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A comparison of the rate of oxygen uptake and the adenosine triphosphate content of routine and experimental BCG preparations
Journa/of Bio/ogica/Standardization ( 1 9 8 3 ) i 1, 2 0 5 - 2 1 2
A c o m p a r i s o n o f t h e rate o f o x y g e n uptake and the adenosine triphosphate content of routine and experimental BCG preparations*
M.
Borremans, t M .
W e c k x t a n d R ~ VerholCstadtt
T h e A T P c o n t e n t a n d o x y g e n u p t a k e r a t e , t w o p a r a m e t e r s o f v i a b i l i t y o f a BCGsuspension, a r e c o m p a r e d . T h e l a c k o f c o r r e l a t i o n b e t w e e n m e a s u r e m e n t s m a d e on fresh ' r o u t i n e p r e p a r a t i o n s i n d i c a t e s t h a t t h e A T P m e a s u r e m e n t is affe~cted by t h e d e g r e e o f d i s p e r s i 0 n o f t h e p r e p a r a t i o n . Experimental preparations made under standard conditions (constant semi-dry weight and s t e p w i s e d i s p e r s i v e g r i n d i n g ) f r o m c u l t u r e s 0 f t h e s a m e a g e as t h e fresh r 0 u t !he c u l t u r e s ( 14 d a y s ) h a d a s m a l l e r A T P c o n t e n t , w h i c h c o r r e l a t e d well with t h e respiration rate. I n e x p e r i m e n t a l p r e p a r a t i o n s f r o m s e v e n - d a y c u l t u r e s t h i s c o r r e l a t i o n was Significant. B e t w e e n d a y s 7 a n d 14 t h e A T P c o n t e n t d e c l i n e d m u c h m o r e r a p i d l y t h a n t h e r e s p i r a t i o n rate.
INTRODUCTION T h e o n l y t e s t for v i a b i l i t y o f a B C G p r e p a r a t i o n r e q u i r e d b y t h e W H O is t h e e n u m e r a t i o n o f t h e c o l o n y f o r m i n g u n i t s ( C F U ) . E v e n so, m o s t p r o d u c e r s u s e s o m e o t h e r v i a b i l i t y test s u c h as t h e o x y g e n u p t a k e r a t e ( O U R ) o r t h e A T P c o n t e n t t o evaluate their BCG preparations. B o t h t h e s e rests h a v e b e e n c o m p a r e d w i t h t h e C F U c o u n t b y several a u t h o r s . L a m e n s a n s & B r e r e y i a n d Milinkovid & Vlajini42 f o u n d a g o o d c o r r e l a t i o n (r = 0 - 8 3 ) b e t w e e n CFLI a n d O U R d e t e r m i n e d b y t h e XVarburg m e t h o d . G o o d c o r r e l a t i o n s b e t w e e n CFLI a n d A T P c o n t e n t w e r e f o u n d b y S u p L e e 3 a n d : b y G h e o r g h i u & L a g r a n d e r i e 4 : r ~-- 0 - 9 8 3 : a n d 0 - 8 8 3 r e s p e c t i v e l y : A l t h o u g h O U R a n d A T P C o n t e n t a r e ° R e c e i v e d for p u b l i c a t i o n ! 1 N o v e m b e r 1982. t B C G P r o d u c i n g L a b o r a t o r y , l n s t i t u u t : P a s t e u r v a n B r a b a n t . Brussels. B e l g i u m . 0 0 9 2 - 1 1 5 7 / 8 3 / 0 3 0 2 0 5 + 08 $03- 00/0
~ ) 1983 The International Association o f Biological Standardization
205
/~!. B O R R E M A N S
ETAL.
b o t h r a p i d m e t h o d s for p r e d i c t i n g t h e v i a b i l i t y o f B C G v a c c i n e , t h e y a p p e a r to be i n f l u e n c e d by t h e d e g r e e o f h o m o g e n e i t y o f t h e B C G s u s p e n s i o n . G h e o r g h i u 6: L a g r a n d e r i e 4 e x t r a c t e d m o r e A T P (2-4 fg o f A T P p e r v i a b l e u n i t ) f r o m B C G v a c c i n e for s c a r i f i c a t i o n ( c o n t a i n i n g l a r g e r c l u m p s ) t h a n f r o m v a c c i n e for i n t r a d e r m a l i n j e c t i o n ( c o n t a i n i n g s m a l l e r c l u m p s ) . A c c o r d i n g l y r e s p i r a t i o n is l p o s i t i v e l y i n f l u e n c e d by a d e c r e a s i n g d e g r e e o f d i s p e r s i o n in t h e B C G s u s p e n s i o n s . T h e m a i n p u r p o s e o f t h i s w o r k was to s t u d y t h e effect o f t h e h o m o g e n e i t y o f B C G s u s p e n s i o n s o n dae r e l a t i o n s h i p b e t w e e n O U R m e a s u r e d by t h e O x y g r a p h m e t h o d a n d A T P c o n t e n t . T h e B C G vaccines e x a m i n e d w e r e r o u t ! n e p r e p a r a t i o n s u s e d in a c l i n i c a l trial o f i m m u n o t h e r a p y a n d e x p e r i m e n t a l p r e p a r a t i o n s o f t h e s a m e ( G L 2 ) s t r a i n b u t o f d i f f e r e n t ages. MATERIALS
AND
METHODS
Cultit'ation of bacteria T h e G L 2 s t r a i n , u s e d for e x p e r i m e n t a l as well as for r o u t i n e p r e p a r a t i o n s , is a v a c c i n e b a t c h t h a t was f r e e z e - d r i e d in s o d i u m g l u t a m a t e in 1974. It is d e r i v e d , via a b a t c h t h a t was f r e e z e - d r i e d in lactose in 1 9 7 0 , f r o m t h e B C G s t r a i n t h a t t h e I n s t i t u u t P a s t e u r van B r a b a n t o b t a i n e d f r o m C a l m e t t e in 1929. T h e c u l t u r e s w e r e g r o w n as surface pellicles o n S a u t o n m e d i u m at 3 7 - 5 + 0 " 5 ° C .
Routine preparations E i g h t 1 4 - d a y - o l d cul t u r e s w e r e passed o v e r a C a l m e r t e a p p a r a t u s . T h e , b a c t e r i a l m a s s w a s p l a c e d i n a f l a t - b o t t o m e d g l a s s f l a s k (25 c m in d i a m e t e r ) c o n t a i n i n g 1-4 k g o f s t a i n l e s s steel b e a d s ( 4 m r n in d i a m e t e r ) a n d 25 m l o f S a u t o n m e d i u m ( d i l u t e d 1/5) a n d h o m o g e n i z e d o n a B C G m i l l ( O l e D i c h , H v i d o v r e , D e n m a r k ) for 10 rain at 15 r rain - I . T h e h o r n o g e n a t e was d i l u t e d w i t h 2 2 5 rnl o f S a u t o n m e d i u m (1/5) a n d s e p a r a t e d f r o m t h e beads o n a s t a i n l e s s steel f u n n e l . A f a r t h e r 2 5 0 nal o f m e d i u m was a d d e d to o b t a i n a final v o l u m e o f 5 0 0 m l o f B C G s u s p e n s i o n c o n t a i n i n g at least 8 0 nag o f B C G m l - * , ready for filling in a r n p o u l e s . T h e m o i s t ( s e m i - d r y ) w e i g h t was d e t e r m i n e d b y c e n t r i f u g a t i o n at 3 5 0 0 r r n i n - l for 10 main.
