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The in vivo effect of vitamin E in experimental porphyria
112
B I O C H I M I C A ET B I O P H Y S I C A ACTA
BBA 26389 T H E I N V I V O E F F E C T OF VITAMIN E IN E X P E R I M E N T A L P O R P H Y R I A PADMANABHAN P. NAIR, HARI S. MURTY AND NEAL R. GROSSMAN* Biochemistry Research Division, Department of 3,Iedicine, Sinai Hospital of Baltimore, Inc., Baltimore, Md. 2r2r 5 (U.S.A.)
(Received March 9th, ~97o)
SUMMARY
The induction of experimental porphyria in rats by allylisopropylacetamide is characterized by elevation of hepatic &aminolevulinate synthetase and dehydratase activities. The administration of vitamin E 6 h prior to initiation of the porphyrinogenic regimen prevented porphyria through a control exercised on the activities of the two hepatic enzymes. The bone marrow of porphyric animals showed enhanced synthetase activity which was prevented by vitamin E. In this paper, evidence is presented to show that vitamin E deficiency increases susceptibility to the induction of &aminolevulinate synthetase by allylisopropylacetamide, indicating that the vitamin is in some way involved in the regulation of the inducible enzyme. Since other studies have shown that the normal constitutive enzyme is vitamin E dependent in vivo, the question is posedwhether the normal and inducible forms represent isozymes of d-aminolevulinate synthetase.
INTRODUCTION Vitamin E, as a member of the group of fat-soluble vitamins, has remained an enigma since its discovery by EVANS AND BISHOP1. A deficiency of this vitamin leads to a variety of species-specific manifestations which could not be explained mechanistically on a unified basis 2. Recent studies have shown the existence of an anemia associated with a lack of this vitamin in man and in primates 3-6. Similarly, our recent studies have demonstrated that vitamin E deficiency in the rat, even in the absence of a demonstiable anemia, leads to a ~ignificant depression in heine synthesis and that the accompanying metabolic detect involving d-aminolevulinate synthetase and dehydratase could be corrected only by the administration ot the vitamin in vivo 7. In contrast, vitamin E in vivo prevents the allylisopropylacetamide-mediated induction of experimental porphyria s as well as causing a remission in the human variety of the same disorder 9. Since both human and experimental porphyrias are characterized by abnormally high activities for hepatic &aminolevulinate synthetase and dehydratase 1°, the present study was undertaken to examine the effect of vitamin E on these "inducible" enzymes. In this paper we have shown that the action of * N. R.G. is a summer research participant. Biochim. Biophys. dcta, 215 (197o) t I 2 II8
VITAMIN E AND EXPERIMENTAL PORPHYRIA
113
vitamin E in preventing the induction of experimental porphyria is effected through a suppression of "inducible" 8-aminolevulinate synthetase and dehydratase. EXPERIMENTAL
Materials Allylisopropylacetamide was a generous gift from Dr. Scott, Hoffmann LaRoche, Nutley, N.J. b-Aminolevulinic acid was purchased from Calbiochem, Los Angeles. All other reagents were obtained trom standard commercial sources.
Methods Induction of experimental porphyria. Experimental porphyria was induced in male Wistar strain rats weighing 15o-2oo g by repeated injections of allylisopropylacetamide s. Groups of 4-5 animals were conditioned in metabolic cages for a period of 3 days and then food was withheld I2 h before the beginning of the experiment. One group of rats served as control, while a second gI oup received orally single doses of ioo mg of vitamin E per rat. Porphyria was induced in two other groups of rats by four successive daily injections of allylisopropylacetamide (400 mg/kg body weight in isotonic saline) subcutaneously. In addition to allylisopropylacetamide, vitamin E was administerea (single Ioo-mg doses per rat) to rats in one of the groups approx. 6 h before the first injection with allylisopropylacetamide. All animals in the control groups received injections of isotonic saline identical in volume to that used for administering allylisopropylacetamide. Porphyria was confirmed in allylisopropylacetamide treated animals b y the detection of 3-aminolevulinate, porphobilinogen and porphyrins in the urine 11,12. All animals were sacrificed under ethel anesthesia by exsanguination from the abdominal a o r ' a 5 days aftei the first injection with allylisopropylacet amide. Establishment of vitamin E deficiency Weanling, male Wistai strain rats were fed a standard vitamin E deficient diet for a period of 4-5 months as described in an earlier publication 13, Vitamin E deficiency was confirmed b y determinations of hepatic and plasma vitamin E levels b y gas liquid chromatogiaphic procedures developed in this laboratory 14-16. A corresponding control group of animals was concurrently reared on the same basal vitamin E deficient diet supplemented with vitamin E, 50 mg/kg diet. Hepatic b-aminolevulinate synthet ase was induced in both vitamin E-deficient and control animals by subcutaneous injections of allylisopropylacetamide, 50 mg/kg body weight. Assay of hepatic b-aminolevulinate synthetaseand dehydratase activities. Hepatic b-aminolevulinate synthetase was assayed according to the procedure of MARVER et al. 17. The assay of hepatic ~-aminolevulinate dehydratase activity was carried out according to the original method of GIBSON et al) s. Assay of bone marrow 8-aminolevulinate synthetase. Femoral bone marrow, aspirated with a hypodermic syringe equipped ~ith a i9-gauge needle, was thoroughly suspended in 2.0 ml of T r i s - E D T A buffer (pH 7.4). The incubation mixture, which consisted of I.O ml ot bone marrow suspension, 400/~moles of glycine, 20/~moles of EDTA, I5o/~moles of Tris-HC1 buffer p H 7.4) and IO/,moles of glucose in a total volume of 3.0 ml, was incubated aerobically for I h at 37 °. The reaction was terminated Biochim. Biophys. dcta, 215 (197 o) 112-118
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114
by the addition of 0.75 ml of 25 % trichloroacetic acid and the 6-aminolevulinate formed was condensed with acetyl acetone to form the corresponding pyrrole, which was then determined with Ehrlich's reagent n. Endogenous 6-aminolevulinate was simultaneously determined in a control incubation mixture to which trichloroacetic acid was added at zero time. Since the erythroid cell population remains unaltered under the experimental conditions, the results are expressed as nmoles of &aminolevulinate formed per 11 per mg protein, the enzymatic activity being linear for this time period. Since sulfhydryl group activators such as glutathione are required for the activation of bone marrow &aminolevulinate dehydratasO s, this enzyme did not interfere with the assay of b-aminolevulinate synthetase activity in this tissue. Furthermore, under our assay conditions, the incorporation of radioactivity from (2J4C]glycine into heine was negligible indicating that &aminolevulinate dehydratase was inactivated in this system. No aminoacetone was detected in the assay system, as shown by the absence of Ehrlich-positive material in the aminoacetone fraction isolated by chromatography on Dowex I-X8 (acetate form).
