141
Biochimica et Biophysica Acta, 421 (1976) 141--152
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27799 TISSUE DISTRIBUTION OF ENDOGENOUS COBALAMINS AND O T H E R CORRINS IN THE RAT, CAT AND GUINEA PIG
EDWARD V. QUADROS, DAVID M. MATTHEWS, IRENE J. WISE and JOHN C. LINNELL ~
Department o f Experimental Chemical Pathology, Vincent Square Laboratories o f Westminster Hospital, 124 Vauxhall Bridge Road, London SW1 V 2 R H (U.K.) (Received July 1st, 1975}
S u m mar y 1. Methylcobalamin, adenosylcobalamin, h y d r o x o c o b a l a m i n and cyanocobalamin have been estimated by a chromato-bioautographic technique in 16 tissues from healthy rats and in five guinea pig tissues. 2. Plasma and e r y t h r o c y t e cobalamins have been estimated in rats, cats and guinea pigs and the results compared with those in man. 3. Unidentified corrins were detected in 8 of the 16 rat tissues and in 3 of the 5 guinea pig tissues analysed, but were not present in tissues from specific pathogen-free rats nor in the standard laboratory diet. 4. Adenosylcobalamin was the major corrin in 8 of the 16 rat tissues. In the remainder h y d r o x o c o b a l a m i n pr edom i na ted or was present in equal proportions with adenosylcobalamin. Methylcobalamin was detected in the majority of rat tissues b u t at levels much lower than those in human tissues. Small amounts of cyanocobalamin were detected also and levels were higher than those o f m e t h y l c o b a l a m i n in 8 of the 16 tissues. 5. In the rat, cat and guinea pig, levels of methylcobalamin and h y d r o x o cobalamin were higher in e r y t h r o c y t e s than in plasma, a pattern almost the c o mp lete reverse of t hat in man.
Introduction Much o f the present knowledge of the distribution of cobalamins in the rat has been drawn from estimates of total cobalamin in the blood and certain organs [1--4] and from the distribution of radioactivity in animals following administration o f labelled h y d r o x o c o b a l a m i n (OH-Cbl) or cyanocobalamin
A b b r e v i a t i o n s : Ado-Cbl, a d e n o s y l c o b a l a m i n ; Me-Cbl, m e t h y l c o b a l a m i n ; CN-Cbl, c y a n o c o b a l a m i n ; OHoCbl, h y d r o x y c o b a l a m i n . * To w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e ~ .
142 {CN-Cbl) [5--7]. Neither of these methods can provide any information abo~t the identity of the individual forms of the vitamin present. Column chromatography has been used to separate radioactive cobalamins from extracts of rat liver and kidney following parenteral administration of labelled CN-Cbl or OHCbl [8] and an enzymic m e t h o d based on the dioldehydrase reaction has allowed the estimation of endogenous deoxyadenosylcobalamin (Ado-Cbl) in rat liver and kidney [ 9 ] . So far as we know other endogenous cobalamins have never been studied in this important laboratory animal. In man the separation of cobalamins was pioneered by Lindstrand and St~hlberg [10] who first detected in plasma a light, sensitive cobalamin later identified as methylcobalamin (Me-Cbl) [11]. More recently a method has been developed which allows estimation of the four cobalamins detectable in human plasma and tissues [12--14]. Results have been reported in control subjects and in patients with pernicious anaemia, folate deficiency and various other disorders [14,15]. Me-Cbl is normally the major form of the vitamin in h u m a n plasma while Ado-Cbl predominates in the tissues. Both of these cobalamins are k n o w n to have coenzyme functions in man. Me-Cbl is involved in the conversion of methyl folate to tetrahydrofolate, and the transmethylation of homocysteine to methionine, while Ado-Cbl is required for the conversion of methylmalonyl CoA to succinyl CoA. OH-Cbl, which is not known to possess any coenzyme function, is probably a precursor of both the cobalamin coenzymes and is found in substantial proportions in many tissues. CN-Cbl is normally only present in traces in h u m a n tissues, but increased proportions have been reported in the plasma of subjects whose dietary intake of cyanide is high, or in whom there is a possible disturbance of cyanide metabolism [16,17]. The distribution of the various cobalamins in small laboratory animals and their relative metabolic importance in each species is quite unknown. The present study provides relevant information about three animal species. Cobalamins have been estimated in 16 tissues from healthy rats and the results compared with those in the cat, guinea pig and man. Some of the results have already been reported in brief [18]. Materials and Methods Six male Wistar rats each weighing between 165 and 185 g were fed on Oxoid diet 41B and given water ad libitum. The animals were killed under ether anaesthesia by exsanguination via cardiac puncture. The autopsy and all subsequent stages up to and including chromatography were carried out in a darkroom by the light of a red photographic lamp or in darkness, to prevent photolytic loss of the cobalamin coenzymes. Blood was collected in heparinised tubes, and the plasma and erythrocytes were separated. The 'buffy coat' layer was rejected. The small bowel was divided 5 cm below the pylorus and a length of approximately 12 cm distal to the point of division taken as 'proximal small intestine'. A second 12 cm length was likewise removed 5 cm above the ileocaecal junction as 'distal small intestine'. Proximal and distal segments were washed out with saline, and the mucosa extruded by stroking with a glass rod. Slices approximately 1 m m thick were cut tangentially from the outer surface of each kidney and assayed as 'kidney cortex'. Further slices approximately
143 5 mm thick containing bot h cortical and medullary regions were cut away and rejected before the remaining inner part was retained as 'kidney medulla'. Cerebrum, cerebellum and brain stem were stored separately, but pituitary glands f r o m all six animals were pooled since each was t oo small to be assayed individually. Skeletal muscle was taken from the gastrocnemius. Other internal organs were r emove d whole. All tissues were wrapped immediately in aluminium foil, sealed in p o l y t h e n e bags and stored a t - - 2 0 ° C . Samples (0.1--0.5 g) of each tissue were homogenised in distilled water (10 ml) using a Potter-Elvehjem homogeniser. Total cobalamin was estimated by a radioisotopic m e t h o d [19], extracting the homogenates, red cells and plasma with a cyanide-containing acetate buffer [ 1 5 ] . Furt her aliquots of the tissue homogenates were extracted with h o t ethanol, desalted and a concentrated extract of cobalamins prepared. Cobalamins were separated by twodimensional ch r o m a t ogr aphy, located bioautographically with an E. coli m u t a n t and the zones quant i t at ed by p h o t o m e t r i c scanning and comparison with standards as previously described [ 1 2 - - 1 4 ] . Reference c o m p o u n d s were kindly supplied by Glaxo Laboratories, Greenford, Middlesex. Differences between the mean results in the various tissues were assessed by Student's 't' test. To determine the light sensitivity of corrins, whose R F values did not correspond with the cobalamin markers, an aliquot of aqueous tissue extract in a microcapillary tube was placed 30 cm from a 40 W tungsten lamp and exposed to light for 30 min. C hr om at ogr a phy and bioautography were then carried o u t as usual. Results
The results of cobalamin estimations in 16 tissues from healthy rats are shown in Table I. Total cobalamin levels were highest in the kidney, and values were significantly higher in the cortex t h a n in the medulla. Unlike the situation in man the total cobalamin level in rat kidney was some eight times higher than t h a t in the liver but the total cobalamin c o n t e n t of each of these organs was very similar (Fig. 1). Of the o t h e r tissues analysed, distal ileal mucosa contained higher total cobalamin levels than proximal ileal mucosa and cardiac muscle contained higher levels than skeletal muscle, though owing to the bulk of the latter (40% of the b o d y weight) the total cobalamin c o n t e n t of this tissue was higher than that o f any ot her (Fig. 1). Difference in total cobalamin between the various regions of the brain were not significant. In plasma and erythrocytes, levels were very similar to those in man. A major finding of the present study was that in all tissues, including plasma and er y th r ocyt es , Ado-Cbl and OH-Cbl together accounted for more than 70% of the total cobalamin. In contrast to the findings in hum an tissues, Me-Cbl appeared as a very minor c o m p o n e n t in the great majority of rat tissues, being undetectable in some and accounting for less than 2% of the total cobalamin in most. E r y t h r o c y t e s and kidney medulla were exceptions however, in that levels o f Me-Cbl were as high as those in the respective tissues in man. CN-Cbl was det ect ed in 13 of the 16 tissues. In most of these it represented only a small p r o p o r t i o n (1--5%) of the total cobalamin, b u t in distal ileal mucosa it ac c ount e d for 10%, twice as much as in the proximal (Fig. 2).