t:_a-perimental preparations F o u r c u l t u r e s , seven or 14 days 01d, w e r e filtered o n a B r e t e y a p p a r a t u s a n d c e n t r i f u g e d f o r 4 0 s at a m a x i m u m o f 2 6 0 0 r r a i n " 1 in a J o u a n c e n t r i f u g e to r e m o v e t h e c u l t u r e l i q u i d . O n l y 5 g ( m o i s t w e i g h t ) o f t h e bacterial m a s s was t r a n s f e r r e d at a x i m e to a flask c o n t a i n i n g s t a i o l e s s steel b e a d s ( I k g o f Zt n a n in d i a m e t e r a n d 1 k g o f 6 tuna in d i a m e t e r ) a n d g r o u n d , s t e p w i s e , on t h e B C G m i l l at 15 r r a i n - l : 30 s d r y m i x i n g , 1 mira w i t h 5 m l o f S a u t o n m e d i u m a n d 1 r a i n w i t h an a d d i t i o n a l 15 m l o f S a u t o n m e d i u m . The- h o m o , e n a t e w a s s e p a r a t e d f r o m t h e beads a n d f u r t h e r d i l u t e d to e x a c t l y 5 nag o f B C G rnl " ~ b y r i n s i n g t h e be-ads w i t h a n a p p r o p r i a t e v o l u m e o f S a u t o n m e d i u m . T h e r e w a s rio f u r t h e r w e i g h t d e t e r m i n a t i o n .
A T P extraction a n d determination o
5
E x t r a c t i o n o f A T P f o l l o w e d t h e m e t h o d o r i g i n a l l y d e s c r i b e d b y T h o r e & A n s 6 h n for t h e d e t e c t i o n o f b a c t e r i u r i a . I n t o 4 - 9 m l o f b o i l i n g T r i s - E D T A b u f f e r ( 2 0 rnM T r i s , 206
02 LIPTAK]~ AND ATP CONT/EbdT OF BCG PREPARATIONS 2 mM E D T A ; p H 7 - 7 5 ) a 100 ltl B C G s a m p l e o f e i t h e r p r e p a r a t i o n ( c o n t a i n i n g 1 m g o f e x t r a c t e d B C G ) was r a p i d l y i n j e c t e d a n d e x t r a c t e d for 5 r a i n (for s e v e n - d a y B C G ) or for 10 rain (for 14-day B C G ) . T h e e x t r a c t i o n was s t o p p e d by a rapid t r a n s f e r to an ice b a t h . A f t e r c o o l i n g , 50 g/l o f e x t r a c t was m i x e d w i t h an a d e q u a t e a m o u n t o f l u c i f e r i n e luciferase r e a c t i o n m i x t u r e ( A T P d e p e n d e n t firefly b i o l u m i n e s c e n t reaction s y s t e m ; M c E l r o y 6) a n d the l i g h t e m i s s i o n m e a s u r e d wi'th a J R B A T P p h o t o m e t e r (SAI T e c h n o l o g y ) as t h e i n t e g r a t e d value o f / ) , , the i n t e n s i t y o f t h e l i g h t e m i t t e d by t h e s a m p l e d u r i n g 6 0 s a f t e r 15 s m i x i n g t i m e . T o t a k e i n t o a c c o u n t the possible effect o f i n r e r f e r i n g c o m p o n e n t s on t h e b i o l u m i n e s cent reaction, the constant addition techniqueS,in which a known a m o u n t of a standard A T P s o l u t i o n is a d d e d to t h e u n k n o w n s a m p l e w,as used. T h e A T P c o n t e n t o f t h e s a m p l e was c a l c u l a t e d f r o m t h e c o u n t p r o v i d e d by t h e s a m p l e w i t h s t a n d a r d A T P Jless t h e c o u n t for t h e s t a n d a r d alone. D u r i n g t h e preparat,/on of;the s u s p e n s i o n s o m e A T P was released into t h e m e d i u m , t h e a m o u n t o f w h i c h was c o u n t e d in the S N F f r o m an u n e x t r a c t e d s a m p l e . T h e i n t r a c e l l u l a r A T P c o n t e n t (of ~I m g o f B C G ) w a s c a l c u l a t e d f r o m t h e total A T P c o n t e n t o f t h e s a m p l e less this free A T P . P r e l i m i n a r y trials to e x t r a c t t h e A T P b y n u c l e o t i d e releasing r e a g e n t for bacterial cells ( N R B ) as s u g g e s t e d by L u m a c S y s t e m A G 4 r e s u l t e d in i n c o m p l e t e e x t r a c t i o n . F i n a l l y T r i s - E D T A e x t r a c t i o n was c h o s e n after a c o m p a r i s o n w i t h c h l o r o f o r m extraction, 7 both giving the same a m o u n t o f extracted ATP.
Oxygen uptake rate T h e O U R was m e a s u r e d by a G i l s o n O x y g r a p h ( G i l s o n M e d i c a l E l e c t r o n i c s , M i d d l e t o n , W i s c o n s i n ) b a s e d on t h e p o l a r o g r a p h i c p r i n c i p l e , a c c o r d i n g to t h e m e ~ h o d o f van H e m e r t & T i e s j e m a . 8 For each d e t e r m i n a t i o n B C G was d i l u t e d in S a u t o n m e d i u m w i t h 0 - 3 % p o l y s o r b a t e 8 0 to exactly 6 m g m1-1 9
Statistics M o i s t w e i g h t, A T P c o n t e n t a n d O U R o f B C G p r e p a r a t i o n s w e r e c o n s i d e r e d . a s variables o f a n o r m a l d i s t r i b u t i o n d e s c r i b e d by t h e m e a n a n d s t a n d a r d d e v i a t i o n o f this mean (SD). For t h e statistica] e v a l u a t i o n o f t h e association b e t w e e n A T P c o n t e n t a n d O U R . t h e s a m p l e c o r r e l a t i o n coefficient (r) was c a l c u l a t e d , l° RESULTS T h e m o i s t w e i g h t s o f 14 r o u t i n e p r e p a r a t i o n s (Table 1) w e r e s o m e w h a t g r e a t e r t h a n t h e p r e d i c t e d 8 0 m g m l - l a n d r a n g e d f r o m 9 0 to 143 m g m l - 1 T h e c a l c u l a t e d w e i g h t s o f e i g h t cultures homogenized at t h e s a m e t i m e varied f r o m 45 to 7 1 . 5 g per flask. N o s i g n i f i c a n t d i f f e r e n c e in A T P c o n t e n t ( 9 - 0 3 4 - 2 . 3 0 n g m g - l ) n o r in O U R ( 1-55 ± 0 - 4 2 /AI O z h - 1 r a g - l) was f o u n d w i t h i n this g r o u p . In s p i t e o f this t h e r e was p o o r c o r r e l a t i o n b e t w e e n A T P c o n t e n t a n d O U R (r ~ 0"33)T h i s lack o f c o r r e l a t i o n lead to m e a s u r e m e n t s o n a set o f e x p e r i m e n t a l p r e p a r a t i o n s f r o m c u l t u r e s o f t h e s a m e a g e ( 14 days) f r o m w h i c h c o n s t a n t w e i g h t s (5 g ) o f B C G cells w e r e h o m o g e n i z e d a n d f u r t h e r t r e a t e d . T o t a k e i n t o a c c o u n t t h e possible i n f l u e n c e o f the a g e o f t h e c u l t u r e s a n d t h u s t h e i r m e t a b o l i c s t a t e u p o n t h e results, A T P c o n t e n t a n d O U R w e r e m e a s u r e d i n p r e p a r a t i o n s o f s e v e n , d a y c u l t u r e s also. A g o o d c o r r e l a t i o n b e t w e e n A T P c o n t e n t a n d O U R was f o u n d in 14-day c u l t u r e s : 207
