- Email: [email protected]
Ascorbic Acid Chelates in Iron Absorption: A Role for Hydrochloric Acid and Bile
Vol. 55, No.1
GASTROENTEROLOGY
Copyright
© 1968 by The Williams
Printed in U.S.A.
& Wilkins Co.
ASCORBIC ACID CHELATES IN IRON ABSORPTION: A ROLE FOR HYDROCHLORIC ACID AND BILE MARCEL E. CoNRAD, M.D., AND STANLEY G. ScHADE, M.D. DepaTtment of Hematology, Walter Reed Army Institute of Research, Washington, D. C.
vitro and measurements of their effect upon iron absorption in animals.
Many investigators have postulated that acid gastric secretions enhanced iron absorption. 1 - 3 This hypothesis was supported by the frequent development of iron deficiency in gastrectomized human patients and decreased absorption of dietary iron by achlorhydric subjects. 4 - 13 Data obtained by radioactive iron studies showed that acid does not enhance the absorption of iron from ferrous salts or hemoglobin iron but might increase the absorption of iron from ferric salts and foodP- 17 Since ionic ferric iron is not absorbed by the intestinal mucosa,1 8 gastrointestinal secretions must reduce or chelate ferric iron to bring this iron into solution and enhance its absorption. Anelli reported that ferrous salts form a colored complex with ascorbic acid. 19 During studies of iron-ascorbate chelates, we found that ascorbic acid forms a soluble chelate with ferric chloride at an acid pH but not with ferric iron precipitates at an alkaline pH. This acid iron chelate was stable and maintained iron in a soluble form when the solution was subsequently alkalinized. Since ascorbic acid is a normal constituent of bile, we believed that studies of the iron-ascorbate chclatcs might provide information on the mechanism by which acid gastric juice enhances iron absorption. This study reports biochemical observations of iron-ascorbate mixtures in
Material and Methods Male albino rats, Walter Reed Carworth Farms strain, weighing 200 to 225 g were used in these studies. The principles of laboratory animal care promulgated by the National Society for Medical Research were observed. Rats were raised in a pathogen-free environment and housed in galvanized wire cages. The standard laboratory diet contained 25% protein and 9 mg of iron per 100 g dry weight. Iron deficiency was induced by bleeding animals 4 ml 1 week before study and feeding them a powdered milk diet. Iron absorption studies were performed in rats fasted overnight. Unopcrated animals were lightly anesthetized with ether for dosing. Oral doses were introduced into the stomach through a 17 -gauge olive tipped end oesophageal needle. Test doses of iron were injected into the intestinal lumen of operated animals with a hypodermic syringe and needle. Operated rats were anesthetized with pentobarbital and their abdomen opened through a midline ventral incision. Intragastric doses were injected through a 23-gauge hypodermic needle. Duodenal doses of iron were administered by inserting a 20~?:auge hypodermic needle into the stomach and throu~?:h the pylorus ; a ligature of umbilical tape was tightened around the pylorus before injection of the test dose. The bile duct of certain rats was transected between silk ligatnres 30 min before administration of the test dose of iron . In experiments testing the effects of bile upon iron absorption, the duodenum wns wnshed with 1 ml of distilled water followed by 1 ml of air injected through a hypodermic needle inserted into the duodenum via the gastropyloric route. The ventral incision of the operated animals described above was closed with metallic surgical clips. These animals were killed 2 hr after administration of the test dose and the clamped unopened gut was excised and discarded. The carcass was
RecPivcd January 23, 1968. Accepted February 19, 1968. Address requests for reprints to: Lieutenant Colonel Marcel E . Conrad, MC, Department of Hematology, Walter Reed Army Institute of Research, Washington, D. C. 20012. The authors are grateful to Harold L. Williams, M.S., William Godfrey, and Robert J. Landman for their technical assistance . 35
36
Vol . 55, No. 1
CONRAD AND SCHADE
saved for measurement of iron absorption. Gastrectomized rats underwent surgery 3 weeks before iron absorption studies were initiated; the stomach was excised and an end-to-end esophagoduodenostomy was constructed. The incision was closed with absorbable suture material. Oral test doses were administered to gastrectomized rats through an endoesophageal polyethylene tubing (PE 90). Iron absorption studies were performed with test solutions containing either ferric 59 chloride (0.5 p.c, New England Nuclear Corporation, 9 me per mg), ferrous'" citrate (0.5 p.c, Abbott Laboratories, 30 me per mg), or ferric .. chloride (50 p.c, New England Nuclear Corporation, 9 me per mg), and 0.25 mg of elemental iron as ferric or ferrous chloride. The pH of test doses was adjusted with HCl or NaOH to 3.5 in experiments in which ascorbic acid was not used and to 8.0 in experiments testing effects of ascorbic acid. Sodium ascorbate or ascorbic acid were added to certain doses to make the solution 0.01 M. Absorption of Fe59 was measured with a small animal whole body liquid scintillation detector (Packard ARMAC) . Rats were placed in vented quart ice cream cartons for measurement of whole body radioactivity. Reference standards were prepared by adding a test dose to a 250-ml water-filled plastic bottle. The radioactivity in animals and standards was measured for 100-second intervals (0.8 Mev to oo ). The quantity of Fe•• absorbed during 2-hr experiments was calculated from measurements of the net radioactivity in the carcass and reference standard. The radioactivity in live animals was measured 3 hr and 7 days after dosing; absorption was calculated by the ratio of net counts 7 days after dosing (rz) to tho8e of the standard (sz) and a similar ratio at 3 hr (r./s.) :•
rx//sx X 100 = percentage Fe 59 absorbed ro So
Absorption of Fe"" was calculated from the ratio of Fe"' to Fe•• detected in specimens of whole blood obtained from rats 10 days after dosing. Measurements were made in a liquid scintillation detector by the method of Mahin and Lofberg (Packard TRICARB) .21 These ratios were multiplied by the percentage Fe59 absorbed as determined from measurements of y-emitting radioactivity in a small animal whole body detector. In vitro experiments were performed with 0.1 M iron and 0.1 M ascorbate solutions to which Fe59 was added. Titration experiments were performed with a calibrated Leeds and Northrup
pH meter and a magnetic stirrer; pH adjustments were made with iron-free HCl and NaOH. Solubility was defined as the percentage of iron remaining in the supernatant fluid of samples centrifuged at 30,000 g for 60 min. The color formed by iron-ascorbate complexes was measured with a Beckman DB spectrophotometer at 540 mp.. The molecular size of the radioiron in the supernatant fractions was measured with Sephadex G-10, G-25, and G-50 gel filtration media (Pharmacia) . The gel was swollen in an isotonic saline solution (pH 7.4) for 24 hr and pH adjusted to that of the test solution before it was used to pack 10 X 0.5-cm columns. The void volume of each column was measured with blue dextran. The quantity of radioiron recovered in the first displacement volume was used to estimate the percentage of iron with a molecular weight greater than 700 (G-10), 4,000 (G-25), or 10,000 (G-50) . 22 Infrared spectroscopy was performed by examination of compressed KBr pellets containing 1 mg of desiccated iron ascorbate precipitates in a Perkins Elmer Model 221 Infrared Spectrophotometer." In these studies, chemicals were reagent grade and solutions were prepared with ironfree distilled water. Mean values in animal experiments were for 8 to 10 rats. Numbers in italics or parentheses are the standard error of the mean. Results
In vitro studies. The chemical reaction of ferric chloride and ascorbic acid must be initiated at an acid pH to maintain solubility of t he iron in aqueous alkaline solutions (fig. 1). The sequence in whi ch ascorbi c acid and sodium hydroxide were added to solutions of ferric chloride determined whether the resultant mixture contained soluble iron or precipitated ferric hydroxide. I. (FeCI.+ ascorbic acid)+ NaOH ~ soluble iron chelate (pH 8) II. (FeCI.+ NaOH) + ascorbic acid~ insoluble iron precipitate (pH 8)
In the first reaction iron displaced hydrogen ions from ascorbate t o form purple colored complexes of variable molecular size that maintained iron in solution over a wide range of pH (2 to 11) (table 1, figs. 2 and 3). Conversely, in the second reaction t he addition of ascorbic acid to al-
37
ACID CHELATES IN IRON ABSORPTION
July 1968
occurred in highly acid solutions (pH 2). Additional evidence of chelate formation was obtained by adding alkali to mixtures of ferric chloride and ascorbic acid; an intense purple color developed in these solutions between pH 4 and 9 and a brownish hue at a more alkaline pH (fig. 3). Preliminary observati ons showed similar reac12 II 10 9
a. pH 7
6 5
4
FIG. 1. Ascorbic acid and sodium hydroxide were added to solutions of ferric chloride in a different sequence. The addition of ascorbic acid to an alkalinized mixture of ferric chloride and NaOH does not dissolve much of the precipitated iron (left). On the other hand, alkalinization of ferri c chloride-ascorbate mixtures does not precipitate iron but the solut ion develops an intense purple color (right). The final pH of both mixtures was pH 8.
kaline precipitates formed by the addition of sodium hydroxid e to ferric chloride produced little chelate formation. Mixing equimolar quantities of pH-adjusted solutions of ascorbic acid and ferric chloride (0.1 M, pH 2.2) made th e resultant solution more acid (pH 1.6) (fig. 2). This displacement of hydrogen ions from ascorbic acid was demonstrated in titration experiments which indicated that complex formation TABLE
......