144 TABLE I COBALAMINS
Tissue *
Liver Kidney (cortex) Kidney (medulla) Spleen Adrenals Pituitary Brain (cerebrum) Brain (cerebellum) Brain (medulla) Heart Muscle Skeletal muscle Testis S m a l l intestinal mucosa (proximal) S m a l l intestinal mucosa (distal)
Plasma Erythrocytes
AND OTHER CORRINS
IN T I S S U E S F R O M H E A L T H Y
Total Cbl
Methyl-Cbl
pg/mg
pg/mg
6 4 ** +- 5.6 551
-+ 6 0
333 46 259
+ 41 + 3.2 +- 1 1 . 8
158
Cyano-Cbl %
-+
0.1
0.9 ± 0.1
0
7.8
±
3.2
1.4+- 0 . 5
8.7
-+ 9 . 1 ± 0.8
9 . 0 + 1.8 4 . 9 ± 1.9 0
13.1 1.7 0
0
0
0
Adenosyl-B-12
Pg/mg
0.6
32 2.2 0
RATS
% 0 +
4.8
116
± 10.8 + 0.1
2.9 -+ 2 . 2 3 . 7 -+ 0 . 4 0 0
133 ± 11.3 1 2 . 2 + 2.6 177 +- 1 3 . 4 54
-+
0.14
2.1 ± 0 . 6
18.9 +
2.2
0.1
1.9 + 0 . 3
26
±
2.0
3.5 + 0.8 1.7 +- 0 . 2
14.5 + 57 +-
2.1 7.4
2 . 3 +- 0 . 6 22 ± 3.6
6 . 5 -+ 11.1 ±
1.5 1.7
1.6
0.07 ±
0.03
0 . 2 -+ 0 . 0 8
0.6
44
±
1.7
0.2
0.16
0.5 + 0.3
0.8
0 . 1 7 -+ 0 . 0 2 1 . 8 -+ 0 . 3
0.7 + 0.2 1.6 + 0 . 1
1.1 2.1
+ ±
0.3 0.2
+ 4.4 + 14.5
+
1.5 ± 0.9
-+
31 126
44
5.0
30
±
pg/mg
±
± 18.5
9.0 ± 27 ±
1.8 2.7
0.05 ± 0
0.02
0.5 ± 0.2 0
0.16 6.0
± ±
0.06 1.3
25
±
3.2
0.3
+
0.1
2.3 ± 0 . 7
1.4
-+
0.2
4 . 8 -+ 0.6
12.1 +
2.6
46
+
6.4
3.6
±
1.2
7.7 ± 2.3
4.5
±
0.9
1 0 . 0 +- 1.6
13.2 ±
2.2
(pg/ml) 2.7 ± 1.5 3 1 . 5 + 21
0.6 ~ 0.3 10.5± 3.8
(pg/ml) 427 ± 29 300 + 54
(pg/ml) 123 + 48 4.8 ± 0.8
28 + 9.3 1.6 ± 0 . 2
(pg/ml) 230 + 21 75 ± 9.8
* n = 6 f o r all t i s s u e s e x c e D t p l a s m a a n d e r y t h r o c y t e s ( w h e r e n = 3 ) a n d p i t u i t a r y ( w h e r e t h e g l a n d s f r o m all 6 a n i m a l s w e r e p o o l e d a n d a s s a y e d as a s i n g l e s a m p l e ) . ** M e a n +- S E M .
The highest p r o p o r t i ons of CN-Cbl were found in plasma and testis, in each of which approximately a quarter of the total cobalamin was in this form. An u n e x p e c t e d finding in the present study was that in addition to the four k n o w n cobalamins, in eight of the 16 rat tissues, one and in some cases two unidentified corrins were detected (Table I). (Corrins contain any one of a n u m ber of possible nucleotides and each may have a different base. Cobalamins are corrins containing a nucleotide with 5,6-dimethylbenziminazole as base). Both unidentified corrins were of low mobility (R F 0.05) in the first solvent system (butan-2-ol/water/0.880 ammonia, 75 : 25 : 2), but in the second solvent (water saturated with benzyl alcohol), one was of medium mobility (RE 0.40) and designated corrin 'p', while the other moved almost with the solvent f r o n t (R F 0.95) and was designated corrin 'q'. Both c o m p o u n d s stimulated the growth of the E. coli m u t a n t used for bioautographic location of the separated zones on the chromatogram. Corrin 'p' was d e t e c t e d only in mucosa from the small intestine, and mainly in the distal region (Fig. 2) where it accounted for almost a fifth of the total cobalamin. Small amounts of corrin 'q'
145
TABLE I
Hydroxo-Cbl % 6 8 ± 2.3
pg/mg
Corrin 'p' %
19.2 ±
pg/mg
Coffin 'q' %
pg/mg
1.8
30
± 2.0
0
0
0.7 135
% ±
0.7
1.3
21 -+ 2.3
291
±
3.4
53
+ 3.1
0
0
± 10.6
24
4 1 ± 1.9 27 ± 6.1 68 ± 2.4 34
99 ± 18.4 ± 82 ± 104
8.9 2.9 4.6
30 39 32 66
± 1.0 ± 3.8 ± 2.4
0 0 0 0
0 0 0 0
54 11.7 0 0
± ±
1 6 . 7 ± 1.9 25 -+ 5 . 0 0 0
62 ± 4.2
10.5 ±
0.7
35
± 3.6
0
0
0
0
59 ± 4 . 2
16.7-+
2.0
38
± 4.7
0
0
0
0
48 ± 3.9 46 ± 2.5
15.2 ± 51 ±
2.6 6.3
48 41
± 3.5 ± 3.3
0 0
0 0
0 13.4
71+ 8.0 40± 4.3
2.0 ± 9.4 ±
0.8 1.0
26 35
± 8.0 ± 3.8
0 0
0 0
0 0.77 +
0.54
0 2.9 ± 2.1
±
6.9 2.4
6.3
± 1.3
0 1 0 . 2 ± 4.1
4827.2
10.2±
1.5
42
±7.4
0.4±0.3
1.2±0.9
0.5
~
0.4
1 . 4 ± 1.2
29±2.6
13.1±
1.0
30
±3.6
8.9±2.3
18.7±3.3
3.3
±
2.1
4.3±3.1
55±8.0 25±4.6
(pg/ml) 72 ± 2.7 189 ±38
1 6 . 9 ± 1.0 63 21.2
(pg/ml) 0 0
0 0
(pg/ml) 0 0
0 0
were d e t e c t e d in t h e ileal m u c o s a and in liver and testis, b u t very m u c h higher levels, c o r r e s p o n d i n g to b e t w e e n 10 and 25% o f the total cobalamin, were f o u n d in spleen (Fig. 3), k i d n e y and heart. T h e highest levels o f corrin ' q ' (135 + 11 pg/mg) were f o u n d in k i d n e y c o r t e x w h e r e it r e p r e s e n t e d a greater p r o p o r tion o f the t o t a l c o b a l a m i n t h a n t h a t o f Ado-Cbl and Me-Cbl t o g e t h e r . Since b o t h Ado-Cbl and Me-Cbl are k n o w n to be very m u c h m o r e sensitive to w h i t e light t h a n are the n o n - c o e n z y m e f o r m s CN-Cbl and OH-Cbl, we d e c i d e d to assess the light sensitivity o f corrins 'p' and 'q'. The results s h o w e d t h a t b o t h corrin ' p ' and ' q ' were m o r e stable t h a n Ado-Cbl or Me-Cbl and r e m a i n e d largely u n a f f e c t e d b y e x p o s u r e to light. It s e e m e d possible t h a t the u n i d e n t i f i e d corrins m i g h t have been derived f r o m the animals' diet {Oxoid 41B pellets). We t h e r e f o r e analysed a sample for cobalamins. T h e results (Table II) s h o w e d t h a t n e i t h e r corrins ' p ' or ' q ' were present. Surprisingly, the bulk o f the total c o b a l a m i n (60%) was in the f o r m of Ado-Cbl which had p r e s u m a b l y n o t b e e n c o n v e r t e d to OH-Cbl by light exp o s u r e d u r i n g m a n u f a c t u r e . The Ado-Cbl spot disappeared f r o m the chro-
146 KEY
~
~
~
300
~ ]
Me-B12
~
CN_B12
~
Ado_B12
~
OH_B12
~
Co~mn
30{
2oo
20,
~oo
q
~
I
.I
?0
i-~ I,:1
~0
i,i .,
•.