Incorporation of radioactivity from i2-14C]glycine into heine by bone marrow cells19. The bone marrow extracted from one femur was suspended in Krebs Ringer phosphate buffer (pH 7-4) centrifuged for 5 rain at 50o × g and reconstituted with the same buffer. The process was repeated twice to obtain the final cell suspension, an appropriate aliquot of which was added to the incubation mixture containing 20 mg of glucose, 20 mg of bovine serum albumin, I.o mg each of penicillin and streptomycin sulfate and lO #C of I2-l~C]glycine (specific activity 21.8 mC/mmole) in a total volume of IO ml. Incubations were carried out aerobically at 37 ° in 5o-ml erlenmeyer flasks for 2 h. At the end of the incubation period, the reaction mixture was chilled to 0 ° and heine was isolated as hemin after the addition of 200 mg of carrier hemoglobin ~9. The radioactivity in the isolated heroin was assayed by liquid scintillation counting. RESULTS AND DISCUSSION
Effect of vitamin E on hepatic &aminolevulinate synthetase and dehydratase Since vitamin R was shown to inhibit the induction of experimental porphyria by allylisopropylacetamide s, its effect on the key enzymatic steps in the early biogenetic sequence leading to heine was studied. A single close of vitamin E, administered to rats approx. 6 tl before initiation of the porphyrinogenic regimen, effectively blocked the induction of both &aminolevulinate synthetase and dehydrase activities (Tables I and II). However, the vitamin, when given alone to control animals, induced a significant rise in the activity of both these hepatic enzymes contrary to our expectations.
Effect of vitamin E on bone marrow &aminolevulinate synthetase and on the incorporation of radioactivity from L2-1~C]glycine into heine The response of &aminolevulinate synthetase in mammalian cells to environmental agents depends not only on the type of cell involved but also on the chemical agents tested 2°. Thus it has been shown that 5fl-H steroids enhanced heine synthesis (as well as b-aminolevulinate synthetase) only in embryonic liver and erythroid cell precursors 21, whereas other nonsteroidal porphyrinogenic chemicals acted exclusively Biochim. Biophys. dcla, 2 I 5 (I97O) I I 2 - I I 8
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TABLE I EFFECT
OF V I T A M I N
E
ON H E P A T I C
(~-AMINOLEVULINATE
ALLYLISOPROPYLACETAMIDE-TREATED
SYNTHETASE
ACTIVITY OF NORMAL
AND
RATS
Results are expressed as nmoles of b-aminolevulinate produced per h. Values represent mean of four individual observations and their S,E. Details of the treatment of the four groups are described under EXPERIMENTAL, Statistical significance of differences in mean 6-aminolevulinate synthetase activities between different groups are: control with respect to control --. vitamin E, P < o . o i and --<0.02; control with respect to allylisopropylacetamide, P < o . o o i and < o . o o i ; control with respect to allylisopropylacetamide + vitamin E, P > o . 3 and >o.7; control + vitamin E with respect to allylisopropylacetamide, P < o . o o i and < o . o i ; control + vitamin E with respect to allylisopropylacetamide + vitamin E, P > o.2 and < o.2 ; allylisopropylacetamide with respect to allylisopropylacetamide + vitamin E, P < o . o o i and < o.ooi. In each pair of P values, the first one corresponds to activity expressed per g wet weight of liver and the second to specific activity. Specific activity is expressed as nmoles of 6-aminolevulinate synthesized per h pcr g protein.
Treatment
6-Aminolevulinate synthetase activity Per g liver
Control Control + vitamin E Allylisopropylacetamide Allylisopropylacetamide + vitamin E
29.94 56.26 234.4 42.58
± ± 4±
Specific activity
4-95 5 .68 23.7 9.5
162. 4 354.3 132o 241.7
± 26.6 ± 23.9 ~_ 72 ± 39.8
TABLE II EFFECT
OF V I T A M I N g
ON H E P A T I C ~ - A M I N O L E V U L I N A T E
ALLYLISOPROPYLACETAMIDE-TREATED
DEHYDRATASE
A C T I V I T Y OF N O R M A L A N D
RATS
Results are expressed as nmoles of porphobilinogen produced per h per g of wet weight tissue. Values represent mean of four individual observations and their S.E. Statistical significance of differences in mean 6-aminolevulinate dehydratase activities between different groups are : control with respect to control + vitamin E, P > o . 3 ; control with respect to allylisopropylacetamide, P < o.ooi ; allylisopropylacetamide with respect to allylisopropylacetamide + vitamin E, P < o.ooi. Experimental details are given in the text.
