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Inhibition of Intestinal Iron Transport Induced by Tetracycline
GASTROENTEROLOGY
Vol. 53, No.4 Printed in U.S.A.
Copyright © 1967 by The William s & Wilkins Co.
INHIBITION OF INTESTINAL IRON TRANSPORT INDUCED BY TETRACYCLINE NORTON J. GREENBERGER, M.D., RICHARD D. RUPPERT, M.D., AND FRANCIS E. CUPPAGE, M.D. Division of Gastroenterology, Department of Medicine and Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio
sorption of radioiron from intestinal loops. Similarly, it was observed that in gut sacs obtained from tetracycline-treated r ats there was a significantly decreased mucosal to serosal transfer of radioiron.
Recent studies have indicated that the intestinal mucosa is important in controlling iron absorption. 1 - 5 However, the actual regulatory mechanisms remain poorly understood. In particular, the nature of mucosa l iron complexes has not been clearly defined. Iron is present in the intestinal mucosa in two forms: (1) non-protein bound iron which is transferred rapidly across the mucosa, a nd (2) protein-bound iron which is transported at a slower rate.2 • 4 • 6 Since a significant amount of iron in the intestinal mucosa is proteinbound, the question arises as to whether such protein-bound iron is important in controlling iron absorption. In the present investigation, studies were carried out to define more clearly the role of protein-bound iron in controlling the intestinal absorption of iron. Antibiotics known to inhibit protein synthesis in m amma lian cells were employed in an attempt to induce a defect in intestinal iron transport. These studies were performed utilizing isolated intestinal loops in the rat and everted duodena l gut sacs in vitro. It was found that treatment with tetracycline and cycloheximide resulted in impaired ab-
Methods Experim ents on iron absorption from isolated intestinal loops. Wistar female rats (Harlan
Received April 19, 1967. Accepted June 10, 1967. Presented in part at the Midwestern Meeting of the American Federation for Clinical Research, Chicago, Illinois, November 2, 1966, (Clin. Res. 14 : 432, 1966 (abstr.) ). Address requests for reprints to: Dr. Norton J . Greenberger, Department of Medicine, Ohio State University Hospital, Columbus, Ohio 43210. This work was supported by Grant AM-1055601 from the National Institu tes of H ealth. We thank Mrs. J acqueline Thomas and Mrs. J ane Funderburg for expert technical assistance.
Farms, Cumberland, Ind.) weighing 140 to 160 g were fasted for 16 hr prior to study. Under light ether anesthesia a laparotomy was performed and a 20- to 25-cm segment of duodenum and jejunum was isolated between double silk ligatures. A needle from a syringe containing Fe59SO, (New England Nuclear Corporation, Boston, Mass.; specific activity, 550 p..c per p..mole) was injected through the stomach and passed into the proximal duodenum, and the animal was given an intraduodenal inj ection of 1.0 ml of a solution containing 138 p..g of ferrous sulfate (50 p..g of iron) and 2.0 X 106 dpm. After varying time intervals (15, 30, or 60 min) the isolated loops were excised and the intestinal contents were washed out with 50 ml of 0.85% N aCl. The intestinal mucosa was removed from the loops by scraping and weighed, and the entire sample was placed in counting tubes. The mucosal scrapings and duplicate aliquots of the loop contents were assayed for radioactivity in a well type scintillation counter along with an appropriate standard. In preliminary studies the completeness of mucosal scraping was evaluated by assaying the muscular and serosal layers of the isolated intestinal loops for radioactivity. In over 50 studies it was found that 0.5 to 1.5% of the injected Fes•so, was recovered in the muscular and serosal layers. Thus, in these and subsequent studies, the radioactivity present in the muscular and serosal layers was considered to represent iron transferred from the mucosa. Experiments with antibiotics. Studies were carried out to determine the effect of inhibiting
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protein synthesis on the absorption of Fe""SO, . Initially, the antibiotics employed were cycloheximide (kindly supplied by Dr. George Savage, Upjohn Laboratories, Detroit, Mich.) and tetracycline (kindly supplied by Mr. James Eberlein, J. B. Roerig & Co., New York, N.Y.), since it had been previously demonstrated that these antibiotics inhibit protein synthesis in mammalian cells.'-'° For purposes of comparison, later studies were carried out utilizing penicillin and chloramphenicol. All antibiotics were given by intraperitoneal injection 4 hr prior to the preparation of isolated intestinal loops. The doses employed in these studies were as follows : cycloheximide, 5 mg per kg; tetracycline hydrochloride, 20, 200, 300, and 400 mg per kg ; potassium penicillin G, 1.8 g per kg; and chloramphenicol sodium succinate, 400 mg per kg. The pH of the tetracycline and cycloheximide solutions ranged from 2.14 to 3.40. To exclude the possibility that changes observed after antibiotic treatment were due to injection of an acidic solution, other studies were carried out in which animals received an int raperitoneal injection of 1.0 ml of a solution containing 0.1 N HCl and 0.15 g of ascorbic acid, the pH being 1.94. Under these conditions, no impairment of iron absorption and no morphological changes in the intestinal mucosa were found. Exp eriments on iron transport by everted duodenal sacs in vitro. Wistar female rats weighing 140 to 160 g were fasted for 16 hr and then injected intraperitoneally with either saline or tetracycline in a dosage of 400 mg per kg. Four hours later t he animals were killed and the small bowel was rinsed in situ with 50 ml of ice-cold 0.85% NaCl. The proximal 15 em of small intestine were excised and everted over a glass rod, and duodenal sacs were prepared according to the method of Wilson and Wiseman" as modified by Schachter and his associates." Two consecutive everted sacs each 6 em long were prepared. Mean wet weights of the proximal and distal sacs were approximately 800 and 600 mg, respectively. Once the intestine was dissected free it was chilled immediately in ice-cold saline and kept at 4 C throughout preparation of the gut sacs. The sacs were filled with 0.5 ml of a medium of the following composition: 0.145 M NaCl, 0.0001 M CaCl", 0.04 M fructose, 0.0008 M sodium ascorbate, freshly prepared, and 0.004 M Tris buffer (tris(hydroxymethyl) aminomethane, pH 7 .4) . Each sac was placed in an incubation flask containing 5.0 ml of the same medium plus 0.0004 M FeSO, and sufficient F e""SO. to give approximately 10,000 cpm per ml in a well type scintillation counter. The elapsed time
from death of the animals to placement of the gut sacs in the incubation flasks was always less than 5 min. The incubations were carried out at 37 C with continuous shaking and gassing with 95% oxygen-5% carbon dioxide for 2Y2 hr. The gut sacs were then washed and blotted dry, and the serosal fluid was drained. Duplicate aliquots of the mucosal and serosal fluids were assayed for radioactivity. In t he present studies the average recovery of serosal fluid was 90% of t he initial volume in proximal sacs and 100% or greater in distal sacs. Studies were also carried out to determine whether tetracycline added directly to the incubation flask would inhibit iron transport by everted duodenal sacs. The gut sacs were obtained from normal rats in the manner described above. In these studies the composition of the incubation media and the media placed within the sacs was identical with that previously described wit h t he exception that 100 /Lg of pure tetracycline powder (kindly supplied by Mr. J ames Eberlein, J . B. Roerig & Co., New York, N. Y .) were added to each flask prior to the start of the incubation. The incubations were carried out in the same manner as described above.