Iol
ol_
SKELETAL MUSCLE TOTAL B~2 600nq
III KIONE:y 500~q
500~q
_
HEART
71~q
TESTIS 58rag
BRAIN
5Stag
SPLEEN 25~9
ADRENALS tOmg
Fig. 1. T o t a l c o n t e n t o f e a c h c o z r i n in v a r i o u s r a t o r g a n s . V a l u e s f o r t h e k i d n e y are c a l c u l a t e d f r o m t h e u n w e i g h t e d m e a n of values for c o r t e x and medulla; those for total skeletal muscle a s s u m e that this tissue a c c o u n t s f o r 40°% o f t h e b o d y w e i g h t .
i
Fig. 2. S e p a r a t i o n o f c o b a l a m i n s a n d c o f f i n ' p ' in a s a m p l e o f r a t d i s t a l ileal m u c o s a . 1. O r i g i n ; 2. A d o - C b l ; 3. OIt-Cbl; 4. C o f f i n ' p ' ; 6. C N - C b h 7. Me-Cbl. In t h i s f i g u r e a n d in Fig. 3. t h e z o n e a d j a c e n t t o t h e o r i g i n p r o b a b l y r e p r e s e n t s u n s e p a r a t e d c o f f i n s a d s o z b e d to t h e c h r o m a t o g r a p h i c l a y e r .
147
2
Fig. 3. S e p a r a t i o n o f c o b a l a m i n s a n d c o r r i n ' q ' in a s a m p l e o f r a t spleen. 1. O r i g i n ; 2. A d o - C b l ; 3. O H - C b l ; 5. C o r r i n ' q ' .
matogram if the ext r a c t were first exposed to white light as described above. To investigate the possibility that either or bot h of the unidentified cortins may have been synthesised by the gut microflora, three specific pathogen free rats, which had been reared and maintained under sterile-barrier conditions were killed in the d a r k r o o m and tissues from each removed by red light in the same manner as previously described. The results (Table III) showed that in spleen and ileal mucosa, tissues containing high levels of corrins 'p' or 'q' in conventionally reared animals, no unidentified corrins were detected. Cobalamins have previously been estimated in the guinea pig using one dimensional c h r o m a t o g r a p h y and bioautography, and results from plasma, liver and small intestinal mucosa have been r e port ed [ 2 0 ] . Ado-Cbl + OH-Cbl were fo u n d to be the p r e d o m i n a n t cobalamins in these tissues, b u t an unidentified corrin o f low RF was also present in substantial amounts. The nature of this c o m p o u n d was n o t f ur t he r investigated. In the present study we noticed that corrins 'p' and 'q' in rat tissues bot h had a n R F Value in the first solvent closely T A B L E II COBALAMINS IN OXOID 41B SMALL ANIMAL DIET Cobalamins
Concentration (pg/mg)
T o t a l Cbl Me-Cbl CN-Cbl Ado-Cbl OH-Cbl Other coffins
14.1 none 2.1 8.5 3.5 none
detected (15%) (60%) (25%) detected
148 T A B L E lII T I S S U E C O B A L A M I N S * IN S P E C I F I C P A T H O G E N F R E E R A T S
Total-Cbl Methyl-Cbl
Cyano-Cbl
Adenosyl-Cbl
Hydroxo-Cbl
Corrin 'p'
Coffin 'q'
(pg/mg)
pg]ml %
pg/ml %
pg/ml %
pg]ml %
pg/ml %
pg/ml %
D i s t a l Ileal Mucosa Rat 1 2 3
65 45 116
Spleen Rat 1 2 3
4940 4030 4950
4 6 1
99 ND ND
5 13 1
13 6 24
21 14 20
16 12 31
25 27 27
32 21 60
49 46 52
None detected None detected None detected
2
1729 1402 1727
35 35 35
1072 983 1223
22 24 25
2040 1644 2000
41 41 40
None detected None detected None detected
* T h e h i g h p r o p o r t i o n o f C N - C b l in t h e s e t i s s u e s is m o s t likely a r e s u l t o f t h e C N - C b l d i e t a r y s u p p l e m e n t which the animals received.
similar to that of the unidentified corrin previously reported in the guinea-pig. Samples of plasma, erythrocytes, kidney, spleen and distal ileal mucosa from a further guinea-pig were therefore collected in the darkroom, and after the usual extraction process cobalamins were estimated by the two dimensional technique. The results (Table IV) confirmed the previous finding of a low RF spot in the first solvent. Two dimensional chromatography showed that this spot represented at least two corrins appearing on the bioautogram just below Ado° Cbl and OH-Cbl with RF values of 0.55 and 0.95, respectively. The faster running spot was thus of identical R F to corrin 'q', but the RF of the other spot (designated corrin 'o') was greater than that of corrin 'p'. Exposure of the ileal mucosal extract to light under conditions similar to that used to test the light sensitivity of rat corrins 'p' and 'q' showed that the unidentified guineapig corrins were likewise more stable to light than Me-Cbl or Ado-Cbl. To extend these studies further, plasma and erythrocyte cobalamins were estimated in five healthy cats and the results compared with those in rats, guinea-pigs and man (Fig. 4). Plasma and erythrocyte levels of total cobalamin were highest in the guinea pig, but in the cat and rat, levels were similar to those in man. The distribution of cobalamins, however, was in all three species, quite different from that in man. Thus in plasma from the rat and guinea pig, AdooCbl was the major cobalamin and only small amounts of Me°Cbl were detected. The pattern was very similar in the cat though Ado-Cbl and OH-Cbl were not separately estimated. Again unlike man, the erythrocytes in all these species contained higher proportions of Me-Cbl than did the plasma and in both rat and guinea pig, higher proportions of OH-Cbl and lower proportions of Ado-Cbl also. In the cat and guinea pig, as in man, the proportions of CN-Cbl were higher in erythrocytes than plasma, but in the rat the situation was strikingly reversed, the proportion of CN-Cbl in the plasma being more than 17 times that in the erythrocytes.
149
~Z
~ ~Z ~ ~ ~ ~ ~-~
~ ~
Z
150
S
H
Z
~
~
1O0
•
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[ Total
B12 :
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1~ U3
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B 12 :
~/ml
.....:
"'" ' " .... . : :2:'.2.