3
0
3
4
6
7
8
9
10
NAOH meq.
Fro. 2. Titration of 50 ml of 0.1 M ferrous and ferric chloride with and without 0.1 M ascorbic acid in aqueous solutions of 0.01 N HCI. Titration was performed by the addition of measured quantities of 0.5 M NaOH. F erric chloride solutions required more NaOH than other chemical mixtures to make the solution alkaline. Less NaOH was required to alkalinize mixtures of ferric chloride and ascorbic acid indicating that most of the iron was reduced to the ferrous state. The addition of ascorbic acid (pH 2.2) to ferric chloride (pH 2.2) made the resultant solution more acid (pH 1.6), indicating that hydrogen ions were released from the ascorbate and suggesting that a chemical combination had occurred. This acidification was not observed in mixtures of ascorbic acid and ferrous chloride at a highly acid pH.
1. Solubility and molecular size of iron in various iron and iTon-ascorbate solutionsa Percentage of Fe at Solutions
FeCI. (pH 7 .5) FeCl~-as co rbate
(pH 7 .5) FeC1 8 -ascorbate (pH 7.5) FeCl 3 -ascorbate (pH 10.5) a
Total soluble iron
34.8 72.4 83.1 81.6
Mol wt <700
Mol wt >700
23 .5 48.2 46.8 32.9
11 .3 24.2 36.3 48 .7
Mol wt >4,000 Mol wt > 10,000
7.1 15 .9 24 .6 32.0
5.6 12 .7 18.9 24 . 2
The total soluble iron is the percentage of Fe 69 remaining in solution after centrifugation at 30,000 X
g for 60 min. The last three columns are the percentage of Fe 59 excluded from GlO, G25, and G50 gel
fil tration columns in the first displacement volume. Solutions were made to contain 0.1 M ferric chloride, ferrous chloride, and ascorbic acid. pH of these solutions and columns was adjusted with N aOH.
38
Vol. 55, No. 1
CONRAD AND SCHADE 100
m
1---NAO.H~
-HCI~
80
"'lL
"'
"'
:0
"
00 .3
60
0
(f)
"'"' 40 E "' ~ "'
0..
.2
20
0 10
8
6
4
4
2
6
8
10
12
pH
FIG. 3. Effect of pH upon ferric chloride with and without ascorbate. An equimolar quantity of ascorbic acid was added to an alkaline mixture of ferric chloride (0.1 M) and NaOH. At pH 10, about 18% of the iron was brought into solution (--) and there was slight color development (shaded area). The addition of HCl to this mixture dissolved the iron below pH 7 so that little iron was centrifuged from solution at pH 2. Realkalinization with NaOH caused little precipitation of iron over a wide range of pH (2 to 11). A visible purple color developed between 4 and 9 which became brown at a more alkaline pH. In the absence of ascorbic acid, ferric chloride was insoluble above pH 5 ( · · · · ). The optical densities ( OD) of 1: 100 dilutions of the supernatant solution were measured with a Beckman DB Spectrophotometer at 540 miL and are illustrated in the shaded areas.
tions between ascorbic acid and the ferric salts of other strong mineral acids but different results with solutions of ascorbic acid and either ferrous or other ferric salts; for example, the color of dilute solutions of ferric nitrate is pale yellow at an acid pH. Upon the addition of ascorbic acid the solution becomes colorless with a decrease in pH. Alkalinization with NaOH produces an intense blue colored complex which remains in solution and becomes brown above pH 9. The addition of ascorbic acid to solutions of ferrous chloride at the slightly alkaline pH encountered in the duodenum increases the amount of iron that remains in solution as a small molecular complex (table 1). Although solutions containing ferrous chloride and ascorbic acid develop a violet colored complex between pH 4 and 9 (fig. 4), this reaction is significantly different from that observed with ferric chloride and ascorbic acid. In the ferrous chloride-ascorbate reaction: (1) complex formation can be initiated at an alkaline pH (fig. 4), (2) titration curves show no displacement of hydrogen ions below pH 4 (fig. 2), and (3) increasing amounts of
ferrous iron are precipitated from ascorbate solutions between pH 6 and 9 to leave no iron in solution at a highly alkaline pH (fig. 4). The pigmented complexes of ascorbic acid and either ferrous or ferric iron were precipitated from aqueous solutions by the addition of four volumes of absolute ethanol. The centrifuged precipitate was washed with successive volumes of ethanol, acetone, and ether and dried in a desiccator with vacuum. Infrared spectroscopy and chemical analyses of ferrous chlorideascorbate precipitates (pH 7) anct ferric chloride ascorbate precipitates isolated at pH 7 and 10 indicated that they each had different chemical structures and composition (table 2 and fig. 5). In vivo studies. Rats absorbed more iron from oral test doses of ferrous chloride than from similar quantities of ferric chloride . 2 ~ The addition of ascorbic acid to solutions of iron salts significantly increased iron absorption. In our experiments as much iron was absorbed from test solutions containing ferric chloride and ascorbic acid as from similarly prepared doses of ferrous chloride (table 3).
39
ACID CHELATES IN IRON ABSORPTION
J uly 1968
100 -H CI~
"'
80
:g"'
60
".!
~NAOH--+
OD .3
0
({)
E'"'
.\\
4o
.2
1!"
.t
\'
20
o~-.~~r-.--.-.-~.--.-.-~,--.-.-r\~···,··~,_,_-,--LO 10
8
6
4
2
4
6
8
10
12
pH
FIG. 4. Effect of pH upon ferrous chloride with and without ascorbate. The addition of ascorbic acid (0.1 M) to alkaline precipitates of ferrous chloride (0.1 M) did not dissolve iron at pH 10. Most of the iron became soluble between pH 7 and 8 and form ed a violet colored complex . At lower pH, the color of the solution faded but the iron remained in solut ion . R ealkalinization of the solution caused precipita tion of the iron from mixtures of ferrous chloride and asco rbic acid (--). Iron in solut ions of ferrous chloride ( · · · ·) was less soluble than iron in mixtures of ferrous chloride and ascorbic acid at the slightly alkaline pH of the duodenum (pH 7 to 8) and it did not develop a visible color in solution. The optical density (OD) of 1:100 dilutions of t he supernatant solution from ferrou s chloride-ascorbate mixtures was measured with a DB Spectrophotometer at 540 ill!' and is illustrated in the shaded areas. T Anr,E 2. Partial percentage composition of precipitated FeCl,-ascorbate and FeCl 3 -ascorbale complexes• Complexes
FeCl , -asco rbate (pH 7) FeCl 3 -ascorbate (pH 10) FeC!,-ascorbate (pH 7)
Hydrogen
Carbon
24 .99 20.41 26 .06
25.47 19.99 26.39
4.26 3.58 3.97
4.79 3.51 4.01
Iron
22.48 29 .53 21.90
24 .26 30 .69 23 . 14
• Res ults of dupli cate a nalyses are reported as perce ntage composition. Measurements were perform ed by Mary H. Aldridge, Ph .D ., American University, Washington, D. C., up on ethanol precipitates which were washed with ethanol, acetone, and ethe r a nd dri ed under vacuum in a desiccator .
