Blockade of vitamin B12-binding sites in gastric juice serum and saliva by analogues and derivatives of vitamin B12 and by antibody to intrinsic factor

560

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 25 806 BLOCKADE OF VITAMIN B~,-BINDING SITES IN GASTRIC JUICE, SERUM AND SALIVA BY ANALOGUES AND DERIVATIVES OF VITAMIN B~z AND BY ANTIBODY TO INTRINSIC FACTOR* CHESTER W. GOTTLIEB, FRANCOIS P. RETIEF**, AND VICTOR H E R B E R T

Department of Hematology, The Mount Sinai Hospital, New York, N.Y. (U.S.A.) (Received November 7th, 1966) (Revised manuscript received January 26th, 1967)

SUMMARY

A variety of vitamin B12 analogues and derivatives (pseudo-vitamin B~, anilide-B~, aquoanilide-B12, cobinamide, cobrynamide, 5,6-dimethylbenzimidazole and five different cobaloxime compounds) were tested for their ability to reduce the unsaturated vitamin Bl~-binding capacity of (I) gastric juice, (2) serum and (3) saliva. The effect of these substances on specific binders in body fluids tested, was determined by rapid DEAE-cellulose chromatography. i. Gastric juice: All vitamin Blz-like substances, except 5,6-dimethylbenzimidazole and cobaloximes, bound well to non-intrinsic factor vitamin B~2 binders in normal gastric iuice. Conversely, only intrinsic factor antibody and CN-B~ blocked radioactive vitamin B~, binding to intrinsic factor. The B~ binders in pernicious anemia gastric iuice were not effectively blocked by either intrinsic factor antibody or analogues. This precludes the use of analogues instead of intrinsic factor antibody for assay in vitro of intrinsic factor in gastric iuice, in the system described. 2. Serum: Unsaturated vitamin B~-binding capacity of normal, chronic myelogenous leukemia and pernicious anemia serum was affected very similarly, and in a pattern resembling that of the non-intrinsic factor binder of normal gastric juice, except that cobaloxime C3, was effective in reducing the serum binding of vitamin B~. Chromatography showed equal reduction of both ~-globulin and r-globulin binders. 3- Analogue interference with vitamin B~z binders in saliva was essentially similar to, but more pronounced than, the results with serum and non-intrinsic factor binders in gastric juice. INTRODUCTION

Many workers have studied the ability of vitamin B12 (B12) analogues and derivatives to compete with cyanocobalamin (CN-B1,) for vitamin Bl~-binding sites of gastric juice and other body fluids. It is known that serum contains at least two distinct vitamin B12 binders: an ~-globulin (to which endogenous vitamin B,2 is * Part of this ~vork has been published in abstract form 1. ** Present address: Department of Medicine, University of Stellenbosch Medical School, P.O Box 53, Bellville, C.P., South Africa.

Biochim. Biophys. Acta, 141 (I967) 560-572

56I

BLOCKADE OF HUMAN B l z BINDERS

primarily bound) and a t-globulin which in normal subjects is relatively devoid of native vitamin B~, and binds the majority of vitamin Bx, added in vitro ~-~. Gastric juice probably contains at least four different vitamin B~z-binding substances, but for practical purposes they may be conveniently grouped into intrinsic factor binder and non-intrinsic factor binders s. BUNGE AND SCHILLING7 reported that the binders of gastric iuice, serum, saliva and colostrum differed in their affinity for vitamin B~. The present study was undertaken to investigate the possibility that some analogues and/or derivatives of vitamin B~, may selectively bind to, and so differentiate between, the various vitamin B ~ binders in body fluids. This was done by (a) testing the ability of a variety of vitamin B~,-like compounds to interfere with radioactive vitamin B~ binding to gastric juice, serum and saliva-binding sites, and (b) by making use of rapid DEAE-cellulose chromatographyS, ss to determine specifically which vitamin B~-binding sites had been "blocked" by those test compounds, causing a reduction in radioactive vitamin B~, binding. METHODS AND MATERIALS

Vitamin B ~ analogues and derivatives* The compounds investigated in the present study differed from CN-B~ (Fig. I) in the following respects: (a) Cobrynamide (cobyric acid, Factor Via): The "nucleotide" and its amino~HZ 0=? N~Hz C~Hz

O=C

CHz "

~

-- R -CN H i

X

~ " II CH~-C~

-H20 ~

~

/

Co ~

~ ' H2C

-OH

CH3CH3 ~

k ~l~[

z~

5'-deoxyade~syl- coenzyme B12 .methyl ......... ,, -CN 3 B~

~C-CH~

~ CH~

~ ~ " O'C-NH21 H~N-C=O "

R-Cobry~omide(Factor Via) " R - Cob~nomide( Fo ctor B )

PERMISSIVENAME cyanocobala rain (vitamin B~2) hydroxocobalamin (vitamin Btaa) oquocobolamin (vitamin B12b)

~ ,

T

/

.<

N

CHz CH2

N

~

.~

H 0 OH x ~ / V~CH~ O=C ~x I ~ I >" ~ 0P N-CHz-CH(CH~)-O-P-O-C--C = / : "'2 ~ .. / X /~ / 5,6-d~methyl-

~

~

"

~-~-~*.o-

" ~ " 'd H H ~ /

Z- propanol ~

~ ~ n~'HZ~

~ 0

~...~,o,.