M. B O R R E M A N S E T A L . TABLF 1.
Pr~xtucrion lot no.
Comparison of moist (semi-dry) w e i g h t , A T P c o n t e n t and O U R in routine preparations o f 14-day BCG Moist w e i g h t (rag m l - )
ATP content (ng ml " l )
OUR (.ttl 0 2 h - l m g
81K09 81K16 81K30 81L07 8ILI4 81L21 82A I l 82A18 82A25 82B01 82B08 82C01 82C08 82D05
103 109 90 102 115 113 119 143 114 119 113 118 109 102
8-37 7" 10 6-40 7"80 8"50 10-24 9"75 7-81 9-31 14-44 13-10 9" 12 6-90 7-56
t "21 1-02 1-41 I" 54 1-51 1-97 1"4 1 2- 19 0-96 1.75 2- 13 1 "30 1- 16 2- 13
Mean SD
1 l 2-07 12-07
9"03 2"3
1-55 0-42
-I)
r ~ coefficient of correlation between ATP content and OUR m 0"33; not significant. r = 0 " 8 6 , s i g n i f i c a n t a t t h e 1% level [ T a b l e 2, F i g . I(a)] as w e l l as in t h e s e v e n - d a y c u l t u r e s : r = 0 " 8 4 , s i g n i f i c a n t a t t h e 0 - 1 % level [ T a b l e 3 , F i g . 1(b)]. A n i n t e r e s t i n g f e a t u r e was t h a t t h e 1 4 - d a y c u l t u r e s w e r e i n o c u l a t e d s i m u l t a n e o u s l y w i t h t h e s e v e n - d a y c u l t u r e s a n d so w e r e i n c u b a t e d o n l y a w e e k l o n g e r . O n e m a y t h u s c o n c l u d e t h a t t h e A T P c o n t e n t d i m i n i s h e d f r o m a m e a n v a l u e o f 6 2 - 9 8 ± 2 4 - 14 n g o f TABLE 2.
C o m p a r i s o n o f A T P c o n t e n t a n d O U R in e x p e r i m e n t a l preparations o f 14-day B C G
Preparation no.
ATP content (ng m g - t )
1 2 3 4 5 6 7 8
7-80 7-75 6-09 6.80 4-52 3;84 2-37 4-60
1-44 1-42 1-44 1-46 1-35 I- 18 1-03 1,12
Mean SD
5"47 1"95
1 "31 0-17
Sample correLation coefficient r ----- 0-86; sigr~ificanr at the 19~- lev, l.
208
OUR (~AI 0 2 h - ! m g
-t)
02 U P T A K E A N D A T P C O N T E N T
-
OF BCG PREPARATIONS
(a')
1.4
~ I-2~ 7 ..¢:
1.0'] I I I ..... ! t t I.. 0 t 2 3 4 5 6 7 S ............................. :. . . . . . . . . . . .
8 : 9
I0
-~ 6 ~- (b) g:
5'
I"
~
Q
i
e
t
I lI l
00
I
I(~
tO
1
20
I
30:40
J
I
50 ATP
I,
I
60 (ng
70
t
I
I
I
BO
90
I00
ItO
mg-I)
Fig. 1. Scatter d!agram of the relationship between A T P content and O U R in experimental prepara tions of (a) 14-day BCG and (b)seven-day BCG. , Regression of A T P content upon OUR; • regression of O U R upon ATP content. Correlation coefficient r = 0"86(a), r = 0-84(b), significant at (a) the 1e.6 and (b) "the 0- l e a levels.
TABLE 3.
C o m p a r i s o n o f A T P c o n t e n t a n d O U R in e x p e r i m e n t a l p r e parations of seven-day BCG ATP content
OUR
(ng mg -1)
( p l 0 2 h - 1 I'ng - I )
2 3 4 5 6 7 8 9 I0 1l 12 13
43- 15 51-50 56-67 68-27 48-03 90-90 100:25 36"90 45-74 83- 1 l 49-84 106-00 38-40
2.5 3-78 3-62 4-48 4-22 5-01 4"26 Z-7 1 2"5.1 4-86 3'77 5;55 3-40
Mean SD
62-98 24.14
3,9 0-96
Preparation
1
no.
Sample correlation coefficient r ---- 0-84, significant at 0- IC~, level.
209
M. B O R R E M A N S
ET AL.
A T P m g -1 of BCG at seven days to 5-47 ---+ 1.15 ng o f A T P m g -1 B C G at 14 days, .
.
.
.
