Biosynthesis of heme in the vitamin E-deficient rat.

Abstract Vitamin E deficiency in the rat leads to decreased activities of bone marrow δ-aminolevulinic acid synthase and hepatic δ-aminolevulinic acid dehydratase. Studies on the incorporation of radioactivity from glycine-2-14C and δ-aminolevulinic acid-4-14C into bone marrow heme show that the defect in this tissue is at the level of the first enzyme, δ-aminolevulinic acid synthase. However, in the liver, the incorporation of δ-aminolevulinic acid-4-14C into microsomal protoheme in vivo was significantly lower than those in the controls, while no differences were observed when porphobilinogen-14C was used. Thus, unlike the bone marrow, in the liver, the defect appears to be at the level of the second enzyme δ-aminolevulinic acid dehydratase. The partial failure to maintain heme synthesis is also reflected in the lowered levels of the hemeprotein enzymes, catalase, and tryptophan pyrrolase as well as in the levels of microsomal cytochrome b5 and cytochrome P-450. The nature of the effect is highly specific as indicated by the observation that nonheme enzymes such as tissue ATPase, mitochondrial ATPase, malate, and isocitrate dehydrogenase, microsomal NADPH-cytochrome c reductase, and cytoplasmic glucose-6-P dehydrogenase remain unaltered in vitamin E deficiency. The results suggest that vitamin E functions as a regulator of heme synthesis at one of the rate-limiting steps in the pathway to heme.

1-L BROOKS, AND P. P. NAIR From the Biochemistry Research Division, Department of Medicine, Sinai Hospital of Baltimore, Inc., Baltimore, Maryland Wi 215 SUMMARY Vitamin E deficiency in the rat leads to decreased activities of bone marrow &aminolevulinic acid synthase and hepatic d-aminolevulinic acid dehydratase. Studies on the incorporation of radioactivity from glycine-2J4C and &arninolevulinic acid-4J4C into bone marrow heme show that the defect in this tissue is at the level of the first enzyme, &aminolevulinic acid synthase.
However, in the liver, the incorporation of 6aminolevulinic acid-4J4C into microsomal protoheme in vivo was significantly lower than those in the controls, while no differences were observed when porphobilinogenJ4C was used.
Thus, unlike the bone marrow, in the liver, the defect appears to be at the level of the second enzyme d-aminolevulinic acid dehydratase.
The partial failure to maintain heme synthesis is also reflected in the lowered levels of the hemeprotein enzymes, catalase, and tryptophan pyrrolase as well as in the levels of microsomal cytochrome b5 and cytochrome P-450. The nature of the effect is highly specific as indicated by the observation that nonheme enzymes such as tissue ATPase, mitochondrial ATPase, malate, and isocitrate dehydrogenase, microsomal NADPH-cytochrome c reductase, and cytoplasmic glucose-6-P dehydrogenase remain unaltered in vitamin E deficiency.
The results suggest that vitamin E functions as a regulator of heme synthesis at one of the rate-limiting steps in the pathway to heme. Biologically, vitamin E or ol-tocopherol represents the most important member of the class of methyl-substituted tocols. A deficiency of this vitamin is generally characterized by species specific syndromes such as fetal resorption, defective spermatogenesis, muscular dystrophy, encephalomalacia, exudative diathesis, and degeneration of skeletal and cardiac muscle (1). Recent studies have shown the existence of an anemia associated with a lack of this vitamin in man and in primates.
The locus of the metabolic defect has been postulated to reside in the biogenetic sequence leading to heme and hemeproteins (2)(3)(4)(5).
* This work wassupported by Grant AM-92131 and General Research Support Grant 5SO-lFR-05478-08 from the United States Public Health Service, National Institut,es of Health, and Contract Number NAS-9 9715 from the National Aeronautics and Space Administration.
Since hemeproteinsl are vital to the maintenance of norma metabolism in the mammalian cell, an aberration in the biogenesis of heme would be expected to give rise to pleomorphic manifestations similar to those described in vitamin E deficiency. The present communication describes experiments indicating the existence of a defect in heme synthesis in the vitamin E-deficient rat.
The results presented here show that vitamin E deficiency leads to a decrease in the ability of the bone marrow to synthesize &aminolevulinic acid. In contrast, the locus of the defect in the liver seems to be at the step involving the formation of porphobilinogen.