To te~t the effect of hydrochloric acid and chelate formation upon iron absorption , we measured th e absorption of Fe 59 from 0.25-mg test doses of iron sa lts to whi ch a::;corbic acid was added either befo re or after the acid iron solutions were brought to pH 8 with a lka li . These test doses were inj ected into the duodenum to prevent reacidification in the stomach. The sequence in which ascorbi c acid and sodium hydroxide were added to ferrous chloride had little effect upon iron absorption but markedly affected th e absorption of iron from test doses of ferric ch loride. More iron was absorbed from the so luble ferric chloride chela te (reaction I) than from the in-
soluble precipitates (reaction II). Iron-deficient rats absorbed more iron from the ferric precipitates than norma l iron-replete rats but less than iron-deficient anima ls dosed with the soluble ferric chelate (table 4) . Similar experim ents were performed with ph armacological doses of ferric chloride. A 2.5-mg dose of iron was select€d to provide a 10-fold increase in the test dose and because this quantity of iron was only slightly greater than the daily dietary consumption of iron by rats. Significantly more iron was a bsorbed from duodenal doses of the ferri c ch loride-ascorbate chelate than from simil ar doses of the precipitate con-
40 2000
Vol . 55, No. 1
CONRAD AND SCHA DE 1500
1000
mixtures passed into the duodenum (fig.
500
6) . FeCiz -ASCORBATE (pH• 7.0 )
Fe CI3 -ASCORBATE (pH•7.0)
FeCI3 -ASCORBATE (pH•IO.O)
Fe CI2
SODIUM ASCORBATE
2000
1500
1000
500
FIG. 5. Infrared spectra of KBr pellets containing 1 mg of desiccated iron-ascorbate precipitates are compared to pellets containing FeCI2 , F eCis , and ascorbic acid. The precipitates were obtained by the addition of ethanol to aqueous solutions. Differences in spectral peaks suggest differences in composition. We believe different biochemical sites on the ascorbate may be bound by the addition of FeCla and FeCI. to ascorbic acid and that the composition of chelates can be altered by changes in pH. Values are reported as em-'.
taining ascorbic acid. On the contrary, similar quantities of iron were absorbed from the two test solutions if test doses were injected into the stomach. Presumably, reacidification of the iron precipitates in the stomach permitted the formation of soluble ferric chloride-ascorbate complexes which enhanced iron absorption when the
Iron absorption of the ferric chlorideascorbate chelate and precipitate was measured in gastrectomized rats. The animals were dosed with Fe 59 -labeled ferric chelate (reaction I) or precipitate (reaction II) on the first day of experimentation and the altern ative preparation labeled with F e55 the followin g day. More iron was absorbed from test doses containing the soluble chelate than from similar oral doses of alkaline ferric precipitates containing ascorbic acid (table 5) . Since bile contains ascorbic acid 2 5 and in vitro addition of bile to ferric chloride inhibits precipitation of the iron by sodium hydroxide (table 6) , we tested the effect of ligation of the bile duct upon the absorption of F e59 from test doses of ferrous and ferric chloride. The bile duct of half of the experimental animals was ligated 30 min before the intragastric inj ection of test doses of labeled iron. The duodenum of each test animal was washed with 1 ml of distilled water, 15 to 30 min before the administration of the radioiron. Ligation of the bile duct significantly decreased iron absorption from test doses of both ferrous and ferric chloride in normal, iron-replete animals. In iron-deficient rats ligation of the bile duc t significantly diminished the absorption of iron from test doses of ferric 3. Absorption of F e 59 fTOm oral test doses of F eCl2 and FeCl3 with and without ascorbic acid
T ABLE
Fe 59 absorbed (% )
Mean SD SE
FeCh
FeCI,
--7 .7 9.3 10 .0 10.8 11 .0 11.3 11 .9 12 .2 15.3 15.3
---
11.5 2.4 0 .7
4.8 5.9 6.9 7.3 7.7 9.1 11 .3 11.7 11 .9 12.6 8.9 2.8 0 .9
FeCh +
FeCI,+
ascorbate
ascorbate
- - - - -11 .6 13 .6 13.8 15.8 16.5 16 .3 18. 4 16. 3 19 .5 18.7 19 .6 19.7 22.4 19 .8 24.7 22.4 25 .6 23.9 29.7 19.1 4.7 1. 6
19 .6 4.7 1.5
TABLE
41
ACID CHELATES IN IRON ABSORPTION
July 1968
4. Absorption of Fe 59 from intradu odenal do ses of FeCl2 and FeCl 3 to which ascorbic acid was added before or after alkalinization: normal and iron-deficient rats (FeCb + NaOH) + ascorba te
(FeCI, + ascorbate) +NaOH
(FeCla + NaOH) + ascorbate
(FeCla + ascorbate ) +N aOH
Normal
Iron deficient
Normal
Iron defici ent
Normal
Iron deficient
Normal
Iron deficient
Fe59 absorbed (%)
1.3 2.0 25 2.8 3.3 5.0 5.8 6.4 7.3
5.7 13.4 13.6 19.8 19.8 20 .0 21.0 22.2 22.4 29 .2
1.4 2.3 3.2 4.2 4.9 5.0 5.3 6.5 7.8
14.2 17 .5 21.6 22.9 23.2 24.5 24.6 25.2 30.5 32 .8
0 .8 1.2 2.1 2.9 3.2 3.4 3.7 4.1 4.9 7.8
21.1 21.5 26.6 32.6 32 .9 33 .5 33.6 37.4 38.4 39.6
5.4 5.8 6.0 6 .8 7.4 7 .7 8.7 9.7 10.4 13.7
33.8 35.9 37 .7 43.2 46 .3 49.9 51.3 52.5 59 .9 60 .5
Mean
4.1 2.2 0.7
18.7 6.4 2.0
4.5 2.0 0.7
23 .7 5.5 1.7
3.4 2.0 0.6
31.7 6.6 2.1
8.2 2.6 0.8
47.1 9.4 3 .0
-
SD SE
5. Gastrectomized1·ats : percentage of 1·adioi1·on absorbed fr om oral test dos es of F eCl 3 in which ascorbic acid was added before or after alkalinization"
TABLE
5
.,., -e 0
"'
.0
Rat no.
d:
(FeCla + NaOH ) + ascorbate
(FeCla + ascorbate) + NaOH
%
"g c:
1 2 3 4 5
2
"Q; 0
11.
I
G
Q L-~~~L-~--~--~~m_--~~L__
Precipitate Chelate Duodenal
Precipitate Chela te Gastric
FIG. 6. Absorption of iron from intraluminal doses of F eCI.. Preparations of F eCI. (2.5 mg), to which 0.1 M ascorbic acid was added before (chelate) or after (precipitate) alkalinization of th e mixtures, were inj ected into either th e duodenum or stomach of rats. Significantly more iron was absorbed from duodenal doses of chelate th an from duodenal doses of precipitate. In contrast , similar quantities of iron were absorbed from gastric doses of either chelate or precipitate. Comparison should not be made between the quantity of iron absorbed from duodenal and intragastric doses because of differences in the method of dosing.
chloride (22.5% ± 2.1 versus 29.2% ± 4.0, P < 0.05) but had little effect upon iron absorption from doses of ferrous chlo-
7 8 Mean SD SE
Fe 5 5
F es9
11.7 2.3 13.1 0 4.0 6.0 2.5 2.7 5.3 4.7 1.7
%
17.0 2.9 8.7 20.7 18.4 42.6 9.2 16.4
Fe ' 9
Fe 55
I
17.0 12.0 4.2
• Iron absorption s tudies of both the chelate a nd precipitate we re performed in ea ch gas trectomized rat to reduce experimental variation caused by surgery . A paired t-test indicated that differences were s ignificant. (P < 0.05 .)