3"0. The average A T P c o n t e n t of the routine preparations (9"03 ± 2"3 ng o f A T P m g - 1 BCG) was h i g h e r than that of the 14-day experimental preparations (5"47 + t" 15 ng of A T P m g - l BCG), a l t h o u g h their O U R did not differ. DISCUSSION Each o f the 14 r o u t i n e preparations was prepared under different conditions. T h e bacterial mass which was h o m o g e n i z e d was a different w e i g h t in the c~se of each preparation (from 45 to 71"5 g per e i g h t cultures). The variable a m o u n t of residual l i q u i d t o g e t h e r w i t h the variable bulk of bacteria gave rise t o different degrees o f dispersion after h o m o g e n i z a t i o n . Additionally, the steel beads, responsible for rather r o u g h m i x i n g , may kill bacteria. Larnensans & B r e t e y l d e m o n s t r a t e d that this effect was inversely related to the bacterial mass: t h e g r e a t e r the mass, the fewer bacteria were killed d u r i n g h o m o g e n i z a t i o n . T h e greater A T P yield from the routine preparations (9-03 ± 2-3 ng o f A T P m g - I BCG) (Table 1) c o m p a r e d with that of the 14,day experimental preparations (5-47 ± I ' 9 5 ng of A T P r a g - l BCG) (Table 2) confirmed this protection from k i l l i n g w i t h i n large bacterial clumps. ~ O n t h e other hand c h a n g i n g the h o m o g e n i z a t i o n conditions did not influence the O U R m e a s u r e m e n t ; the respiration rate of both preparations may be considered to be equal: 1-55 ~1 of O2 per h per m g (Table 1) and 1-31/~1 of O2 h - 1 r a g - l (Table 2, last column). This brings us to the p r o b l e m raised by Chappelle e t a / . l ~. 12 w h o were aware that any m e t h o d o f q u a n t i f y i n g bacteria based on the m e a s u r e m e n t of t h e intracellular concentration of a particular m e t a b o l i t e did not necessarily distinguish viable cells from dead cells. Periodic m e a s u r e m e n t o f A T P and C F U d u r i n g 15 days g r o w t h o f an E s c h e r i c h i a co/i culture were welt correlated b u t it was shown, too, that A T P levels were s'asrained and measured w i t h i n those cells chat were still intact but metaboIically inactive. T h e lack of correlation between A T P c o n t e n t and O U R in the routine preparations (Table 1) m a y be d u e to the more g e n t l e h o m o g e n i z a t i o n that renders s o m e of the bacteria in t h e c l u m p s metabolically inactive but leaves t h e m morphologically intact. W h e n the A T P content is d e t e r m i n e d i m m e d i a t e l y after preparation the A T P w i t h i n such cells is measured a l t h o u g h their respiration is zero. Accordingly, rougher h o m o g e n i z a t i o n (for t h e seven- and 14-day experimental preparations) breaks the c l u m p s and releases A T P from a certain proportion of the bacteria into t h e m e d i u m g i v i n g a smaller intracellular A T P yield (5"47 ~ 1.95 ng o f A T P r a g - l o f BCG) (Table 2), w h i c h correlates well w i t h the O U R of the B C G suspension (r -~ 0"86) [Fig. l(a)]. F i n d i n g a good statistical correlation between A T P content and O U R in both sets o f experimental preparations (r ~ ' 0"83 for seven days and r -~--0 - 8 6 for 14 days) (Fig- 1) indicates t h e possibility o f p r o d u c i n g several vaccines w i t h comparable m e t a b o l i c activity. In fact the statistical correlation found b e t w e e n A T P c o n t e n t and O U R can be explained as a metabolic correlation. O x y g e n and A T P are related to each other in the respiratory chain phosphorylation. I3 In this pathway, in which oxygen acts as an u l t i m a t e electron acceptor for the oxidation o f a substrate, A T P is formed. This phosphorylation is n o t the only metabolic pathway leading to the formation o f A T P and 210
02 UPTAKE A N D ATP C O N T E N T OF BC~3 PREPARATIONS A T P is c o n s u m e d in several b i o s y n t h e t i c reactions. *~ T h e r e f o r e t h e statistical c o r r e l a t i o n b e t w e e n A T P c o n t e n t a n d O U R p r o b a b l y c a n n o t be i m p r o v e d . T h e lack o f a c o m p l e t e c o r r e l a t i o n o n l y p a r t l y e x p l a i n s t h e d i f f e r e n t d e c l i n i n g rates o f A T P a n d O U R ( 1 1' 5 t i m e s a n d 3 - 0 t i m e s respectively). A full e x p l a n a t i o n is p r o b a b l y g i v e n b y analysis o f t h e g r o w t h c u r v e o f B C G . A c c o r d i n g to S u p Lee 3 B C G in d e e p c u l t u r e ceases to g r o w h e x p o n e n t i a l l y a t d a y n i n e , g i v i n g m a x i m u m A T P c o n t e n t a n d d r y w e i g h t pe/" m i l l i l i t r e . G r o w t h then enters t h e declining p h a s e u n t i l d a y I 3 , associated w i t h a c o n s t a n t d r y w e i g h t b u t w i t h d e c l i n i n g A T P c o n t e n t . T h e r e s p i r a t i o n rate, h o w e v e r , reaches a m a x i m u m a f t e r seven d a y s in c u l t u r e s g r o w n as surface pellicles 1 or a f t e r five to six days in d e e p c u l t u r e . 9 A l t h o u g h it m a y be a s s u m e d t h a t for e a c h g r o w t h e x p e r i m e n t m e n t i o n e d a d i f f e r e n t B C G s t r a i n was e m p l o y e d , t h i s c o u l d i n d i c a t e t h a t A T P c o n c e n t r a t i o n a n d r e s p i r a t i o n rate d o n o t r e a c h m a x i m a o n t h e s a m e day. T h e g r o w t h c u r v e o f t h e G L 2 strain o f B C G has not b e e n d e t e r m i n e d . I f t h e r e s p i r a t i o n rate h a s n o t a t t a i n e d its m a x i m u m after seven days o f g r o w t h w h i l s t A T P c o n c e n t r a t i o n has, it is o b v i o u s t h a t s i m p l y c a l c u l a t i n g t h e r e s p i r a t i o n ratio or t h e A T P c o n t e n t ratio b e t w e e n seven- a n d 1 4 - d a y s u s p e n s i o n s does n o t reflect t h e exact d e c l i n e . Also it m u s t n o t be f o r g o r t e n t h a t t h e m e a s u r e m e n t s w e r e d o n e on B C G p r e p a r a t i o n s r a t h e r t h a n o n u n t r e a t e d cells, s a m p l e d f r o m c u l t u r e s . R e s p i r a t i o n rate a n d A T P c o n t e n t m e a s u r e d in a B C G s u s p e n s i o n p r e p a r e d u n d e r s t a n d a r d c o n d i t i o n s b o t h g i v e a first i m p r e s s i o n o f t h e v i a b i l i t y o f t h e p r e p a r a t i o n . N e v e r t h e l e s s O U R m e a s u r e d w i t h t h e G i l s o n o x y g r a p h offers s o m e a d v a n t a g e . T h e m e a s u r e m e n t can be d o n e o n a s a m p l e as s u c h a n d is n o t i n f l u e n c e d b y t h e d e g r e e o f d i s p e r s i o n o f b a c t e r i a as t h e s a m p l e is constantly mixed.
Acknowledgements The a u t h o r s t h a n k / ~ l r A. Vanonckelen and M r Marlier for technical assistance. They are indebted also to Professor Dr D. D e k e g e l for his objective judgements.