Treatment
Control Control + vitamin E Allylisopropylacetamide Allylisopropylacetamide + vitamin E
d-A minolevulinate dehydratase activity 57 r.8-L 627.4-c 2o66 ± 562.9 -[_
33-4 36.4 125 82.3
o n n o n e r y t h r o i d h e p a t i c cells22,2% H o w e v e r , i n e x p e r i m e n t a l p o r p h y r i a i n d u c e d b y a l l y l i s o p r o p y l a c e t a m i d e , w e o b s e r v e d a s m a l l b u t s i g n i f i c a n t r i s e in b o n e m a r r o w & a m i n o l e v u l i n a t e s y n t h e t a s e , p r e v e n t a b l e b y p r i o r a d m i n i s t r a t i o n of v i t a m i n E ( T a b l e I I I ) . U n l i k e liver, t h e b o n e m a r r o w of c o n t r o l a n i m a l s w h i c h r e c e i v e d t h e vitamin did not exhibit a response. S i n c e t h e rise in b o n e m a r r o w & a m i n o l e v u l i n a t e s y n t h e t a s e i n d u c e d b y allylisopropylacetamide was minimal, the treatment with allylisopropylacetamide was not a c c o m p a n i e d b y an increase in h e m e synthesis. On t h e c o n t r a r y , t h e r e was a decrease in t h e i n c o r p o r a t i o n of r a d i o a c t i v i t y f r o m g l y c i n e i n t o h e m e (Table IV). V i t a m i n E
Biochim. Biophys. dcta, 215 (197o) 112-1 i8
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t'. P. x.-\tR et al.
TABLE III EFFECT AND
OF VITAMIN
g
O N B O N E MARRO\X," ( ~ - A M I N O L E V U L I N A T E S Y N T H E T A S E
ALLYLISOPROPYLACETAMIDE-TREATED
A C T I V I T Y OF N O R M A L
RATS
Results are expressed as nmoles of d-anfinolevulinate produced per h per m g proteiu. Values represent m e a n of four individual o b s e r v a t i o n s and their S.E. Statistical significance of differences in m e a n 6-aminolevulinate s y n t h e t a s e activities between different groups are : control with respect to allylisopropylacetamide, / ~ < o . o o i ; control - v i t a m i n E w i t h respect to allylisopropylacetamide, P < o . o 5 ; allylisopropylacetamide with respect to allylisopropylacetamide + v i t a m i n E, P < o . i . E x p e r i m e n t a l details are given in the text.
Treatment
6- d minolevulinate synthetase activity
Control Control + v i t a m i n E Allylisopropylacetamide Allylisopropylacetamide + v i t a m i n E
32. 4 39.6 6i.i 4o.2
~_ 2.6 ~h 7.4 :~ 3.4 -- 8. 5
T A B L E 1V EFFECT INTO
OF V I T A M I N
HEME
BY BONE
E
ON T H E
INCORPORATION
in vitro OF
RADIOACTIVITY
FROM
[2-14C3GLYCINE
MARROXV
Results are m e a n s of observations from two animals in each group, expressed as disint./min per mg of bone m a r r o w protein. E x p e r i m e n t a l details are given in the text. Treatment
Activity (Disint./min × ±o-4)
Control Control -i- v i t a m i n E Allylisopropylacetamide Allylisopropylaeetamide + v i t a m i n E
3.97 4.05 2.2 i 2.64
p a r t i a l l y c o u n t e r a c t e d this defect. However, t h e a c t i o n of a l l y l i s o p r o p y l a c e t a m i d e on t h e bone m a r r o w is n o t clear a t t h e m o m e n t .
The effect of allylisopropylacetamide on the induction of hepatic b-aminolevulinate synthetase in vitamin E-deficient rats Since vitamin E blocks the induction of &aminolevulinate synthetase in the livers of allylisopropylacetamide-treated animals, experiments were conducted to compare the degree of induction of this enzyme in vitamin E-deficient animals and the corresponding controls. Table V presents data showing that with identical doses of allylisopropylacetamide, synthetase activities in vitamin E-deficient livers were approx. 5-fold higher than those in vitamin E-fed control livers. In experiments not reported here, addition in vitro of vitamin E or liver homogenates of vitamin E-treated rats did not alter the activity of the induced enzyme in homogenates of porphyric rat liver. This observation rules out a direct inhibitory action of vitamin E on 8-aminolevulinate synthetase.
Role of vitamin E in the regulation of heine synthesis W e h a v e shown in a previous c o m m u n i c a t i o n t h a t in t h e v i t a m i n E-deficient r a t , e v e n in t h e a b s e n c e of a d e m o n s t r a b l e
Biochim. Biophys. Acta, 215 (197 o) 112-118
a n e m i a , t h e r e is a d i s t i n c t d e p r e s s i o n i n
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TABLE V COMPARISON OF ALLYLISOPROPYLACETAMIDE-MEDIATEDINDUCTION OF HEPATIC d-AMINOLEVULINATE SYNTHETASE IN NORMAL AND VITAMIN E-DEFICIENT RATS A l l y l i s o p r o p y l a c e t a m i d e d i s s o l v e d in p h y s i o l o g i c a l saline w a s i n j e c t e d s u b c u t a n e o u s l y (5 ° m g / k g b o d y weig ht) i n t o n o r m a l a n d v i t a m i n E - d e f i c i e n t a n i m a l s . The s e a n i m a l s w e re sacrificed 3 h l a t e r a n d t h e i r h e p a t i c d - a m i n o l e v u l i n a t e s y n t h e t a s e a s s a y e d . R e s u l t s are e x p r e s s e d as n m o l e s of d - a m i n o l e v u l i n a t e f o r m e d per g of t i s s u e p e r h a n d r e p r e s e n t s m e a n of four i n d i v i d u a l o b s e r v a t i o n s a n d t h e i r s t a n d a r d error of m e a n .