~ H

~-Cobomide

R-Cob~amm

Fig. I. The vitamin Blz molecule (see ref. 36). * Cobinamide was obtained from Dr. D. PERLMAN, Squibb Institute, New Brunswick, N. J., as an ionopherigram (I rag). Cobrynamide (powder, L 6o4o37-o-i), pseudo-vitamin BI~ (powder, L 562 413-o-2) and 5,6-dimethylbenzimidazole (powder L 488165-o-4) were obtained from Merck, Sharp and Dohme. Anilide-Bla and aquoanilide-Bla (in powder form) was supplied by Dr. E.L~STER S~alTH, Glaxo Laboratories, Great Britain, and cobaloximes were supplied by Dr. G. N. SCI~RAIJZER, whose current address is Department of Chemistry, University of California at San Diego.

l~iochim. Biophys, Acla, 141 (1967) 560-572

562

C . W . GOTTLIEB, F. P. RETIEF, V. HERBERT

propanol linkage are absent from the vitamin BI~ molecule, the D-ring propionamide terminating in a carboxyl residue. (b) Cobinamide (Factor B): Cobrynamide molecule with I)-I-amino-propanol-2 replacing the carboxyl group of the D-ring propionamide. (c) Pseudo-vitamin Bx~: The 5,6-dimethylbenzimidazole is replaced by adenine. (d) 5,6-Dimethylbenzimidazole. (e) Anilide-B~z and aquoanilide-B~: cyanocobalamin (CN-Ba~) and aquocobalamin (aquo-Bl~) molecules with a substituted amide (NH.CnHs) attached to a corrin nucleus propionamide (probably the C-ring side-chain). (f) Cobaloximes (CI-C5): Bis-(dimethylglyoximato) cobalt complexes~ which partially resemble the corrin nucleus of vitamin B~z (Fig. 2). The B- mad R-radicals attached to Co in each of the five compounds, were the following: CI: HeO. Co-CH3; C2: pyridine.Co.(CH~)3CHn; C3: HzO.Co.(CH~)2COOCH~; C4: pyridine.Co. CH~CH~; C5 : pyridine. Co- CN. /~H~ 0" 0

c.,\/\I

R I

/1%/t CH~

co

i

I

~CH~ 1

O ~ H../O Fi~. ~. ~ e b~siccob~lo~me st.~c~e ~. Cobinamide was eluted from an ionopherigram strip in o.oi % KCN, and made up to a working solution of IOO ng/ml. All other compounds were obtained in powdered form and dissolved in o. 9 % NaC1 and prepared as solutions containing IO or ioo ng/ml. In view of the photosensitivity of most vitamin B~z analogues ~° the compounds were stored in the dark at 4°; in addition, light exposure was minimized during their use. It is recommended that fresh cobaloxime solutions be prepared daily.

Serum, gastric juice and saliva Serum, obtained from normal persons and patients with chronic myelogenous leukemia and pernicious anemia in relapse, was stored at --20 °. Serum from patients with pernicious anemia known to have high titers of autoantibody to intrinsic factor was saturated with an amount of non-radioactive CN-BI~ in excess of its unsaturated vitamin B l~-binding capacity, incubated for 15 rain at 37 ° and then shaken with 5° mg of hemoglobin-coated charcoal 11 to remove all unbound vitamin BI~. The sera so treated had no measurable unsaturated vitamin B~z-binding capacity; the ability of antibody to intrinsic factor to block the subsequent binding of added radioactive vitamin BI~ was not affected~2. Gastric juice, obtained from subjects with and without pernicious anemia, after maximal histamine stimulation, was depepsinized and stored at pH 7.o at --20 ° (ref. 13). Saliva, obtained from normal persons was stored at --20 °. Biochim. Biophys. Acta, 141 (1967) 56o-572