REFERENCES 1. Lamensans A, B r e t e y J . Sur une m6thode de contr61e rapide des prdparations de BCG. Ann Inst Pasteur, 1967; i 12: 342--357. 2. Milinkovi6 M , Vlajini6 S. Relationship between oxygen consumption and colony count test. Sym Ser I m m u n b i o l Stand 1971; 17: 2 2 7 - 2 3 2 ; 3. Sup Lee. M e a s u r e m e n t of viability of BCG vaccine by analysis of A T P content. In: Borun GA, ed. A T P Methodology Seminar; San Diego: SAI Technology Co, 1975: 3 1 6 - 3 3 9 . 4. Gheorghiu M, Lagranderie M. Mesure rapide de la viabilit~ du BCG par dosage d'ATP. Ann Microbiol 1979; 130: 147--156. 5. Thore A, Ans6hn A, L u n d i n A, Bergman S. Detection ofbacteriuria by luci~erase assay of adenosine triphosphate. J Clin Microbiol 1975; 1: 1-8. 6. McElroy W D . Properties of the reaction utilizing adenosinetriphosphate for bioluminescence. J. BioI C h e m 1951; 191: 5 4 7 - 5 5 7 . 7. Dhople AIM, H a n k s j H . Quantitative extraction o f adenosine triphosphate from cultivable and host-grown microbes: calculation o f adenosine triphosphate pools. Appl Microbiol 1973; 26: 399--4038. van H e m e r t PA, Tiesjema R H . Possible use o f the oxygen uptake rate in the evaluation of BCG vaccines. J Biol Stand 1977; 5: 121-129. 21:1
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9. van den Hetavel N, Tiesjema R H , van H e m e r t PA. Optimization measurements o f oxygen uptake rate o f BCG with the Gilson oxygraph. Antonie van I.eeuwenhoek. J . Microbiol Serol 1981; 47: 185- 186. 10. Snedecor GXV. Statistical m e t h ~ s . Ames: The Iowa Stare College Press, 1 9 5 6 : 1 6 1 - - ! 9 3 . I ~1. Chappelle E\X/, Picciolo GL, Okrend H , "l~homas R R , Deming J w , Nibley DA. Significance o f luminescence assays for characterizing bacteria. In: Borun GA, ed. Second bi-annual ATP/~lethoclology Symposium. San Diego: SAI Technolo~gy Co, 1 9 7 7 : 6 1 1 629. 12. Chappelle E\W, Picciolo GtL, Oeming J\~'. Determination of bacterial content in fluids. Meth Enzymol 1978; 37: 6 5 - 7 2 . 13. Stouthamer A H . Patterns in progress. Yield studies in microorganisms. England: Meadowfield Press, 1976. 14. Chapman A, Atkinson D. Adenosine nucleotide concentration and turnover rates. Their correlation with biological activity in bacteria and yeasts. Ad'¢ Microbiol Physiol 1977; 15: 253-306.
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A c o m p a r i s o n o f t h e rate o f o x y g e n uptake and the adenosine triphosphate content of routine and experimental BCG preparations*
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Borremans, t M .
W e c k x t a n d R ~ VerholCstadtt
T h e A T P c o n t e n t a n d o x y g e n u p t a k e r a t e , t w o p a r a m e t e r s o f v i a b i l i t y o f a BCGsuspension, a r e c o m p a r e d . T h e l a c k o f c o r r e l a t i o n b e t w e e n m e a s u r e m e n t s m a d e on fresh ' r o u t i n e p r e p a r a t i o n s i n d i c a t e s t h a t t h e A T P m e a s u r e m e n t is affe~cted by t h e d e g r e e o f d i s p e r s i 0 n o f t h e p r e p a r a t i o n . Experimental preparations made under standard conditions (constant semi-dry weight and s t e p w i s e d i s p e r s i v e g r i n d i n g ) f r o m c u l t u r e s 0 f t h e s a m e a g e as t h e fresh r 0 u t !he c u l t u r e s ( 14 d a y s ) h a d a s m a l l e r A T P c o n t e n t , w h i c h c o r r e l a t e d well with t h e respiration rate. I n e x p e r i m e n t a l p r e p a r a t i o n s f r o m s e v e n - d a y c u l t u r e s t h i s c o r r e l a t i o n was Significant. B e t w e e n d a y s 7 a n d 14 t h e A T P c o n t e n t d e c l i n e d m u c h m o r e r a p i d l y t h a n t h e r e s p i r a t i o n rate.
INTRODUCTION T h e o n l y t e s t for v i a b i l i t y o f a B C G p r e p a r a t i o n r e q u i r e d b y t h e W H O is t h e e n u m e r a t i o n o f t h e c o l o n y f o r m i n g u n i t s ( C F U ) . E v e n so, m o s t p r o d u c e r s u s e s o m e o t h e r v i a b i l i t y test s u c h as t h e o x y g e n u p t a k e r a t e ( O U R ) o r t h e A T P c o n t e n t t o evaluate their BCG preparations. B o t h t h e s e rests h a v e b e e n c o m p a r e d w i t h t h e C F U c o u n t b y several a u t h o r s . L a m e n s a n s & B r e r e y i a n d Milinkovid & Vlajini42 f o u n d a g o o d c o r r e l a t i o n (r = 0 - 8 3 ) b e t w e e n CFLI a n d O U R d e t e r m i n e d b y t h e XVarburg m e t h o d . G o o d c o r r e l a t i o n s b e t w e e n CFLI a n d A T P c o n t e n t w e r e f o u n d b y S u p L e e 3 a n d : b y G h e o r g h i u & L a g r a n d e r i e 4 : r ~-- 0 - 9 8 3 : a n d 0 - 8 8 3 r e s p e c t i v e l y : A l t h o u g h O U R a n d A T P C o n t e n t a r e ° R e c e i v e d for p u b l i c a t i o n ! 1 N o v e m b e r 1982. t B C G P r o d u c i n g L a b o r a t o r y , l n s t i t u u t : P a s t e u r v a n B r a b a n t . Brussels. B e l g i u m . 0 0 9 2 - 1 1 5 7 / 8 3 / 0 3 0 2 0 5 + 08 $03- 00/0
~ ) 1983 The International Association o f Biological Standardization
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b o t h r a p i d m e t h o d s for p r e d i c t i n g t h e v i a b i l i t y o f B C G v a c c i n e , t h e y a p p e a r to be i n f l u e n c e d by t h e d e g r e e o f h o m o g e n e i t y o f t h e B C G s u s p e n s i o n . G h e o r g h i u 6: L a g r a n d e r i e 4 e x t r a c t e d m o r e A T P (2-4 fg o f A T P p e r v i a b l e u n i t ) f r o m B C G v a c c i n e for s c a r i f i c a t i o n ( c o n t a i n i n g l a r g e r c l u m p s ) t h a n f r o m v a c c i n e for i n t r a d e r m a l i n j e c t i o n ( c o n t a i n i n g s m a l l e r c l u m p s ) . A c c o r d i n g l y r e s p i r a t i o n is l p o s i t i v e l y i n f l u e n c e d by a d e c r e a s i n g d e g r e e o f d i s p e r s i o n in t h e B C G s u s p e n s i o n s . T h e m a i n p u r p o s e o f t h i s w o r k was to s t u d y t h e effect o f t h e h o m o g e n e i t y o f B C G s u s p e n s i o n s o n dae r e l a t i o n s h i p b e t w e e n O U R m e a s u r e d by t h e O x y g r a p h m e t h o d a n d A T P c o n t e n t . T h e B C G vaccines e x a m i n e d w e r e r o u t ! n e p r e p a r a t i o n s u s e d in a c l i n i c a l trial o f i m m u n o t h e r a p y a n d e x p e r i m e n t a l p r e p a r a t i o n s o f t h e s a m e ( G L 2 ) s t r a i n b u t o f d i f f e r e n t ages. MATERIALS
AND
METHODS
Cultit'ation of bacteria T h e G L 2 s t r a i n , u s e d for e x p e r i m e n t a l as well as for r o u t i n e p r e p a r a t i o n s , is a v a c c i n e b a t c h t h a t was f r e e z e - d r i e d in s o d i u m g l u t a m a t e in 1974. It is d e r i v e d , via a b a t c h t h a t was f r e e z e - d r i e d in lactose in 1 9 7 0 , f r o m t h e B C G s t r a i n t h a t t h e I n s t i t u u t P a s t e u r van B r a b a n t o b t a i n e d f r o m C a l m e t t e in 1929. T h e c u l t u r e s w e r e g r o w n as surface pellicles o n S a u t o n m e d i u m at 3 7 - 5 + 0 " 5 ° C .