Groups
Control Vitamin E deficient
d-A minolevulinate synthetase
4o.7 ± 8.1 251
4- io
the ability of the bone marrow and liver to synthesize heme, the defect residing at one or both of the two enzymatic steps, d-aminolevulinate synthetase and &aminolevulinate dehydratase 7. The mechanism b y which vitamin E prevents experimental porphyria s and also causes a remission in human porphyria 9 probably involves the blocking of the induction of &aminolevulinate synthetase and secondarily that of &aminolevulinate dehydratase as seen in this study. Since the response to vitamin E, in vivo, of &aminolevulinate synthetase in the vitamin E-deficient and porphyric animals are mutually divergent, it appears that &aminolevulinate synthetase might exist as isozymes representing the constitutive and inducible forms. Similarly, LABBE et al. 24 suggested the existence of isozymes b y showing that the inducible forms of both succinyl-CoA and &aminolevulinate synthetases possessed characteristics which were different from those of their corresponding constitutive enzymes. Although vitamin E deficiency does not manifest itself in the form of porphyria, the levels of &aminolevulinate synthetase induced in these animals by porphyrinogenic agents are distinctly higher than those in their corresponding controls. This increased susceptibility to induction suggests a possible role for this vitamin at a repressor site for &aminolevulinate synthetase. On the other hand, a chemical agent such as ethionine when fed to rats produces not only symptoms of vitamin E deftciency ~5 but also induces porphyria ~6 without an associated rise in the activity of &aminolevulinate synthetase. According to current concepts, the biosynthesis of heme could be regulated b y (a) the rate of generation of succinyl-CoA, (b) end product inhibition or repression of O-aminolevulinate synthetase b y free heme or (c) control of the synthesis of globin which functions as a scavenger of free heme ~4,~v,a°. Among these mechanisms, the one involving a primary control on the level of succinyl-CoA generation has been questioned on the basis that less than 1 % of the total succinyl-CoA formed in the cell enters the pathway to heine. In this context, it is also important to point out t h a t an impairment of terminal oxidation involving NAD-linked respiratory activity and an accumulation of succinate is a feature common to both porphyria and vitamin E deficiency 31-83. Whether vitamin E regulates heme synthesis indirectly through control on terminal oxidation or b y directly participating in the control of macroBiochim. Biophys. Acta, 215 (i97 o) II2--II8
II8
i'. P. NAIR cl a[.
molecular synthesis is not known at the moment. Further work is in progress to elucidate these mechanisms. ACKNOWLEDGMENTS
This work was supported by grants from the United States Public Health Service, National Institutes of Health, AM-o213I and General Research Support Grant, 5S0-IFR-o5478~o8, and from the National Aeronautics and Space Administration, contract No NAS-9-9715. N. R. G. is a summer research participant.
REFERENCES I H. M. EVANS AND Is[. S. BISHOP, Science, 56 (I922) 650. 2 0 . A. ROELS, Nutr. Rev., 25 (I967) 33. 3 A. S. ]~{AJAJ, J. S. DINNING, S. A. AZZAM AND xcV. J. DARBY, Am. J. Clin. Nutr,, 12 (I963) 374. 4 F. S. PORTER AND C. D. FITCH, Seand. J. Hematol., 3 (1966) 175. 5 J- A. YVHITTAKER, E. G. FORT, S. \FIMOKESANT .AND J. S. DINNING, A m . J. Clin. Nutr., 20 (1967) 783 . 6 \V. J. DARBY, Vitamins Hormones, 26 (1968) 685, 7 H. S. MURTY AND P. P. NAIR, in p r e p a r a t i o n . 8 H . S. MURTY AND P. P. NAIR, Nature, 223 (1969) 200. 9 H . S. MURTY, A. PINELLI, P. t9. NAIR AND A. [. MENDELOFF, Clin. Res., 17 (1969) 474IO F. D E MATTEIS, Seminars Hematol., 5 (I968) 417 . I I H. S..'~{ARVEP,, D. P. TSCHUDY, .~I. G. PERLROTH, A. COLLINS AND G. HUNTER, JR., Anal. Biochem., 14 (1966) 53. 12 J. D. BAUER, P. G. ACKERMAN AND G. TORO, in \V. E. BRAY, Clinical Laboratory Methods, C. V. M o s b y , St. L o u i s , 1968, p. 43. 13 \V. B. \VEGLICKI, W . REICHEL AND P. P. NAIR, J. Gerontol., 23 (1968) 469 . 14 P. P. NAIR, I. SARLOS AND J. MACHIZ, Arch. Biochem. Biophys., 114 (1966) 488. 15 P. P. NAIR AND J. MACHIZ, Biochim. Biophys. Aeta, 144 (1967) 446. 16 P. P. NAIR AND Z. LUNA, Arch. Biochem. Biophys., 127 (1968) 413 . 17 H . S. MARVER, D. P. TSCHUDY, M. G. PERLROTH AND A. COLLINS, J. Biol. Chem., 241 (I966) 2803 . 