Results
Effect of common bile duct ligation on the absorption of radioiron from intestinal loops. Since bile accumulated in the closed intestinal loops during the absorption period, it was considered important to determine the effect of bile duct ligation on iron absorption (table 1). This was determined by measuring the mucosal uptake and mucosal transfer of radioiron. Mucosal uptake (A) was determined by subtracting the amount of Fe 59 S0 4 recovered in the loop contents from the amount injected. The amount of Fe 59S0 4 remaining in the mucosa (B) was determined directly. Mucosal transfer represents the difference between mucosal uptake (A) and the amount remaining in the mucosa (B) . In essence, therefore, mucosal transfer (A - B) reflects net absorption. It can be seen that there was no signific~nt difference in the mucosal uptake and transfer of radioiron between control animals and animals in which the common bile duct had been ligated either 30 min or 48 hr prior to study. Since bile duct liga-
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TABLE
Vol. 53, No . 4
1. Effect of common bile duct ligation on the absorption of Feo'SQ,a f1·om isolated intestinal loopsb Administered radioactivity
No. of experiments
Group
..... . . . .. . . Control . . .. . . . . . . Bile duc t ligated 30 min before ex. . .. .... . ... periment .. Bile duc t ligated 48 h r before experiment . . . .. . . a 6 c
20
Recovered in loop contents
(A) Mucosal uptake
%
%
(B) Remaining 1n mucosa
(A - B) Mucosal transfer
%
35.2 ± 11. 4• 64.8 ± 11 .4
%
27 .8 ± 9.3
37.0 ± 9.6
7
37. 0 ± 14.5
63. 0 ± 14 .5
30.9 ± 10 .8
32. 1 ± 18 .7
5
38.2 ± 7.3
61. 8 ± 7.3
27.6 ± 9.6
34.2 ± 7.9
138 l'g (50 l'g as iron) containing 2.0 X 10 6 dpm. Loops removed 30 min a fte r injection of radi oiron. Mean± 1 sD .
TABLE
2. Effect of graded doses of letmcycline on the absorption of Fe 59 SQ ,a from isolated intestinal loops 6 Administered Radioacti v ity
Group
No. of expcriments
B/ A (A) Mucosal uptake
Reco vered in loop contents
(B) Remaining m mucosa
(A - B) Mucosal tran sfe r
-%
.. Cont rol. ... T etracycli ne (20 mg/ kg ) . Te t racycline (200 mg/ kg) T et racy cline (300 mg/ kg ) . Tetracycline (400 mg/kg) T et racyclined (400 mg/kg) a
6 c d
20 6 8 6 5 8
35.2 39.6 43 .6 55.3 64.2 56.5
± ± ± ± ± ±
%
11 .4• 8.2 10.9 9.7 3 .1 8.9
64.8 ± 60. 4 ± 56.4 ± 44 .7 ± 35 .8± 43.6 ±
%
11. 4 8.2 10. 9 9 .7 3 1 8.9
27.8 22.5 21.9 23.5 18 .5 23.7
± ± ± ± ± ±
%
%
9.3 6.4 4. 1 9.4 3.0 9.3
37.0 37.9 34.5 21.2 17 .3 19.9
± ± ± ± ± ±
9.6 11. 8 9.9 10 .8 3.9 7 .7
42 ± 37 ± 39 ± 53± 52± 54±
10.0 12.4 5.4 13. 1 7 .8 10.5
138 l'g (50 l'g as iron ) co n taining 2.0 X 10 6 dpm. Loops removed 30 min afte r injection of radioiron. Mean± 1 SD. Common bile duct ligated 24 hr prior to study.
tion did not appear to affect iron absorption, the bile duct was not ligated for any of the subsequent studies with only one exception, described below.
Effect of tetracycline on the absorption of radioiron from intestinal loops. In table 2 are shown data on the effect of graded doses of tetracycline on the absorption of Fe 59 S0 4 from intestinal loops. In rats previously given tetracycline in a dose of 20 mg per kg, there was no difference in the mucosal uptake and transfer of radioiron as compared with controls. With injection of larger doses of tetracycline, there was a progressive decrease in iron absorption. In r ats injected with tetracycline in a dosage of 400 mg per kg, there was a significantly decreased mucosal uptake (36 versus 65%) and transfer (17 versus 37%) of radioiron as compared with controls.
The amount of radioiron remaining in the mucosa divided by the mucosal uptake, B j A, also reflects the interference in iron transport induced by treatment with t etracycline (table 2) . In control animals, 42% of the iron taken up by the mucosa was still present in the mucosa after 30 min as compared with a value of 52% in r ats treated with tetracycline in a dosage of 400 mg per kg (t = 2.07; P = <0.05). Studies were also carried out in which the absorption of -radioiron was determined in tetracycline-treated rats in which the common bile duct was ligated 24 hr prior to study (table 2). This was done in order to exclude the possibility that the inhibitory effects of t etracycline on iron transport were due to tetracycline being secreted via the common bile duct into the proximal small bowel and chelating iron
present in the lumen. It was found that there was no significant difference in the mucosal transfer of radioiron in tetracycline-treated rats with intact bile ducts as compared with animals with ligated bile ducts (17 versus 20%; P = >0.5). Thus, ligation of the common bile duct did not affect the inhibition of iron absorption from intestinal loops caused by treatment with tetracycline. To confirm that tetracycline blocks iron absorption, the absorption of iron was compared in control and tetracycline-treated rats at various time intervals. Isolated intestinal loops were removed 15, 30, or 60 min after injection of radioiron, and the mucosal uptake and transfer were determined as described above. The amount of iron absorbed was calculated on the basis of the mucosal transfer and the dose of radioiron (50 ,ug). These data are shown in figure 1. In control animals, iron absorption progressively increased and 23.5 ,ug were absorbed in 60 min. In contrast, in tetracycline-treated rats, there was a minimal increase in iron absorption over a 60min period with only 8.5 ,ug being absorbed. Iron transport by duodenal sacs in vitro. To document further that tetracycline inhibits iron transport, we carried out in vitro studies utilizing everted duodenal sacs obtained from control and tetracycline-treated rats (table 3). In studies using proximal gut sacs from control animals,
3. Effect of prior tetracycline" on iron transport by everted duodenal sacsb in vitro
T A BLE
Net iron transfer to serosal medium
Group Proximal sac
24 20 IRON 16
p.gm
8 4
15
30
45
60
MINUTES
FIG. 1. Absorption of Fe"'SO, (50 p.g of iron containing 2 X 10" dpm) from isolated intestinal loops in control and tetracycline-treated rats. The values shown represent the mean ± 1 so.
I
Distal sac
mp.moles/g'lft sac
Control. .... .. .. . . Tetracycline
P value.
35.6 ± 11 .4< 17.3 ± 10.0 (25) (26) 20.9 ± 11 .8 11. 8 ± 5.0 (30) (32) <0.001 <0.01
• The dose of tetracycline was 400 mg per kg given intraperitoneall y 4 hr prior to study. Figures in parentheses represent the number of determinations performed. b Two gut sacs were prepared from the proximal 12 em of rat small intestine and results a re shown for the more proximal and adjacent distal sac. Each sac was filled with 0.5 ml of a medium of the following composition: 0.145 M NaCl, 0.0001 lii CaCh , 0.04 M fructose, 0.0008 M sodium asco rbate, and 0.004 M Tris buffer. The sacs were placed in incubation flasks containing 5.0 ml of the same medium plus 0.0004 M FeS04 and sufficient Fe 59 S04 to give approximately 10,000 cpm per ml. The incubations were carried out at 37 C with continuous shaking and gassing with 95% oxygen and 5% carbon dioxide for 2.5 hr. c Mean± 1 so.
