Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX

Biochimica et Biophysica Acta 928 (1987) 83-91

83

Elsevier BBA 11919

Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX Mohamed

M . R . E 1 D e i b a, C h a r l o t t e D . P a r k e r c a n d A r n o l d A. W h i t e a,b

John M. Dalton Research Center and Departments of h BiochemisttT and ': Microbiology, University of Missouri-Columbia, Columbia, MO (U.S.A.)

(Received 1 September 1986)

Key words: Guanylate cyclase; Enterotoxin; Hemin; Heme; Protoporphyrin IX; (Pig intestine mucosa)

Mg2+-dependent activity of intestinal brush border guanylate cyclase was stimulated 4-5-fold by 50-100 p M hemin. Higher concentrations were inhibitory. In the presence of 25% dimethyl sulfoxide, which stimulated activity 9-times, 50 /tM hemin further increased activity 1.7-fold. However, when activity was stimulated 32-fold by the Escherichia coil heat-stable enterotoxin, or 26-fold by Lubroi PX, hemin produced only concentration-dependent inhibition. The first type of activation was more sensitive to hemin than the second. Reduction of hemin by dithiothreitol eliminated stimulation of basal activity, while inhibition of Lubrol PX-stimulated activity remained. Protoporphyrin IX also had no effect on basal activity, however, it inhibited enterotoxin- and Lubrol PX-stimulated activities similarly, but only to half the extent of hemin. Substitution of Mn 2+ for Mg 2+ elevated basal activity 15-fold, and this Mn2+-dependent activity was inhibited by hemin. Mn2+-dependent activity was stimulated (43%) by enterotoxin, however, the stimulated activity was more sensitive to hemin inhibition than the basal Mn2+-dependent activity and both inhibition curves were congruent above 5 0 / t M heroin. Heroin inhibition of Lubrol PX-stimulated activity was much less with Mn 2+ than with Mg 2+. These results were interpreted as suggesting two sites of hemin inhibition; on an inhibitory regulator and on the enzyme. We also found that the secretory effect of enterotoxin in the suckling mouse bioassay was reduced 56% by the oral administration of hemin.

Introduction Ever since the discovery that activation of soluble guanylate cyclase by NO required heme, and the suggestion that NO-heme was the activating species [1], there has been increasing interest in the role of porphyrins and metalloporphyrins in guanylate cyclase regulation [2,3]. Protoporphyrin IX and closely related porphyrins were shown to activate purified soluble enzyme while certain structurally modified porphyrins and metalloporphyrins were inhibitors. Activation by N O or Correspondence: A.A. White, Dalton Research Center, University of Missouri, Columbia, Missouri 65211, U.S.A.

by compounds capable of releasing NO, was explained as resulting from its binding to heme iron and displacing it from the plane of the porphyrin ring. The opposite surface of the ring would then resemble protoporphyrin IX and hence be capable of interacting with and stimulating guanylate cyclase [3]. The situation with respect to the particulate guanyalte cyclase is not as well defined and it was observed very early that it was usually much less responsive to NO than was the soluble enzyme [4,5]. However, a well-washed particulate preparation from rat liver has been reported to be activated 4-fold by NO [6], and a more purified plasma membrane preparation from the same tissue was reported to be activated 9-fold by proto-

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

84

porphyrin IX, and to a variable extent by closely related porphyrins [7]. Recently, Waldman et al. [8] found that hemin would stimulate (3-10-fold), the activity of guanylate cyclase in membranes prepared from homogenates of rat lung, C 6 rat glioma cells or B103 rat neuroblastoma cells. While the tung membrane might be contaminated with soluble enzyme, activation was occurring in a hemin concentration range (50-100 ffm) which was known to inhibit soluble enzyme. The other two membranes were known to possess only particulate guanylate cyclase. Reduction of hemin to heme largely eliminated activation, and protoporphyrin IX was also inactive. We decided to perform similar experiments with a brush border membrane preparation from pig intestinal mucosa [9]. Mucosal guanylate cyclase is virtually all particulate, and the larger part of this activity is in the brush border [10]. The brush border enzyme is also specifically activated (30-50-fold) by the Escherichia coli heat-stable enterotoxin [11,13], We found that hemin would stimulate this particulate guanylate cyclase, while heme and protoporphyrin IX did not. However, when the enzyme was stimulated with enterotoxin or with Lubrol PX, all three inhibited activity. Materials and Methods