ride (23.3 % ± 4.5 versus 25.2% ± 2.3) (table7). Discussion
For many years, investigators postulated that intestinal secretions affect iron
42
CONRAD AND SCHADE
hance absorption.13 The present study suggested that gastric and biliary secretions have a combinational effect upon ferric iron which permits the formation of soluble iron complexes that enhance iron absorption. The singular reaction of acidified ferric chloride and ascorbate provides a role for hydrochloric acid and bile in iron absorption and an explanation for the conflicting results reported from many studies of iron absorption in gastrectomized and achlorhydri c patients. Previous observations that ascorbic acid enhances iron absorption from food but not from hemoglobin suggest that significant quantities of nonheme iron are avai lable in food which may be affected by these intestinal secretions.15· 30 That our studies with iron salts have more than pharmacological significance is indicated by the effects of ascorbic acid upon the absorption of iron incorporated into foods. 3 o The chemical reaction of ferric chloride and ascorbic acid must be initiated at an acid pH to ensure solubility of the iron at the pH of the duodenum. In t his reaction, iron displaces hydrogen ions from ascorbate to form purple colored complexes which are soluble over a wide range of pH. If ascorbic acid is added to alkalinized ferric
absorption. Gastrectomized patients develop iron deficiency, 4 - 10 siderosis is observed with pancreatic insufficiency, 26 • 27 and animals with biliary fistulas become iron deficient. 28 Recent studies indicated that pancreatic secretions may cause decreased absorption of iron because bicarbonate combines with iron to form precipitates and macromolecules. 29 Previous investigators believed t hat acid gastric secretions released iron from food to enT ABLE
6. Solubility of various amounts of FeCla in 1 ml of bile at pH 7.5• FeCla added
Fe" in supernatant fluid after centrifugation
mg
%
14 100 100 100 100
1.0 0.1 0.01 0.001 0.0001
a Bile was collected from rats by inserting a polyethylene catheter (10 PE) into the bile duct. Measured quantities of Fe 59 -labeled FeCla were added to aliquots from pooled collections of bile . The pH was adjusted to 7.5 with NaOH . Radioactivity in these mixtures was compared to radioiron in t he supernatant fluid after centrifugation at 30,000 g for 60 min.
TABLE
Vol. 55, No . 1
7. Effect of ligation of the bile duct upon the absorption of Fe 59 from test doses of FeClz and FeCla in normal and iron -deficient rats Ferrous ch loride
Ligated
Patent Iron deficient
N ormal
Ferric chloride
Normal
Patent
Iron deficient
Normal
Ligated
Iron deficient
Normal
Iron defi cient
%
%
%
%
%
%
%
%
Fe 59 absorbed
4.3 5 .3 8.6 14.2 16.1 17 . 1 17. 6 19.5 20.9 23 .3
13 .4 18.1 19.5 22 .7 23.6 25.0 29 .5 30.9 31.8 37 .6
1.9 1.9 2.2 3.2 3.6 8.9 9.8 15. 9 16. 3
2.6 11.4 14.0 16 .5 17 .4 21.4 24.1 37 .0 41.6 46 .9
2.1 3 .6 3.9 5.7 6 .6 9.5 9.6 10 .8 11.2 14 .2
9 .5 14 .8 19 .8 24.4 26.0 27 . 2 38 .7 40.8 44 .9 45 .8
0.1 0 .1 0.6 0.7 1.1 1.7 2.6 2 .6 3. 9 5.4
10 .8 18 .2 19. 9 21.4 22 .5 22 .6 22.7 24.9 25.8 36 .6
Mea n
14 .6 6 .5 2.1
25.2 7.3 2.3
6 .8 6.2 2.1
23 .3 14 .2 4.5
7.7 4.0 1.3
29 .2 12.7 4.0
1.9 1.7 0.6
22.5 6.5 2.1
SD SE
I
July 1968
ACID CHELATES IN IRON ABSORPTION
chloride, most of the iron hydroxide precipitate is not brought into solution or chelated. In contrast, ferrous chloride and ascorbic acid form violet colored complexes at the alkaline pH of the duodenum. This chelate produces a modest increase in the quantity of low molecular weight iron that remains in solution between pH 7 and 8. Biochemical observations indicate that the chelates formed by adding ascorbic acid to ferrous chloride and ferric chloride are different compounds. These include differences in solubility, infrared spectroscopy, and perhaps chemical composition. The ferric iron may bind to different biochemical sites on the ascorbate in the process of undergoing reduction. The similarity between the sequential requirements for reaction of ferric chloride and ascorbate and the successive conditions encountered by food in the normal gut suggested that it might be physiologically significant. To test this hypothesis, we injected pH-adjusted doses (pH 8) of the iron chelate and precipitate into the duodenum of rats. There was poorer absorption of iron from the insoluble precipitate than from the soluble chelate. If the preparations were injected into the stomach, there was no significant difference in iron absorption because the hydrochloric acid converted the precipitate into a chelate. The biochemical reaction and physiological response of animals to chemical mixtures of ferrous chloride and ascorbic acid were less striking. The ferrous chloride-ascorbate chelate increased the solubility of iron at the slightly alkaline pH of the duodenum. Probably the chelat e increases solubility by interfering with the form ation of macromolecular iron in which one iron molecule binds to others through water bridges. 29 Likewise, it may diminish the effect of precipitating chelates in the diet (phytates, phosphates, carbonates, and oxalates ) or in pancreatic secretions (bicarbonate) upon dietary iron. 29 • 31 • 32 The capability of iron-deficient rats to absorb increased amounts of iron from various iron preparations in the absence of either bile or hydrochloric acid indicated that these intraluminal factors were less important regulators of iron absorption
43
than the body requirement for iron. This would provide an explanation for the slow development of iron deficiency in gastrectomized patients and the relatively mild state of iron deficiency observed in both achlorhydric and gastrectomized subjects. We studied iron-ascorbate complexes because vitamin C is a common ingredient in the diet, a normal constituent of bile, and an effective substance for increasing iron absorption. Similar to sugars and polyols, 33 • 34 ascorbic acid forms soluble chelates with iron which enhance iron absorption. Unlike the sugars and polyols, ascorbate complexes iron in equimolar concentrations. Both the efficient chelation of iron by ascorbic acid and its capability to reduce ferric iron may provide an explanation for its greater effect upon iron absorption. Previous observations indicated that only ferrous iron is absorbed by the intestinal epithelium of rats and man. 18 The studies reported in this article do not permit a different conclusion because ferric iron is reduced by the addition of ascorbic acid. However, our observations in combination with those of Charley et al. suggest that the primary effect of valence upon iron a bsorption is the change in solubility at the alkaline pH of the duodenum. 29 • 3 5 Rodents may absorb more iron from dietary ferric chloride than man because they are not scorbutic animals and they may secrete greater quantities of ascorbate in their bile. 3 G Summary
The chemical reaction of ferric chloride a nd ascorbic acid must be initiated at an acid pH to insure solubility of the iron at the pH of th e duodenum: (FeCI.+ ascorbic acid) +base-> soluble iron chelate (FeCla + base ) +ascorbic acid-> insoluble iron precipitate
In this reaction, iron displaces hydrogen ions from ascorbate to form purple colored complexes that can be isolated by alcoholic precipitation and which hold iron in solution over a wide range of pH (2 to 11). In contrast, FeCb combines with ascorbate at the pH of the duodenum making HCl less
44
CONRAD AND SCHADE
important for iron absorption from this compound. Biochemical differences were found which suggested that FeCb and FeCla ascorbate complexes were different compounds. The similarity between the sequence of the FeC1 3 and ascorbic acid reaction and successive conditions encountered by food in the intact gut suggested that it might be physiologically significant. To test this hypothesis, we introduced pH-adjusted doses of the iron chelate and precipitate into the duodenum of normal rats and administered oral doses to gastrectomized animals. There was poorer absorption of iron from the insoluble precipitate than from the soluble chelate. If the preparations were administered through the stomach, there was no significant difference in iron absorption because acid converted the precipitate into a chelate. Ligation of the bile duct decreased the absorption of iron from test doses of FeC1 3 . This may be caused by the ascorbate content of the bile; in vitro bile dissolves iron from FeC1 3 . These data provide information of the importance of an intact gastrointestinal tract in maintaining iron equilibrium. Gastrectomized patients might become iron deficient because of an inability to utilize ascorbate from the diet or bile to bring dietary ferric iron into solution and make it available for absorption. REFERENCES 1. Mettier, S. R., and G. R. Minot. 1931. The
effect of iron on blood formation as influ enced by changing the acidity of the gastroduod0nal contents in certain cases of anemia. Amer. J. Med . Sci. 181: 25-36. 2. MPttier, S. R., F. Kellogg, and J. F. Rinehart . 1933. Chronic idiopathic hy pochromic anemia. Etiologic relationship of achlorhydria to the anemia, with sper-ial reference to the effect of large doses of iron, organic (dietary) iron and of predigested food upon formation of erythro cytes. Amer. J . Med. Sci. 1S6 : 694-704. 3. K ellogg, F ., and S. R. Mettier. 1936. Effect of alkaline therapy for peptic ulcer on utilization of dietary iron in the regeneration of hemoglobin. Arch. Intern. Med. (Chicago) 58: 278-284. 4. Leroux, R., and E. Vermes. 1939. Anemies agas triques. Sang. 13: 241-267.