36 m,umoles of F eS0 4 were transferred from the mucosal to the serosal medium in 2Y2 hr. In contrast, with proximal sacs obtained from tetracycline-treated rats, only 21 m,umoles were transferred, a 40% decrease (P < 0.001). Using distal sacs from tetracycline-treated rats, there was also a significantly decreased transfer of FeS0 4 as compared with controls, 11.8 versus 17.3 m,umoles (P = <0.01). To evaluate the possibility that the inhibitory effect of tetracycline on iron transport by everted gut sacs might be due to nonspecific divalent cation binding, studies were carried out in which tetracycline was added to flasks containing gut sacs from normal rats (table 4). In studies utilizing proximal sacs, the addition of tetracycline to the media resulted in a 20% decrease in the mucosal to serosal transfer of FeS0 4 , 28 m,umoles as compared with 36 m,umoles (P = <0.025). However, with distal sacs, there was no significant differ-
=
28
ABSORBED
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ence between control fl asks and flasks containing tetracycline in the transfer of FeS0 4 , 17 versus 19 m~-tmoles (P = > 0.5). Effect of various antibiotics on iron absorption. To determine whether the inhibitory effect of tetracycline on iron a bsorption was specific for tetracycline, studies were carried out in which the absorption of Fe 59 S0 4 was measured in isolated intestinal loops in animals treated with massive do ses of different antibiotics (table 5). There was no significant difference in the mucosal uptake and transfer of Fe 59S0 4 in animals treated with penicillin (1800 mg per kg) and chloramphenicol (400 mg per kg) as 4. E:O"ect of tetracycline" added to the media on iron transport by everted duodenal sacs in vitro
TABLE
Net iron transfer to serosal medium
Group Proximal sacb
I
Distal sac
mp.molesfgut sac
Control. Control + tetra cycline". P value.
35 .6 ± 11.4' (25)
17. 3 ± 10.0 (26)
27.7 ± 9.7 (22) < 0.025
19.0 ± 6.6 (22) >0.5
a Tetracycline, 100 /J.g , was added to eac h fl ask at t he start of t he incubation. Figures in parentheses represent the number of dete rmin ations performed. b The preparation of the gut sacs an d the condi tions of the expe riment we re s imila r to t hose described in table 3. 'Mean± 1 sn.
T A BLE
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compared with controls. Similarly, the BI A ratio in these 3 groups of rats was the same. In contrast, in rats treated with tetracycline (400 mg per kg ) or cycloheximide (5 mg per k g) , there was a marked decrease in the mucosal uptake and transfer of Fe 59S0 4 • In addition, there was an increase in the B I A ratio refl ecting significant impairment in iron transport. Effect of tetracy cline on intestinal histology. Sections of jejunum were t ak en for histological examination 4 hr after treatment with tetracycline. The surface epithelium, villi, lamina propria, and crypts all appeared norma l upon examination by light microscopy (fig. 2) . Electron microscopy of the lumin al absorptive surface (fig. 3) and subapical cytoplasm (fig. 4) of jejunal epithelial cells revealed normal microvilli, mitochondria, and endoplasmic reticulum. In earlier studies, it was demonstrated that treatment of rats with cycloheximide was not associated with significant histological changes in the intestinal mucosa. 13 Discussion
It has been established that, after the administration of a physiological dose of iron to the rat, a significant amount of iron in the intestinal mucosa is proteinbound during the initial 60 to 90 min that iron absorption is taking place. 2 • 4 To evaluate the possibility that such protein-bindin g is important in the regulation of iron a bsorption, the antibiotics cycloheximide
5. Effect of various antibiotics on the absorption of F e 59 SO 4" from isolated intestinal loopsb Administered radioactivity
Group
N o. of experiments
Recovered in loop con tents
%
Con trol. - .... P enicillin (1800 mg/kg). Chloramphenicol (400 mg/ kg) .. T etracycline (400 mg / kg). Cycloheximide (5 mg/kg).
(A) Mucosal uptake
(B ~ R emaining m mucosa
(A - B) Mucosal transfer
%
%
%
20 5
35.2 ± 11.4' 38 .7 ± 13.0
64.8 ± 11.4 27.8 ± 9.3 37 .0 ± 9.6 42 61.3 ± 13 .0 24.1 ± 11.0 37.2 ± 13 . 0 39
6 5 18
39 .1 ± 8.6 64.2 ± 3.1 71.2 ± 14 . 1
60.9 ± 8.6 25.1±6 .1 35.8 ± 3. 1 18.5 ± 3 .0 28.8 ± 14 . 1 17.0 ± 6.8
138 /J.g (50 /J.g as iron) containing 2.0 X 10 6 dpm. Loops removed 30 min after injection of radioiron. 'Mean± 1 so.
a
b
B/A
%
± 10.0 ± 12 .0
35.8 ± 10.0 39 ± 9.0 17. 3 ± 3.9 52 ± 7.8 11.8 ± 8.2 59 ± 13.0
Frc. 2. Photomicrograph of ra t jejunum 4 hr after tetracycline, 400 mg per kg. The surface epithelium, villi, crypts, and lamina propria all appear normal (X 100) .
Frc . 3. Electron micrograph of th e luminal absorptive surface of th e small intestine with normal appearing organelles. A, amorphous coat; Mv, microvilli ; TW, terminal web area ; C, cent riole; R, clustered ribosomes; lvf, mitochondrion. Dow epoxy resin embedded. Stained with uranyl acetate and lead citrate ( X 27,000).
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and tetracycline were utilized because they It appears unlikely that the observed have been shown to inhibit protein synthe- effects of tetracycline were due to nonsis in mammalian cells. 7 - 10 It was found specific mucosal damage since tetracycline that treatment of rats with these drugs treatment was not associated with evident results in impaired a bsorption of radioiron structural changes in the intestinal mucosa from isolated intestinal loops (table 2; upon examination .by light and electron Fig. 1). The inhibitory effects of t etracy- microscopy (Figs. 2 to 4). However, this cline were found to be dose-dependent. The structural integrity does not rule out the observation that there was still some mu- · possibility of biochemical defects in the cosal uptake and transfer of radioiron after cell other than protein synthetic mechatreatment with massive doses of tetracy- nisms; for example, it has been demoncline indicates that this t reatment did not strated that treatment of rats with tetracompletely block iron transport. cycline may result in impaired fat abThe absorption of radioiron in tetracy- sorption.15 Thus, the possibility that the cline-treated rats was similar to that in inhibition of iron transport by t etracycline controls during the first 15 min but did not was due to nonspecific effects on the intesincrease appreciably thereafter (fig. 1). tinal mucosa cannot be excluded. One possible interpretation of this observaIt has been demonstrated that the tetration is that tetracycline treatment did not cycline drugs can bind a number of divainterfere with the absorption of the rapidly lent cations, including ferrous iron.16· 17 It transported or non-protein bound iron but has also been reported that tetracycline did impair the transport of protein-bound could bind extensively to both nucleic acids iron. During the initial 30 to 60 min that and proteins, with the binding being mediiron absorption is occurring, progressively ated by divalent cations such as zinc, calmore iron in the mucosa is present as pro- cium, magnesium, and manganese. 18 These tein-bound iron. 2 • 4 and other observations have led to the sugAdditional evidence that tetracycline gestion that the biological effects of the treatment induces a defect in iron trans- tetracycline drugs might be secondary to a port was obtained in the in vitro studies. It chelation phenomenon. 18 Thus, it would was found t hat there was a significantly appear reasonable to expect tetracycline to decreased mucosal t o serosal transfer of bind ferrous iron. However, there are ferrous sulfate utilizing everted duodenal several lines of evidence which suggest that sacs obtained from tetracycline-treated the impaired transport of iron observed in rats (table 3). The values that we ob- the present studies was due to other factained for the mucosal t o serosal transfer tors in addition to the presumed binding of of radioiron in proximal and distal sacs ferrous iron by tetracycline. from control animals are quite comparable First, in studies employing rats treated to those reported by Manis and Schachter. 14 with tetracycline and bile duct ligation, The data obtained in the present study there was no difference in the mucosal are in agreement with studies recently re- transfer of iron as compared to rats ported by Y eh and Shils. 15 These investi- treated with tetracycline alone (table 2) . gators demonstrated that there was a de- These studies exclude the possibility that creased absorption of radioiron in rats t he inhibitory effects of tetracycline were given an intramuscular or intraperitoneal due to chelation of intraluminal iron. Secinjection of tetracycline (250 mg per kg). ond, inhibitory effects on iron transport Three mechanisms whereby treatment with similar to those obtained with tetracycline tetracycline results in impaired iron a b- were observed with cycloheximide. We sorption may be considered: (1) non- are not aware of any reports indicating specific damage to the intestinal mucosa; that cycloheximide chelates divalent cat(2) binding of divalent cations such as ions. Third, it will be recalled that, in ferrous iron; and (3) interference with studies in which pure tetracycline powder specific transport mechanisms. was added to incubation fl asks containing
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Fm. 4. Electron micrograph of the subapical cytoplasm of absorptive intestinal epithelial cells. D, desmosome at intercellular margin; ER, rough surfaced endoplasmic reticulum; R, clustered ribosomes; M, mitochondrion; I, inclusion, possibly a reabsorption droplet. Dow epoxy resin embedded. Stained with uranyl acetate and lead citrate ( X 18,000.)