18 K. D. GIBSON, A. NEUBERGER AND J. j . SCOTT, Biochem. J., 61 (1955) 618. 19 H . S. MURTY AND P. P. NAIR, Anal. Biochem., 31 (1969) I. 20 A. KAPPAS, C. S. SONG, R. D. LEVERE, R. A. SACHSON AND S. GRANICK, Proc. Natl. Acad. Sci. U . S . , 61 (1968) 509 . 21 R. D. LEVERE, A. I~.APPAS AND S. GRANICK, Proc. Natl. Acad. Sei. U.S., 58 (i967) 985. 22 S. GRANICK AND G. URATA, .]. Biol. Chem., 238 (1963) 821. 23 H . S. ~'~IARVER, A. COLLINS, I). P. TSCHUDY AND ~I. RECHCmL, JR., J . Biol. Chem., 241 (1966) 432324 R. F. LABBE, T. KURUMADA AND J. ONISAWA, Biochim. Biophys. Acta, 111 (1965) 403 . 25 L. GOLDBERG AND J. P. SMITH, Am. J. Pathol., 36 (196o) 125 . 26 A. PALMA-CARLOS, L. PALMA-CARLOS, ~{. GAJDOS-T(SROK AND A. GAJDOS, Nature, 211 (1966) 97727 F. DE MATTEIS, Pharmacol. Rev., 19 (1967) 523. 28 A. KAPPAS, R . 1). LEVERE aND S. GRANICK, Seminars Hematol., 5 (1968) 323 . 29 D. KARIBIAN AND I. M. LONDON, Bioehem. Biophys. Res. Commun., 18 (1965) 243. 3 ° A. I. GRA'ZZFL, J. E . FUHR AND I. M. LONDON, Biochem. Biophys. Res. Commun., 28 (1967) 705 . 31 M. L. COWGER, R. F. LABBE AND M. SEWELL, Arch. Bioehem. Biophys., i o i (1963) 96. 32 L. M. CORWIN AND K. SCH\VARZ, J. Biol. Chem., 234 (1959) 191. 33 L. M. CORWlN, J. Biol. Chem., 240 (1965) 34-
Biochim. Biophys. A cta, 215 ( 197 o) I 12-118
B I O C H I M I C A ET B I O P H Y S I C A ACTA
BBA 26389 T H E I N V I V O E F F E C T OF VITAMIN E IN E X P E R I M E N T A L P O R P H Y R I A PADMANABHAN P. NAIR, HARI S. MURTY AND NEAL R. GROSSMAN* Biochemistry Research Division, Department of 3,Iedicine, Sinai Hospital of Baltimore, Inc., Baltimore, Md. 2r2r 5 (U.S.A.)
(Received March 9th, ~97o)
SUMMARY
The induction of experimental porphyria in rats by allylisopropylacetamide is characterized by elevation of hepatic &aminolevulinate synthetase and dehydratase activities. The administration of vitamin E 6 h prior to initiation of the porphyrinogenic regimen prevented porphyria through a control exercised on the activities of the two hepatic enzymes. The bone marrow of porphyric animals showed enhanced synthetase activity which was prevented by vitamin E. In this paper, evidence is presented to show that vitamin E deficiency increases susceptibility to the induction of &aminolevulinate synthetase by allylisopropylacetamide, indicating that the vitamin is in some way involved in the regulation of the inducible enzyme. Since other studies have shown that the normal constitutive enzyme is vitamin E dependent in vivo, the question is posedwhether the normal and inducible forms represent isozymes of d-aminolevulinate synthetase.
INTRODUCTION Vitamin E, as a member of the group of fat-soluble vitamins, has remained an enigma since its discovery by EVANS AND BISHOP1. A deficiency of this vitamin leads to a variety of species-specific manifestations which could not be explained mechanistically on a unified basis 2. Recent studies have shown the existence of an anemia associated with a lack of this vitamin in man and in primates 3-6. Similarly, our recent studies have demonstrated that vitamin E deficiency in the rat, even in the absence of a demonstiable anemia, leads to a ~ignificant depression in heine synthesis and that the accompanying metabolic detect involving d-aminolevulinate synthetase and dehydratase could be corrected only by the administration ot the vitamin in vivo 7. In contrast, vitamin E in vivo prevents the allylisopropylacetamide-mediated induction of experimental porphyria s as well as causing a remission in the human variety of the same disorder 9. Since both human and experimental porphyrias are characterized by abnormally high activities for hepatic &aminolevulinate synthetase and dehydratase 1°, the present study was undertaken to examine the effect of vitamin E on these "inducible" enzymes. In this paper we have shown that the action of * N. R.G. is a summer research participant. Biochim. Biophys. dcta, 215 (197o) t I 2 II8
VITAMIN E AND EXPERIMENTAL PORPHYRIA
113
vitamin E in preventing the induction of experimental porphyria is effected through a suppression of "inducible" 8-aminolevulinate synthetase and dehydratase. EXPERIMENTAL
Materials Allylisopropylacetamide was a generous gift from Dr. Scott, Hoffmann LaRoche, Nutley, N.J. b-Aminolevulinic acid was purchased from Calbiochem, Los Angeles. All other reagents were obtained trom standard commercial sources.