Vol. 55, No. 1
5. Lyngar, E. 1950. Blood changes after partial
gastrectomy for ulcer. Acta Med. Scand ., sup pl. 247 : 5-71. 6. Owren, P. A. 1952. The pathogenesis and treatment of iron deficiency anemia after partial gastrectomy. Acta Chir. Scand. 104: 206-214.
7. Baird, I. MeL., D. A. Podmore, and G. M. Wilson. 1957. Changes in iron metabolism following gastrectomy and other surgical operations. Clin. Sci. 16: 463-473 . 8. Stevens, A. R., Jr., G. Pirzio-Biroli, H . N. Harkins, L. M. Nyhus, and C. A. Finch. 1959. Iron metabolism in patients after partial gastrectomy. Ann. Surg. 149: 534538. 9. Choudhury, M . R., and J. Williams. 1959.
10.
11.
12.
13.
Iron absorption and gastric operations. Clin . Sci.18: 527-532. Hines, J. D., A. V. Hoffbrand, and D. L. Mollin. 1967. The hematologic complications following partial gastrectomy. Amer. J . Med. 43: 555-569. Barer, A. P., and W. M. Fowler. 1937. Influence of gastric acidity and degree of anemia on iron retention. Arch. Intern. Med. (Chicago) 59: 785-792. Goldberg, A., A. C. Lockhead, and J. H. Dagg. 1963. Histamine fast achlorhydria and iron absorption. Lancet 1: 848-850 . Cook, J. D., G. M. Brown, and L. S. Valberg. 1964. The effect of achylia gastrica on iron absorption. J. Clin. Invest. 43: 1185-
1191. 14. Dubach, R. S., T. E. Callender, and C. V. Moore. 1948. StudiPs in iron transporta-
tion and metabo lism. VI. Absorption of radioactive iron in patients with fever and with anemias of varied etiology. Blood 3 : 526-540. 15. Calil'nder, S. T., B. J. Mallett, and M. D. Smith. 1957. Absorption of haemoglobin iron. Brit. J. Haemat. 3: 186-192. 16. Willia ms, J. 1959. The effect of ascorbic acid
on iron absorption in postgastrectomy anemia and achlorhydria. Clin . Sci. 18: 521525 . 17. Charlton, R. W., P. J acobs, H. Stefel, and T. H. Bothwell. 1964. Effect of alcohol on iron absorption. Brit. Med. J. 2: 1427-1429. 18. Venkatachalam, P . S., I. Brading, E. P. George, and R. J. Walsh. 1956. An experi-
ment in rats to determine whether iron is absorbed only in the ferrous state. Aust. J. Exp. Biol. Med. Sci. 34: 389--393. 19. Anelli , J. 1958. Combinations of iron and ascorbic acid. Acta Cient. Venez. 9: 105114.
July 1968
ACID CHELATES IN IRON ABSORPTION
20. Forrester, R. H ., M. E . Conrad, Jr., and W. H. Crosby. 1962. Measurement of total body iron 59 in anima ls using whole-body liquid scintillation detectors. Proc . Soc. Exp. Bioi. M ed . 111 : 115-119. 21. M ahin, D. T., and R. T. Lofberg. 1966. A simplified method of sample preparation for determination of tritrium, carbon-14 or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. Biochem. 16: 500-509. 22. Gelotte, B. 1960. Studies on gel filtration sorption properties of the bed material sephadex. J . Chromatogr. 3: 330-342. 23. Stimson, M. M., and M . J. O'Donnell. 1952. The infrared and ultraviolet absorption spec tra of cytosine and isocytosine in the solid state . J. Amer. Chern . Soc. 74: 18051808. 24 . Groen, J., W. A. Van D en Broek, and H. Veldman . 1947. Absorption of iron compounds from the small intestine of the rat. Biochim. Biophys. Acta 1: 315-326. 25. Bockus, H. L. 1965. Gas troenterology, Vol. 3, p. 576-577. W. B. Saunders Company, Philadelphia. 26. Kinney, T. D., C. A. Finch, N. K aufman, D . M . H cgsted, and P. F. Part hington. 1950. The relationship of the pancreas to the absorption of iron. Amer. J. Path. 26: 746. 27. Tonz, 0., S. Weiss, H . W. Strahm, and E. Rossi. 1965. Iron absorption in cystic fibrosis. Lancet 2 : 1096-1099. 28. H awkins. W. B .. F. S. Robschertt-Robbins,
29.
30. 31.
32.
33.
34.
35.
36.
45
and H. Whipple. 1938. H emoglobin production in anem ia as influenced by bile fistula. J . Exp. Med. 67 : 89-110. Benj amin, B. I., S. Cortell, and M. E. Conrad. 1967. Bicarbonate-induced iron complexes and iron absorption : one effect of pancreatic secretions. Gastroenterology 53 : 389-396. Moore, C. V. 1961. Iron metabolism and nutrition. H arvey Lect. 55: 67-101. Chaberek, S., and A. E. M artell. 1959. Organic s2questering agents. A discussion of the chemical behavior and applications of metal chelate compounds in aqueous systems, p. 187-198. John Wiley & Sons, Inc., New York. Bailar, J. C., Jr. 1956. Chemistry of the coordination compounds, p. 114-125. Rheinhold, New York. Charley, P. J ., B. Sarkar, C. F. Stitt, and P . Saltman . 1963. Chelation of iron by sugars. Biochim . Biophy ~ . Acta 69 : 313-321. Pollack, S ., R. M . K aufman, and W . H. Crosby . 1964. Iron absorption: effects of sugars and reducing agents. Blood 24: 577581. Charley, P . J., C. Stitt, E. Shore, and P. Saltman. 1963. Studies in the regulation of intestinal iron absorption. J. Lab. Clin . Med. 61: 397-410 . Farris, :K J., and J . W. Griffith, Jr. 1949. The rat in laborato ry in vestigation , p. 137. J. B. Lippincott Company, Philadelphia.
GASTROENTEROLOGY
Copyright
© 1968 by The Williams
Printed in U.S.A.
& Wilkins Co.
ASCORBIC ACID CHELATES IN IRON ABSORPTION: A ROLE FOR HYDROCHLORIC ACID AND BILE MARCEL E. CoNRAD, M.D., AND STANLEY G. ScHADE, M.D. DepaTtment of Hematology, Walter Reed Army Institute of Research, Washington, D. C.
vitro and measurements of their effect upon iron absorption in animals.