everted duodenal sacs, there was no decrease in the transfer of radioiron by distal sacs (table 5). If tetracycline did bind significantly to ferrous sulfate, one would have expected impaired transport of iron in distal sacs as well as proximal sacs. The amount of tetracycline added to the media was equivalent to that found in the small intestine 4 hr after tetracycline was injected in a dosage of 400 mg per kg (N. J. Greenberger, R. D. Ruppert, and R. L. Perkins, unpublished observations). Thus, there was sufficient tetracycline present to bind t heoretically much of the iron in the media. The failure to observe a defect in iron transport in distal sacs under these conditions suggests that factors in addition to chelation are important in the tetracycline-induced impairment of iron transport. The divergent data in proximal and
distal sacs might possibly be accounted for by different mechanisms of iron transport in the duodenum (proximal sacs) as compared to jejunum (distal sacs). It has been demonstrated both in vivo and in vitro that there is decreased incorporation of C14 -labeled amino acids into intestinal mucosal proteins in tetracyclinetreated rats as compared with controls. 9 • 10 Furthermore, this inhibition of protein synthesis occurred within 4 hr after the administration of tetracycline. 10 On the basis of these observations and the data obtained in the present study, it could be proposed that treatment of rats with tetracycline resulted in inhibition of protein synthesis in the intestinal mucosa with decreased synthesis of a carrier substance that is important in the mucosal transport of iron. Such a protein or polypeptide carrier might
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also be important in the entry of iron into t he mucosal cell. An alternative hypothesis is that the carrier substance may bind or hold iron that has entered the mucosal cell. As a result of treatment with agents that in hi bit protein synthesis, there might be decreased amounts of a binding protein available. Consequently, iron entering the mucosal cell might not be retained within the cell and could diffuse back into the intestinal lumen . Thus, the postulate that there was lack of a binding protein could also account for the decreased mucosal uptake and transfer of radioiron observed after treatment with tetracycline and cycloheximide. R ecent preliminary studies have suggested that there is a high molecular ·weight protein in gastric juice which can bind iron and t hus may influence iron absorpt ion.19 There is additiona l indirect evidence in support of the concept of an iron-manipulating protein in the intestinal mucosa. It has been demonstrat ed in in vitro studies that the intestinal mucosa is capable of synthesizing proteins and lipoproteins.20 Furthermore, treatment of rats with agents that inhibit protein synthesis has been shown t o result in a defect in fat transport.21· 22 This is t hought t o be due to impaired synthesis of lipoproteins by the intestinal mucosa which in turn results in defective formation of chylomicrons. A defect in fat transport can be demonstrated ·within 4 hr after the administration of agents such as acetoxycycloheximide and puromycin. 21 • 22 The data obtained in the present study clearly indicate that treatment of rats wit h tetracycline results in impaired intestinal iron transport, both in vivo and in vitro. The mechanism of this inhibitory effect has not been defined by these studies. It is possible that impaired iron transport was due at least in part to sequelae of protein synthesis inhibition, either direct effects or secondary, such as acidosis or azotemia. However, at present these two phenomena, inhibition of protein synthesis and inhibition of iron transport, are linked by only indirect evidence. Further studies
are needed to clarify t he nature of the inhibitory effect of tetracycline on iron transport. Su1n1nary
A series of studies was carried out to define more clearly t he role of protein-bound iron in controlling intestinal iron absorption. Antibiotics known to inhibit protein synthesis were employed in an attempt to induce a defect in iron absorption. These studies were performed utilizing isolated intestinal loops in the rat and everted duodenal gut sacs. It was found that treatment of r ats with tetracycline and cycloheximide resulted in impaired absorption of radioiron from intestinal loops. Similarly, it was observed that in gut sacs obtained from tetracycline-treated rats, there -\vas a signifi cantly decreased mucosal to serosal transfer of radioiron. The inhibit ory effects of tetracycline were found t o be dose-dependent and were observed only after large doses. Treatment with tetracycline was not associated with evident structural changes in the intestinal mucosa upon examination by light and electron microscopy. The data obtained suggest that t he inhibitory effects of tetracycline were due t o other factors in addition t o binding of iron by tetracycline. It is proposed t hat these effects were due at least in part to sequelae of protein synthesis inhibition and this postulate is discussed. REFERENCES 1. Manis, J. G ., and D. Schachter. 1962. Actire transport of iron by in testine: features of the two-step mechanism. Amer. J. Physiol. 203 : 73-80 . 2. Brown, E. B ., and lVI. L. Rother. 1963. Studies of the mechanism of iron abso rption: I. Iron uptake by t he normal rat. J. Lab. Clin . Med. 62: 357-373. 3. Wheby, M. S., L. G . J ones, and W. H. Crosby. 1964. Studies on iron abso rption. I ntestinal regulatory mechanisms . J. Clin. Invest. 43 : 1433-1442. 4. Charlton, R. vV., P . J acobs, J . D. Torrance, and T. H. Bo thwell. 1965. The role of the in testinal m ucosa in iron abso r ption. J. Clin. Invest. 44: 543-554.
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5. Conrad, M. E., L. R. Weintraub, and W. H. Crosby. 1964. The role of the intestine in iron kinetics. J. Clin. Invest. 43 : 963-974. fi. Manis, J. G., and D . Schachter. 1964. Active transpo rt of iron by intestine : mucosal iron pools. Amer. J. Physiol. 207: 893-900. 7. Young, C. W., P. F. Robinson, and B. Sacktor. 1963. Inhibi tion of the synthesis of protein in intact animals by acetoxycycloheximide and a metabolic derangement concomitant with th is blockage. Biochem. Pharmacol. 12: 855-865. 8. Nikolo v, T . K., and A. T. Ilkov . 1961. Effects of chlortetracycline on methionine-35S incorporation into macroorganism proteins. In Abstracts of the Fifth International Congress of Biochemistry, Vol., 9, p. 102. 9. Yeh, S. D. J., and M. E. Shils, 1966. T etracycline and inco rporation of a mino acids in to proteins of rat tissue. Proc. Soc. Exp . Biol. Med. 121: 729-734. 10. Greenberger, N. J . 1967. T etracycline-induced inhibition of protein synthesis in rat intestinal slices. Nature (London) 214: 702703. 11. Wilson, T. H ., and G. Wiseman. 1954. The
use of sacs of everted small intestine for t he study of transference of substances from the mucosal to serosal surface. J. Physiol. (London) 123 : 116- 125. 12. Dowdle, E. B ., D. Schachter, and H . Schenker. 1960. Active t ransport of Fe 59 by everted segments of rat duodenum. Amer. J. Physiol. 198: 609-613 . 13. Greenberger , N. J., and R. D . Ruppert. 1966. Inhibi tion of protein synthesis : a mecha-
14.