Routine preparations E i g h t 1 4 - d a y - o l d cul t u r e s w e r e passed o v e r a C a l m e r t e a p p a r a t u s . T h e , b a c t e r i a l m a s s w a s p l a c e d i n a f l a t - b o t t o m e d g l a s s f l a s k (25 c m in d i a m e t e r ) c o n t a i n i n g 1-4 k g o f s t a i n l e s s steel b e a d s ( 4 m r n in d i a m e t e r ) a n d 25 m l o f S a u t o n m e d i u m ( d i l u t e d 1/5) a n d h o m o g e n i z e d o n a B C G m i l l ( O l e D i c h , H v i d o v r e , D e n m a r k ) for 10 rain at 15 r rain - I . T h e h o r n o g e n a t e was d i l u t e d w i t h 2 2 5 rnl o f S a u t o n m e d i u m (1/5) a n d s e p a r a t e d f r o m t h e beads o n a s t a i n l e s s steel f u n n e l . A f a r t h e r 2 5 0 nal o f m e d i u m was a d d e d to o b t a i n a final v o l u m e o f 5 0 0 m l o f B C G s u s p e n s i o n c o n t a i n i n g at least 8 0 nag o f B C G m l - * , ready for filling in a r n p o u l e s . T h e m o i s t ( s e m i - d r y ) w e i g h t was d e t e r m i n e d b y c e n t r i f u g a t i o n at 3 5 0 0 r r n i n - l for 10 main.
t:_a-perimental preparations F o u r c u l t u r e s , seven or 14 days 01d, w e r e filtered o n a B r e t e y a p p a r a t u s a n d c e n t r i f u g e d f o r 4 0 s at a m a x i m u m o f 2 6 0 0 r r a i n " 1 in a J o u a n c e n t r i f u g e to r e m o v e t h e c u l t u r e l i q u i d . O n l y 5 g ( m o i s t w e i g h t ) o f t h e bacterial m a s s was t r a n s f e r r e d at a x i m e to a flask c o n t a i n i n g s t a i o l e s s steel b e a d s ( I k g o f Zt n a n in d i a m e t e r a n d 1 k g o f 6 tuna in d i a m e t e r ) a n d g r o u n d , s t e p w i s e , on t h e B C G m i l l at 15 r r a i n - l : 30 s d r y m i x i n g , 1 mira w i t h 5 m l o f S a u t o n m e d i u m a n d 1 r a i n w i t h an a d d i t i o n a l 15 m l o f S a u t o n m e d i u m . The- h o m o , e n a t e w a s s e p a r a t e d f r o m t h e beads a n d f u r t h e r d i l u t e d to e x a c t l y 5 nag o f B C G rnl " ~ b y r i n s i n g t h e be-ads w i t h a n a p p r o p r i a t e v o l u m e o f S a u t o n m e d i u m . T h e r e w a s rio f u r t h e r w e i g h t d e t e r m i n a t i o n .
A T P extraction a n d determination o
5
E x t r a c t i o n o f A T P f o l l o w e d t h e m e t h o d o r i g i n a l l y d e s c r i b e d b y T h o r e & A n s 6 h n for t h e d e t e c t i o n o f b a c t e r i u r i a . I n t o 4 - 9 m l o f b o i l i n g T r i s - E D T A b u f f e r ( 2 0 rnM T r i s , 206
02 LIPTAK]~ AND ATP CONT/EbdT OF BCG PREPARATIONS 2 mM E D T A ; p H 7 - 7 5 ) a 100 ltl B C G s a m p l e o f e i t h e r p r e p a r a t i o n ( c o n t a i n i n g 1 m g o f e x t r a c t e d B C G ) was r a p i d l y i n j e c t e d a n d e x t r a c t e d for 5 r a i n (for s e v e n - d a y B C G ) or for 10 rain (for 14-day B C G ) . T h e e x t r a c t i o n was s t o p p e d by a rapid t r a n s f e r to an ice b a t h . A f t e r c o o l i n g , 50 g/l o f e x t r a c t was m i x e d w i t h an a d e q u a t e a m o u n t o f l u c i f e r i n e luciferase r e a c t i o n m i x t u r e ( A T P d e p e n d e n t firefly b i o l u m i n e s c e n t reaction s y s t e m ; M c E l r o y 6) a n d the l i g h t e m i s s i o n m e a s u r e d wi'th a J R B A T P p h o t o m e t e r (SAI T e c h n o l o g y ) as t h e i n t e g r a t e d value o f / ) , , the i n t e n s i t y o f t h e l i g h t e m i t t e d by t h e s a m p l e d u r i n g 6 0 s a f t e r 15 s m i x i n g t i m e . T o t a k e i n t o a c c o u n t the possible effect o f i n r e r f e r i n g c o m p o n e n t s on t h e b i o l u m i n e s cent reaction, the constant addition techniqueS,in which a known a m o u n t of a standard A T P s o l u t i o n is a d d e d to t h e u n k n o w n s a m p l e w,as used. T h e A T P c o n t e n t o f t h e s a m p l e was c a l c u l a t e d f r o m t h e c o u n t p r o v i d e d by t h e s a m p l e w i t h s t a n d a r d A T P Jless t h e c o u n t for t h e s t a n d a r d alone. D u r i n g t h e preparat,/on of;the s u s p e n s i o n s o m e A T P was released into t h e m e d i u m , t h e a m o u n t o f w h i c h was c o u n t e d in the S N F f r o m an u n e x t r a c t e d s a m p l e . T h e i n t r a c e l l u l a r A T P c o n t e n t (of ~I m g o f B C G ) w a s c a l c u l a t e d f r o m t h e total A T P c o n t e n t o f t h e s a m p l e less this free A T P . P r e l i m i n a r y trials to e x t r a c t t h e A T P b y n u c l e o t i d e releasing r e a g e n t for bacterial cells ( N R B ) as s u g g e s t e d by L u m a c S y s t e m A G 4 r e s u l t e d in i n c o m p l e t e e x t r a c t i o n . F i n a l l y T r i s - E D T A e x t r a c t i o n was c h o s e n after a c o m p a r i s o n w i t h c h l o r o f o r m extraction, 7 both giving the same a m o u n t o f extracted ATP.