Many investigators have postulated that acid gastric secretions enhanced iron absorption. 1 - 3 This hypothesis was supported by the frequent development of iron deficiency in gastrectomized human patients and decreased absorption of dietary iron by achlorhydric subjects. 4 - 13 Data obtained by radioactive iron studies showed that acid does not enhance the absorption of iron from ferrous salts or hemoglobin iron but might increase the absorption of iron from ferric salts and foodP- 17 Since ionic ferric iron is not absorbed by the intestinal mucosa,1 8 gastrointestinal secretions must reduce or chelate ferric iron to bring this iron into solution and enhance its absorption. Anelli reported that ferrous salts form a colored complex with ascorbic acid. 19 During studies of iron-ascorbate chelates, we found that ascorbic acid forms a soluble chelate with ferric chloride at an acid pH but not with ferric iron precipitates at an alkaline pH. This acid iron chelate was stable and maintained iron in a soluble form when the solution was subsequently alkalinized. Since ascorbic acid is a normal constituent of bile, we believed that studies of the iron-ascorbate chclatcs might provide information on the mechanism by which acid gastric juice enhances iron absorption. This study reports biochemical observations of iron-ascorbate mixtures in
Material and Methods Male albino rats, Walter Reed Carworth Farms strain, weighing 200 to 225 g were used in these studies. The principles of laboratory animal care promulgated by the National Society for Medical Research were observed. Rats were raised in a pathogen-free environment and housed in galvanized wire cages. The standard laboratory diet contained 25% protein and 9 mg of iron per 100 g dry weight. Iron deficiency was induced by bleeding animals 4 ml 1 week before study and feeding them a powdered milk diet. Iron absorption studies were performed in rats fasted overnight. Unopcrated animals were lightly anesthetized with ether for dosing. Oral doses were introduced into the stomach through a 17 -gauge olive tipped end oesophageal needle. Test doses of iron were injected into the intestinal lumen of operated animals with a hypodermic syringe and needle. Operated rats were anesthetized with pentobarbital and their abdomen opened through a midline ventral incision. Intragastric doses were injected through a 23-gauge hypodermic needle. Duodenal doses of iron were administered by inserting a 20~?:auge hypodermic needle into the stomach and throu~?:h the pylorus ; a ligature of umbilical tape was tightened around the pylorus before injection of the test dose. The bile duct of certain rats was transected between silk ligatnres 30 min before administration of the test dose of iron . In experiments testing the effects of bile upon iron absorption, the duodenum wns wnshed with 1 ml of distilled water followed by 1 ml of air injected through a hypodermic needle inserted into the duodenum via the gastropyloric route. The ventral incision of the operated animals described above was closed with metallic surgical clips. These animals were killed 2 hr after administration of the test dose and the clamped unopened gut was excised and discarded. The carcass was
RecPivcd January 23, 1968. Accepted February 19, 1968. Address requests for reprints to: Lieutenant Colonel Marcel E . Conrad, MC, Department of Hematology, Walter Reed Army Institute of Research, Washington, D. C. 20012. The authors are grateful to Harold L. Williams, M.S., William Godfrey, and Robert J. Landman for their technical assistance . 35
36
Vol . 55, No. 1
CONRAD AND SCHADE
saved for measurement of iron absorption. Gastrectomized rats underwent surgery 3 weeks before iron absorption studies were initiated; the stomach was excised and an end-to-end esophagoduodenostomy was constructed. The incision was closed with absorbable suture material. Oral test doses were administered to gastrectomized rats through an endoesophageal polyethylene tubing (PE 90). Iron absorption studies were performed with test solutions containing either ferric 59 chloride (0.5 p.c, New England Nuclear Corporation, 9 me per mg), ferrous'" citrate (0.5 p.c, Abbott Laboratories, 30 me per mg), or ferric .. chloride (50 p.c, New England Nuclear Corporation, 9 me per mg), and 0.25 mg of elemental iron as ferric or ferrous chloride. The pH of test doses was adjusted with HCl or NaOH to 3.5 in experiments in which ascorbic acid was not used and to 8.0 in experiments testing effects of ascorbic acid. Sodium ascorbate or ascorbic acid were added to certain doses to make the solution 0.01 M. Absorption of Fe59 was measured with a small animal whole body liquid scintillation detector (Packard ARMAC) . Rats were placed in vented quart ice cream cartons for measurement of whole body radioactivity. Reference standards were prepared by adding a test dose to a 250-ml water-filled plastic bottle. The radioactivity in animals and standards was measured for 100-second intervals (0.8 Mev to oo ). The quantity of Fe•• absorbed during 2-hr experiments was calculated from measurements of the net radioactivity in the carcass and reference standard. The radioactivity in live animals was measured 3 hr and 7 days after dosing; absorption was calculated by the ratio of net counts 7 days after dosing (rz) to tho8e of the standard (sz) and a similar ratio at 3 hr (r./s.) :•
rx//sx X 100 = percentage Fe 59 absorbed ro So
Absorption of Fe"" was calculated from the ratio of Fe"' to Fe•• detected in specimens of whole blood obtained from rats 10 days after dosing. Measurements were made in a liquid scintillation detector by the method of Mahin and Lofberg (Packard TRICARB) .21 These ratios were multiplied by the percentage Fe59 absorbed as determined from measurements of y-emitting radioactivity in a small animal whole body detector. In vitro experiments were performed with 0.1 M iron and 0.1 M ascorbate solutions to which Fe59 was added. Titration experiments were performed with a calibrated Leeds and Northrup
pH meter and a magnetic stirrer; pH adjustments were made with iron-free HCl and NaOH. Solubility was defined as the percentage of iron remaining in the supernatant fluid of samples centrifuged at 30,000 g for 60 min. The color formed by iron-ascorbate complexes was measured with a Beckman DB spectrophotometer at 540 mp.. The molecular size of the radioiron in the supernatant fractions was measured with Sephadex G-10, G-25, and G-50 gel filtration media (Pharmacia) . The gel was swollen in an isotonic saline solution (pH 7.4) for 24 hr and pH adjusted to that of the test solution before it was used to pack 10 X 0.5-cm columns. The void volume of each column was measured with blue dextran. The quantity of radioiron recovered in the first displacement volume was used to estimate the percentage of iron with a molecular weight greater than 700 (G-10), 4,000 (G-25), or 10,000 (G-50) . 22 Infrared spectroscopy was performed by examination of compressed KBr pellets containing 1 mg of desiccated iron ascorbate precipitates in a Perkins Elmer Model 221 Infrared Spectrophotometer." In these studies, chemicals were reagent grade and solutions were prepared with ironfree distilled water. Mean values in animal experiments were for 8 to 10 rats. Numbers in italics or parentheses are the standard error of the mean. Results
In vitro studies. The chemical reaction of ferric chloride and ascorbic acid must be initiated at an acid pH to maintain solubility of t he iron in aqueous alkaline solutions (fig. 1). The sequence in whi ch ascorbi c acid and sodium hydroxide were added to solutions of ferric chloride determined whether the resultant mixture contained soluble iron or precipitated ferric hydroxide. I. (FeCI.+ ascorbic acid)+ NaOH ~ soluble iron chelate (pH 8) II. (FeCI.+ NaOH) + ascorbic acid~ insoluble iron precipitate (pH 8)
In the first reaction iron displaced hydrogen ions from ascorbate t o form purple colored complexes of variable molecular size that maintained iron in solution over a wide range of pH (2 to 11) (table 1, figs. 2 and 3). Conversely, in the second reaction t he addition of ascorbic acid to al-
37
ACID CHELATES IN IRON ABSORPTION
July 1968
occurred in highly acid solutions (pH 2). Additional evidence of chelate formation was obtained by adding alkali to mixtures of ferric chloride and ascorbic acid; an intense purple color developed in these solutions between pH 4 and 9 and a brownish hue at a more alkaline pH (fig. 3). Preliminary observati ons showed similar reac12 II 10 9
a. pH 7
6 5
4
FIG. 1. Ascorbic acid and sodium hydroxide were added to solutions of ferric chloride in a different sequence. The addition of ascorbic acid to an alkalinized mixture of ferric chloride and NaOH does not dissolve much of the precipitated iron (left). On the other hand, alkalinization of ferri c chloride-ascorbate mixtures does not precipitate iron but the solut ion develops an intense purple color (right). The final pH of both mixtures was pH 8.
kaline precipitates formed by the addition of sodium hydroxid e to ferric chloride produced little chelate formation. Mixing equimolar quantities of pH-adjusted solutions of ascorbic acid and ferric chloride (0.1 M, pH 2.2) made th e resultant solution more acid (pH 1.6) (fig. 2). This displacement of hydrogen ions from ascorbic acid was demonstrated in titration experiments which indicated that complex formation TABLE
......
3
0
3
4
6
7
8
9
10
NAOH meq.
Fro. 2. Titration of 50 ml of 0.1 M ferrous and ferric chloride with and without 0.1 M ascorbic acid in aqueous solutions of 0.01 N HCI. Titration was performed by the addition of measured quantities of 0.5 M NaOH. F erric chloride solutions required more NaOH than other chemical mixtures to make the solution alkaline. Less NaOH was required to alkalinize mixtures of ferric chloride and ascorbic acid indicating that most of the iron was reduced to the ferrous state. The addition of ascorbic acid (pH 2.2) to ferric chloride (pH 2.2) made the resultant solution more acid (pH 1.6), indicating that hydrogen ions were released from the ascorbate and suggesting that a chemical combination had occurred. This acidification was not observed in mixtures of ascorbic acid and ferrous chloride at a highly acid pH.