Weanling male rats of the Wistar strain were fed a standard vitamin E-deficient diet for 15 to 18 weeks, unless otherwise stated, before being used for the experiments (6). The corresponding control animals were fed the basal vitamin E-deficient diet supplemented with vitamin E, 200 mg per kg of diet. Rats were killed by exsanguination from the abdominal aorta under mild ether anesthesia.
Vitamin E was dissolved in alcohol and emulsified with a solution of bovine serum albumin to give the appropriate final concentration of the vitamin (7). A similar solution of the vehicle alone was prepared to serve as control.
In an alternate method, in studies on microsomal heme synthesis, vitamin E was dissolved in 0.3 ml of ethanol and made up to 10 ml with propylene glycol for intraperitoneal administration. Porphobilinogen-14C was prepared enzymatically by the action of rat liver cytoplasmic d-aminolevulinic acid dehydratase on b-aminolevulinic acid-4-1%. Rat liver (15.5 g) was homogenized in 3 volumes of 1.15% KC1 and centrifuged at 10,000 X g for 30 min. The supernatant fluid was made up to a volume of 40 ml with 1.15% KC1 and distributed equally between two Erlenmeyer flasks. The incubation mixture in each flask consisted of 40 ml of 0.15 M phosphate buffer, pH 6.8; &aminolevulinic acid-4-l%, 45 PCi dissolved in 5 ml of phosphate buffer; nonradioactive b-aminolevulinic acid, 1.0 pmole, and glutathione, 200 1 The term hemeprotein is used here to represent collect.ively various molecular forms such as hemoglobin, myoglobulin, cytochromes, catalase, and other heme-containing enzymes.
Issue of October 25, 1970 lkfurty, Caasi, Brooks, and Nair 5499 pmoles. The mixture was incubated under nitrogen at 37" for 2 hours and the reaction was terminated by the addition of 20 ml of 10% trichloracetic acid. The precipitated protein was removed by centrifugation, washed once with 5 ml of distilled water, and the combined supernatants were adjusted to pH 5 to 6 with KOH.
Porphobilinogen was purified by chromatoggraphy on Dowex 2- X8 (8, 9), and eluted from the column with 25 ml of 1 N acetic acid. Its concentration was determined on an aliquot of the eluate with modified Ehrlich's reagent (8). The rest of the solution was lyophilized to dryness and the residue dissolved in 7 ml of 0.9% sodium chloride solution.
This preparation of porphobilinogen had a specific radioactivity of 26.2 mCi per mM.

Methods
Determination of Tissue Tocopherol Levels-Tissue tocopherol levels were determined by gas-liquid chromatographic procedures developed in this laboratory .
Assay of Hepatic and Bone Marrow &Aminolewulinic Acid Synthase Activities-Hepatic b-aminolevulinic acid synthase was assayed by the procedure of Marver et al. (13). Rat liver was homogenized in 3 volumes of Tris-EDTA buffer, pH 7.4. The incubation mixture containing 2.5 ml of the homogenate, 1 mmole of glycine, 100 pmoles of EDTA, and 750 pmoles of Tris-HCl buffer, pH 7.4, in a total volume of 10 ml was incubated aerobically in a shaker water bath for 1 hour at 37". The reaction was terminated by the addition of 2.5 ml of 25% trichloracetic acid, and b-aminolevulinic acid was determined in the supernatant fluid following its isolation from a Dowex l-X8 column (14). Endogenous &aminolevulinic acid was measured simultaneously in a control mixture to which trichloracetic acid was added at zero time. Results are expressed as millimicromoles of &aminolevulinic acid formed per hour per g of tissue. Femoral bone marrow, aspirated with a hypodermic syringe equipped with a 19-gauge needle, was suspended in 2 ml of Tris-EDTA buffer, pH 7.4. &Aminolevulinic acid synthase activity was measured as described earlier (15). Results are expressed as millimicromoles of &aminolevulinic acid formed per hour per mg of protein.
Protein was measured by the Folin-Ciocalteau procedure.
Assay of Hepatic 6-Aminolevulinic Acid Dehydratase-Hepatic d-aminolevulinic acid dehydratase was assayed by the procedure of Gibson,Neuberger,and Scott (16). Results are expressed as millimicromoles of porphobilinogen formed per hour per g of tissue.