Oxygen uptake rate T h e O U R was m e a s u r e d by a G i l s o n O x y g r a p h ( G i l s o n M e d i c a l E l e c t r o n i c s , M i d d l e t o n , W i s c o n s i n ) b a s e d on t h e p o l a r o g r a p h i c p r i n c i p l e , a c c o r d i n g to t h e m e ~ h o d o f van H e m e r t & T i e s j e m a . 8 For each d e t e r m i n a t i o n B C G was d i l u t e d in S a u t o n m e d i u m w i t h 0 - 3 % p o l y s o r b a t e 8 0 to exactly 6 m g m1-1 9
Statistics M o i s t w e i g h t, A T P c o n t e n t a n d O U R o f B C G p r e p a r a t i o n s w e r e c o n s i d e r e d . a s variables o f a n o r m a l d i s t r i b u t i o n d e s c r i b e d by t h e m e a n a n d s t a n d a r d d e v i a t i o n o f this mean (SD). For t h e statistica] e v a l u a t i o n o f t h e association b e t w e e n A T P c o n t e n t a n d O U R . t h e s a m p l e c o r r e l a t i o n coefficient (r) was c a l c u l a t e d , l° RESULTS T h e m o i s t w e i g h t s o f 14 r o u t i n e p r e p a r a t i o n s (Table 1) w e r e s o m e w h a t g r e a t e r t h a n t h e p r e d i c t e d 8 0 m g m l - l a n d r a n g e d f r o m 9 0 to 143 m g m l - 1 T h e c a l c u l a t e d w e i g h t s o f e i g h t cultures homogenized at t h e s a m e t i m e varied f r o m 45 to 7 1 . 5 g per flask. N o s i g n i f i c a n t d i f f e r e n c e in A T P c o n t e n t ( 9 - 0 3 4 - 2 . 3 0 n g m g - l ) n o r in O U R ( 1-55 ± 0 - 4 2 /AI O z h - 1 r a g - l) was f o u n d w i t h i n this g r o u p . In s p i t e o f this t h e r e was p o o r c o r r e l a t i o n b e t w e e n A T P c o n t e n t a n d O U R (r ~ 0"33)T h i s lack o f c o r r e l a t i o n lead to m e a s u r e m e n t s o n a set o f e x p e r i m e n t a l p r e p a r a t i o n s f r o m c u l t u r e s o f t h e s a m e a g e ( 14 days) f r o m w h i c h c o n s t a n t w e i g h t s (5 g ) o f B C G cells w e r e h o m o g e n i z e d a n d f u r t h e r t r e a t e d . T o t a k e i n t o a c c o u n t t h e possible i n f l u e n c e o f the a g e o f t h e c u l t u r e s a n d t h u s t h e i r m e t a b o l i c s t a t e u p o n t h e results, A T P c o n t e n t a n d O U R w e r e m e a s u r e d i n p r e p a r a t i o n s o f s e v e n , d a y c u l t u r e s also. A g o o d c o r r e l a t i o n b e t w e e n A T P c o n t e n t a n d O U R was f o u n d in 14-day c u l t u r e s : 207
M. B O R R E M A N S E T A L . TABLF 1.
Pr~xtucrion lot no.
Comparison of moist (semi-dry) w e i g h t , A T P c o n t e n t and O U R in routine preparations o f 14-day BCG Moist w e i g h t (rag m l - )
ATP content (ng ml " l )
OUR (.ttl 0 2 h - l m g
81K09 81K16 81K30 81L07 8ILI4 81L21 82A I l 82A18 82A25 82B01 82B08 82C01 82C08 82D05
103 109 90 102 115 113 119 143 114 119 113 118 109 102
8-37 7" 10 6-40 7"80 8"50 10-24 9"75 7-81 9-31 14-44 13-10 9" 12 6-90 7-56
t "21 1-02 1-41 I" 54 1-51 1-97 1"4 1 2- 19 0-96 1.75 2- 13 1 "30 1- 16 2- 13
Mean SD
1 l 2-07 12-07
9"03 2"3
1-55 0-42
-I)
r ~ coefficient of correlation between ATP content and OUR m 0"33; not significant. r = 0 " 8 6 , s i g n i f i c a n t a t t h e 1% level [ T a b l e 2, F i g . I(a)] as w e l l as in t h e s e v e n - d a y c u l t u r e s : r = 0 " 8 4 , s i g n i f i c a n t a t t h e 0 - 1 % level [ T a b l e 3 , F i g . 1(b)]. A n i n t e r e s t i n g f e a t u r e was t h a t t h e 1 4 - d a y c u l t u r e s w e r e i n o c u l a t e d s i m u l t a n e o u s l y w i t h t h e s e v e n - d a y c u l t u r e s a n d so w e r e i n c u b a t e d o n l y a w e e k l o n g e r . O n e m a y t h u s c o n c l u d e t h a t t h e A T P c o n t e n t d i m i n i s h e d f r o m a m e a n v a l u e o f 6 2 - 9 8 ± 2 4 - 14 n g o f TABLE 2.
C o m p a r i s o n o f A T P c o n t e n t a n d O U R in e x p e r i m e n t a l preparations o f 14-day B C G
Preparation no.
ATP content (ng m g - t )
1 2 3 4 5 6 7 8
7-80 7-75 6-09 6.80 4-52 3;84 2-37 4-60
1-44 1-42 1-44 1-46 1-35 I- 18 1-03 1,12
Mean SD
5"47 1"95
1 "31 0-17
Sample correLation coefficient r ----- 0-86; sigr~ificanr at the 19~- lev, l.
208
OUR (~AI 0 2 h - ! m g
-t)
02 U P T A K E A N D A T P C O N T E N T
-
OF BCG PREPARATIONS
(a')
1.4
~ I-2~ 7 ..¢:
1.0'] I I I ..... ! t t I.. 0 t 2 3 4 5 6 7 S ............................. :. . . . . . . . . . . .
8 : 9
I0
-~ 6 ~- (b) g:
5'
I"
~
Q
i
e
t
I lI l
00
I
I(~
tO
1
20
I
30:40
J
I
50 ATP
I,
I
60 (ng
70
t
I
I
I
BO
90
I00
ItO
mg-I)
Fig. 1. Scatter d!agram of the relationship between A T P content and O U R in experimental prepara tions of (a) 14-day BCG and (b)seven-day BCG. , Regression of A T P content upon OUR; • regression of O U R upon ATP content. Correlation coefficient r = 0"86(a), r = 0-84(b), significant at (a) the 1e.6 and (b) "the 0- l e a levels.
TABLE 3.
C o m p a r i s o n o f A T P c o n t e n t a n d O U R in e x p e r i m e n t a l p r e parations of seven-day BCG ATP content
OUR
(ng mg -1)
( p l 0 2 h - 1 I'ng - I )
2 3 4 5 6 7 8 9 I0 1l 12 13
43- 15 51-50 56-67 68-27 48-03 90-90 100:25 36"90 45-74 83- 1 l 49-84 106-00 38-40
2.5 3-78 3-62 4-48 4-22 5-01 4"26 Z-7 1 2"5.1 4-86 3'77 5;55 3-40
Mean SD
62-98 24.14
3,9 0-96
Preparation
1
no.
Sample correlation coefficient r ---- 0-84, significant at 0- IC~, level.
209
M. B O R R E M A N S
ET AL.
A T P m g -1 of BCG at seven days to 5-47 ---+ 1.15 ng o f A T P m g -1 B C G at 14 days, .
.
.
.