BLOCKADE OF HUMAN B ~ BINDERS

563

Unsaturated vitamin B~z-binding capacity The capacity of I.O ml serum, o.I ml gastric juice and o.I ml saliva was determined by batch separation using coated charcoal, as previously described l~. The ability of vitamin B~e analogues to compete for vitamin B~e-binding sites was assessed by incubating these compounds in the test fluids under investigation for I5 rain at 37 ° prior to adding I~°Colvitamin B ~ (specific activity approx. I/~C//~g). Analogues were added in amounts identical by weight to subsequently added radioactive vitamin B~2. o.I ml of serum containing antibody to intrinsic factor was similarly preincubated with test fluids; the antibody to intrinsic factor used blocks the vitamin Bt2-binding capacity of up to 15 ng of vitamin-B~e binding substance in o.I ml of gastric juice. A small DEAE-cellulose column s,~ was used for chromatographic separation of vitamin BI~ binders in serum, gastric juice and saliva. I ml serum, o.I ml gastric juice and o.I ml saliva were saturated with an excess of [~Colvitamin B ~ (specific activity 2o-4o #C/#g) ; total volume of final sample was adjusted with o. 9 % saline to 1.25-i.75 ml. Unbound [5~Colvitamin Bt~ was removed by shaking with a 50 mg hemoglobin-coated charcoal pellet and dialyzing the sample overnight against 0.2 M phosphate buffer (pH 6.3) before application to the DEAE-cellulose column. The effect of vitamin Bx2 analogues and antibody to intrinsic factor on elution patterns was assessed by preincubating test fluids with analogues and derivatives (as described above) or with o. i ml serum containing antibody to intrinsic factor. RESULTS

Serum

Unsaturated B12-binding capacity: The effect of antibody to intrinsic factor and vitamin B~2 analogues and derivatives on the unsaturated vitamin Bl~-binding capacity of normal, pernicious anemia and chronic myelogenous leukemia serum is is represented in Table [. An essentially similar binding pattern was evident in all three conditions. Pseudo-vitamin B,~, anilide-B~, aquoanilide-B~ and cobaloxime C3 produced greatest interference with radioactive vitamin BI~ binding, whereas cobinamide and cobrynamide causes approx, one-third reduction of normal serum unsaturated vitamin B~-binding capacity Antibody to intrinsic factor, dimethylbenzimidazole and cobaloximes CI, C2, C4 and C5 had no significant blocking effect on vitamin B ~ binding. The profile of inhibition of these compounds on the unsaturated vitamin B~2-binding capacity is presented in bar graph form in Fig. 3. DEAE-cdlulose chromatography: In Fig. 4, the elution pattern of normal serum saturated with [~Co~vitamin B ~ is compared with that of serum pretreated with vitamin B~2 analogues, derivatives and antibody to intrinsic factor. The two peaks of radioactivity, representing the/~- and ~-globulins in serum which bind vitamin B ~ (refs. 8, 35) were similarly affected by those compounds capable of interfering with radioactive vitamin B ~ binding. Gastric juice Normal gastric juice Unsaturated vitamin B12-binding capacity: The effect of various analogues, derivatives and antibody to intrinsic factor in blocking radioactive vitamin B12 binding Biochim. Biophys. Acta, 14i (i967) 56o-572

I

OF

UNSATURATED

VITAMIN

B12-BINDING

CAPACITY

BIOLOGICAL

FLUIDS

BY

VARIOUS

INHIBITORS

97.i

.

99.1

98. 4

41.8

__

--

--

C o b a l o x i m e C2

Cobaloxime C3

.

C o b a l o x i m e C4

Cobaloximo C5

IF Ab

I F A b plus a n i l i d e - B l ~

I F A b plus a q u o a n i l i d e - B a z

A n i l i d e - B ~ plus a q u o a n i l i d e - B i ~ 55.8

2.4

2. 7

45,6

--

--

--

--

--

63.9

.

.

--

--

__

39-3

--

--

--

--

>IOO

773.6

75,5

7° . 8

65,0

73.6

o

lOO

No. 3

96.6

--

--

__

io.o

98.2

>ioo

.

>ioo

98.3

97.9

93. I

97 .2

92'9

95. x

8.2

ioo

No. 4

>IOO

93.1

91.6

82. 4

95-4

>IOO

>1oo

96.9

56'5

48"I

93.I

65.6

ioo

PA

:>IOO

97.3

99-4

13,6

>ioo

>ioo

98. 5

62, 3

62. 4

I3.6

25. 5

5 .2

6.5

IOO

Normal

Serum

--

97-7

98.6

5. i

97.2

>IOO

--

--

--

4,7

2 I .o

--

3.3

IOO

CML

--

98.6

98.6

13.9

>ioo

>ioo

--

--

--

7.5

36.9

--

6.I

ioo

.PA

>Ioo

>1oo

99.9

--

>ioo

99-7

98. 5

I7.6

17.0

6.8

4.2

7.7

3.6

ioo

Saliva

~ U n s a t u r a t e d v i t a m i n B l ~ - b i n d i n g c a p a c i t y e x p r e s s e d a s a p e r c e n t a g e of u n t r e a t e d u n s a t u r a t e d v i t a m i n B i z b i n d i n g c a p a c i t y t a k e n a s IOO % . Values > ioo % do not represent significant v a r i a t i o n f r o m the u n t r e a t e d value.