Enterotoxin purification. E. coli strain 431 was cultured as previously described [9], and the culture supernatants were carried through absorption to and elution from an Amberlite XAD-2 column, and acetone fractionation to remove protein, as described by Staples et al. [14]. The subsequent steps in the purification were adapted from the following previously described procedures: chromatography on a 2.5 x 82 cm Sephadex LH-20 column, eluted with 99% methanol, 1% acetic acid [11]; extraction of the neutral aqueous solution with c h l o r o f o r m / m e t h a n o l (1 : 1) to remove color and lipid [15]; chromatography on a 2.5 x 90 cm Bio-Gel P-4 column, eluted with 5% acetic acid [16]; chromatography on a Pharmacia Mono Q H R 5 / 5 anion exchange H P L C column [17]; reverse-phase H P L C on a Waters /~Bondapak C18 celumn [16]. Enterotoxin was located by guanylate cyclase activation, and the two principal peaks of

activity eluting from the last column were separately collected and lyophilized. The second peak was larger, and this was used for the present work. The protein content of this preparation was determined by the fluorescamine method [18], using bovine serum albumin as standard and was found to underestimate by 11% the enterotoxin present as determined by enzyme-linked immunosorbent assay [19]. We present the enterotoxin used in terms of fluorescamine protein, and found that 1.6 ng produced 50% of maximal guanylate cyclase activation. Brush border membranes. These were prepared from everted segments of weanling pig jejunum, as described earlier [9]. The segments were placed in 750 ml of ice-cold solution A [20] (1.5 mM KC1, 96 m M NaC1, 15 mM EDTA, 8 m M K H z P O 4, 0.02% N a N 3, p H 6.8), and were vibrated for 30 min at full speed with a Vibro-Mixer E~ (Chemapec, Inc., Woodbury, NY) fitted with a blade stirrer. The segments were removed from the resulting cell suspension by filtration through nylon netting, and the vibration and filtration was repeated twice with fresh solution A. The remainder of the procedure was essentially as described by Gerke and Weber [20]. Solution A and the homogenizing solution contained 50 fig of phenylmethylsulfonyl fluoride per ml. The final pellet was resuspended in 12 ml of 20% glycerol (and is referred to here as BBMV) and stored in liquid N 2 in small aliquots. Membrane protein was determined by the bicinchoninic acid method of Smith et al. [21], using reagents provided by Pierce Chemical Company (Rockford, IL). With bovine plasma albumin as standard, this method gives values for membrane protein which are only 6-7% lower than those found by the method of Lowry et al. [22]. Guanylate cyclase determination. The reaction mixture contained 50 mM Tris-HCl (pH 7.6) 1.2 m M [a-32p]GTP (approx. 1 #Ci), 3 mM MgC12, 10 mM sodium azide, 15 m M creatine phosphate, 10 units creatine phosphokinase, 0.2 m M E G T A and different amounts of porphyrin, in a final volume of 75 #1. Reactions were initiated by adding ice-cold membranes (approx. 5 fig protein) to the reaction mixture contained in a siliconized 10 × 75 m m tube and prewarmed to 30 ° C. After a 5 min incubation 10 /~1 of water was added

85 containing either nothing, 5 ng enterotoxin, or 1.5% Lubrol PX and the reaction continued for another 10 min. The reactions were terminated by adding 0.15 ml of 1 M perchloric acid and the cyclic [3zp]GMP formed was purified and counted as previously described [23]. All determinations were performed in triplicate and presented are means _ S.D. of a representative experiment. Each experiment was performed two or three times. The figures have bars representing the S.D. except where this was smaller than the symbol. Further details of the determination were described earlier [91. The experiment on the activating effect of nitrosylhemoglobin (Table I), in general, followed the procedure of Tsai et al. [24] for preparation of a solution of deoxyhemoglobin. The concentration of this solution was determined as heme [25], after which it was diluted to 0.5 m M and a portion was converted to nitrosylhemoglobin [24]. Activation was commenced by the addition of enzyme (5-6 /~g protein) to ice-cold reaction tubes containing deoxyhemoglobin or nitrosylhemoglobin in reaction mixture containing 2 m M dithiothreitol but without substrate. After 2 min, 10 /~1 of [ct32p]GTP was added and the tubes were placed in the 3 0 ° C water both for a 15 rain reaction. Solutions of hemin and protoporphyrin IX were prepared by adding 0.2 ml of 0.2 M N a O H to 8 - 1 0 mg of the compound in an ultracentrifuge tube. After vortexing for 5-10 s, 1.8 ml of water was added and the p H was adjusted to approx. 7.6 with 0.1 M HC1. After centrifugation at 102 900 × g for 15 rain at 4 ° C, the supernatant solution was removed and the concentration of the hemin determined as pyridine hemochrome [25], (E557 = 34.4 m M 1 . c m - 1 ) [26]. The concentration of protoporphyrin IX solution was determined from the absorbance of its dication in 2.7 M HC1 (E408 = 262 m M 1. c m - 1 ) [26]. Suckling mouse bioassay. Secretory activity was determined as fluid accumulation in the intestine of 3-5-day-old suckling mice (CFW strain, SwissWebster) essentially as described by Giannella [27]. They were injected intragastrically with 0.1 ml of test solution and after 90 min the fluid accumulation ratio of each animal calculated as the ratio of the weight of the entire intestine to that of the rest of the body. 1 mouse unit (MU) is