3"0. The average A T P c o n t e n t of the routine preparations (9"03 ± 2"3 ng o f A T P m g - 1 BCG) was h i g h e r than that of the 14-day experimental preparations (5"47 + t" 15 ng of A T P m g - l BCG), a l t h o u g h their O U R did not differ. DISCUSSION Each o f the 14 r o u t i n e preparations was prepared under different conditions. T h e bacterial mass which was h o m o g e n i z e d was a different w e i g h t in the c~se of each preparation (from 45 to 71"5 g per e i g h t cultures). The variable a m o u n t of residual l i q u i d t o g e t h e r w i t h the variable bulk of bacteria gave rise t o different degrees o f dispersion after h o m o g e n i z a t i o n . Additionally, the steel beads, responsible for rather r o u g h m i x i n g , may kill bacteria. Larnensans & B r e t e y l d e m o n s t r a t e d that this effect was inversely related to the bacterial mass: t h e g r e a t e r the mass, the fewer bacteria were killed d u r i n g h o m o g e n i z a t i o n . T h e greater A T P yield from the routine preparations (9-03 ± 2-3 ng o f A T P m g - I BCG) (Table 1) c o m p a r e d with that of the 14,day experimental preparations (5-47 ± I ' 9 5 ng of A T P r a g - l BCG) (Table 2) confirmed this protection from k i l l i n g w i t h i n large bacterial clumps. ~ O n t h e other hand c h a n g i n g the h o m o g e n i z a t i o n conditions did not influence the O U R m e a s u r e m e n t ; the respiration rate of both preparations may be considered to be equal: 1-55 ~1 of O2 per h per m g (Table 1) and 1-31/~1 of O2 h - 1 r a g - l (Table 2, last column). This brings us to the p r o b l e m raised by Chappelle e t a / . l ~. 12 w h o were aware that any m e t h o d o f q u a n t i f y i n g bacteria based on the m e a s u r e m e n t of t h e intracellular concentration of a particular m e t a b o l i t e did not necessarily distinguish viable cells from dead cells. Periodic m e a s u r e m e n t o f A T P and C F U d u r i n g 15 days g r o w t h o f an E s c h e r i c h i a co/i culture were welt correlated b u t it was shown, too, that A T P levels were s'asrained and measured w i t h i n those cells chat were still intact but metaboIically inactive. T h e lack of correlation between A T P c o n t e n t and O U R in the routine preparations (Table 1) m a y be d u e to the more g e n t l e h o m o g e n i z a t i o n that renders s o m e of the bacteria in t h e c l u m p s metabolically inactive but leaves t h e m morphologically intact. W h e n the A T P content is d e t e r m i n e d i m m e d i a t e l y after preparation the A T P w i t h i n such cells is measured a l t h o u g h their respiration is zero. Accordingly, rougher h o m o g e n i z a t i o n (for t h e seven- and 14-day experimental preparations) breaks the c l u m p s and releases A T P from a certain proportion of the bacteria into t h e m e d i u m g i v i n g a smaller intracellular A T P yield (5"47 ~ 1.95 ng o f A T P r a g - l o f BCG) (Table 2), w h i c h correlates well w i t h the O U R of the B C G suspension (r -~ 0"86) [Fig. l(a)]. F i n d i n g a good statistical correlation between A T P content and O U R in both sets o f experimental preparations (r ~ ' 0"83 for seven days and r -~--0 - 8 6 for 14 days) (Fig- 1) indicates t h e possibility o f p r o d u c i n g several vaccines w i t h comparable m e t a b o l i c activity. In fact the statistical correlation found b e t w e e n A T P c o n t e n t and O U R can be explained as a metabolic correlation. O x y g e n and A T P are related to each other in the respiratory chain phosphorylation. I3 In this pathway, in which oxygen acts as an u l t i m a t e electron acceptor for the oxidation o f a substrate, A T P is formed. This phosphorylation is n o t the only metabolic pathway leading to the formation o f A T P and 210
02 UPTAKE A N D ATP C O N T E N T OF BC~3 PREPARATIONS A T P is c o n s u m e d in several b i o s y n t h e t i c reactions. *~ T h e r e f o r e t h e statistical c o r r e l a t i o n b e t w e e n A T P c o n t e n t a n d O U R p r o b a b l y c a n n o t be i m p r o v e d . T h e lack o f a c o m p l e t e c o r r e l a t i o n o n l y p a r t l y e x p l a i n s t h e d i f f e r e n t d e c l i n i n g rates o f A T P a n d O U R ( 1 1' 5 t i m e s a n d 3 - 0 t i m e s respectively). A full e x p l a n a t i o n is p r o b a b l y g i v e n b y analysis o f t h e g r o w t h c u r v e o f B C G . A c c o r d i n g to S u p Lee 3 B C G in d e e p c u l t u r e ceases to g r o w h e x p o n e n t i a l l y a t d a y n i n e , g i v i n g m a x i m u m A T P c o n t e n t a n d d r y w e i g h t pe/" m i l l i l i t r e . G r o w t h then enters t h e declining p h a s e u n t i l d a y I 3 , associated w i t h a c o n s t a n t d r y w e i g h t b u t w i t h d e c l i n i n g A T P c o n t e n t . T h e r e s p i r a t i o n rate, h o w e v e r , reaches a m a x i m u m a f t e r seven d a y s in c u l t u r e s g r o w n as surface pellicles 1 or a f t e r five to six days in d e e p c u l t u r e . 9 A l t h o u g h it m a y be a s s u m e d t h a t for e a c h g r o w t h e x p e r i m e n t m e n t i o n e d a d i f f e r e n t B C G s t r a i n was e m p l o y e d , t h i s c o u l d i n d i c a t e t h a t A T P c o n c e n t r a t i o n a n d r e s p i r a t i o n rate d o n o t r e a c h m a x i m a o n t h e s a m e day. T h e g r o w t h c u r v e o f t h e G L 2 strain o f B C G has not b e e n d e t e r m i n e d . I f t h e r e s p i r a t i o n rate h a s n o t a t t a i n e d its m a x i m u m after seven days o f g r o w t h w h i l s t A T P c o n c e n t r a t i o n has, it is o b v i o u s t h a t s i m p l y c a l c u l a t i n g t h e r e s p i r a t i o n ratio or t h e A T P c o n t e n t ratio b e t w e e n seven- a n d 1 4 - d a y s u s p e n s i o n s does n o t reflect t h e exact d e c l i n e . Also it m u s t n o t be f o r g o r t e n t h a t t h e m e a s u r e m e n t s w e r e d o n e on B C G p r e p a r a t i o n s r a t h e r t h a n o n u n t r e a t e d cells, s a m p l e d f r o m c u l t u r e s . R e s p i r a t i o n rate a n d A T P c o n t e n t m e a s u r e d in a B C G s u s p e n s i o n p r e p a r e d u n d e r s t a n d a r d c o n d i t i o n s b o t h g i v e a first i m p r e s s i o n o f t h e v i a b i l i t y o f t h e p r e p a r a t i o n . N e v e r t h e l e s s O U R m e a s u r e d w i t h t h e G i l s o n o x y g r a p h offers s o m e a d v a n t a g e . T h e m e a s u r e m e n t can be d o n e o n a s a m p l e as s u c h a n d is n o t i n f l u e n c e d b y t h e d e g r e e o f d i s p e r s i o n o f b a c t e r i a as t h e s a m p l e is constantly mixed.
Acknowledgements The a u t h o r s t h a n k / ~ l r A. Vanonckelen and M r Marlier for technical assistance. They are indebted also to Professor Dr D. D e k e g e l for his objective judgements.
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