96, 4

Cobaloxime CI

--

--

--

Cobrynamide

Cobinamide

5,6-Dimethylbenzimidazole

--

57.5

--

Aqnoanilide-Bl~

63.0

55-3

Anilide-Bi ~

--

--

ioo

--

1.9

IOO

Pseudo-Bi~

CN-Ba2

(Untreated) *

No. 2

No. I

Gastric juice

~

Inhibitor

Normal (4 samples)

?

1N

Abbreviations: CML, c h r o n i c m y e l o g e n o u s l e u k e m i a ; P A , p e r n i c i o u s a n e m i a ; I F A b , a n t i b o d y t o i n t r i n s i c f a c t o r .

BLOCKAGE

TABLE

~

~

~-

~

~. :~ ~. '

~

~

t~ ~

m

~

.~

t~ .~

~

.~

t*

~

~

BLOCKADE OF HUMAN B I ~ BINDERS

I1

565

Non-radioactive CN-B 12

[

Human intrinsic factor auto-antibody

[

I

[ U [

Anil]de - B,2 H20 - anilide - B~2 Pseudo-B

[

1

Cobrino mide

l

Cobinamide

[

]

5,6- Dim ethyl -benzimida z ole

J~

H20 (CO)(CH2)2 cOaCH 3

(

H20 (CO)CH3 Pyridine (CO)(CH2)3 CH3 l Coba Ioximes Pyrldlne (CO) C2H5

[

Pyridine (CO) CN

L

0

20

I

40

I0

6

I

80

100%

Fig. 3. T h e effect of v i t a m i n B l i a n a l o g u e s a n d derivatives, a n d a n t i b o d y to intrinsic factor on t h e u n s a t u r a t e d v i t a m i n B12-binding c a p a c i t y of n o r m a l s e r u m . T h e solid b a r r e p r e s e n t s t h e u n s a t u r a t e d v i t a m i n Bt~-binding c a p a c i t y of n o r m a l serum, e x p r e s s e d as i o o % . T h e open b a r s r e p r e s e n t t h e r e m a i n i n g u n s a t u r a t e d v i t a m i n Bx~-binding c a p a c i t y after i n c u b a t i o n of s e r u m for 15 m i n a t 37 ~ w i t h t h e c o m p o u n d s s h o w n in t h e r i g h t h a n d m a r g i n . H u m a n I F a u t o - a n t i b o d y = a n t i b o d y to intrinsic factor f r o m a p a t i e n t with pernicious a n e m i a .

1800 I60C 140(

~120( cT ~ooo .c_

Eo 800

t-~ 60(2 m

tOlL,_ :.t~al-m~

.

. --. .

123456 78910

DEAE-cellulose column eluates:tubes 1-10 Fig. 4. T h e effect of v i t a m i n BI~ analogues, d e r i v a t i v e s a n d intrinsic factor a n t i b o d y on s m a l l D E A E - c e l l u l o s e c o l u m n elution p a t t e r n s of two r a d i o a c t i v e v i t a m i n B~=-saturated n o r m a l sera; t h e t h r e e g r a p h s to t h e r i g h t were o b t a i n e d f r o m s a m e s e r u m . Q - - O , S e r u m ; . - - . , s e r u m p r e t r e a t e d w i t h a n t i b o d y to intrinsic factor; C)--C), s e r u m p r e t r e a t e d w i t h CN-BI~ ; , - - , , serum p r e t r e a t e d w i t h aquoanilide-Bl~; [ ] - - ~ , s e r u m p r e t r e a t e d w i t h anilide-Bx~; x - × , s e r u m pret r e a t e d w i t h pseudo-Bza; & - - & , s e r u m p r e t r e a t e d w i t h c o b i n a m i d e ; ~ - - ~ . , s e r u m p r e t r e a t e d with cobrynamide; ~-- ~, serum pretreated with 5,6-dimethylbenzimidazate.

Biockim. Biophys. Acta, I 4 I (1967) 56o-572

566

c.w.