a fluid accumulation ratio of 0.09. Significance between sample means was evaluated using Student's t-distribution [28]. Materials. H e m i n (Type III) and protoporphyrin IX were purchased from Sigma, as were other unspecified biochemicals. Reagents for the guanylate cyclase assay were obtained as described previously [23]. Resets

Brush border m e m b r a n e guanylate cyclase was not activated by nitric oxide (NO) or NO-heme nor by reagents giving rise to N O such as sodium azide, sodium nitroprusside or N-methyl-N-nitroN-nitrosoguanidine. While such a lack of response has been described for other particulate guanylate cyclase [4,5] we decided to attempt activation with NO-hemoglobin, since Tsai et al. [24] found that it would activate purified rat liver soluble guanylate cyclase, although this preparation was unresponsive to N O or NO-heme. Table I shows results from one such experiment and demonstrates a doubling of activity by NO-hemoglobin in the concentration range used (0.5-8.0 /~M). In other experiments we found strong inhibition above 8 /~M. In view of the lack of stimulation by reagents giving rise to N O [3], and because of the extensive washing of these membranes during their purification, it appeared doubtful that these results were due to contamination by soluble guanylate cyclase [29], particularly since virtually all of the guanylate cyclase in intestinal epithelium is particulate [101. TABLE I ACTIVATION BY NO-HEMOGLOBIN Hemoglobin or NO-hemoglobin concentration (~M)

Guanylate cyclase activity (pmol cGMP/min per mg protein) with with hemoglobin NO-hemoglobin

0 0.5 1.0 2.0 4.0 6.0 8.0

33±0.8 32+0.4 29±1 29±1 30±2 29±1 30±2

33±0.8 57±3 62±2 64±2 65+5 67+3 61±3

86

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shifted to the left, however, trials with hemin concentrations below 10 ffM resulted only in small inhibitions. Removal of azide from the reaction mixture did not prevent hemin activation of basal activity nor hemin inhibition of enterotoxin- or Lubrol PX-stimulated activity. Thus, the presence of an agent that complexed with the coordinated iron had no effect on the results [8]. Furthermore, 100 ffM free Fe 3+ (in excess of the 0.2 mM E G T A ) as FeC13, had no effect on basal activity and inhibited enterotoxin- and Lubrol PX-stimulated activity only 6-7%. Although 100 /~M methemoglobin was found to have no effect on basal activity or on activity stimulated by enterotoxin, it inhibited Lubrol PX-stimulated activity by 47%. We concluded that the detergent was probably unfolding the methemoglobin so as to release the hemin for association with the enzyme. In order to learn if the valance state of the hemin iron was a factor, we determined the effect of reducing it to Fe 2+ (heme). It was necessary to

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While the results with NO-hemoglobin were not quantitatively impressive as compared with the activation induced by enterotoxin [9], they were reproducible, and this induced us to attempt activation with a different porphyrin derivative. H e m i n had been reported to activate other particulate guanylate cyclases [8] and Fig. 1 shows that hemin activated brush border guanylate cyclase to a comparable extent (4.6-fold at 75/~M) and in the same concentration range (50-100 #M) as previously reported [8]. As in that report, higher concentrations inhibited activity. However, Fig. 1 also shows that when brush border guanylate cyclase was stimulated by either enterotoxin or Lubrol PX, hemin produced only a concentration-dependent inhibition. At 150 /~M hemin, enterotoxin-stimulated activity approached basal activity while Lubrol PX-stimulated activity was decreased to 4.5-times basal. We considered the possibility that activated guanylate cyclase might have a stimulatory response to hemin that was