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560
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Fig. 4. Plasma
and
erythrocyte
not separately
estimated
~ g
:.: ~::
•
:...
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".'('."::
•
g
,
$
I
~ ~ , ~
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cobalamins
in the cat plasma
.~
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200 pg/ml in
t h e
guinea-pig,
or erythrocytes.
rat, cat and man.
,, M e - C b l ;
• CN-Cbl;
Ado-Cbl
and
~ Ado-Cbl;
OH-Cbl
w e r e
~ OH-Cbl.
Discussion
In man, recent studies have shown that Me-Cbl is normally the major cobalamin in plasma, while Ado-Cbl predominates in the tissues [14,15]. Some organs contain in addition to AdooCbl and OH-Cbl substantial amounts of Me-Cbl, but very little if any CN-Cbl has been detected in the normal tissues so far studied (liver, kidney, spleen, brain, pituitary, cardiac and skeletal muscle). The present investigation shows that in the rat, though total cobalamin levels in many tissues are similar to those in man, the distribution of individual cobalamins is very different. The main differences concern Me-Cbl, CN-Cbl and the unidentified corrins 'p' and 'q'. The extremely low proportion of Me-Cbl in many rat tissues suggests either that this coenzyme is of little importance in the methylation of homocysteine in this species which may proceed mainly via a cobalamin-independent pathway, or perhaps that Me-Cbl is only produced in the cell as required and then rapidly demethylated, when it would likely be detected as OH-Cbl by the technique used. Some support for this suggestion is provided by the finding that while in many rat tissues the proportion of Me-Cbl is very low, that of
151 OH-Cbl is high, and indeed may even be the major form as it is in kidney cortex, spleen and pituitary. In the rat, the distribution of plasma cobalamins resembles that in cobalamin-deficient man. Not only is Me-Cbl very low but the proportion of CN-Cbl is relatively high as in h u m a n pernicious anaemia [12,14,15]. An increase in plasma CN-Cbl has been reported in patients with tropical ataxic neuropathy [17], in tobacco amplyopia and in various inherited optic atrophies [16,21] and probably results either from an increased exposure to cyanide or to a disturbance in cyanide metabolism. A similar increase in CN-Cbl has recently been observed in plasma and tissues from baboons which were given cyanide and OH-Cbl [22]. In the rat, plasma cyanide and thiocyanate levels are normally higher than in man (ref. 23 and Quadros, E.V. and Dastur, D.K., unpublished) and it is possible that some cyanide combines with cobalamin to form CN-Cbl. This might explain the appearance of small amounts of CN-Cbl in many of the tissues in the present study, although the high proportion of CN-Cbl in the testis cannot satisfactorily be explained in this way. The distribution of cobalamins in the rat varies considerably between organs and may even, as in the kidney, vary within the same organ. The reason for this is not certain, but since Me-Cbl and Ado-Cbl are each known to participate in quite separate reactions it is possible that the cobalamin distribution in a tissue reflects its particular metabolic activity. Another question raised by the present study is why does the cobalamin pattern in a tissue vary so much between species? The normally high plasma Me-Cbl and low erythrocyte Me-Cbl in man for example, makes a striking contrast to the pattern in rat plasma and erythrocytes, where the distribution is almost completely reversed. Likewise in the guinea pig, Me-Cbl is low in the plasma and higher in erythrocytes. The high proportions of Ado-Cbl and possibly of CN-Cbl also in rat plasma may be a reflection of the levels of these cobalamins in the diet, though it is unlikely that the high plasma CN-Cbl arises entirely in this way. Recent studies have shown that in baboons the distribution of cobalamins between plasma and erythrocytes more closely resembles that in man [22], while in the cat the pattern appears to be intermediate between that in rodents and primates. The high level of corrin 'p' in mucosa from the distal small intestine of conventionally fed rats and its absence from this tissue in specific pathogen free animals adds weight to the suggestion that the gut microflora may be responsible for its synthesis, as may occur in the guinea pig also. Microorganisms known to inhabit the lower rat ileum include various species of coliforms, fusiforms, enterococci, bacteroides and lactobacilli [24], many of which are capable of synthesising cobalamin analogues. Porter [25] detected seven such analogues in rat faeces but traces of only one in the liver, which suggests that unlike CN-Cbl most cobalamin analogues are poorly absorbed in this species. It is n o t clear why in the present study corrin 'p' was detected only in the ileal mucosa while corrin 'q' was f o u n d in kidney and other tissues. Although it is possible that corrin 'p' may be converted to corrin 'q' either during absorption or in the kidney it is more likely that of the corrins produced by the microflora only corrin 'q' is absorbed in appreciable amounts by the rat intestine. The importance of this to the rat is not known though it is possible that the animal may
152
augment its intake of corrins in this way, as do ruminants. This could help. to explain why it is so difficult to produce u n d o u b t e d haematological signs of cobalamin deficiency in the rat, by dietary restriction alone.
Acknowledgements We thank Professor J.B. Cavanagh for kindly providing samples of cat blood. Financial support from the Wellcome Trust is gratefully acknowledged. One of us (EVQ) is the holder of a Commonwealth Tropical Medicine Award from the Ministry of Overseas Development.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