GOTTLIEB, F. P. RETIEF, V. HERBERT

of normal gastric juice is shown in Table I. Four gastric juice specimens with varying intrinsic factor content are compared. The reduction in unsaturated vitamin BI~binding capacity caused by antibody to intrinsic factor appears to be complementary to that caused by pseudo-vitamin B~, anilide-B~, aquoanilide-Bl~ , and even cobinamide and cobrynamide. The other compounds had no effect (data for cobaloxime C~ is not shown since it was probably inactive prior to its use in the gastric juice and saliva experiments and failed to reduce the unsaturated vitamin B~-binding capacity of those media). When gastric juice was preincubated with simultaneously added antibody to intrinsic factor, anilide-Bla and aquoanilide-B~ caused no greater unsaturated vitamin Bl~-binding capacity reduction than preincubation with either of the analogues alone. These findings suggest that the analogues bound primarily to the non-intrinsic factor fraction of normal gastric juice. The reason for failure of normal gastric juice sample No. 4 to be significantly altered by the compounds studied is unclear. The profile of inhibition of these compounds on the unsaturated vitamin B~2-binding capacity is presented in bar graph form in Fig. 5.

CN-B12 Numan intrinsic factor auto-antibod~ Anilide - 1312 H20- onilide-I312

Pseudo-B Cobrynamide Cobina micle J 5,6 Dimethyl-benzirnidezole I

H20 (Co) (CH2)2 COOCH 3

I ~so (Co) c~s

J

Pyridine(Co) (CH2)3CH 3

Cobaloxirnes

J Pyrid]rle (Co)C2H 5

J Pyridine(Co)CN I

;:)

2'0

10

60

80

I

100%

Fig. 5. T h e effect of v i t a m i n ]312 a n a l o g u e s a n d d e r i v a t i v e s a n d a n t i b o d y to intrinsic factor on t h e u n s a t u r a t e d v i t a m i n B12-binding c a p a c i t y of n o r m a l gastric juice. T h e solid b a r r e p r e s e n t s t h e u n s a t u r a t e d v i t a m i n B l z - b i n d i n g c a p a c i t y of n o r m a l gastric juice e x p r e s s e d as IOO ~/o. T h e open b a r s r e p r e s e n t t h e r e m a i n i n g u n s a t u r a t e d v i t a m i n B l ~ - b i n d i n g c a p a c i t y after i n c u b a t i o n of gastric juice for 15 rain at 37 ° with t h e c o m p o u n d s s h o w n in t h e r i g h t h a n d m a r g i n . H u m a n I F a u t o a n t i b o d y = a n t i b o d y to intrinsic factor from a p a t i e n t w i t h pernicious a n e m i a ,

DEAE-cellulose chromatography of normal gastric juice results in the separation af two peaks of radioactivity (Fig. 6). Preincubation with antibody to intrinsic factor results in abolition of the first peak, suggesting that this fraction contains predominantly intrinsic factor. The compounds which were capable of decreasing the unsaturated vitamin B12-binding capacity of gastric juice affected only the second binder. This tends to confirm the finding of blocking of different vitamin BI~ binders in gastric juice by antibody to intrinsic factor and vitamin B12 analogues. B$ochira. Biophys. Acta, 141 (1967) 560-572

BLOCKADE OF HUMAN BI~ BINDERS

567

24001 i

2200 2000 i

1600 1400

I~00

~ I000 (3. l~ 800

rn

"~

600

.~ > 400 ~

2oo

12345678910

12345678910

DEAE-cellulose c o l u m n e l u o t e s t u b e s 1-10

Fig. 6. The effect of vitamin BI~ analogues, derivatives and intrinsic factor antibody on small DEAE-cellulose column elution patterns of two radioactive vitamin B12-saturated normal gastric juices; the three graphs on the right were obtained from same gastric juice. 0 - - 0 , Gastric juice; o--., gastric juice pretreated with antibody to intrinsic factor; ©--O, gastric juice pretreated with CN-B12; 1 - - ~ , gastric juice pretreated with aquoanilide-B~; E]--~, gastric juice pretreated with anilide-B~2; × - - × , gastric juice pretreated with pseudo-Bt~; &--&, gastric juice pretreated with cobinamide; I , - - ~ , gastric juice pretreated with cobrynamide; V--V, gastric juice pretreated with 5,6-dimethylbenzimidazole.

Pernicious anemia gastric juice Antibody to intrinsic factor had no effect on the unsaturated vitamin BI~binding capacity of pernicious anemia gastric juice while anilide-B~ and aquoanilideBI~ caused some reduction in the unsaturated vitamin Bl~-binding capacity (Table I). However, the interpretation of these findings is clouded by the peculiar inability of CN-BI~ to abolish subsequent binding b y radioactive vitamin ]31~ (Table I).