1. Solubility and molecular size of iron in various iron and iTon-ascorbate solutionsa Percentage of Fe at Solutions
FeCI. (pH 7 .5) FeCl~-as co rbate
(pH 7 .5) FeC1 8 -ascorbate (pH 7.5) FeCl 3 -ascorbate (pH 10.5) a
Total soluble iron
34.8 72.4 83.1 81.6
Mol wt <700
Mol wt >700
23 .5 48.2 46.8 32.9
11 .3 24.2 36.3 48 .7
Mol wt >4,000 Mol wt > 10,000
7.1 15 .9 24 .6 32.0
5.6 12 .7 18.9 24 . 2
The total soluble iron is the percentage of Fe 69 remaining in solution after centrifugation at 30,000 X
g for 60 min. The last three columns are the percentage of Fe 59 excluded from GlO, G25, and G50 gel
fil tration columns in the first displacement volume. Solutions were made to contain 0.1 M ferric chloride, ferrous chloride, and ascorbic acid. pH of these solutions and columns was adjusted with N aOH.
38
Vol. 55, No. 1
CONRAD AND SCHADE 100
m
1---NAO.H~
-HCI~
80
"'lL
"'
"'
:0
"
00 .3
60
0
(f)
"'"' 40 E "' ~ "'
0..
.2
20
0 10
8
6
4
4
2
6
8
10
12
pH
FIG. 3. Effect of pH upon ferric chloride with and without ascorbate. An equimolar quantity of ascorbic acid was added to an alkaline mixture of ferric chloride (0.1 M) and NaOH. At pH 10, about 18% of the iron was brought into solution (--) and there was slight color development (shaded area). The addition of HCl to this mixture dissolved the iron below pH 7 so that little iron was centrifuged from solution at pH 2. Realkalinization with NaOH caused little precipitation of iron over a wide range of pH (2 to 11). A visible purple color developed between 4 and 9 which became brown at a more alkaline pH. In the absence of ascorbic acid, ferric chloride was insoluble above pH 5 ( · · · · ). The optical densities ( OD) of 1: 100 dilutions of the supernatant solution were measured with a Beckman DB Spectrophotometer at 540 miL and are illustrated in the shaded areas.
tions between ascorbic acid and the ferric salts of other strong mineral acids but different results with solutions of ascorbic acid and either ferrous or other ferric salts; for example, the color of dilute solutions of ferric nitrate is pale yellow at an acid pH. Upon the addition of ascorbic acid the solution becomes colorless with a decrease in pH. Alkalinization with NaOH produces an intense blue colored complex which remains in solution and becomes brown above pH 9. The addition of ascorbic acid to solutions of ferrous chloride at the slightly alkaline pH encountered in the duodenum increases the amount of iron that remains in solution as a small molecular complex (table 1). Although solutions containing ferrous chloride and ascorbic acid develop a violet colored complex between pH 4 and 9 (fig. 4), this reaction is significantly different from that observed with ferric chloride and ascorbic acid. In the ferrous chloride-ascorbate reaction: (1) complex formation can be initiated at an alkaline pH (fig. 4), (2) titration curves show no displacement of hydrogen ions below pH 4 (fig. 2), and (3) increasing amounts of
ferrous iron are precipitated from ascorbate solutions between pH 6 and 9 to leave no iron in solution at a highly alkaline pH (fig. 4). The pigmented complexes of ascorbic acid and either ferrous or ferric iron were precipitated from aqueous solutions by the addition of four volumes of absolute ethanol. The centrifuged precipitate was washed with successive volumes of ethanol, acetone, and ether and dried in a desiccator with vacuum. Infrared spectroscopy and chemical analyses of ferrous chlorideascorbate precipitates (pH 7) anct ferric chloride ascorbate precipitates isolated at pH 7 and 10 indicated that they each had different chemical structures and composition (table 2 and fig. 5). In vivo studies. Rats absorbed more iron from oral test doses of ferrous chloride than from similar quantities of ferric chloride . 2 ~ The addition of ascorbic acid to solutions of iron salts significantly increased iron absorption. In our experiments as much iron was absorbed from test solutions containing ferric chloride and ascorbic acid as from similarly prepared doses of ferrous chloride (table 3).
39
ACID CHELATES IN IRON ABSORPTION
J uly 1968
100 -H CI~
"'
80
:g"'
60
".!
~NAOH--+
OD .3
0
({)
E'"'
.\\
4o
.2
1!"
.t
\'
20
o~-.~~r-.--.-.-~.--.-.-~,--.-.-r\~···,··~,_,_-,--LO 10
8
6
4
2
4
6
8
10
12
pH
FIG. 4. Effect of pH upon ferrous chloride with and without ascorbate. The addition of ascorbic acid (0.1 M) to alkaline precipitates of ferrous chloride (0.1 M) did not dissolve iron at pH 10. Most of the iron became soluble between pH 7 and 8 and form ed a violet colored complex . At lower pH, the color of the solution faded but the iron remained in solut ion . R ealkalinization of the solution caused precipita tion of the iron from mixtures of ferrous chloride and asco rbic acid (--). Iron in solut ions of ferrous chloride ( · · · ·) was less soluble than iron in mixtures of ferrous chloride and ascorbic acid at the slightly alkaline pH of the duodenum (pH 7 to 8) and it did not develop a visible color in solution. The optical density (OD) of 1:100 dilutions of t he supernatant solution from ferrou s chloride-ascorbate mixtures was measured with a DB Spectrophotometer at 540 ill!' and is illustrated in the shaded areas. T Anr,E 2. Partial percentage composition of precipitated FeCl,-ascorbate and FeCl 3 -ascorbale complexes• Complexes
FeCl , -asco rbate (pH 7) FeCl 3 -ascorbate (pH 10) FeC!,-ascorbate (pH 7)
Hydrogen
Carbon
24 .99 20.41 26 .06
25.47 19.99 26.39
4.26 3.58 3.97
4.79 3.51 4.01
Iron
22.48 29 .53 21.90
24 .26 30 .69 23 . 14
• Res ults of dupli cate a nalyses are reported as perce ntage composition. Measurements were perform ed by Mary H. Aldridge, Ph .D ., American University, Washington, D. C., up on ethanol precipitates which were washed with ethanol, acetone, and ethe r a nd dri ed under vacuum in a desiccator .
To te~t the effect of hydrochloric acid and chelate formation upon iron absorption , we measured th e absorption of Fe 59 from 0.25-mg test doses of iron sa lts to whi ch a::;corbic acid was added either befo re or after the acid iron solutions were brought to pH 8 with a lka li . These test doses were inj ected into the duodenum to prevent reacidification in the stomach. The sequence in which ascorbi c acid and sodium hydroxide were added to ferrous chloride had little effect upon iron absorption but markedly affected th e absorption of iron from test doses of ferric ch loride. More iron was absorbed from the so luble ferric chloride chela te (reaction I) than from the in-
soluble precipitates (reaction II). Iron-deficient rats absorbed more iron from the ferric precipitates than norma l iron-replete rats but less than iron-deficient anima ls dosed with the soluble ferric chelate (table 4) . Similar experim ents were performed with ph armacological doses of ferric chloride. A 2.5-mg dose of iron was select€d to provide a 10-fold increase in the test dose and because this quantity of iron was only slightly greater than the daily dietary consumption of iron by rats. Significantly more iron was a bsorbed from duodenal doses of the ferri c ch loride-ascorbate chelate than from simil ar doses of the precipitate con-
40 2000
Vol . 55, No. 1
CONRAD AND SCHA DE 1500
1000
mixtures passed into the duodenum (fig.