Incorporation of Radioactivity from Glycine-2-W and &Aminolevulinic Acid-Q-W into Heme by Bone Marrow Cells-The marrow from one femur suspended in Krebs-Ringer phosphate buffer, pH 7.4, was centrifuged for 5 min at 500 X 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, 1 mg each of penicillin and streptomycin sulfate, and either 10 PCi of glycine-2-Y (specific radioactivity, 21.8 mCi per mM) or 5 @Zi of d-aminolevulinic acid-4-W (specific radioactivity, 45 mCi per mu) in a total volume of 10 ml. Incubations were carried out aerobically at 37" in 50-ml Erlenmeyer flasks for 2 hours. At the end of the incubation period, the reaction mixture was chilled to 0" and heme was isolated as hemin after the addition of 200 mg of carrier hemoglobin (17). The radioactivity in heme was assayed by liquid scintillation counting. Assay of Catalase and Tryptophan Pyrrolase Activities in Rat Liver HornogenatesHepatic catalase (hydrogen peroxide: hydrogen peroxide oxidoreductase, EC 1.11.1.6) activity was measured as described by Luck (18). One gram of tissue was homogenized with 10 ml of 0.007 M phosphate buffer, pH 7.0, and centrifuged for 10 min at 2000 x g. The pellet was homogenized again in phosphate buffer and recentrifuged.
The supernatant fluids from both steps were combined and appropriate dilutions were made for spectrophotometric assay. One unit of catalase activity is defined as that amount of enzyme which liberates 50'% of peroxide oxygen from a given concentration of HzOz in 100 see at 25'.
Hepatic tryptophan pyrrolase (n-tryptophan: oxygen oxidoreductase, EC 1.13.1.12) was assayed according to a modified procedure of Knox (19) in which the amount of kynurenine formed in 60 min at 37" in the presence of n-tryptophan was measured.
Livers were homogenized in 7 volumes of 0.14 M KC1 containing 2.5 mM NaOH and 1.0 mM EDTA (20). Duplicate l-ml portions of this homogenate (equivalent to 125 mg of liver) were incubated at 37" under 02 for 60 min in the presence of 1 ml of 0.2 M phosphate buffer, pH 7.0, 4 pM of hematin (21), and 9 PM of ntryptophan in a total volume of 4 ml. At the end of the incubation period, the mixture was deproteinized with 2 ml of 15% metaphosphoric acid and the amount of kynurenine formed was measured spectrophotometrically at 365 rnp (19). The results are expressed as micromoles of kynurenine formed per hour per g of liver, wet weight.
Preparation of Rat Liver Fractions-Vitamin E-deficient rats and the corresponding control animals were killed under ether anesthesia by exsanguination from the abdominal aorta. The livers were rapidly excised, rinsed, and homogenized in a Potter-Elvehjem homogenizer with 10 volumes of cold 0.25 M sucrose containing 1 mM disodium EDTA, pH 7.4. The homogenate was fractionated by differential centrifugation to give the mitochondrial, microsomal, and soluble fractions as described by Baron and Tephly (22).
In studies involving measurement of the incorporation in vivo of labeled precursors into protoheme, microsomal preparations were suspended in a volume of 1.15% KC1 equal to that of the original postmitochondrial supernatant and centrifuged at 105,000 x g for 30 min. The purified microsomal pellet which was free of hemoglobin (22), was suspended in 0.1 M phosphate buffer, pH 7.4, and used for the extraction of protoheme.
Enzymatic Assays on Rat Liver Fractions-The mitochondrial preparation as well as an aliquot of the original tissue homogenate was assayed for ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity according to the method of Masoro, Korchak, and Porter (23). Assays of pyridine nucleotide-linked dehydrogenases were based on the extinction coefficient of the difference between reduced and oxidized pyridine nucleotides at 340 rnp. The results are expressed as micromoles of NAD or NADP reduced per mg of protein per min. Mitochondria were also used for the measurement of isocitrate dehydrogenase (threo-n,-isocitrate:-NADP oxidoreductase, EC 1.1.1.42) (24) and malate dehydrogenase (n-malate :NAD oxidoreductase, EC 1.1.1.37) (25) activities.