E~ect of antibody In Table I I the effect of antibody to intrinsic factor and pseudo-vitamin BI~ in reducing the .unsaturated vitamin B~-binding capacity of a series of pernicious anemia and non-pernicious anemia gastric juice specimens, is compared. The nonpernicious anemia samples included two specimens which revealed temporary lack of demonstrable intrinsic factor secretion in a patient with chronic pancreatitis and with polycythemia vera, respectively. In confirmation of the above findings, antibody to intrinsic factor and pseudovitamin BI~ showed reasonable complementary "blocking" of vitamin BI~ binders in non-pernicious anemia gastric juice. This was also evident in the gastric iuice of the two patients temporarily lacking intrinsic factor. Pernicious anemia gastric juice was totally unaffected b y either antibody to intrinsic factor or pseudo-vitamin B~.

Biochim. Biophys. Acta, 141 (I967) 56o-572

568

C. W. GOTTLIEB, F. P. RETIEF, V. HERBERT

T A B L E II E F F E C T O1~ I N S T R I N S I C F A C T O R A N T I B O D Y A N D P S E U D o - B I 2 CAPACITY OF PERNICIOUS ANEMIA AND NON-PERNICIOUS

Gastric juice

ON UNSATURATED ANEMIA GASTRIC

Unsaturated vitamin Bi~-binding capacity (%)* Antibody to intrinsic factor

I 2 3 4 5 6 7 8 i 2 I 2 3 4 5

Normal Normal Normal Normal Normal Normal Normal Normal Non-pernicious a n e m i a I w i t h intrinsic Non-pernicious a n e m i a ~ factor lack*** Pernicious a n e m i a Pernicious a n e m i a Pernicious a n e m i a Pernicious a n e m i a Pernicious a n e m i a

VITAMIN JUIC]~

66.0 17. 4 2. 7 4.6 82.2 75.o 38.6 IO.O >ioo ~IOO 98.6 >ioo >ioo >ioo > ioo

Bla-BINDING

Intrinsic factor (%)**

Pseudo-Bx~

34.4 >ioo >ioo 95.4 26. 5 38.8 93.6 95.I 6. 7 4.1 97.9 90.8 >ioo 32.1 > ioo

34 .0 82.6 97.3 95.4 17.8 25.0 61.4 9o.o o o 1.4 o o o o

* U n s a t u r a t e d v i t a m i n B l z - b i n d i n g c a p a c i t y e x p r e s s e d as a p e r c e n t a g e of u n t r e a t e d u n s a t u r a t e d v i t a m i n Bl~-binding capacity, t a k e n as i o o % , after i n c u b a t i o n w i t h a n t i b o d y to intrinsic factor or p s e u d o - B l v Values > lOO% do n o t r e p r e s e n t significant v a r i a t i o n f r o m t h e u n t r e a t e d value. ** I n t r i n s i c factor e x p r e s s e d as a p e r c e n t a g e of t h e t o t a l u n s a t u r a t e d v i t a m i n B l z - b i n d i n g c a p a c i t y w h e n a n t i b o d y to intrinsic factor is u s e d for a s s a y x4. *** Gastric juice o b t a i n e d fl:om non-pernicious a n e m i a p a t i e n t s s h o w i n g intrinsic factor lack in b a s a l gastric secretion. B o t h p a t i e n t s h a d n o r m a l intrinsic factor secretion after h i s t a m i n e stimulation.

300

200

v

c 100 .~, .~ r-~ I~

,~,

O

~ 123456789i0 I 2 345678910 DEAE-cellulose column eluotes:tubes 1-10

Fig. 7. T h e effect of v i t a m i n Blz a n a l o g u e s a n d derivatives on s m a l l D E A E - c e l l u l o s e c o l u m n elution p a t t e r n s of r a d i o a c t i v e v i t a m i n B l ~ - s a t u r a t e d n o r m a l saliva. 0 - - 0 , Saliva; O - - O , saliva p r e t r e a t e d w i t h CN-B12; ~ - - [ ~ , saliva p r e t r e a t e d w i t h anilide-Bxz; × - - × , saliva pret r e a t e d w i t h pseudo-Bl~; A - - A , saliva p r e t r e a t e d w i t h cobinamide.

Biochim. Biophys. Acta, 141 (1967) 56o-572

BLOCKADE OF HUMAN BI~ BINDERS

569

Saliva Prior incubation of saliva with pseudo-vitamin B ~2, anilide-B ~2, aquoanilide-B~ 2, combinamide and cobrynamide produce marked reduction in the subsequent binding of added radioactive vitamin BI~ (Table I). DEAE-cdlulose chromatography of normal saliva produced a major second peak of radioactivity (corresponding to the non-intrinsic factor binder of normal gastric juice) with an insignificant first peak. Pretreatment with pseudo-vitamin Bt2, anilideB~2 and cobinamide resulted in marked flattening of the graph (Fig. 7) as would be expected from the unsaturated vitamin B~,-binding capacity experiment, demonstrating saturation of vitamin B~2-binding substance in saliva which may be similar to non-intrinsic factor vitamin B ~ binders in gastric iuice. DISCUSSION