B r a e k k a n , O., Njaa, L . R . a n d U t n e , F. ( 1 9 5 7 ) A c t a P h a r m a c o l . ( K b h ) . 13, 2 2 8 - - 2 3 2 N a t h , N. a n d N a t h , M.C. ( 1 9 6 7 ) P r o c . Soc. E x p . Biol. ( N e w Y o r k ) 1 2 4 , 2 1 0 - - 2 1 3 G r o s s o w i c z , N., J a b l o n s k a , M., I z a k , G. a n d R a c h m i l e w i t z , M. ( 1 9 7 0 ) A m . J. Clin. N u t r . 2 3 , 1 2 7 - - 1 3 1 W i l h a m s , D.L. a n d S p r a y , G . H . ( 1 9 7 3 ) Brit. J. N u t r . 2 9 , 5 7 - - 6 3 N e w m a n , G . E . , O ' B r i e n , J . R . P . , S p r a y , G . H . a n d Witts, L.J. ( 1 9 6 2 ) in V i t a m i n B-12 u n d I n t r i n s i c F a c t o r . 2. Europ~/isches S y m p o s i o n , H a m b u r g ( H e i n r i c h , H.C., e d . ) , p p . 4 2 4 - - 4 3 2 , F. E n k e , S t u t t g a r t U c h i n o , H., Yagiri, Y., Y o s h i n o , T., K o n d o , M. a n d W a k i s a k a , G. ( 1 9 6 5 ) N a t u r e 2 0 5 , 1 7 6 - - 1 7 7 N e w m a r k , P.A. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 6 1 , 8 5 - - 9 3 Yagiri, Y. ( 1 9 6 7 ) J. V i t a m i n o l . 1 3 , 2 2 8 - - 2 3 8 T o o h e y , J.I. a n d B a r k e r , H . A . ( 1 9 6 1 ) J. Biol. C h e m . 2 3 6 , 5 6 0 - - 5 6 3 L i n d s t r a n d , K. a n d S t h h l b e r g , K-G. ( 1 9 6 3 ) A c t a Med. S c a n & 1 7 4 , 6 6 5 ~ 6 6 9 L i n d s t r a n d , K. ( 1 9 6 4 ) N a t u r e 2 0 4 , 1 8 8 - - 1 8 9 L i n n e l l , J.C., M a c k e n z i e , H., Wilson, J. a n d M a t t h e w s , D.M. ( 1 9 6 9 ) J. Clin. P a t h . 2 2 , 5 4 5 - - 5 5 0 L i n n e l l , J . C . , H u s s e i n , H . A - A . a n d M a t t h e w s , D.M. ( 1 9 7 0 ) J. Clin. P a t h . 23, 8 2 0 - 8 2 1 L i n n e l l , J . C . , H o f f b r a n d , A.V., P e t e r s , T.J. a n d M a t t h e w s , D.M. ( 1 9 7 1 ) Clin. Sci. 4 0 , 1 - - 1 6 Linnell, J.C., H o f f b r a n d , A.V., H u s s e i n , H . A - A . , Wise, I.J. a n d M a t t h e w s , D.M. ( 1 9 7 4 ) Clin. Sci. Mol. Med. 4 6 , 1 6 3 - - 1 7 2 Wilson, J., L i n n e l l , J.C. a n d M a t t h e w s , D.M. ( 1 9 7 1 ) L a n c e t 1, 2 5 9 - - 2 6 1 O s u n t o k u n , B.O., M a t t h e w s , D.M., H u s s e i n , H . A - A . , Wise, I.J. a n d L i n n e l l , J.C. ( 1 9 7 4 ) Clin. Sci. Mol. Med. 46, 563--567 Q u a d r o s , E.V., Wise, I.J., M a t t h e w s , D.M. a n d L i n n e l l , J.C. ( 1 9 7 5 ) Cliff. Sci. Mol. Med. 4 8 , 4 - - 5 P M a t t h e w s , D.M., G u n a s e g a r a m , R. a n d L i n n e l l , J . C . ( 1 9 6 7 ) J. Clin. P a t h . 2 0 , 6 8 3 - - 6 8 6 Peters, T.J., L i n n e l l , J . C . , M a t t h e w s , D.M. a n d H o f f b r a n d , A.V. ( 1 9 7 1 ) Brit. J. H a e m a t . 2 0 , 2 9 9 - - 3 0 5 Wilson, J. a n d M a t t h e w s , D.M. ( 1 9 7 0 ) T r a n s . O p h t h a l . S o c . U.K. 9 0 , 7 3 3 - - 7 3 7 C r a m p t o n , R . F , , G a u n t , I., LinneIl, J.C., M a t t h e w s , D.M., Mollin, D.L., S m i t h , W.T., Waters, A . H . a n d Wilson, J. in p r e p a r a t i o n S m i t h , A.D.M. a n d D u c k e t t , S. ( 1 9 6 5 ) Brit. J. E x p . P a t h . 4 6 , 6 1 5 - - 6 2 2 R a i b a u d , P., D i c k i n s o n , A.B., S a c q u e t , E., C h a r l i e r , H. a n d M o c q u o t , G. ( 1 9 6 6 ) A n n . Inst. P a s t e u r 111, 193--210 P o r t e r , J . W . G . ( 1 9 5 7 ) in V i t a m i n B-12 u n d I n t r i n s i c F a c t o r , 1. Europ~iisches S y m p o s i o n ( H e i n r i c h , H.C., ed.), p p . 4 3 - - 5 5 , F. E n k e , S t u t t g a x t
Biochimica et Biophysica Acta, 421 (1976) 141--152
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27799 TISSUE DISTRIBUTION OF ENDOGENOUS COBALAMINS AND O T H E R CORRINS IN THE RAT, CAT AND GUINEA PIG
EDWARD V. QUADROS, DAVID M. MATTHEWS, IRENE J. WISE and JOHN C. LINNELL ~
Department o f Experimental Chemical Pathology, Vincent Square Laboratories o f Westminster Hospital, 124 Vauxhall Bridge Road, London SW1 V 2 R H (U.K.) (Received July 1st, 1975}
S u m mar y 1. Methylcobalamin, adenosylcobalamin, h y d r o x o c o b a l a m i n and cyanocobalamin have been estimated by a chromato-bioautographic technique in 16 tissues from healthy rats and in five guinea pig tissues. 2. Plasma and e r y t h r o c y t e cobalamins have been estimated in rats, cats and guinea pigs and the results compared with those in man. 3. Unidentified corrins were detected in 8 of the 16 rat tissues and in 3 of the 5 guinea pig tissues analysed, but were not present in tissues from specific pathogen-free rats nor in the standard laboratory diet. 4. Adenosylcobalamin was the major corrin in 8 of the 16 rat tissues. In the remainder h y d r o x o c o b a l a m i n pr edom i na ted or was present in equal proportions with adenosylcobalamin. Methylcobalamin was detected in the majority of rat tissues b u t at levels much lower than those in human tissues. Small amounts of cyanocobalamin were detected also and levels were higher than those o f m e t h y l c o b a l a m i n in 8 of the 16 tissues. 5. In the rat, cat and guinea pig, levels of methylcobalamin and h y d r o x o cobalamin were higher in e r y t h r o c y t e s than in plasma, a pattern almost the c o mp lete reverse of t hat in man.
Introduction Much o f the present knowledge of the distribution of cobalamins in the rat has been drawn from estimates of total cobalamin in the blood and certain organs [1--4] and from the distribution of radioactivity in animals following administration o f labelled h y d r o x o c o b a l a m i n (OH-Cbl) or cyanocobalamin
A b b r e v i a t i o n s : Ado-Cbl, a d e n o s y l c o b a l a m i n ; Me-Cbl, m e t h y l c o b a l a m i n ; CN-Cbl, c y a n o c o b a l a m i n ; OHoCbl, h y d r o x y c o b a l a m i n . * To w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e ~ .
142 {CN-Cbl) [5--7]. Neither of these methods can provide any information abo~t the identity of the individual forms of the vitamin present. Column chromatography has been used to separate radioactive cobalamins from extracts of rat liver and kidney following parenteral administration of labelled CN-Cbl or OHCbl [8] and an enzymic m e t h o d based on the dioldehydrase reaction has allowed the estimation of endogenous deoxyadenosylcobalamin (Ado-Cbl) in rat liver and kidney [ 9 ] . So far as we know other endogenous cobalamins have never been studied in this important laboratory animal. In man the separation of cobalamins was pioneered by Lindstrand and St~hlberg [10] who first detected in plasma a light, sensitive cobalamin later identified as methylcobalamin (Me-Cbl) [11]. More recently a method has been developed which allows estimation of the four cobalamins detectable in human plasma and tissues [12--14]. Results have been reported in control subjects and in patients with pernicious anaemia, folate deficiency and various other disorders [14,15]. Me-Cbl is normally the major form of the vitamin in h u m a n plasma while Ado-Cbl predominates in the tissues. Both of these cobalamins are k n o w n to have coenzyme functions in man. Me-Cbl is involved in the conversion of methyl folate to tetrahydrofolate, and the transmethylation of homocysteine to methionine, while Ado-Cbl is required for the conversion of methylmalonyl CoA to succinyl CoA. OH-Cbl, which is not known to possess any coenzyme function, is probably a precursor of both the cobalamin coenzymes and is found in substantial proportions in many tissues. CN-Cbl is normally only present in traces in h u m a n tissues, but increased proportions have been reported in the plasma of subjects whose dietary intake of cyanide is high, or in whom there is a