The activity of microsomal NSDPH-cytochrome c reductase was measured as the rate of increase in absorbance at 550 rnp produced by the reduction of cytochrome c (22). The results Heme Xynthesis in Vitamin E Dejkiency Vol. 245,No. 20 are expressed as millimicromoles of cytochrome c reduced per mg of protein per min with the extinction coefficient 19.7 cm+ rnM-1 for the difference in absorbance between reduced and oxidized cytochrome c at 550 rnp. The activity of glucose-6-P dehydrogenase (n-glucose-6phosphate:NADP oxidoreductase, EC 1.1.1.49) was measured in the cytoplasmic fraction according to the method of Glock and McLean (26).
Microsomal Cytochrome bs and Cytochrome P-45&Cytochrome bs was determined from the difference spectrum between NADHreduced and air-saturated microsomes.
A value of 163 cm-l rnM-1 for the increment in extinction coefficient between 424 and 409 rnp was used in computing the concentration of cytochrome bs (27). The results are expressed as millimicromoles of cytochrome b6 per mg of protein.
Cytochrome P-450 was determined from the CO difference spectrum of dithionite-reduced microsomes with a molar extinction difference of 91 cm-l rnr@ between 450 and 490 rnp (28). The results are expressed as millimicromoles of cytochrome P-450 per mg of protein.
Incorporation in liivo of Radioactivity from &Aminolevulinic AC&-&Q-W and Porphobilinogen-l4C into Microsomal Protoheme-Experimental animals fasted for a period of 16 hours received a pulse-dose of either 10 PCi of &aminolevulinic acid-4-14C (specific radioactivity, 45 mCi per mM) or approximately 6.4 X lo5 dpm of porphobilinogen-14C (specific radioactivity, 26.2 mCi per mM) prepared enzymat.ically in this laboratory. The animals were killed exactly 1 hour after the administration of the radioactively labeled precursor and liver microsomes were prepared An aliquot of the microsomal suspension in 0.1 M phosphate buffer, pH 7.4, was centrifuged at 105,000 X g for 15 min, and the pellet was extracted with 2 to 3 volumes of acetone containing 4% HCl. The precipitated protein was centrifuged and re-extracted with the same solvent.
The combined extracts were diluted with an equal volume of water and protoheme was extracted with peroxide-free ether (29).
An appropriate aliquot of the protoheme solution was evaporated in a liquid scintillation vial under a stream of dry nitrogen and dissolved in a mixture of 3 ml of ethyleneglycol monobutyl ether and 12 ml of diluted "liquifluor" (Pilot Chemical, Watertown, Massachusetts) for liquid scintillation counting (30). Radioactivity Measurements-All radioactive fractions were counted in a Packard Tri-Carb automatic liquid scintillation counter (model 3314) equipped with an automatic external standardization device. The counts from each sample were corrected for quenching and expressed as disintegrations per min as described earlier (31).

RESULTS AND DISCUSSION
Hepatic Levels of Vitamin E-In vitamin E-deficient animals the livers have a mean vitamin E concentration of 0.7 pg per g of tissue compared to about 150 pg per g of tissue in control animals.
&Aminolevulinic Acid Synthase and &Aminolevulinic Acid Dehydratase Activities-Since &aminolevulinic acid synthase and &aminolevulinic acid dehydratase are possibly rate-determining enzymes in the heme biosynthetic pathway, their levels were determined in tissues from vitamin E-deficient animals (Tables I  and II).
The activity of b-aminolevulinic acid synthase in bone marrow from deficient animals was only one-half that of the control group (Table I). However, the corresponding hepatic enzyme exhibited only a marginal decrease in its activity, since both control and experimental values were within the normal range reported for the hepatic enzyme (13). Hepatic b-aminolevulinic acid dehydratase on the other hand was significantly lower than that from control animals (Table II).
&Aminolevulinic acid dehydratase activities in bone marrow were not reported since existing methods for the assay of this enzyme in hematopoietic tissues were found to be inadequate. V&-Isotopic experiments with labeled precursors such as glycine-2-% and 6-aminolevulinic acid-4J4C were conducted on bone marrows of deficient animals to determine whether the decrease in &aminolevulinic acid synthase activity represented a specific defect at this step. The ability of deficient bone marrow to incorporate radioactivity from glycine-2J4C into heme i n vitro was approximately one-sixth that of the control bone marrow as seen in Table III. Furthermore, in the same experiment when &aminolevulinic acid-4-W was used as the labeled precursor, no differences were observed in the incorporation of radioactivity into heme in both deficient and control animals. This experiment indicates that in the vitamin E-deficient bone marrow, the defect in heme synthesis is at the step involving the formation of &aminolevulinic acid. Effects, in Vitro and in Vivo, of Vitamin E-Since several other metabolic aberrations in vitamin E deficiency are ascribed partly to lipid peroxidation it was necessary to examine whether the observed decrease in enzymatic activities as well as in the incorporation of radioactivity into heme is the result of peroxide inactivation in vitro of preformed enzyme protein, or whether it is the direct result of a failure in the protein-synthesizing machinery. In the former instance, addition of vitamin E in vitro to the incubation mixture would be expected to restore enzymatic activity.