Vitamin B~2 analogues and derivatives may either behave like vitamin B1, physiologically, be metabolically inert, or act as vitamin B ~, antagonists ~°. Some analogues may appear inactive because they are poorly absorbed; when such compounds are administered parenterally they often show normal physiological function15, ~6. BISI~OI, et al. ~ showed that binding of vitamin B~2 to gastric juice was essential for optimal absorption. Gastric juice binding by vitamin BI~ analogues Although vitamin B12 binds to intrinsic factor in normal gastric juice, prior to absorption in man, workers have shown that there is no absolute correlation between vitamin Bx2 binding to gastric juice (because of its variable non-intrinsic factor content) and its subsequent absorptionV, 19. Boiled gastric iuice will, for instance, retain binding capacity but lose the ability to facilitate vitamin B12 absorption 7. Although BUNGE AND SCHILLING7 reported that gastric iuice showed no preferential binding of CN-B12 over sulfato-B~2, nitro-B~2 and chloro-Bx2 , ROSENBLUM et al. ~5,~6 found decreased cobalamin absorption in man and the rat when the CN radical was replaced by chloride, sulfate, nitrate, or thiocyanate groups. In the chick, these analogues, when fed by mouth gave a growth rate similar to that for CN-B~2 (ref. 20). However, these authors could not exclude possible intestinal transformation of analogues due to bacterial and/or enzymatic action or a basic difference in the absorption mechanism of different species; this problem is inherent in most absorption studies involving these compounds. HERBERT AND SULLIVAN~1 noted possible decreased absorption of coenzyme B ~ as compared to CN-B~2. In the present work CN-substituted analogues of CN-B12 were not investigated. ROSENBLUM, DAVIS AND CHOW17 studied a group of vitamin B~2 analogues with altered substituents in the benzimidazole moiety and suggested that while the 5,6disubstitution appeared essential for adequate absorption, 5,6-dichlorobenzimidazoleB~2 was absorbed half as well as CN-BI~ with a 5,6-dimethyl arrangement. HEINRICH19 confirmed this finding. However, BLACKBURN et al. 22 reported good absorption of desdimethyl-B12 in man, and LATNER AND RAINE2~ showed that this analogue competed with CN-B~2 in the rat. Pseudo-vitamin B~z (5,6-dimethylbenzimidazole replaced by adenine in the vitamin B12 molecule) does not interfere with CN-B~z absorption in man 24 or the rat 23 suggesting that it does not block intrinsic factor Biochim. Biophys. Acta, 141 (1967) 560-572

57 °

C . W . GOTTLIEB, F. P. RETIEF, V. HERBERT

binding by CN-B~.. BUNGE, SCHLOESSER AND SCHILLING~6, GREGORY AND HOLDSWORTH2v and TOPOREK et al. ~ found that gastric juice bound CN-BI~ in preference to pseudo-vitamin B~, (ref. 28) but chicken proventricular extract does not selectively bind CN-B1, in preference to the analogues, as does human gastric juice and pig gastric mucosa ~z. The vitamin B~2-absorption mechanism in the chick is possibly less intrinsic factor-dependent than in man. In the present study pseudo-vitamin B ~ bound selectively to the non-intrinsic factor fraction of gastric juice. One would thus expect it not to interfere with intrinsic factor-mediated vitamin B ~ absorption. BUNGE, SCHLOESSER AND SCHILLING~6 found 5,6-dimethylbenzimidazole inert as a blocker of vitamin B ~ absorption and we could similarly demonstrate no binding to gastric juice. Cobinamide, which lacks the vitamin B~2 "nucleotide" side-chain, does not interfere significantly with CN-B~2 absorption 2~, and GREGORY AND I-IOLDSWORTH ~v reported poor binding to gastric juice. We found cobinamide and cobrynamide fairly efficient binders of the non-intrinsic factor of gastric juice. HEI~RICH A ~ GABBE~ investigated the alkanolamine analogues characterized by substitutions at the aminopropanol linkage of the "nucleotide" radical. 2-Methyl-2-aminopropanol-B~ was shown to be an efficient blocker of vitamin B12 absorption in man and may thus be one of the few analogues capable of binding to intrinsic factor. I)L-I-Phenyl-2aminoethanol-Bl~ competed less with vitamin ]312 absorption, but both compounds are active vitamin B ~ antimetabolites in man. Monoethylamide-Bx~, an analogue with a substituted amide on a corrin nucleus propionamide, was shown to compete with vitamin B ~ absorption in the rat 2~. The related analogues, anilide-Bx~ and aquoanilide-B~ do not bind intrinsic factor in our experience, but rather may compete with vitamin B ~ for non-intrinsic factor binding sites in gastric juice. The cobaloximes were inert binders. Lactone-Bxz, with structural alteration of the ]3-ring in the corrin nucleus, does not affect vitamin Bx~ absorption in the rat ~3. Serum binding by vitamin B12 analogues BUI~GE AND SCHILLING~ showed that gastric juice bound CN-Blo in preference to 5,6-dimethylbenzimidazole-substituted analogues, pseudo-vitamin B~, lactone-B~ and lactam-B~, while serum bound most of these compounds as efficiently as CN-B~, with the exception of lactone- and lactam-Bl~. MEYER et al. 3° also found that lactamBx2 blocked CN-B~ binding less completely than other analogues tested such as hydroxo-B~2 and a spectrum of propionamide-substituted compounds. Hydroxo-Bx~ bound slightly better than CN-B~2. Our studies confirmed maximai binding by anilide-Bl~, aquoanilide-Bl~, pseudo-vitamin BI~ and cobaloxime C3, with poorer binding by cobinamide and cobrynamide. 5,6-Dimethylbenzimidazole and cobaloximes CI, C2, C4 and C5 were ineffective. It is perhaps of interest, that cobaloxime Cs has a side-chain (-CH~CH2COOCH3) structurally similar to the propionamide radical (-CH,CH,CONH2) of vitamin Blv Saliva: Previous workers ~ reported that pseudo-vitamin ]3~,, lactam-Bj 2, lactone-Bl~ and a variety of 5,6-dimethylbenzimidazole-substituted vitamin B ~ analogues bound to saliva as effectively as did CN-B~v Of the compounds tested by us, only 5,6-dimethylbenzimidazole and the cobaloximes were unable to block vitamin B1,binding sites. Biochim. Biophys. Acta, 14I (I967) 560-572