possible disturbance of cyanide metabolism [16,17]. The distribution of the various cobalamins in small laboratory animals and their relative metabolic importance in each species is quite unknown. The present study provides relevant information about three animal species. Cobalamins have been estimated in 16 tissues from healthy rats and the results compared with those in the cat, guinea pig and man. Some of the results have already been reported in brief [18]. Materials and Methods Six male Wistar rats each weighing between 165 and 185 g were fed on Oxoid diet 41B and given water ad libitum. The animals were killed under ether anaesthesia by exsanguination via cardiac puncture. The autopsy and all subsequent stages up to and including chromatography were carried out in a darkroom by the light of a red photographic lamp or in darkness, to prevent photolytic loss of the cobalamin coenzymes. Blood was collected in heparinised tubes, and the plasma and erythrocytes were separated. The 'buffy coat' layer was rejected. The small bowel was divided 5 cm below the pylorus and a length of approximately 12 cm distal to the point of division taken as 'proximal small intestine'. A second 12 cm length was likewise removed 5 cm above the ileocaecal junction as 'distal small intestine'. Proximal and distal segments were washed out with saline, and the mucosa extruded by stroking with a glass rod. Slices approximately 1 m m thick were cut tangentially from the outer surface of each kidney and assayed as 'kidney cortex'. Further slices approximately
143 5 mm thick containing bot h cortical and medullary regions were cut away and rejected before the remaining inner part was retained as 'kidney medulla'. Cerebrum, cerebellum and brain stem were stored separately, but pituitary glands f r o m all six animals were pooled since each was t oo small to be assayed individually. Skeletal muscle was taken from the gastrocnemius. Other internal organs were r emove d whole. All tissues were wrapped immediately in aluminium foil, sealed in p o l y t h e n e bags and stored a t - - 2 0 ° C . Samples (0.1--0.5 g) of each tissue were homogenised in distilled water (10 ml) using a Potter-Elvehjem homogeniser. Total cobalamin was estimated by a radioisotopic m e t h o d [19], extracting the homogenates, red cells and plasma with a cyanide-containing acetate buffer [ 1 5 ] . Furt her aliquots of the tissue homogenates were extracted with h o t ethanol, desalted and a concentrated extract of cobalamins prepared. Cobalamins were separated by twodimensional ch r o m a t ogr aphy, located bioautographically with an E. coli m u t a n t and the zones quant i t at ed by p h o t o m e t r i c scanning and comparison with standards as previously described [ 1 2 - - 1 4 ] . Reference c o m p o u n d s were kindly supplied by Glaxo Laboratories, Greenford, Middlesex. Differences between the mean results in the various tissues were assessed by Student's 't' test. To determine the light sensitivity of corrins, whose R F values did not correspond with the cobalamin markers, an aliquot of aqueous tissue extract in a microcapillary tube was placed 30 cm from a 40 W tungsten lamp and exposed to light for 30 min. C hr om at ogr a phy and bioautography were then carried o u t as usual. Results
The results of cobalamin estimations in 16 tissues from healthy rats are shown in Table I. Total cobalamin levels were highest in the kidney, and values were significantly higher in the cortex t h a n in the medulla. Unlike the situation in man the total cobalamin level in rat kidney was some eight times higher than t h a t in the liver but the total cobalamin c o n t e n t of each of these organs was very similar (Fig. 1). Of the o t h e r tissues analysed, distal ileal mucosa contained higher total cobalamin levels than proximal ileal mucosa and cardiac muscle contained higher levels than skeletal muscle, though owing to the bulk of the latter (40% of the b o d y weight) the total cobalamin c o n t e n t of this tissue was higher than that o f any ot her (Fig. 1). Difference in total cobalamin between the various regions of the brain were not significant. In plasma and erythrocytes, levels were very similar to those in man. A major finding of the present study was that in all tissues, including plasma and er y th r ocyt es , Ado-Cbl and OH-Cbl together accounted for more than 70% of the total cobalamin. In contrast to the findings in hum an tissues, Me-Cbl appeared as a very minor c o m p o n e n t in the great majority of rat tissues, being undetectable in some and accounting for less than 2% of the total cobalamin in most. E r y t h r o c y t e s and kidney medulla were exceptions however, in that levels o f Me-Cbl were as high as those in the respective tissues in man. CN-Cbl was det ect ed in 13 of the 16 tissues. In most of these it represented only a small p r o p o r t i o n (1--5%) of the total cobalamin, b u t in distal ileal mucosa it ac c ount e d for 10%, twice as much as in the proximal (Fig. 2).
144 TABLE I COBALAMINS
Tissue *
Liver Kidney (cortex) Kidney (medulla) Spleen Adrenals Pituitary Brain (cerebrum) Brain (cerebellum) Brain (medulla) Heart Muscle Skeletal muscle Testis S m a l l intestinal mucosa (proximal) S m a l l intestinal mucosa (distal)
Plasma Erythrocytes
AND OTHER CORRINS
IN T I S S U E S F R O M H E A L T H Y
Total Cbl
Methyl-Cbl
pg/mg
pg/mg
6 4 ** +- 5.6 551
-+ 6 0
333 46 259
+ 41 + 3.2 +- 1 1 . 8
158
Cyano-Cbl %
-+
0.1
0.9 ± 0.1
0
7.8
±
3.2
1.4+- 0 . 5
8.7
-+ 9 . 1 ± 0.8
9 . 0 + 1.8 4 . 9 ± 1.9 0
13.1 1.7 0
0
0
0
Adenosyl-B-12
Pg/mg
0.6
32 2.2 0
RATS
% 0 +
4.8
116
± 10.8 + 0.1
2.9 -+ 2 . 2 3 . 7 -+ 0 . 4 0 0
133 ± 11.3 1 2 . 2 + 2.6 177 +- 1 3 . 4 54
-+
0.14
2.1 ± 0 . 6
18.9 +
2.2
0.1
1.9 + 0 . 3
26
±
2.0
3.5 + 0.8 1.7 +- 0 . 2
14.5 + 57 +-
2.1 7.4
2 . 3 +- 0 . 6 22 ± 3.6
6 . 5 -+ 11.1 ±
1.5 1.7
1.6
0.07 ±
0.03
0 . 2 -+ 0 . 0 8
0.6
44
±
1.7
0.2
0.16
0.5 + 0.3
0.8
0 . 1 7 -+ 0 . 0 2 1 . 8 -+ 0 . 3
0.7 + 0.2 1.6 + 0 . 1
1.1 2.1
+ ±
0.3 0.2
+ 4.4 + 14.5
+
1.5 ± 0.9
-+
31 126
44
5.0
30
±
pg/mg
±
± 18.5
9.0 ± 27 ±
1.8 2.7
0.05 ± 0
0.02
0.5 ± 0.2 0
0.16 6.0
± ±
0.06 1.3
25
±
3.2
0.3
+
0.1
2.3 ± 0 . 7
1.4
-+
0.2
4 . 8 -+ 0.6
12.1 +
2.6
46
+
6.4
3.6
±
1.2
7.7 ± 2.3
4.5
±
0.9
1 0 . 0 +- 1.6
13.2 ±
2.2
(pg/ml) 2.7 ± 1.5 3 1 . 5 + 21
0.6 ~ 0.3 10.5± 3.8
(pg/ml) 427 ± 29 300 + 54
(pg/ml) 123 + 48 4.8 ± 0.8
28 + 9.3 1.6 ± 0 . 2
(pg/ml) 230 + 21 75 ± 9.8
* n = 6 f o r all t i s s u e s e x c e D t p l a s m a a n d e r y t h r o c y t e s ( w h e r e n = 3 ) a n d p i t u i t a r y ( w h e r e t h e g l a n d s f r o m all 6 a n i m a l s w e r e p o o l e d a n d a s s a y e d as a s i n g l e s a m p l e ) . ** M e a n +- S E M .