Incorporation of Labeled Precursors into Bone Marrow Heme, in
The results presented in Table IV show that vitamin E had no effect on the activity of hepatic and bone marrow enzymes as well as on the incorporation of glycine-2JC into bone marrow.
In contrast, vitamin E restored to normal levels all the parameters related to the biosynthesis of heme when it was administered to deficient rats prior to killing ( Table V) . The relatively higher radioactivities obtained for deficient bone marrow in this experiment is caused by the addition of isologous serum which is known to enhance heme synthesis, nonspecifically.
Effect of Vitamin E Dejicieny on Hepatic Catalase and Tryptophan Pyrrolase Activities-Our observations in earlier experiments showed that the liver in vitamin E deficiency exhibited a lowered level of &aminolevulinic acid dehydratase activity. It appeared that this enzymatic step could become rate limiting for the synthesis of heme in this tissue and since both catalase and tryptophan pyrrolase are hemeproteins, their activities in the liver would reflect the levels of available heme. h expected, the activities of these two enzymes in vitamin E-deficient livers were significantly lower than those in the corresponding controls (Table VI).

Microsomal
Cytochromes bg and P-450--In vitamin E deficiency, the hemeprotein components of the microsomal drug-metabolizing enzyme systems were also depressed as shown in Table VII.
The levels of  VI   TABLE   IX Effect of vitamin E deJiciency on hepatic catalase and tryptophan pyrrolase activities Enzymatic assays were performed as described under "Experimental Procedure." The experimental animals were maintained on a vitamin E-deficient diet for a period of 18 weeks. Values represent means f SE of observations from five animals. p Values <0.05 were considered significant.

Response
in speci$c radioactivity of pulse-labeled hepatic microsomal protoheme after administration of vitamin E to dejkient animals Groups of vitamin E-deficient animals, fasted overnight, were injected with vitamin E, 1.0 mg per animal, and killed at various time intervals.
Each animal received a pulse-dose of 10 pCi of d-aminolevulinic acid-4-W 1 hour before being killed. Concentrations of microsomal protoheme were measured spectrophotometrically as its alkaline pyridine-hemochromogen. Details are described under "Experimental Procedure." Results are means of three animals in each group. acid-&-W or porphobilinogen-YY In this experiment rats were maintained on a vitamin Edeficient diet for a period of 17 weeks. After 16 hours fasting, each animal received a pulse-dose of either 10 pCi, &aminolevulinic acid-4-W, or 6.4 X lo5 dpm, porphobilinogen-W, and hepatic microsomes were isolated 1 hour after injection of the isotope. Concentrations of microsomal protoheme were measured spectrophotometrically as its alkaline pyridine-hemochromogen.
Details are described under "Experimental Procedure." Results are mean of three animals. Protoheme-Since &aminolevulinic acid synthase activity was not depressed to the same extent as b-aminolevulinic acid dehydratase in the livers of deficient animals, the results were suggestive of an aberration at the level of the second enzyme. In order to test this hypothesis, microsomal protoheme was pulselabeled in vivo with either b-aminolevulinic acid-4J4C or porphobilinogen-W, these being the substrate and product for the enzymatic reaction catalyzed by d-aminolevulinic acid dehydratase. The results showed that in deficient animals the incorporation of radioactivity from &aminolevulinic acid-4-Y into protoheme was only about 7% of that in the control animals whereas the difference in the incorporation of label from porphobilinogen-14C into protoheme was not significant (Table VIII). E$ect of Vitamin E on Specific Radioactivity of Pulse-labeled Hepatic iMicrosoma1 Protoheme in Dejkient Animals-When deficient animals were injected with vitamin E, within an hour there was a dramatic rise in the specific radioactivity of hepatic microsomal protoheme, pulse-labeled with Saminolevulinic acid-4-14C (Table IX).