15.
16.
17.
18.
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nism for the production of impaired uon absorption. Science 153: 315-316. M anis, J., and D . Schachter. 1966. Active transport of iron by intestine: effect of erythropoiesis stimulated by phenylhydrazine. Nature (London) 209 : 1356. Yeh, S. D. J., and M. E. Shils. 1966. Effect of tetracycline on intestinal absorption of various nutrients by the rat. Proc. Soc. Exp . Biol. Med. 123: 367-370 . Albert, A. 1953. Avidity of terramycin and aureomycin for metallic cations. Nature (London) 172: 201. Albert, A., and C. W. Rees. 1956. Avidity of the tetracyclines for the cations of metals. Nature (London) 177: 433-434. Kohn, K. W. 1961. Mediation of divalent metal ions in the binding of tetracycline to macromolecules. Nature (London) 191:
1156-1158. 19. Davis, P. S., C. G. Luke, and D. J. Deller. 1966. Reduction of gastric iron-binding protein in haemochromatosis. Lancet 2 : 143 11433. 20 . I sselbacher, K. J., and D. Budz. 1963. Syn-
thesis of lipoproteins by rat intestinal mucosa. Nature (London) 200: 364-365. 21. Sabesin, S. M., and K. J. I sselbacher. 1965. Pro tein synthesis inhibition: mechanism for the production of impaired fat absorption. Science 147: 1149-1151. 22. Greenberger, N. J ., J . B. Rodgers, and K . J. I sselbacher. 1966. Absorption of medium and long chain triglycerides: factors influ encing their hydro lysis and transport. J. Clin. Invest. 45: 217-227.
Vol. 53, No.4 Printed in U.S.A.
Copyright © 1967 by The William s & Wilkins Co.
INHIBITION OF INTESTINAL IRON TRANSPORT INDUCED BY TETRACYCLINE NORTON J. GREENBERGER, M.D., RICHARD D. RUPPERT, M.D., AND FRANCIS E. CUPPAGE, M.D. Division of Gastroenterology, Department of Medicine and Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio
sorption of radioiron from intestinal loops. Similarly, it was observed that in gut sacs obtained from tetracycline-treated r ats there was a significantly decreased mucosal to serosal transfer of radioiron.
Recent studies have indicated that the intestinal mucosa is important in controlling iron absorption. 1 - 5 However, the actual regulatory mechanisms remain poorly understood. In particular, the nature of mucosa l iron complexes has not been clearly defined. Iron is present in the intestinal mucosa in two forms: (1) non-protein bound iron which is transferred rapidly across the mucosa, a nd (2) protein-bound iron which is transported at a slower rate.2 • 4 • 6 Since a significant amount of iron in the intestinal mucosa is proteinbound, the question arises as to whether such protein-bound iron is important in controlling iron absorption. In the present investigation, studies were carried out to define more clearly the role of protein-bound iron in controlling the intestinal absorption of iron. Antibiotics known to inhibit protein synthesis in m amma lian cells were employed in an attempt to induce a defect in intestinal iron transport. These studies were performed utilizing isolated intestinal loops in the rat and everted duodena l gut sacs in vitro. It was found that treatment with tetracycline and cycloheximide resulted in impaired ab-
Methods Experim ents on iron absorption from isolated intestinal loops. Wistar female rats (Harlan
Received April 19, 1967. Accepted June 10, 1967. Presented in part at the Midwestern Meeting of the American Federation for Clinical Research, Chicago, Illinois, November 2, 1966, (Clin. Res. 14 : 432, 1966 (abstr.) ). Address requests for reprints to: Dr. Norton J . Greenberger, Department of Medicine, Ohio State University Hospital, Columbus, Ohio 43210. This work was supported by Grant AM-1055601 from the National Institu tes of H ealth. We thank Mrs. J acqueline Thomas and Mrs. J ane Funderburg for expert technical assistance.
Farms, Cumberland, Ind.) weighing 140 to 160 g were fasted for 16 hr prior to study. Under light ether anesthesia a laparotomy was performed and a 20- to 25-cm segment of duodenum and jejunum was isolated between double silk ligatures. A needle from a syringe containing Fe59SO, (New England Nuclear Corporation, Boston, Mass.; specific activity, 550 p..c per p..mole) was injected through the stomach and passed into the proximal duodenum, and the animal was given an intraduodenal inj ection of 1.0 ml of a solution containing 138 p..g of ferrous sulfate (50 p..g of iron) and 2.0 X 106 dpm. After varying time intervals (15, 30, or 60 min) the isolated loops were excised and the intestinal contents were washed out with 50 ml of 0.85% N aCl. The intestinal mucosa was removed from the loops by scraping and weighed, and the entire sample was placed in counting tubes. The mucosal scrapings and duplicate aliquots of the loop contents were assayed for radioactivity in a well type scintillation counter along with an appropriate standard. In preliminary studies the completeness of mucosal scraping was evaluated by assaying the muscular and serosal layers of the isolated intestinal loops for radioactivity. In over 50 studies it was found that 0.5 to 1.5% of the injected Fes•so, was recovered in the muscular and serosal layers. Thus, in these and subsequent studies, the radioactivity present in the muscular and serosal layers was considered to represent iron transferred from the mucosa. Experiments with antibiotics. Studies were carried out to determine the effect of inhibiting
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protein synthesis on the absorption of Fe""SO, . Initially, the antibiotics employed were cycloheximide (kindly supplied by Dr. George Savage, Upjohn Laboratories, Detroit, Mich.) and tetracycline (kindly supplied by Mr. James Eberlein, J. B. Roerig & Co., New York, N.Y.), since it had been previously demonstrated that these antibiotics inhibit protein synthesis in mammalian cells.'-'° For purposes of comparison, later studies were carried out utilizing penicillin and chloramphenicol. All antibiotics were given by intraperitoneal injection 4 hr prior to the preparation of isolated intestinal loops. The doses employed in these studies were as follows : cycloheximide, 5 mg per kg; tetracycline hydrochloride, 20, 200, 300, and 400 mg per kg ; potassium penicillin G, 1.8 g per kg; and chloramphenicol sodium succinate, 400 mg per kg. The pH of the tetracycline and cycloheximide solutions ranged from 2.14 to 3.40. To exclude the possibility that changes observed after antibiotic treatment were due to injection of an acidic solution, other studies were carried out in which animals received an int raperitoneal injection of 1.0 ml of a solution containing 0.1 N HCl and 0.15 g of ascorbic acid, the pH being 1.94. Under these conditions, no impairment of iron absorption and no morphological changes in the intestinal mucosa were found. Exp eriments on iron transport by everted duodenal sacs in vitro. Wistar female rats weighing 140 to 160 g were fasted for 16 hr and then injected intraperitoneally with either saline or tetracycline in a dosage of 400 mg per kg. Four hours later t he animals were killed and the small bowel was rinsed in situ with 50 ml of ice-cold 0.85% NaCl. The proximal 15 em of small intestine were excised and everted over a glass rod, and duodenal sacs were prepared according to the method of Wilson and Wiseman" as modified by Schachter and his associates." Two consecutive everted sacs each 6 em long were prepared. Mean wet weights of the proximal and distal sacs were approximately 800 and 600 mg, respectively. Once the intestine was dissected free it was chilled immediately in ice-cold saline and kept at 4 C throughout preparation of the gut sacs. The sacs were filled with 0.5 ml of a medium of the following composition: 0.145 M NaCl, 0.0001 M CaCl", 0.04 M fructose, 0.0008 M sodium ascorbate, freshly prepared, and 0.004 M Tris buffer (tris(hydroxymethyl) aminomethane, pH 7 .4) . Each sac was placed in an incubation flask containing 5.0 ml of the same medium plus 0.0004 M FeSO, and sufficient F e""SO. to give approximately 10,000 cpm per ml in a well type scintillation counter. The elapsed time
from death of the animals to placement of the gut sacs in the incubation flasks was always less than 5 min. The incubations were carried out at 37 C with continuous shaking and gassing with 95% oxygen-5% carbon dioxide for 2Y2 hr. The gut sacs were then washed and blotted dry, and the serosal fluid was drained. Duplicate aliquots of the mucosal and serosal fluids were assayed for radioactivity. In t he present studies the average recovery of serosal fluid was 90% of t he initial volume in proximal sacs and 100% or greater in distal sacs. Studies were also carried out to determine whether tetracycline added directly to the incubation flask would inhibit iron transport by everted duodenal sacs. The gut sacs were obtained from normal rats in the manner described above. In these studies the composition of the incubation media and the media placed within the sacs was identical with that previously described wit h t he exception that 100 /Lg of pure tetracycline powder (kindly supplied by Mr. J ames Eberlein, J . B. Roerig & Co., New York, N. Y .) were added to each flask prior to the start of the incubation. The incubations were carried out in the same manner as described above.