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87 include the reducing agent (2 mM dithiothreitol) in the reaction mixture, in order to prevent autoxidation of the heme, however, since dithiothreitol destroys the biological activity of this. enterotoxin [14], we could only use Lubrol PX to activate the enzyme. As reported by Waldman et al. [8], the stimulatory effect of heroin on basal activity was erased by reduction to heme, however, reduction did not prevent its inhibition of Lubrol PX-stimulated activity. Although the shape of the hemin dose-response curve was changed, the maximal inhibition achieved was actually increased when the iron was reduced. The final factor to be considered was the iron itself, and Fig. 3 shows results obtained with protoporphyrin IX. This compound exerted comparable inhibitory effects on both enterotoxin- and Lubrol PX-stimulated activity, although the maximal inhibition at 150 /~M was about half that reached by hemin or heme. As in the case of heme, protoporphyrin IX did not stimulate basal activity [8].

Guanylate cyclase basal activity measured in the presence of Mn 2+ is 10-20-fold higher than Mg2+-dependent activity, however, the latter is more responsive to enzyme activation than the former, so that on activation the two approach equivalence [30]. Fig 4 shows the effects of hemin on Mn2+-dependent activity of BBMV. This was a different enzyme preparation than that used for the other figures, so the absolute specific activities are not directly comparable, however, the Mn 2+dependent basal activity was 15-fold higher than the Mg2+-dependent basal activity in this preparation. There was no hemin stimulation of Mn 2 +-dependent basal activity, instead, increasing hemin exerted only a progressive inhibitory effect that reached 64% at 150/xM. Enterotoxin was able to stimulate Mn2+-dependent activity only 43% and this increased activity was more sensitive to hemin inhibition than was the basal activity; the two curves becoming congruent above 50 /~M hemin. In the presence of Lubrol PX, hemin was less

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88

effective as an inhibitor of Mn2+-dependent activity than it was of Mg2+-dependent activity (compare with Fig. 1); inhibition of the first by 150/~M heroin was 21% of the second. Moreover, increasing hemin produced an essentially linear inhibition of Lubrol PX-stimulted MnZ+-dependent activity, which was unique. In previous work, we showed that dimethyl sulfoxide exerted a concentration-dependent inhibition of enterotoxin-stimulated activity, while at the same time stimulating basal activity [9]. We concluded that this was probably due to an inhibition of enterotoxin binding to its receptor plus an interference with receptor-cyclase coupling. Table II shows the effect of an increasing concentration of hemin in the absence and presence of 25% dimethyl sulfoxide, at which concentration enterotoxin receptor-mediated activation is essentially ehminated [9]. While 25% dimethyl sulfoxide increased basal activity 9.5-fold, this increase was not inhibited by hemin. Rather, hemin exerted an additional stimulatory effect, although the percent increase was less than with the control and the activity peak shifted to a lower hemin concentration. In order to determine if the hemin (heme) inhibition of enterotoxin-stimulated guanylate cyclase activity might be of physiological importance, we attempted to inhibit the biological effect of enterotoxin in the suckling mouse bioassay. Solutions of hemin and of enterotoxin were injected perorally, both separately and mixed together. Table III shows results from that study, where the addition of 75 nmol of hemin to the TABLE II A C T I V A T I O N BY H E M I N IN THE P R E S E N C E OF DIMETHYL SULFOXIDE Heroin concentration

0 10 25 50 75 100

Guanylate cyclase activity (pmol c G M P / m i n per mg protein) control

+ 25% dimethyl sulfoxide

36_+4 47+5 93.+5 151_+6 166 + 3 142 + 6

344.+ 11 382.+15 474+ 8 593+ 2 467 + 21 403 _+17

TABLE III H E M I N I N H I B I T I O N OF T H E SECRETORY E F F E C T OF STa IN I N F A N T MIC E Treatment

Fluid accumulation ratio

Control heroin (75 nmol) STa (5 MU) Heroin + STa

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~,h,~ There were l 0 13 mice in each treatment group. Fluid accumulation values represent the mean for that group ± S,E. Values with the same letters are significantly different, P < 0.005.