The highest p r o p o r t i ons of CN-Cbl were found in plasma and testis, in each of which approximately a quarter of the total cobalamin was in this form. An u n e x p e c t e d finding in the present study was that in addition to the four k n o w n cobalamins, in eight of the 16 rat tissues, one and in some cases two unidentified corrins were detected (Table I). (Corrins contain any one of a n u m ber of possible nucleotides and each may have a different base. Cobalamins are corrins containing a nucleotide with 5,6-dimethylbenziminazole as base). Both unidentified corrins were of low mobility (R F 0.05) in the first solvent system (butan-2-ol/water/0.880 ammonia, 75 : 25 : 2), but in the second solvent (water saturated with benzyl alcohol), one was of medium mobility (RE 0.40) and designated corrin 'p', while the other moved almost with the solvent f r o n t (R F 0.95) and was designated corrin 'q'. Both c o m p o u n d s stimulated the growth of the E. coli m u t a n t used for bioautographic location of the separated zones on the chromatogram. Corrin 'p' was d e t e c t e d only in mucosa from the small intestine, and mainly in the distal region (Fig. 2) where it accounted for almost a fifth of the total cobalamin. Small amounts of corrin 'q'
145
TABLE I
Hydroxo-Cbl % 6 8 ± 2.3
pg/mg
Corrin 'p' %
19.2 ±
pg/mg
Coffin 'q' %
pg/mg
1.8
30
± 2.0
0
0
0.7 135
% ±
0.7
1.3
21 -+ 2.3
291
±
3.4
53
+ 3.1
0
0
± 10.6
24
4 1 ± 1.9 27 ± 6.1 68 ± 2.4 34
99 ± 18.4 ± 82 ± 104
8.9 2.9 4.6
30 39 32 66
± 1.0 ± 3.8 ± 2.4
0 0 0 0
0 0 0 0
54 11.7 0 0
± ±
1 6 . 7 ± 1.9 25 -+ 5 . 0 0 0
62 ± 4.2
10.5 ±
0.7
35
± 3.6
0
0
0
0
59 ± 4 . 2
16.7-+
2.0
38
± 4.7
0
0
0
0
48 ± 3.9 46 ± 2.5
15.2 ± 51 ±
2.6 6.3
48 41
± 3.5 ± 3.3
0 0
0 0
0 13.4
71+ 8.0 40± 4.3
2.0 ± 9.4 ±
0.8 1.0
26 35
± 8.0 ± 3.8
0 0
0 0
0 0.77 +
0.54
0 2.9 ± 2.1
±
6.9 2.4
6.3
± 1.3
0 1 0 . 2 ± 4.1
4827.2
10.2±
1.5
42
±7.4
0.4±0.3
1.2±0.9
0.5
~
0.4
1 . 4 ± 1.2
29±2.6
13.1±
1.0
30
±3.6
8.9±2.3
18.7±3.3
3.3
±
2.1
4.3±3.1
55±8.0 25±4.6
(pg/ml) 72 ± 2.7 189 ±38
1 6 . 9 ± 1.0 63 21.2
(pg/ml) 0 0
0 0
(pg/ml) 0 0
0 0
were d e t e c t e d in t h e ileal m u c o s a and in liver and testis, b u t very m u c h higher levels, c o r r e s p o n d i n g to b e t w e e n 10 and 25% o f the total cobalamin, were f o u n d in spleen (Fig. 3), k i d n e y and heart. T h e highest levels o f corrin ' q ' (135 + 11 pg/mg) were f o u n d in k i d n e y c o r t e x w h e r e it r e p r e s e n t e d a greater p r o p o r tion o f the t o t a l c o b a l a m i n t h a n t h a t o f Ado-Cbl and Me-Cbl t o g e t h e r . Since b o t h Ado-Cbl and Me-Cbl are k n o w n to be very m u c h m o r e sensitive to w h i t e light t h a n are the n o n - c o e n z y m e f o r m s CN-Cbl and OH-Cbl, we d e c i d e d to assess the light sensitivity o f corrins 'p' and 'q'. The results s h o w e d t h a t b o t h corrin ' p ' and ' q ' were m o r e stable t h a n Ado-Cbl or Me-Cbl and r e m a i n e d largely u n a f f e c t e d b y e x p o s u r e to light. It s e e m e d possible t h a t the u n i d e n t i f i e d corrins m i g h t have been derived f r o m the animals' diet {Oxoid 41B pellets). We t h e r e f o r e analysed a sample for cobalamins. T h e results (Table II) s h o w e d t h a t n e i t h e r corrins ' p ' or ' q ' were present. Surprisingly, the bulk o f the total c o b a l a m i n (60%) was in the f o r m of Ado-Cbl which had p r e s u m a b l y n o t b e e n c o n v e r t e d to OH-Cbl by light exp o s u r e d u r i n g m a n u f a c t u r e . The Ado-Cbl spot disappeared f r o m the chro-
146 KEY
~
~
~
300
~ ]
Me-B12
~
CN_B12
~
Ado_B12
~
OH_B12
~
Co~mn
30{
2oo
20,
~oo
q
~
I
.I
?0
i-~ I,:1
~0
i,i .,
•.
Iol
ol_
SKELETAL MUSCLE TOTAL B~2 600nq
III KIONE:y 500~q
500~q
_
HEART
71~q
TESTIS 58rag
BRAIN
5Stag
SPLEEN 25~9
ADRENALS tOmg
Fig. 1. T o t a l c o n t e n t o f e a c h c o z r i n in v a r i o u s r a t o r g a n s . V a l u e s f o r t h e k i d n e y are c a l c u l a t e d f r o m t h e u n w e i g h t e d m e a n of values for c o r t e x and medulla; those for total skeletal muscle a s s u m e that this tissue a c c o u n t s f o r 40°% o f t h e b o d y w e i g h t .
i
Fig. 2. S e p a r a t i o n o f c o b a l a m i n s a n d c o f f i n ' p ' in a s a m p l e o f r a t d i s t a l ileal m u c o s a . 1. O r i g i n ; 2. A d o - C b l ; 3. OIt-Cbl; 4. C o f f i n ' p ' ; 6. C N - C b h 7. Me-Cbl. In t h i s f i g u r e a n d in Fig. 3. t h e z o n e a d j a c e n t t o t h e o r i g i n p r o b a b l y r e p r e s e n t s u n s e p a r a t e d c o f f i n s a d s o z b e d to t h e c h r o m a t o g r a p h i c l a y e r .
147
2
Fig. 3. S e p a r a t i o n o f c o b a l a m i n s a n d c o r r i n ' q ' in a s a m p l e o f r a t spleen. 1. O r i g i n ; 2. A d o - C b l ; 3. O H - C b l ; 5. C o r r i n ' q ' .
matogram if the ext r a c t were first exposed to white light as described above. To investigate the possibility that either or bot h of the unidentified cortins may have been synthesised by the gut microflora, three specific pathogen free rats, which had been reared and maintained under sterile-barrier conditions were killed in the d a r k r o o m and tissues from each removed by red light in the same manner as previously described. The results (Table III) showed that in spleen and ileal mucosa, tissues containing high levels of corrins 'p' or 'q' in conventionally reared animals, no unidentified corrins were detected. Cobalamins have previously been estimated in the guinea pig using one dimensional c h r o m a t o g r a p h y and bioautography, and results from plasma, liver and small intestinal mucosa have been r e port ed [ 2 0 ] . Ado-Cbl + OH-Cbl were fo u n d to be the p r e d o m i n a n t cobalamins in these tissues, b u t an unidentified corrin o f low RF was also present in substantial amounts. The nature of this c o m p o u n d was n o t f ur t he r investigated. In the present study we noticed that corrins 'p' and 'q' in rat tissues bot h had a n R F Value in the first solvent closely T A B L E II COBALAMINS IN OXOID 41B SMALL ANIMAL DIET Cobalamins
Concentration (pg/mg)
T o t a l Cbl Me-Cbl CN-Cbl Ado-Cbl OH-Cbl Other coffins
14.1 none 2.1 8.5 3.5 none
detected (15%) (60%) (25%) detected
148 T A B L E lII T I S S U E C O B A L A M I N S * IN S P E C I F I C P A T H O G E N F R E E R A T S
Total-Cbl Methyl-Cbl
Cyano-Cbl
Adenosyl-Cbl
Hydroxo-Cbl
Corrin 'p'
Coffin 'q'
(pg/mg)
pg]ml %
pg/ml %
pg/ml %
pg]ml %
pg/ml %
pg/ml %
D i s t a l Ileal Mucosa Rat 1 2 3
65 45 116
Spleen Rat 1 2 3
4940 4030 4950
4 6 1
99 ND ND
5 13 1
13 6 24
21 14 20
16 12 31
25 27 27
32 21 60
49 46 52
None detected None detected None detected
2
1729 1402 1727
35 35 35
1072 983 1223
22 24 25
2040 1644 2000
41 41 40
None detected None detected None detected
* T h e h i g h p r o p o r t i o n o f C N - C b l in t h e s e t i s s u e s is m o s t likely a r e s u l t o f t h e C N - C b l d i e t a r y s u p p l e m e n t which the animals received.