Results
Effect of common bile duct ligation on the absorption of radioiron from intestinal loops. Since bile accumulated in the closed intestinal loops during the absorption period, it was considered important to determine the effect of bile duct ligation on iron absorption (table 1). This was determined by measuring the mucosal uptake and mucosal transfer of radioiron. Mucosal uptake (A) was determined by subtracting the amount of Fe 59 S0 4 recovered in the loop contents from the amount injected. The amount of Fe 59S0 4 remaining in the mucosa (B) was determined directly. Mucosal transfer represents the difference between mucosal uptake (A) and the amount remaining in the mucosa (B) . In essence, therefore, mucosal transfer (A - B) reflects net absorption. It can be seen that there was no signific~nt difference in the mucosal uptake and transfer of radioiron between control animals and animals in which the common bile duct had been ligated either 30 min or 48 hr prior to study. Since bile duct liga-
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TABLE
Vol. 53, No . 4
1. Effect of common bile duct ligation on the absorption of Feo'SQ,a f1·om isolated intestinal loopsb Administered radioactivity
No. of experiments
Group
..... . . . .. . . Control . . .. . . . . . . Bile duc t ligated 30 min before ex. . .. .... . ... periment .. Bile duc t ligated 48 h r before experiment . . . .. . . a 6 c
20
Recovered in loop contents
(A) Mucosal uptake
%
%
(B) Remaining 1n mucosa
(A - B) Mucosal transfer
%
35.2 ± 11. 4• 64.8 ± 11 .4
%
27 .8 ± 9.3
37.0 ± 9.6
7
37. 0 ± 14.5
63. 0 ± 14 .5
30.9 ± 10 .8
32. 1 ± 18 .7
5
38.2 ± 7.3
61. 8 ± 7.3
27.6 ± 9.6
34.2 ± 7.9
138 l'g (50 l'g as iron) containing 2.0 X 10 6 dpm. Loops removed 30 min a fte r injection of radi oiron. Mean± 1 sD .
TABLE
2. Effect of graded doses of letmcycline on the absorption of Fe 59 SQ ,a from isolated intestinal loops 6 Administered Radioacti v ity
Group
No. of expcriments
B/ A (A) Mucosal uptake
Reco vered in loop contents
(B) Remaining m mucosa
(A - B) Mucosal tran sfe r
-%
.. Cont rol. ... T etracycli ne (20 mg/ kg ) . Te t racycline (200 mg/ kg) T et racy cline (300 mg/ kg ) . Tetracycline (400 mg/kg) T et racyclined (400 mg/kg) a
6 c d
20 6 8 6 5 8
35.2 39.6 43 .6 55.3 64.2 56.5
± ± ± ± ± ±
%
11 .4• 8.2 10.9 9.7 3 .1 8.9
64.8 ± 60. 4 ± 56.4 ± 44 .7 ± 35 .8± 43.6 ±
%
11. 4 8.2 10. 9 9 .7 3 1 8.9
27.8 22.5 21.9 23.5 18 .5 23.7
± ± ± ± ± ±
%
%
9.3 6.4 4. 1 9.4 3.0 9.3
37.0 37.9 34.5 21.2 17 .3 19.9
± ± ± ± ± ±
9.6 11. 8 9.9 10 .8 3.9 7 .7
42 ± 37 ± 39 ± 53± 52± 54±
10.0 12.4 5.4 13. 1 7 .8 10.5
138 l'g (50 l'g as iron ) co n taining 2.0 X 10 6 dpm. Loops removed 30 min afte r injection of radioiron. Mean± 1 SD. Common bile duct ligated 24 hr prior to study.
tion did not appear to affect iron absorption, the bile duct was not ligated for any of the subsequent studies with only one exception, described below.
Effect of tetracycline on the absorption of radioiron from intestinal loops. In table 2 are shown data on the effect of graded doses of tetracycline on the absorption of Fe 59 S0 4 from intestinal loops. In rats previously given tetracycline in a dose of 20 mg per kg, there was no difference in the mucosal uptake and transfer of radioiron as compared with controls. With injection of larger doses of tetracycline, there was a progressive decrease in iron absorption. In r ats injected with tetracycline in a dosage of 400 mg per kg, there was a significantly decreased mucosal uptake (36 versus 65%) and transfer (17 versus 37%) of radioiron as compared with controls.
The amount of radioiron remaining in the mucosa divided by the mucosal uptake, B j A, also reflects the interference in iron transport induced by treatment with t etracycline (table 2) . In control animals, 42% of the iron taken up by the mucosa was still present in the mucosa after 30 min as compared with a value of 52% in r ats treated with tetracycline in a dosage of 400 mg per kg (t = 2.07; P = <0.05). Studies were also carried out in which the absorption of -radioiron was determined in tetracycline-treated rats in which the common bile duct was ligated 24 hr prior to study (table 2). This was done in order to exclude the possibility that the inhibitory effects of t etracycline on iron transport were due to tetracycline being secreted via the common bile duct into the proximal small bowel and chelating iron
present in the lumen. It was found that there was no significant difference in the mucosal transfer of radioiron in tetracycline-treated rats with intact bile ducts as compared with animals with ligated bile ducts (17 versus 20%; P = >0.5). Thus, ligation of the common bile duct did not affect the inhibition of iron absorption from intestinal loops caused by treatment with tetracycline. To confirm that tetracycline blocks iron absorption, the absorption of iron was compared in control and tetracycline-treated rats at various time intervals. Isolated intestinal loops were removed 15, 30, or 60 min after injection of radioiron, and the mucosal uptake and transfer were determined as described above. The amount of iron absorbed was calculated on the basis of the mucosal transfer and the dose of radioiron (50 ,ug). These data are shown in figure 1. In control animals, iron absorption progressively increased and 23.5 ,ug were absorbed in 60 min. In contrast, in tetracycline-treated rats, there was a minimal increase in iron absorption over a 60min period with only 8.5 ,ug being absorbed. Iron transport by duodenal sacs in vitro. To document further that tetracycline inhibits iron transport, we carried out in vitro studies utilizing everted duodenal sacs obtained from control and tetracycline-treated rats (table 3). In studies using proximal gut sacs from control animals,
3. Effect of prior tetracycline" on iron transport by everted duodenal sacsb in vitro
T A BLE
Net iron transfer to serosal medium
Group Proximal sac
24 20 IRON 16
p.gm
8 4
15
30
45
60
MINUTES
FIG. 1. Absorption of Fe"'SO, (50 p.g of iron containing 2 X 10" dpm) from isolated intestinal loops in control and tetracycline-treated rats. The values shown represent the mean ± 1 so.
I
Distal sac
mp.moles/g'lft sac
Control. .... .. .. . . Tetracycline
P value.