Methods Induction of experimental porphyria. Experimental porphyria was induced in male Wistar strain rats weighing 15o-2oo g by repeated injections of allylisopropylacetamide s. Groups of 4-5 animals were conditioned in metabolic cages for a period of 3 days and then food was withheld I2 h before the beginning of the experiment. One group of rats served as control, while a second gI oup received orally single doses of ioo mg of vitamin E per rat. Porphyria was induced in two other groups of rats by four successive daily injections of allylisopropylacetamide (400 mg/kg body weight in isotonic saline) subcutaneously. In addition to allylisopropylacetamide, vitamin E was administerea (single Ioo-mg doses per rat) to rats in one of the groups approx. 6 h before the first injection with allylisopropylacetamide. All animals in the control groups received injections of isotonic saline identical in volume to that used for administering allylisopropylacetamide. Porphyria was confirmed in allylisopropylacetamide treated animals b y the detection of 3-aminolevulinate, porphobilinogen and porphyrins in the urine 11,12. All animals were sacrificed under ethel anesthesia by exsanguination from the abdominal a o r ' a 5 days aftei the first injection with allylisopropylacet amide. Establishment of vitamin E deficiency Weanling, male Wistai strain rats were fed a standard vitamin E deficient diet for a period of 4-5 months as described in an earlier publication 13, Vitamin E deficiency was confirmed b y determinations of hepatic and plasma vitamin E levels b y gas liquid chromatogiaphic procedures developed in this laboratory 14-16. A corresponding control group of animals was concurrently reared on the same basal vitamin E deficient diet supplemented with vitamin E, 50 mg/kg diet. Hepatic b-aminolevulinate synthet ase was induced in both vitamin E-deficient and control animals by subcutaneous injections of allylisopropylacetamide, 50 mg/kg body weight. Assay of hepatic b-aminolevulinate synthetaseand dehydratase activities. Hepatic b-aminolevulinate synthetase was assayed according to the procedure of MARVER et al. 17. The assay of hepatic ~-aminolevulinate dehydratase activity was carried out according to the original method of GIBSON et al) s. Assay of bone marrow 8-aminolevulinate synthetase. Femoral bone marrow, aspirated with a hypodermic syringe equipped ~ith a i9-gauge needle, was thoroughly suspended in 2.0 ml of T r i s - E D T A buffer (pH 7.4). The incubation mixture, which consisted of I.O ml ot bone marrow suspension, 400/~moles of glycine, 20/~moles of EDTA, I5o/~moles of Tris-HC1 buffer p H 7.4) and IO/,moles of glucose in a total volume of 3.0 ml, was incubated aerobically for I h at 37 °. The reaction was terminated Biochim. Biophys. dcta, 215 (197 o) 112-118
P.P. NAIR et al.
114
by the addition of 0.75 ml of 25 % trichloroacetic acid and the 6-aminolevulinate formed was condensed with acetyl acetone to form the corresponding pyrrole, which was then determined with Ehrlich's reagent n. Endogenous 6-aminolevulinate was simultaneously determined in a control incubation mixture to which trichloroacetic acid was added at zero time. Since the erythroid cell population remains unaltered under the experimental conditions, the results are expressed as nmoles of &aminolevulinate formed per 11 per mg protein, the enzymatic activity being linear for this time period. Since sulfhydryl group activators such as glutathione are required for the activation of bone marrow &aminolevulinate dehydratasO s, this enzyme did not interfere with the assay of b-aminolevulinate synthetase activity in this tissue. Furthermore, under our assay conditions, the incorporation of radioactivity from (2J4C]glycine into heine was negligible indicating that &aminolevulinate dehydratase was inactivated in this system. No aminoacetone was detected in the assay system, as shown by the absence of Ehrlich-positive material in the aminoacetone fraction isolated by chromatography on Dowex I-X8 (acetate form).
Incorporation of radioactivity from i2-14C]glycine into heine by bone marrow cells19. The bone marrow extracted from one femur was suspended in Krebs Ringer phosphate buffer (pH 7-4) centrifuged for 5 rain at 50o × g and reconstituted with the same buffer. The process was repeated twice to obtain the final cell suspension, an appropriate aliquot of which was added to the incubation mixture containing 20 mg of glucose, 20 mg of bovine serum albumin, I.o mg each of penicillin and streptomycin sulfate and lO #C of I2-l~C]glycine (specific activity 21.8 mC/mmole) in a total volume of IO ml. Incubations were carried out aerobically at 37 ° in 5o-ml erlenmeyer flasks for 2 h. At the end of the incubation period, the reaction mixture was chilled to 0 ° and heine was isolated as hemin after the addition of 200 mg of carrier hemoglobin ~9. The radioactivity in the isolated heroin was assayed by liquid scintillation counting. RESULTS AND DISCUSSION
Effect of vitamin E on hepatic &aminolevulinate synthetase and dehydratase Since vitamin R was shown to inhibit the induction of experimental porphyria by allylisopropylacetamide s, its effect on the key enzymatic steps in the early biogenetic sequence leading to heine was studied. A single close of vitamin E, administered to rats approx. 6 tl before initiation of the porphyrinogenic regimen, effectively blocked the induction of both &aminolevulinate synthetase and dehydrase activities (Tables I and II). However, the vitamin, when given alone to control animals, induced a significant rise in the activity of both these hepatic enzymes contrary to our expectations.
