d also d i m e t h y l a m i n o e t h y l a c e t a t e g a v e precipit a t e s with t h e iodine r e a g e n t u n d e r t h e above conditions. Solutions of t h e s e precipitates in chloroform showed in each case an absorption p e a k at 365 m/~. T h e m o l a r extinction coefficients of t h e choline derivatives did n o t differ significantly from t h a t of t h e iodine c o m p l e x of choline. I m p u r e p r e p a r a t i o n s of ovolecithin a n d h u m a n brain s p h i n g o m y e l i n also f o r m e d insoluble c o m plexes with iodine which, w h e n dissolved in chloroform, a b s o r b e d m a x i m a l l y at 365 m/*. HAvAzsrII AND KORNBERG 3 reported similar findings for lecithin. Several o t h e r s u b s t a n c e s are k n o w n to give precipitates with t h e triiodide r e a g e n t 1. In a g r e e m e n t with APPLETON et al. 2, p u r e solutions of p h o s p h o r y l choline, prepared b y t h e m e t h o d of RZLEY4, a n d glycerophosphoryl choline g a v e no precipitate. Q u a n t i t a t i v e recovery of a d d e d choline was o b t a i n e d from b r a i n h o m o g e n a t e s , a n d from e m u l s i o n s of ovolecithin, after addition of a n e q u a l v o l u m e of IO % trichloroacetic acid, s u g g e s t i n g t h a t precipitation of phospholipids by this c o n c e n t r a t i o n of trichloroacetic acid was essentially complete. E x p e r i m e n t s were carried o u t in which p h o s p h o r y l choline was h y d r o l y s e d by a n acetonedried p r e p a r a t i o n of h u m a n cerebral cortex at p H 8.9 u n d e r t h e conditions described b y STRICKLAND, THOraPSON AND WEESTERs, followed b y e s t i m a t i o n of b o t h free choline a n d inorganic p h o s p h a t e in trichloroacetic acid filtrates of t h e i n c u b a t i o n media. T h e free c h o l i n e : p h o s p h a t e ratio found in these e x p e r i m e n t s a v e r a g e d 1.3o i n s t e a d of t h e theoretical v a l u e of r.o. I n this s y s t e m , however, t h e m o l a r c o n c e n t r a t i o n of u n h y d r o l y s e d p h o s p h o r y l choline at t h e e n d of t h e e x p e r i m e n t was 6 - 7 t i m e s greater t h a n t h a t of t h e free choline. W h e n e x p e r i m e n t s were carried o u t u s i n g s t a n d a r d s containing choline a n d p h o s p h o r y l choline in t h e s e proportions, t h e free c h o l i n e : p h o s p h a t e ratio was reduced to z.o 5. I t appears, therefore, t h a t a l t h o u g h p h o s p h o r y l choline itself does n o t form a n insoluble c o m p l e x with iodine, possibly because of its acidic nature, its presence influences t h e degree of precipitation of t h e iodine c o m p l e x of choline. T h e presence of a relative excess of glycerophosphoryl choline also caused a slight increase in t h e a p p a r e n t a m o u n t of choline e s t i m a t e d . It was concluded from these investigations t h a t p r o v i d e d care is t a k e n to use s t a n d a r d s

VOL. 20 (1956)

PRELIMINARY NOTES

433

c o n t a i n i n g a m o u n t s of p h o s p h o r y l choline a n d g l y c e r o p h o s p h o r y l choline similar to t h o s e in t h e s o l u t i o n s to be a s s a y e d , a n d a p p r o p r i a t e b l a n k s a n d controls, t h e t e c h n i q u e outlined above offers a m e a n s o f e s t i m a t i n g , chemically, a m o u n t s of choline b e t w e e n 0.o5-o. 5/~mole w h i c h could be applied to t h e s t u d y of t h e e n z y m i c b r e a k d o w n of choline-containing p h o s p h o I p i d s b y tissue preparations. T h e a u t h o r is grateful to Professor R. H. S. THOMPSON, in w h o s e l a b o r a t o r y this work w a s carried out, for his i n t e r e s t a n d h e l p f u l advice, to Professor G. PAYLING WRIGHT for t h e s p e c i m e n of h u m a n b r a i n s p h i n g o m y e l i n , a n d to Professor C. S. MCARTHUR of t h e U n i v e r s i t y of S a s k a t c h e w a n for t h e gift of p u r e g l y c e r o p h o s p h o r y l choline. D i m e t h y l a m i n o e t h y l a c e t a t e was p r e p a r e d b y Dr. K. P. STRICKLAND in t h i s l a b o r a t o r y . T h a n k s are also due to t h e Medical R e s e a r c h Council for a g r a n t t o w a r d s t h e e x p e n s e s of t h i s work.