35.6 ± 11 .4< 17.3 ± 10.0 (25) (26) 20.9 ± 11 .8 11. 8 ± 5.0 (30) (32) <0.001 <0.01
• The dose of tetracycline was 400 mg per kg given intraperitoneall y 4 hr prior to study. Figures in parentheses represent the number of determinations performed. b Two gut sacs were prepared from the proximal 12 em of rat small intestine and results a re shown for the more proximal and adjacent distal sac. Each sac was filled with 0.5 ml of a medium of the following composition: 0.145 M NaCl, 0.0001 lii CaCh , 0.04 M fructose, 0.0008 M sodium asco rbate, and 0.004 M Tris buffer. The sacs were placed in incubation flasks containing 5.0 ml of the same medium plus 0.0004 M FeS04 and sufficient Fe 59 S04 to give approximately 10,000 cpm per ml. The incubations were carried out at 37 C with continuous shaking and gassing with 95% oxygen and 5% carbon dioxide for 2.5 hr. c Mean± 1 so.
36 m,umoles of F eS0 4 were transferred from the mucosal to the serosal medium in 2Y2 hr. In contrast, with proximal sacs obtained from tetracycline-treated rats, only 21 m,umoles were transferred, a 40% decrease (P < 0.001). Using distal sacs from tetracycline-treated rats, there was also a significantly decreased transfer of FeS0 4 as compared with controls, 11.8 versus 17.3 m,umoles (P = <0.01). To evaluate the possibility that the inhibitory effect of tetracycline on iron transport by everted gut sacs might be due to nonspecific divalent cation binding, studies were carried out in which tetracycline was added to flasks containing gut sacs from normal rats (table 4). In studies utilizing proximal sacs, the addition of tetracycline to the media resulted in a 20% decrease in the mucosal to serosal transfer of FeS0 4 , 28 m,umoles as compared with 36 m,umoles (P = <0.025). However, with distal sacs, there was no significant differ-
=
28
ABSORBED
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ence between control fl asks and flasks containing tetracycline in the transfer of FeS0 4 , 17 versus 19 m~-tmoles (P = > 0.5). Effect of various antibiotics on iron absorption. To determine whether the inhibitory effect of tetracycline on iron a bsorption was specific for tetracycline, studies were carried out in which the absorption of Fe 59 S0 4 was measured in isolated intestinal loops in animals treated with massive do ses of different antibiotics (table 5). There was no significant difference in the mucosal uptake and transfer of Fe 59S0 4 in animals treated with penicillin (1800 mg per kg) and chloramphenicol (400 mg per kg) as 4. E:O"ect of tetracycline" added to the media on iron transport by everted duodenal sacs in vitro
TABLE
Net iron transfer to serosal medium
Group Proximal sacb
I
Distal sac
mp.molesfgut sac
Control. Control + tetra cycline". P value.
35 .6 ± 11.4' (25)
17. 3 ± 10.0 (26)
27.7 ± 9.7 (22) < 0.025
19.0 ± 6.6 (22) >0.5
a Tetracycline, 100 /J.g , was added to eac h fl ask at t he start of t he incubation. Figures in parentheses represent the number of dete rmin ations performed. b The preparation of the gut sacs an d the condi tions of the expe riment we re s imila r to t hose described in table 3. 'Mean± 1 sn.
T A BLE
Vol. 53, iVa. 4
compared with controls. Similarly, the BI A ratio in these 3 groups of rats was the same. In contrast, in rats treated with tetracycline (400 mg per kg ) or cycloheximide (5 mg per k g) , there was a marked decrease in the mucosal uptake and transfer of Fe 59S0 4 • In addition, there was an increase in the B I A ratio refl ecting significant impairment in iron transport. Effect of tetracy cline on intestinal histology. Sections of jejunum were t ak en for histological examination 4 hr after treatment with tetracycline. The surface epithelium, villi, lamina propria, and crypts all appeared norma l upon examination by light microscopy (fig. 2) . Electron microscopy of the lumin al absorptive surface (fig. 3) and subapical cytoplasm (fig. 4) of jejunal epithelial cells revealed normal microvilli, mitochondria, and endoplasmic reticulum. In earlier studies, it was demonstrated that treatment of rats with cycloheximide was not associated with significant histological changes in the intestinal mucosa. 13 Discussion
It has been established that, after the administration of a physiological dose of iron to the rat, a significant amount of iron in the intestinal mucosa is proteinbound during the initial 60 to 90 min that iron absorption is taking place. 2 • 4 To evaluate the possibility that such protein-bindin g is important in the regulation of iron a bsorption, the antibiotics cycloheximide
5. Effect of various antibiotics on the absorption of F e 59 SO 4" from isolated intestinal loopsb Administered radioactivity
Group
N o. of experiments
Recovered in loop con tents
%
Con trol. - .... P enicillin (1800 mg/kg). Chloramphenicol (400 mg/ kg) .. T etracycline (400 mg / kg). Cycloheximide (5 mg/kg).
(A) Mucosal uptake
(B ~ R emaining m mucosa
(A - B) Mucosal transfer
%
%
%
20 5
35.2 ± 11.4' 38 .7 ± 13.0
64.8 ± 11.4 27.8 ± 9.3 37 .0 ± 9.6 42 61.3 ± 13 .0 24.1 ± 11.0 37.2 ± 13 . 0 39
6 5 18
39 .1 ± 8.6 64.2 ± 3.1 71.2 ± 14 . 1
60.9 ± 8.6 25.1±6 .1 35.8 ± 3. 1 18.5 ± 3 .0 28.8 ± 14 . 1 17.0 ± 6.8
138 /J.g (50 /J.g as iron) containing 2.0 X 10 6 dpm. Loops removed 30 min after injection of radioiron. 'Mean± 1 so.
a
b
B/A
%
± 10.0 ± 12 .0
35.8 ± 10.0 39 ± 9.0 17. 3 ± 3.9 52 ± 7.8 11.8 ± 8.2 59 ± 13.0
Frc. 2. Photomicrograph of ra t jejunum 4 hr after tetracycline, 400 mg per kg. The surface epithelium, villi, crypts, and lamina propria all appear normal (X 100) .
Frc . 3. Electron micrograph of th e luminal absorptive surface of th e small intestine with normal appearing organelles. A, amorphous coat; Mv, microvilli ; TW, terminal web area ; C, cent riole; R, clustered ribosomes; lvf, mitochondrion. Dow epoxy resin embedded. Stained with uranyl acetate and lead citrate ( X 27,000).