Effect of vitamin E on bone marrow &aminolevulinate synthetase and on the incorporation of radioactivity from L2-1~C]glycine into heine The response of &aminolevulinate synthetase in mammalian cells to environmental agents depends not only on the type of cell involved but also on the chemical agents tested 2°. Thus it has been shown that 5fl-H steroids enhanced heine synthesis (as well as b-aminolevulinate synthetase) only in embryonic liver and erythroid cell precursors 21, whereas other nonsteroidal porphyrinogenic chemicals acted exclusively Biochim. Biophys. dcla, 2 I 5 (I97O) I I 2 - I I 8
VITAMIN E AND EXPERIMENTAL PORPHYRIA
115
TABLE I EFFECT
OF V I T A M I N
E
ON H E P A T I C
(~-AMINOLEVULINATE
ALLYLISOPROPYLACETAMIDE-TREATED
SYNTHETASE
ACTIVITY OF NORMAL
AND
RATS
Results are expressed as nmoles of b-aminolevulinate produced per h. Values represent mean of four individual observations and their S,E. Details of the treatment of the four groups are described under EXPERIMENTAL, Statistical significance of differences in mean 6-aminolevulinate synthetase activities between different groups are: control with respect to control --. vitamin E, P < o . o i and --<0.02; control with respect to allylisopropylacetamide, P < o . o o i and < o . o o i ; control with respect to allylisopropylacetamide + vitamin E, P > o . 3 and >o.7; control + vitamin E with respect to allylisopropylacetamide, P < o . o o i and < o . o i ; control + vitamin E with respect to allylisopropylacetamide + vitamin E, P > o.2 and < o.2 ; allylisopropylacetamide with respect to allylisopropylacetamide + vitamin E, P < o . o o i and < o.ooi. In each pair of P values, the first one corresponds to activity expressed per g wet weight of liver and the second to specific activity. Specific activity is expressed as nmoles of 6-aminolevulinate synthesized per h pcr g protein.
Treatment
6-Aminolevulinate synthetase activity Per g liver
Control Control + vitamin E Allylisopropylacetamide Allylisopropylacetamide + vitamin E
29.94 56.26 234.4 42.58
± ± 4±
Specific activity
4-95 5 .68 23.7 9.5
162. 4 354.3 132o 241.7
± 26.6 ± 23.9 ~_ 72 ± 39.8
TABLE II EFFECT
OF V I T A M I N g
ON H E P A T I C ~ - A M I N O L E V U L I N A T E
ALLYLISOPROPYLACETAMIDE-TREATED
DEHYDRATASE
A C T I V I T Y OF N O R M A L A N D
RATS
Results are expressed as nmoles of porphobilinogen produced per h per g of wet weight tissue. Values represent mean of four individual observations and their S.E. Statistical significance of differences in mean 6-aminolevulinate dehydratase activities between different groups are : control with respect to control + vitamin E, P > o . 3 ; control with respect to allylisopropylacetamide, P < o.ooi ; allylisopropylacetamide with respect to allylisopropylacetamide + vitamin E, P < o.ooi. Experimental details are given in the text.
Treatment
Control Control + vitamin E Allylisopropylacetamide Allylisopropylacetamide + vitamin E
d-A minolevulinate dehydratase activity 57 r.8-L 627.4-c 2o66 ± 562.9 -[_
33-4 36.4 125 82.3
o n n o n e r y t h r o i d h e p a t i c cells22,2% H o w e v e r , i n e x p e r i m e n t a l p o r p h y r i a i n d u c e d b y a l l y l i s o p r o p y l a c e t a m i d e , w e o b s e r v e d a s m a l l b u t s i g n i f i c a n t r i s e in b o n e m a r r o w & a m i n o l e v u l i n a t e s y n t h e t a s e , p r e v e n t a b l e b y p r i o r a d m i n i s t r a t i o n of v i t a m i n E ( T a b l e I I I ) . U n l i k e liver, t h e b o n e m a r r o w of c o n t r o l a n i m a l s w h i c h r e c e i v e d t h e vitamin did not exhibit a response. S i n c e t h e rise in b o n e m a r r o w & a m i n o l e v u l i n a t e s y n t h e t a s e i n d u c e d b y allylisopropylacetamide was minimal, the treatment with allylisopropylacetamide was not a c c o m p a n i e d b y an increase in h e m e synthesis. On t h e c o n t r a r y , t h e r e was a decrease in t h e i n c o r p o r a t i o n of r a d i o a c t i v i t y f r o m g l y c i n e i n t o h e m e (Table IV). V i t a m i n E
Biochim. Biophys. dcta, 215 (197o) 112-1 i8
116
t'. P. x.-\tR et al.
TABLE III EFFECT AND
OF VITAMIN
g
O N B O N E MARRO\X," ( ~ - A M I N O L E V U L I N A T E S Y N T H E T A S E
ALLYLISOPROPYLACETAMIDE-TREATED
A C T I V I T Y OF N O R M A L
RATS
Results are expressed as nmoles of d-anfinolevulinate produced per h per m g proteiu. Values represent m e a n of four individual o b s e r v a t i o n s and their S.E. Statistical significance of differences in m e a n 6-aminolevulinate s y n t h e t a s e activities between different groups are : control with respect to allylisopropylacetamide, / ~ < o . o o i ; control - v i t a m i n E w i t h respect to allylisopropylacetamide, P < o . o 5 ; allylisopropylacetamide with respect to allylisopropylacetamide + v i t a m i n E, P < o . i . E x p e r i m e n t a l details are given in the text.
Treatment
6- d minolevulinate synthetase activity
Control Control + v i t a m i n E Allylisopropylacetamide Allylisopropylacetamide + v i t a m i n E
32. 4 39.6 6i.i 4o.2
~_ 2.6 ~h 7.4 :~ 3.4 -- 8. 5
T A B L E 1V EFFECT INTO
OF V I T A M I N
HEME
BY BONE
E
ON T H E
INCORPORATION
in vitro OF
RADIOACTIVITY
FROM
[2-14C3GLYCINE
MARROXV
Results are m e a n s of observations from two animals in each group, expressed as disint./min per mg of bone m a r r o w protein. E x p e r i m e n t a l details are given in the text. Treatment
Activity (Disint./min × ±o-4)
Control Control -i- v i t a m i n E Allylisopropylacetamide Allylisopropylaeetamide + v i t a m i n E
3.97 4.05 2.2 i 2.64
p a r t i a l l y c o u n t e r a c t e d this defect. However, t h e a c t i o n of a l l y l i s o p r o p y l a c e t a m i d e on t h e bone m a r r o w is n o t clear a t t h e m o m e n t .