enterotoxin solution (5 MU), reduced the secretory effect by 56%. In preliminary trials we were not able to increase the effect by pretreating the mice with heroin. Discussion We have optimized conditions for the assay of porcine intestinal brush border guanylate cyclase [9]. For example, the results depicted in Fig. 1 show a 32-fold stimulation by enterotoxin and a 26-fold stimulation by Lubrol PX, and with other brush border preparations we have demonstrated 60- to 70-fold activations by the same amount of enterotoxin. Comparable stimulations of soluble guanylate cyclase have been demonstrated with NO-heme and NO-hemoglobin [1,24,33]. It has therefore been our inclination to view the doubling of activity we found with NO-hemoglobin (Table I), as resulting from contamination of the membranes with a different guanylate cyclase, perhaps associated with the small amount of basal lateral membranes known to be present [31]. De Jonge [32] found kinetic differences between guanylate cyclase in brush border and microsomal preparations from rat small intestinal villous cells. The microsomal preparation, resulting mainly from basal lateral membranes, resembled soluble guanylate cyclase in kinetic properties, while the brush border enzyme had characteristics in comm o n with other particulate enzymes. Ignarro et al. [33] have provided evidence for the transfer of NO-heme from NO-hemoglobin and other NOhemoproteins, to heme-deficient purified guany-

89 late cyclase, with consequent enzyme activation. This type of activation was therefore a special case of the general mechanism for activation of soluble guanylate cyclase by NO-heme [3]. We will have to defer conclusion as to the significance of the small activation of brush border enzyme by NOhemoglobin, until we have studied the guanylate cyclase activity in purified basal lateral membranes. In contrast, the effects of hemin were obvious. Its biphasic effect on basal activity (Figs. 1, 2) implied that the maximum achieved at approx. 50 /~M was a resultant of both stimulatory and inhibitory effects. This concept was reinforced by the rapid decrease in stimulated activity (particularly enterotoxin stimulated) induced by relatively low concentrations of hemin. The implication was that stimulation of activity by either enterotoxin or Lubrol PX had relieved (or reset) one component of the basal biphasic response, so that only the inhibitory response to hemin was observed. Hemin stimulation of basal activity was Mg 2 +-dependent and was eliminated in the presence of Mn 2+ [8] (Fig. 4). Mn2-dependent basal activity was 15-fold higher than Mg2÷-dependent activity and here again hemin was only inhibitory (Fig. 4). The component involved in heroin stimulation required a greater specificity of structure than did the inhibitory component, since the porphyrin had to contain iron in the ferric state (Figs. 2 and 3). This was the same requirement found for activation of guanylate cyclase in certain other membranes [8], but contrasts with the report that protoporphyrin IX and related metal-free porphyrins stimulated activity in rat liver plasma membranes [7]. An important distinction between the effects of hemin on brush border as compared with other particulate guanylate cyclases is the difference in response when Lubrol PX was present. Hemin was able to further stimulate activity in rat lung membranes that was already elevated by Lubrol PX, and this stimulation was maintained after purification of the Lubrol-solubilized enzyme [8]. In contrast, treatment of BBMV with Lubrol PX resulted only in heroin inhibition (Figs. 1, 2). Since porphyrin regulation of purified guanylate cyclase both soluble [3] and particulate [8], implied a direct interaction with the enzyme, we assumed a similar direct interaction with brush