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V ol. 53, No.4
and tetracycline were utilized because they It appears unlikely that the observed have been shown to inhibit protein synthe- effects of tetracycline were due to nonsis in mammalian cells. 7 - 10 It was found specific mucosal damage since tetracycline that treatment of rats with these drugs treatment was not associated with evident results in impaired a bsorption of radioiron structural changes in the intestinal mucosa from isolated intestinal loops (table 2; upon examination .by light and electron Fig. 1). The inhibitory effects of t etracy- microscopy (Figs. 2 to 4). However, this cline were found to be dose-dependent. The structural integrity does not rule out the observation that there was still some mu- · possibility of biochemical defects in the cosal uptake and transfer of radioiron after cell other than protein synthetic mechatreatment with massive doses of tetracy- nisms; for example, it has been demoncline indicates that this t reatment did not strated that treatment of rats with tetracompletely block iron transport. cycline may result in impaired fat abThe absorption of radioiron in tetracy- sorption.15 Thus, the possibility that the cline-treated rats was similar to that in inhibition of iron transport by t etracycline controls during the first 15 min but did not was due to nonspecific effects on the intesincrease appreciably thereafter (fig. 1). tinal mucosa cannot be excluded. One possible interpretation of this observaIt has been demonstrated that the tetration is that tetracycline treatment did not cycline drugs can bind a number of divainterfere with the absorption of the rapidly lent cations, including ferrous iron.16· 17 It transported or non-protein bound iron but has also been reported that tetracycline did impair the transport of protein-bound could bind extensively to both nucleic acids iron. During the initial 30 to 60 min that and proteins, with the binding being mediiron absorption is occurring, progressively ated by divalent cations such as zinc, calmore iron in the mucosa is present as pro- cium, magnesium, and manganese. 18 These tein-bound iron. 2 • 4 and other observations have led to the sugAdditional evidence that tetracycline gestion that the biological effects of the treatment induces a defect in iron trans- tetracycline drugs might be secondary to a port was obtained in the in vitro studies. It chelation phenomenon. 18 Thus, it would was found t hat there was a significantly appear reasonable to expect tetracycline to decreased mucosal t o serosal transfer of bind ferrous iron. However, there are ferrous sulfate utilizing everted duodenal several lines of evidence which suggest that sacs obtained from tetracycline-treated the impaired transport of iron observed in rats (table 3). The values that we ob- the present studies was due to other factained for the mucosal t o serosal transfer tors in addition to the presumed binding of of radioiron in proximal and distal sacs ferrous iron by tetracycline. from control animals are quite comparable First, in studies employing rats treated to those reported by Manis and Schachter. 14 with tetracycline and bile duct ligation, The data obtained in the present study there was no difference in the mucosal are in agreement with studies recently re- transfer of iron as compared to rats ported by Y eh and Shils. 15 These investi- treated with tetracycline alone (table 2) . gators demonstrated that there was a de- These studies exclude the possibility that creased absorption of radioiron in rats t he inhibitory effects of tetracycline were given an intramuscular or intraperitoneal due to chelation of intraluminal iron. Secinjection of tetracycline (250 mg per kg). ond, inhibitory effects on iron transport Three mechanisms whereby treatment with similar to those obtained with tetracycline tetracycline results in impaired iron a b- were observed with cycloheximide. We sorption may be considered: (1) non- are not aware of any reports indicating specific damage to the intestinal mucosa; that cycloheximide chelates divalent cat(2) binding of divalent cations such as ions. Third, it will be recalled that, in ferrous iron; and (3) interference with studies in which pure tetracycline powder specific transport mechanisms. was added to incubation fl asks containing
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597
Fm. 4. Electron micrograph of the subapical cytoplasm of absorptive intestinal epithelial cells. D, desmosome at intercellular margin; ER, rough surfaced endoplasmic reticulum; R, clustered ribosomes; M, mitochondrion; I, inclusion, possibly a reabsorption droplet. Dow epoxy resin embedded. Stained with uranyl acetate and lead citrate ( X 18,000.)
everted duodenal sacs, there was no decrease in the transfer of radioiron by distal sacs (table 5). If tetracycline did bind significantly to ferrous sulfate, one would have expected impaired transport of iron in distal sacs as well as proximal sacs. The amount of tetracycline added to the media was equivalent to that found in the small intestine 4 hr after tetracycline was injected in a dosage of 400 mg per kg (N. J. Greenberger, R. D. Ruppert, and R. L. Perkins, unpublished observations). Thus, there was sufficient tetracycline present to bind t heoretically much of the iron in the media. The failure to observe a defect in iron transport in distal sacs under these conditions suggests that factors in addition to chelation are important in the tetracycline-induced impairment of iron transport. The divergent data in proximal and
distal sacs might possibly be accounted for by different mechanisms of iron transport in the duodenum (proximal sacs) as compared to jejunum (distal sacs). It has been demonstrated both in vivo and in vitro that there is decreased incorporation of C14 -labeled amino acids into intestinal mucosal proteins in tetracyclinetreated rats as compared with controls. 9 • 10 Furthermore, this inhibition of protein synthesis occurred within 4 hr after the administration of tetracycline. 10 On the basis of these observations and the data obtained in the present study, it could be proposed that treatment of rats with tetracycline resulted in inhibition of protein synthesis in the intestinal mucosa with decreased synthesis of a carrier substance that is important in the mucosal transport of iron. Such a protein or polypeptide carrier might
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Vo l. 53, No.4
GREENBERGER E'l' AL.
also be important in the entry of iron into t he mucosal cell. An alternative hypothesis is that the carrier substance may bind or hold iron that has entered the mucosal cell. As a result of treatment with agents that in hi bit protein synthesis, there might be decreased amounts of a binding protein available. Consequently, iron entering the mucosal cell might not be retained within the cell and could diffuse back into the intestinal lumen . Thus, the postulate that there was lack of a binding protein could also account for the decreased mucosal uptake and transfer of radioiron observed after treatment with tetracycline and cycloheximide. R ecent preliminary studies have suggested that there is a high molecular ·weight protein in gastric juice which can bind iron and t hus may influence iron absorpt ion.19 There is additiona l indirect evidence in support of the concept of an iron-manipulating protein in the intestinal mucosa. It has been demonstrat ed in in vitro studies that the intestinal mucosa is capable of synthesizing proteins and lipoproteins.20 Furthermore, treatment of rats with agents that inhibit protein synthesis has been shown t o result in a defect in fat transport.21· 22 This is t hought t o be due to impaired synthesis of lipoproteins by the intestinal mucosa which in turn results in defective formation of chylomicrons. A defect in fat transport can be demonstrated ·within 4 hr after the administration of agents such as acetoxycycloheximide and puromycin. 21 • 22 The data obtained in the present study clearly indicate that treatment of rats wit h tetracycline results in impaired intestinal iron transport, both in vivo and in vitro. The mechanism of this inhibitory effect has not been defined by these studies. It is possible that impaired iron transport was due at least in part to sequelae of protein synthesis inhibition, either direct effects or secondary, such as acidosis or azotemia. However, at present these two phenomena, inhibition of protein synthesis and inhibition of iron transport, are linked by only indirect evidence. Further studies
are needed to clarify t he nature of the inhibitory effect of tetracycline on iron transport. Su1n1nary
A series of studies was carried out to define more clearly t he role of protein-bound iron in controlling intestinal iron absorption. Antibiotics known to inhibit protein synthesis were employed in an attempt to induce a defect in iron absorption. These studies were performed utilizing isolated intestinal loops in the rat and everted duodenal gut sacs. It was found that treatment of r ats with tetracycline and cycloheximide resulted in impaired absorption of radioiron from intestinal loops. Similarly, it was observed that in gut sacs obtained from tetracycline-treated rats, there -\vas a signifi cantly decreased mucosal to serosal transfer of radioiron. The inhibit ory effects of tetracycline were found t o be dose-dependent and were observed only after large doses. Treatment with tetracycline was not associated with evident structural changes in the intestinal mucosa upon examination by light and electron microscopy. The data obtained suggest that t he inhibitory effects of tetracycline were due t o other factors in addition t o binding of iron by tetracycline. It is proposed t hat these effects were due at least in part to sequelae of protein synthesis inhibition and this postulate is discussed. REFERENCES 1. Manis, J. G ., and D. Schachter. 1962. Actire transport of iron by in testine: features of the two-step mechanism. Amer. J. Physiol. 203 : 73-80 . 2. Brown, E. B ., and lVI. L. Rother. 1963. Studies of the mechanism of iron abso rption: I. Iron uptake by t he normal rat. J. Lab. Clin . Med. 62: 357-373. 3. Wheby, M. S., L. G . J ones, and W. H. Crosby. 1964. Studies on iron abso rption. I ntestinal regulatory mechanisms . J. Clin. Invest. 43 : 1433-1442. 4. Charlton, R. vV., P . J acobs, J . D. Torrance, and T. H. Bo thwell. 1965. The role of the in testinal m ucosa in iron abso r ption. J. Clin. Invest. 44: 543-554.
October 1967
Il\TTESTINAL IRON TRANSPORT
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