The effect of allylisopropylacetamide on the induction of hepatic b-aminolevulinate synthetase in vitamin E-deficient rats Since vitamin E blocks the induction of &aminolevulinate synthetase in the livers of allylisopropylacetamide-treated animals, experiments were conducted to compare the degree of induction of this enzyme in vitamin E-deficient animals and the corresponding controls. Table V presents data showing that with identical doses of allylisopropylacetamide, synthetase activities in vitamin E-deficient livers were approx. 5-fold higher than those in vitamin E-fed control livers. In experiments not reported here, addition in vitro of vitamin E or liver homogenates of vitamin E-treated rats did not alter the activity of the induced enzyme in homogenates of porphyric rat liver. This observation rules out a direct inhibitory action of vitamin E on 8-aminolevulinate synthetase.
Role of vitamin E in the regulation of heine synthesis W e h a v e shown in a previous c o m m u n i c a t i o n t h a t in t h e v i t a m i n E-deficient r a t , e v e n in t h e a b s e n c e of a d e m o n s t r a b l e
Biochim. Biophys. Acta, 215 (197 o) 112-118
a n e m i a , t h e r e is a d i s t i n c t d e p r e s s i o n i n
VITAMIN E AND EXPERIMENTAL PORPHYRIA
117
TABLE V COMPARISON OF ALLYLISOPROPYLACETAMIDE-MEDIATEDINDUCTION OF HEPATIC d-AMINOLEVULINATE SYNTHETASE IN NORMAL AND VITAMIN E-DEFICIENT RATS A l l y l i s o p r o p y l a c e t a m i d e d i s s o l v e d in p h y s i o l o g i c a l saline w a s i n j e c t e d s u b c u t a n e o u s l y (5 ° m g / k g b o d y weig ht) i n t o n o r m a l a n d v i t a m i n E - d e f i c i e n t a n i m a l s . The s e a n i m a l s w e re sacrificed 3 h l a t e r a n d t h e i r h e p a t i c d - a m i n o l e v u l i n a t e s y n t h e t a s e a s s a y e d . R e s u l t s are e x p r e s s e d as n m o l e s of d - a m i n o l e v u l i n a t e f o r m e d per g of t i s s u e p e r h a n d r e p r e s e n t s m e a n of four i n d i v i d u a l o b s e r v a t i o n s a n d t h e i r s t a n d a r d error of m e a n .
Groups
Control Vitamin E deficient
d-A minolevulinate synthetase
4o.7 ± 8.1 251
4- io
the ability of the bone marrow and liver to synthesize heme, the defect residing at one or both of the two enzymatic steps, d-aminolevulinate synthetase and &aminolevulinate dehydratase 7. The mechanism b y which vitamin E prevents experimental porphyria s and also causes a remission in human porphyria 9 probably involves the blocking of the induction of &aminolevulinate synthetase and secondarily that of &aminolevulinate dehydratase as seen in this study. Since the response to vitamin E, in vivo, of &aminolevulinate synthetase in the vitamin E-deficient and porphyric animals are mutually divergent, it appears that &aminolevulinate synthetase might exist as isozymes representing the constitutive and inducible forms. Similarly, LABBE et al. 24 suggested the existence of isozymes b y showing that the inducible forms of both succinyl-CoA and &aminolevulinate synthetases possessed characteristics which were different from those of their corresponding constitutive enzymes. Although vitamin E deficiency does not manifest itself in the form of porphyria, the levels of &aminolevulinate synthetase induced in these animals by porphyrinogenic agents are distinctly higher than those in their corresponding controls. This increased susceptibility to induction suggests a possible role for this vitamin at a repressor site for &aminolevulinate synthetase. On the other hand, a chemical agent such as ethionine when fed to rats produces not only symptoms of vitamin E deftciency ~5 but also induces porphyria ~6 without an associated rise in the activity of &aminolevulinate synthetase. According to current concepts, the biosynthesis of heme could be regulated b y (a) the rate of generation of succinyl-CoA, (b) end product inhibition or repression of O-aminolevulinate synthetase b y free heme or (c) control of the synthesis of globin which functions as a scavenger of free heme ~4,~v,a°. Among these mechanisms, the one involving a primary control on the level of succinyl-CoA generation has been questioned on the basis that less than 1 % of the total succinyl-CoA formed in the cell enters the pathway to heine. In this context, it is also important to point out t h a t an impairment of terminal oxidation involving NAD-linked respiratory activity and an accumulation of succinate is a feature common to both porphyria and vitamin E deficiency 31-83. Whether vitamin E regulates heme synthesis indirectly through control on terminal oxidation or b y directly participating in the control of macroBiochim. Biophys. Acta, 215 (i97 o) II2--II8
II8
i'. P. NAIR cl a[.
molecular synthesis is not known at the moment. Further work is in progress to elucidate these mechanisms. ACKNOWLEDGMENTS
This work was supported by grants from the United States Public Health Service, National Institutes of Health, AM-o213I and General Research Support Grant, 5S0-IFR-o5478~o8, and from the National Aeronautics and Space Administration, contract No NAS-9-9715. N. R. G. is a summer research participant.
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