Department o/Chemical Pathology, Guy's Hospital Medical School, London (England)

G. R. WEBSTER

1 C. H. BEsT A N D C. C, L u c A s , Vitamins and Hormones, I (1943) 1. I H . D. APPLETON, B. I~T.LA DU, Jr., B. B, LEVY, J. M. STEELE AND B. B. BRODIE, Jr. Biol. Chem. 2o5 (I953) 803a O. HAYAISHI AND A. KORNBERG, jr. Biol. Chem., 206 (1954) 647. • R. F. RILEY, J. Am. Chem. Sot., 66 (i944) 512. 6 K . P. STRICKLAND, R. a . S. THOMPSON AND G. R. WEBSTER, J. Neurol. Neurosurg. Ps~,chiat., (1956) (in press). Received J a n u a r y 25th, I956

Preliminary Notes

The action of enterokinase on trypsinogen T r y p s i n o g e n is a c t i v a t e d to t r y p s i n either b y t r y p s i n itself or b y e n t e r o k i n a s e z. In t h e former case, a p e p t i d e is s p i t off f r o m t h e a m i n o - e n d of t r y p s i n o g e n , t h e a m o u n t of p e p t i d e split off being proportional to t h e t r y p t i c a c t i v i t y developed as s u g g e s t e d b y ROVERY, FABRE AND DESNUELLE 2 a n d r e c e n t l y confirmed b y DAVIE AND NEURATHs. However, no s t u d i e s h a v e been m a d e of t h e c h e m i c a l c h a n g e s t a k i n g place d u r i n g t h e a c t i v a t i o n b y e n t e r o k i n a s e w h i c h s e e m s to be h i g h l y specific for t r y p s i n o g e n . I n t h i s c o m m u n i c a t i o n , it is s h o w n t h a t isoleucine is t h e N - t e r m i n a l a m i n o acid of t r y p s i n developed f r o m t r y p s i n o g e n b y e n t e r o k i n a s e a c t i v a t i o n . T r y p s i n o g e n w a s p r e p a r e d f r o m beef p a n c r e a s according to t h e m e t h o d of NORTHROP AND KUNXT# t h r o u g h crystallization a n d f u r t h e r purification b y trichloracetic acid. E n t e r o k i n a s e of a h i g h degree of p u r i t y w a s p r e p a r e d f r o m swine d u o d e n a l fluid c o n t e n t s according to t h e a u t h o r ' s methodS, 6. T h e a c t i v a t i o n was carried o u t a t o ° C a n d p H 5.6 u s i n g o.i M t r i e t h y l a m m o n i u m a c e t a t e buffer, 5 m g of t r y p s i n o g e n a n d 0.8 m g of e n t e r o k i n a s e c o n t a i n i n g zooo u n i t s per m g b e i n g dissolved in 12.5 m l of t h e a c t i v a t i o n m i x t u r e . U n d e r t h e s e conditions, 5 0 % a c t i v a t i o n could be o b t a i n e d w i t h i n 8 m i n u t e s . T h e reaction w a s essentially of first order kinetics t h r o u g h o u t t h e whole a c t i v a t i o n w i t h o u t a n y appreciable signs of a u t o a c t i v a t i o n or a u t o d i g e s t i o n d u e to t r y p s i n . T h e a c t i v a t i o n w a s s t o p p e d b y t h e a d d i t i o n of l.O m l of I N HCI a n d aliquots were t a k e n for t h e m e a s u r e m e n t of t r y p t i c a c t i v i t y {hemoglobin m e t h o d ) a n d t h e rest w a s s u b m i t t e d to t h e d e t e r m i n a t i o n on N - t e r m i n a l a m i n o acids. EDMAN'S m e t h o d 7 w a s u s e d for t h i s purpose. T h u s , a n e q u a l v o l u m e of p y r i d i n e c o n t a i n i n g p h e n y l i s o t h i o c y a n a t e in a c o n c e n t r a t i o n of 5o/~l p e r m l w a s a d d e d to t h e a c t i v a t i o n m i x t u r e a n d t h e p H w a s a d j u s t e d to 9.o b y t h e a d d i t i o n of a few d r o p s of t r i e t h y l a m i n e . T h e activities of e n t e r o k i n a ~ a n d of t r y p s i n were a l m o s t i n s t a n t a n e o u s l y blocked d u r i n g t h i s t r e a t m e n t . A f t e r t h e c o m p l e t i o n of t h e coupling, t h e m i x t u r e was w a s h e d b y b e n z e n e a n d t h e a q u e o u s solution w a s t h e n lyophilized. T h e dried m a t e r i a l w a s h y d r o l y z e d b y i N HCI a t I o o ° C for one h o u r a n d t h e p h e n y l ~ h i o h y d a n t o i n d e r i v a t i v e s of a m i n o acids were e x t r a c t e d b y e t h y l acetate. T h e aliquots were