similar to that of the unidentified corrin previously reported in the guinea-pig. Samples of plasma, erythrocytes, kidney, spleen and distal ileal mucosa from a further guinea-pig were therefore collected in the darkroom, and after the usual extraction process cobalamins were estimated by the two dimensional technique. The results (Table IV) confirmed the previous finding of a low RF spot in the first solvent. Two dimensional chromatography showed that this spot represented at least two corrins appearing on the bioautogram just below Ado° Cbl and OH-Cbl with RF values of 0.55 and 0.95, respectively. The faster running spot was thus of identical R F to corrin 'q', but the RF of the other spot (designated corrin 'o') was greater than that of corrin 'p'. Exposure of the ileal mucosal extract to light under conditions similar to that used to test the light sensitivity of rat corrins 'p' and 'q' showed that the unidentified guineapig corrins were likewise more stable to light than Me-Cbl or Ado-Cbl. To extend these studies further, plasma and erythrocyte cobalamins were estimated in five healthy cats and the results compared with those in rats, guinea-pigs and man (Fig. 4). Plasma and erythrocyte levels of total cobalamin were highest in the guinea pig, but in the cat and rat, levels were similar to those in man. The distribution of cobalamins, however, was in all three species, quite different from that in man. Thus in plasma from the rat and guinea pig, AdooCbl was the major cobalamin and only small amounts of Me°Cbl were detected. The pattern was very similar in the cat though Ado-Cbl and OH-Cbl were not separately estimated. Again unlike man, the erythrocytes in all these species contained higher proportions of Me-Cbl than did the plasma and in both rat and guinea pig, higher proportions of OH-Cbl and lower proportions of Ado-Cbl also. In the cat and guinea pig, as in man, the proportions of CN-Cbl were higher in erythrocytes than plasma, but in the rat the situation was strikingly reversed, the proportion of CN-Cbl in the plasma being more than 17 times that in the erythrocytes.
149
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~ ~
Z
150
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H
Z
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~
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[ Total
B12 :
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~
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2300
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B 12 :
~/ml
.....:
"'" ' " .... . : :2:'.2.
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Fig. 4. Plasma
and
erythrocyte
not separately
estimated
~ g
:.: ~::
•
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. ' •
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,
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t h e
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rat, cat and man.
,, M e - C b l ;
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and
~ Ado-Cbl;
OH-Cbl
w e r e
~ OH-Cbl.
Discussion
In man, recent studies have shown that Me-Cbl is normally the major cobalamin in plasma, while Ado-Cbl predominates in the tissues [14,15]. Some organs contain in addition to AdooCbl and OH-Cbl substantial amounts of Me-Cbl, but very little if any CN-Cbl has been detected in the normal tissues so far studied (liver, kidney, spleen, brain, pituitary, cardiac and skeletal muscle). The present investigation shows that in the rat, though total cobalamin levels in many tissues are similar to those in man, the distribution of individual cobalamins is very different. The main differences concern Me-Cbl, CN-Cbl and the unidentified corrins 'p' and 'q'. The extremely low proportion of Me-Cbl in many rat tissues suggests either that this coenzyme is of little importance in the methylation of homocysteine in this species which may proceed mainly via a cobalamin-independent pathway, or perhaps that Me-Cbl is only produced in the cell as required and then rapidly demethylated, when it would likely be detected as OH-Cbl by the technique used. Some support for this suggestion is provided by the finding that while in many rat tissues the proportion of Me-Cbl is very low, that of
151 OH-Cbl is high, and indeed may even be the major form as it is in kidney cortex, spleen and pituitary. In the rat, the distribution of plasma cobalamins resembles that in cobalamin-deficient man. Not only is Me-Cbl very low but the proportion of CN-Cbl is relatively high as in h u m a n pernicious anaemia [12,14,15]. An increase in plasma CN-Cbl has been reported in patients with tropical ataxic neuropathy [17], in tobacco amplyopia and in various inherited optic atrophies [16,21] and probably results either from an increased exposure to cyanide or to a disturbance in cyanide metabolism. A similar increase in CN-Cbl has recently been observed in plasma and tissues from baboons which were given cyanide and OH-Cbl [22]. In the rat, plasma cyanide and thiocyanate levels are normally higher than in man (ref. 23 and Quadros, E.V. and Dastur, D.K., unpublished) and it is possible that some cyanide combines with cobalamin to form CN-Cbl. This might explain the appearance of small amounts of CN-Cbl in many of the tissues in the present study, although the high proportion of CN-Cbl in the testis cannot satisfactorily be explained in this way. The distribution of cobalamins in the rat varies considerably between organs and may even, as in the kidney, vary within the same organ. The reason for this is not certain, but since Me-Cbl and Ado-Cbl are each known to participate in quite separate reactions it is possible that the cobalamin distribution in a tissue reflects its particular metabolic activity. Another question raised by the present study is why does the cobalamin pattern in a tissue vary so much between species? The normally high plasma Me-Cbl and low erythrocyte Me-Cbl in man for example, makes a striking contrast to the pattern in rat plasma and erythrocytes, where the distribution is almost completely reversed. Likewise in the guinea pig, Me-Cbl is low in the plasma and higher in erythrocytes. The high proportions of Ado-Cbl and possibly of CN-Cbl also in rat plasma may be a reflection of the levels of these cobalamins in the diet, though it is unlikely that the high plasma CN-Cbl arises entirely in this way. Recent studies have shown that in baboons the distribution of cobalamins between plasma and erythrocytes more closely resembles that in man [22], while in the cat the pattern appears to be intermediate between that in rodents and primates. The high level of corrin 'p' in mucosa from the distal small intestine of conventionally fed rats and its absence from this tissue in specific pathogen free animals adds weight to the suggestion that the gut microflora may be responsible for its synthesis, as may occur in the guinea pig also. Microorganisms known to inhabit the lower rat ileum include various species of coliforms, fusiforms, enterococci, bacteroides and lactobacilli [24], many of which are capable of synthesising cobalamin analogues. Porter [25] detected seven such analogues in rat faeces but traces of only one in the liver, which suggests that unlike CN-Cbl most cobalamin analogues are poorly absorbed in this species. It is n o t clear why in the present study corrin 'p' was detected only in the ileal mucosa while corrin 'q' was f o u n d in kidney and other tissues. Although it is possible that corrin 'p' may be converted to corrin 'q' either during absorption or in the kidney it is more likely that of the corrins produced by the microflora only corrin 'q' is absorbed in appreciable amounts by the rat intestine. The importance of this to the rat is not known though it is possible that the animal may
152
augment its intake of corrins in this way, as do ruminants. This could help. to explain why it is so difficult to produce u n d o u b t e d haematological signs of cobalamin deficiency in the rat, by dietary restriction alone.
Acknowledgements We thank Professor J.B. Cavanagh for kindly providing samples of cat blood. Financial support from the Wellcome Trust is gratefully acknowledged. One of us (EVQ) is the holder of a Commonwealth Tropical Medicine Award from the Ministry of Overseas Development.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
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