border guanylate cyclase. To test this assumption, we studied the effect of hemin on Mn2+-depen dent activity, expecting an analogy with the better defined adenylate cyclase system. The catalytic subunit of adenylate cyclase uses MnATP preferentially as substrate, although MgATP is used as effectively when the catalytic subunit is associated with the GTP-binding protein mediating stimulation [34]. We expected that if hemin was interacting directly with the catalytic subunit of guanylate cyclase, the enterotoxin- or Lubrol PXstimulated Mn2+-dependent activity should be inhibited essentially as had Mg2+-dependent activity. The results in Fig. 4 were very different from those expected, since hemin was much less effective as an inhibitor of Mn2+-dependent activity (particularly Lubrol PX-stimulated) than of Mg2+-dependent activity. If the analogy with adenylate cyclase was valid, this suggested the existence of a separate factor mediating hemin inhibition. It appears that in addition to substituting for Mg 2+ in combination with GTP, Mn 2+ can activate guanylate cyclase. Braughler [35] proposed that changes in the oxidation state of excess Mn 2÷ induced similar changes in the rat liver enzyme, while Gerzer et al. [36] working with the purified bovine lung soluble enzyme suggested that excess Mn 2÷ changed the properties of the enzyme to a partially activated form. Since reduced hemin (heme) did not activate basal brush border activity (Fig. 2) it is possible that an oxidative event is also involved in hemin activation of this guanylate cyclase. DeJonge et al. [37] have a paper in the press in which they suggest that critical enzyme sulfhydryl groups are involved in activation. They found activation of the BBMV enzyme not only by hemin, but also by certain bivalent metal ions (Cu 2+, Zn 2+, Cd 2+, pb2+). Dithiothreitol eliminated both hemin and Cd z + activation. An oxidative event cannot be involved in the inhibitory effects of hemin since heme has similar effects. This inhibition appears to involve two sites of action. The first site may be related to enterotoxin activation, since Fig. 4 shows that such activation of Mn2+-dependent activity was suppressed by relatively low concentration of hemin, while the second site may be on the catalytic subunit. If we examine the curves for hemin

90 inhibition of Mn2+-dependent activity (Fig .4), it m a y be noted that the slopes of all three curves are similar at hemin concentration above 100/~M. Since the inhibition curve of Lubrol PX-stimulated activity is linear, this implies that it diagrams the inhibitory response of a single c o m p o n e n t c o m m o n to all three activity states, the catalytic activity (site 2). The linear response of the one curve also suggests that one or more additional factors necessary for an enhanced response to hemin (site 1 inhibition) were functionally eliminated by Lubrol PX plus M n 2+. Lubrol PX [38] as well as M n 2+ [39] have been proposed to induce a functional blockade of the inhibitory regulatory c o m p o n e n t of adenylate cyclase, and their combined effects on brush border guanylate cyclase m a y result in a complete blockade of a similar inhibitory component(s). The presence of such a factor was suggested by the observations of Waldm a n et al. [39], that there was a decrease in the ratio of MnZ+-dependent to MgZ+-dependent activities during purification of the Lubrol PXsolubilized particulate guanylate cyclase from rat lung. Such a decrease would occur if a Mg2+-de pendent inhibitor was being removed. Considering Mg2+-dependent activity in these terms, p r o t o p o r p h y r i n IX inhibition may represent inhibition primarily at site 2 (Fig. 3), while inhibition by hemin and heme was effected at both sites (Figs. 1,2). Reduction of hemin to heme decreased inhibition at site 1 while increasing inhibition at site 2 (Fig. 2). Site 1 inhibition might involve the binding of heroin or heme to the inhibitor, with a consequent increase in the affinity of the inhibitor for the catalytic unit. A difference in the binding affinities of hemin and heme would, therefore, result in a difference in inhibition. O u r demonstration that hemin inhibited the biological effect of the enterotoxin in the suckling mouse biassay (Table III), provides evidence that hemin (or heme) m a y regulate guanylate cyclase activity in vivo. However, we have not excluded the possibility of hemin inhibition at other steps in stimulus-secretion coupling, e.g., cyclic G M P - d e p e n d e n t phosphorylation or the activation of C1 and K + channels. Although it has been d e m o n strated that hemin is absorbed intact by the intestine, there is a species difference in the percent

absorption [41]. Mice were shown to absorb only about 1% of an oral dose, as c o m p a r e d with 8-9% for guinea pigs. This m a y be part of the explanation for our inability to completely prevent enterotoxin-induced secretion in the mouse. Another relates to the fact that hemin (or heme) solutions contain polymers of high molecular weight which are poorly absorbed. The addition to these solutions of c o m p o u n d s which complex with hemin and maintain it in the monomeric form, e.g., histidine or niacin, enhance absorption [42]. Therefore, our future efforts in this direction will be to correlate enhanced absorption with in vivo efficacy.

Acknowledgements This work was supproted by the U.S. Department of Agriculture under Agreement 82-CRST-22049, D H H S Biomedical Research Support grant No. S07 RR05387 and the John M. Dalton Research Center. We are grateful to Dr. Michael R. T h o m p s o n , University of Cincinnati, for the enzyme linked i m m u n o s o r b e n t assay of our enterotoxin.

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