BLOCKADE OF HUMAN

BI~

BINDERS

571

It was thus evident that pretreatment with pseudo-vitamin B12, anilide-B12, aquoanilide-B1, and to a lesser extent cobinamide and cobrynamide, blocked subsequent binding of radioactive vitamin B1, to serum, saliva and non-intrinsic factor of normal gastric juice. None of the compounds tested affected the vitamin B~2-binding capacity of intrinsic factor, except CN-B, ~ and antibody to intrinsic factor. The finding that certain analogues could block the non-intrinsic factor binders in gastric juice suggested that these substances may be acceptable alternative in assays for intrinsic factor in vitro, in a manner akin to the use of antibody to intrinsic factor14-3L However, while gastric juice binder of two non-pernicious anemia patients with temporary intrinsic factor lack could be blocked by pseudo-vitamin BI~, pernicious anemia gastric juice, which contained no intrinsic factor, bound vitamin B,z analogues poorly and radioactive Vitamin B i ~binding was only partially blocked by pretreatment with nonradioactive vitamin B,~ (Table II). GLASSa2 postulated that an acid gastric juice contains "tertiary" vitamin BI~ binders distinct from the "primary" and "secondary" binders of normal gastric juice. The "tertiary" binder was thought to correspond with the "rapid" or R-binder of GR.~SBECK, SIMONS AND SINKKONEN~:~. GLASSa~ theorized that it may derive from ingested saliva, mucosal transudation of serum binders, or from mucus secreted by the atrophic gastric mucosa. Our evidence that pseudo-vitamin 812 binds well to serum and salivary vitamin BI, binders but not to pernicious anemia gastric juice, would count against the first two suggestions. More recently SIMONS~4 differentiated between a so-called F-binder, which in his experience often constituted the bulk of vitamin Bl~-binding material in pernicious anemia gastric juice but was ahnost absent from normal gastric juice, and the non-intrinsic factor R-binder of GRXSBECK, SIMONS AND SINKKONENaa. It is tempting to suggest that the F-binder may be responsible for analogue-resistance of pernicious anemia gastric juice. Little is known about the vitamin B1,-binding characteristics of bile and duodenal juice ~*, and it is possible that regurgitated duodenal secretions may be responsible for the vitamin Bi2-binding aberration of pernicious anemia gastric juice, as well as in other disorders of gastric secretion. ACKNOWLEDGEMENTS

This investigation was supported by U.S. Public Health Service Research Grants AM 09062 and AM 09564; and by the Albert A. List, Frederick Machlin, and Anna Ruth Lowenberg Funds. F.P.R. is a U.S. Public Health Service International Postdoctoral Research Fellow; award No. FO5TW 918. V.H. is a City of New York Health Research Council Career Scientist; award No. 1-435. The authors are indebted to Misses LE TENG GO, MELODY LEE and Mr. J. FARRELLY for technical assistance. REFERENCES

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Biochim. Biophys. Acta, 141 (I967) 560-572