Maximum stimulation of incorporation of radioactivity was obtained at approximately 3 hours following the administration of vitamin E. Between 6 and 9 hours after Vitamin E, the rate of heme synthesis declined to that of controls.
Locus of Defect in Heme Synthesis-The results presented in this paper indicate that vitamin E deficiency in the rat results in a partial loss in the ability of cells to synthesize heme. However, the locus of the defect in hematopoietic cells such as the bone marrow appears to be distinctly different from that in nonhematopoietic cells of the adult liver. In the following simplified outline of the biosynthesis of heme, respectively. Since deficient bone marrow cells exhibit a low level of &aminolevulinic acid synthase activity in parallel with a specific loss in their ability to incorporate radioactivity from glycine-2J4C into heme without any significant change in the pattern of labeling of heme from d-aminolevulinic acid-4-14C, it is inferred that in hematopoietic cells, vitamin E deficiency resultsin a defect at the &aminolevulinic acid synthase locus. On the other hand, in the deficient liver, b-aminolevulinic acid dehydratase, the second enzyme of the heme biosynthetic pathway is lowered. This is compatible with the finding that the concomitant decrease in the incorporation of radioactivity into hepatic microsomal protoheme is observed only when &-aminolevulinic acid&W is used as the labeled precursor. The labeling pattern with porphobilinogen-1% remains unaltered under these conditions.
Therefore, it appears that in nonhematopoietic cells, such as in the liver, the defect is at the level of the &aminolevulinic acid dehydratase locus.

Suspected
Role of Lipid Peroxidation in Vitro in Inactivation of Enzymes-Although the rapid deterioration in the activity of rat liver microsomal drug-metabolizing enzymes in vitro has been suspected to be caused by the effects of lipid peroxides, Gram and Fouts (32) found that prevention of lipid peroxidation by addition in vitro of vitamin E had no effect on the loss of drug-metabolizing activity.
Likewise, our inability to reverse the partial loss in heme-synthesizing ability of deficient cells by the addition in vitro of vitamin E shows that the reduction in biosynthetic activity is not attributable to lipid peroxidation in vitro.

Role of Vitamin E in Heme
Synthesis-In 1967, Carpenter (33) reported that oxidative demethylation of codeine and aminopyrine by microsomes from vitamin E-deficient rats is significantly lower than that of the corresponding control animals. She also indicated that while vitamin E or other antioxidants were without any effect in vitro, feeding deficient animals a diet supplemented with the vitamin restored the drug-metabolizing activity.
Because actinomycin D blocked this action of vitamin E, she postulated a regulatory function for the vitamin at the level of the synthesis of the drug-metabolizing enzymes. Since, in this study, the microsomal hemeproteins, cytochrome bg and cytochrome P-450, which are functional in drug metabolism have been shown to be regulated by the vitamin E-dependent hemesynthesizing system, it appears that the alterations in drug metabolism reported by Carpenter (33) are secondary to the decreased rate of heme synthesis.
The development of anemia is one of the manifestations of vitamin E deficiency in man and in primates (2-5).
It is also known that in the deficient primate, the anemia is in part the result of a reduced synthesis cf porphyrins and heme (3, 34). Similar anemias have been described in the pig (35) and in the rat (36) which were reared on a diet deficient in both protein and vitamin E. A failure to synthesize adequate amounts of heme precursors could be expected to result in several other abnormalities. The iron toxicity described in vitamin E deficiency (37,38) is probably caused by the limited production of protoporphyrin IX which is otherwise normally available for scavenging iron.
Our present studies have shown that in the Vitamin E-deficient rat, even in the absence of a demonstrable anemia, there is a distinct depression in the biosynthesis of heme. In the bone marrow the defect resides at the level of the first enzyme, 6aminolevulinic acid synthase involved in the formation of 6aminolevulinic acid, while in liver, it seems to be at the level of the second enzyme &aminolevulinic acid dehydratase which synthesizes porphobilinogen. There is an increasing body of evidence that the fat-soluble vitamins unlike the water soluble vitamins participate in the regulation of the synthesis of macromolecules (39)(40)(41)(42)(43).
A similar mechanism for vitamin E action is strongly favored by our recent studies showing that vitamin E prevents the induction of experimental porphyria (44) and also causes a remission in human porphyria (45), the former through a mechanism specific for vitamin E, involving the regulation of "inducible" hepatic &aminolevulinic acid synthase (15,46).