500
6) . FeCiz -ASCORBATE (pH• 7.0 )
Fe CI3 -ASCORBATE (pH•7.0)
FeCI3 -ASCORBATE (pH•IO.O)
Fe CI2
SODIUM ASCORBATE
2000
1500
1000
500
FIG. 5. Infrared spectra of KBr pellets containing 1 mg of desiccated iron-ascorbate precipitates are compared to pellets containing FeCI2 , F eCis , and ascorbic acid. The precipitates were obtained by the addition of ethanol to aqueous solutions. Differences in spectral peaks suggest differences in composition. We believe different biochemical sites on the ascorbate may be bound by the addition of FeCla and FeCI. to ascorbic acid and that the composition of chelates can be altered by changes in pH. Values are reported as em-'.
taining ascorbic acid. On the contrary, similar quantities of iron were absorbed from the two test solutions if test doses were injected into the stomach. Presumably, reacidification of the iron precipitates in the stomach permitted the formation of soluble ferric chloride-ascorbate complexes which enhanced iron absorption when the
Iron absorption of the ferric chlorideascorbate chelate and precipitate was measured in gastrectomized rats. The animals were dosed with Fe 59 -labeled ferric chelate (reaction I) or precipitate (reaction II) on the first day of experimentation and the altern ative preparation labeled with F e55 the followin g day. More iron was absorbed from test doses containing the soluble chelate than from similar oral doses of alkaline ferric precipitates containing ascorbic acid (table 5) . Since bile contains ascorbic acid 2 5 and in vitro addition of bile to ferric chloride inhibits precipitation of the iron by sodium hydroxide (table 6) , we tested the effect of ligation of the bile duct upon the absorption of F e59 from test doses of ferrous and ferric chloride. The bile duct of half of the experimental animals was ligated 30 min before the intragastric inj ection of test doses of labeled iron. The duodenum of each test animal was washed with 1 ml of distilled water, 15 to 30 min before the administration of the radioiron. Ligation of the bile duct significantly decreased iron absorption from test doses of both ferrous and ferric chloride in normal, iron-replete animals. In iron-deficient rats ligation of the bile duc t significantly diminished the absorption of iron from test doses of ferric 3. Absorption of F e 59 fTOm oral test doses of F eCl2 and FeCl3 with and without ascorbic acid
T ABLE
Fe 59 absorbed (% )
Mean SD SE
FeCh
FeCI,
--7 .7 9.3 10 .0 10.8 11 .0 11.3 11 .9 12 .2 15.3 15.3
---
11.5 2.4 0 .7
4.8 5.9 6.9 7.3 7.7 9.1 11 .3 11.7 11 .9 12.6 8.9 2.8 0 .9
FeCh +
FeCI,+
ascorbate
ascorbate
- - - - -11 .6 13 .6 13.8 15.8 16.5 16 .3 18. 4 16. 3 19 .5 18.7 19 .6 19.7 22.4 19 .8 24.7 22.4 25 .6 23.9 29.7 19.1 4.7 1. 6
19 .6 4.7 1.5
TABLE
41
ACID CHELATES IN IRON ABSORPTION
July 1968
4. Absorption of Fe 59 from intradu odenal do ses of FeCl2 and FeCl 3 to which ascorbic acid was added before or after alkalinization: normal and iron-deficient rats (FeCb + NaOH) + ascorba te
(FeCI, + ascorbate) +NaOH
(FeCla + NaOH) + ascorbate
(FeCla + ascorbate ) +N aOH
Normal
Iron deficient
Normal
Iron defici ent
Normal
Iron deficient
Normal
Iron deficient
Fe59 absorbed (%)
1.3 2.0 25 2.8 3.3 5.0 5.8 6.4 7.3
5.7 13.4 13.6 19.8 19.8 20 .0 21.0 22.2 22.4 29 .2
1.4 2.3 3.2 4.2 4.9 5.0 5.3 6.5 7.8
14.2 17 .5 21.6 22.9 23.2 24.5 24.6 25.2 30.5 32 .8
0 .8 1.2 2.1 2.9 3.2 3.4 3.7 4.1 4.9 7.8
21.1 21.5 26.6 32.6 32 .9 33 .5 33.6 37.4 38.4 39.6
5.4 5.8 6.0 6 .8 7.4 7 .7 8.7 9.7 10.4 13.7
33.8 35.9 37 .7 43.2 46 .3 49.9 51.3 52.5 59 .9 60 .5
Mean
4.1 2.2 0.7
18.7 6.4 2.0
4.5 2.0 0.7
23 .7 5.5 1.7
3.4 2.0 0.6
31.7 6.6 2.1
8.2 2.6 0.8
47.1 9.4 3 .0
-
SD SE
5. Gastrectomized1·ats : percentage of 1·adioi1·on absorbed fr om oral test dos es of F eCl 3 in which ascorbic acid was added before or after alkalinization"
TABLE
5
.,., -e 0
"'
.0
Rat no.
d:
(FeCla + NaOH ) + ascorbate
(FeCla + ascorbate) + NaOH
%
"g c:
1 2 3 4 5
2
"Q; 0
11.
I
G
Q L-~~~L-~--~--~~m_--~~L__
Precipitate Chelate Duodenal
Precipitate Chela te Gastric
FIG. 6. Absorption of iron from intraluminal doses of F eCI.. Preparations of F eCI. (2.5 mg), to which 0.1 M ascorbic acid was added before (chelate) or after (precipitate) alkalinization of th e mixtures, were inj ected into either th e duodenum or stomach of rats. Significantly more iron was absorbed from duodenal doses of chelate th an from duodenal doses of precipitate. In contrast , similar quantities of iron were absorbed from gastric doses of either chelate or precipitate. Comparison should not be made between the quantity of iron absorbed from duodenal and intragastric doses because of differences in the method of dosing.
chloride (22.5% ± 2.1 versus 29.2% ± 4.0, P < 0.05) but had little effect upon iron absorption from doses of ferrous chlo-
7 8 Mean SD SE
Fe 5 5
F es9
11.7 2.3 13.1 0 4.0 6.0 2.5 2.7 5.3 4.7 1.7
%
17.0 2.9 8.7 20.7 18.4 42.6 9.2 16.4
Fe ' 9
Fe 55
I
17.0 12.0 4.2
• Iron absorption s tudies of both the chelate a nd precipitate we re performed in ea ch gas trectomized rat to reduce experimental variation caused by surgery . A paired t-test indicated that differences were s ignificant. (P < 0.05 .)
ride (23.3 % ± 4.5 versus 25.2% ± 2.3) (table7). Discussion
For many years, investigators postulated that intestinal secretions affect iron
42
CONRAD AND SCHADE
hance absorption.13 The present study suggested that gastric and biliary secretions have a combinational effect upon ferric iron which permits the formation of soluble iron complexes that enhance iron absorption. The singular reaction of acidified ferric chloride and ascorbate provides a role for hydrochloric acid and bile in iron absorption and an explanation for the conflicting results reported from many studies of iron absorption in gastrectomized and achlorhydri c patients. Previous observations that ascorbic acid enhances iron absorption from food but not from hemoglobin suggest that significant quantities of nonheme iron are avai lable in food which may be affected by these intestinal secretions.15· 30 That our studies with iron salts have more than pharmacological significance is indicated by the effects of ascorbic acid upon the absorption of iron incorporated into foods. 3 o The chemical reaction of ferric chloride and ascorbic acid must be initiated at an acid pH to ensure solubility of the iron at the pH of the duodenum. In t his reaction, iron displaces hydrogen ions from ascorbate to form purple colored complexes which are soluble over a wide range of pH. If ascorbic acid is added to alkalinized ferric
absorption. Gastrectomized patients develop iron deficiency, 4 - 10 siderosis is observed with pancreatic insufficiency, 26 • 27 and animals with biliary fistulas become iron deficient. 28 Recent studies indicated that pancreatic secretions may cause decreased absorption of iron because bicarbonate combines with iron to form precipitates and macromolecules. 29 Previous investigators believed t hat acid gastric secretions released iron from food to enT ABLE
6. Solubility of various amounts of FeCla in 1 ml of bile at pH 7.5• FeCla added
Fe" in supernatant fluid after centrifugation
mg
%
14 100 100 100 100
1.0 0.1 0.01 0.001 0.0001
a Bile was collected from rats by inserting a polyethylene catheter (10 PE) into the bile duct. Measured quantities of Fe 59 -labeled FeCla were added to aliquots from pooled collections of bile . The pH was adjusted to 7.5 with NaOH . Radioactivity in these mixtures was compared to radioiron in t he supernatant fluid after centrifugation at 30,000 g for 60 min.
TABLE
Vol. 55, No . 1
7. Effect of ligation of the bile duct upon the absorption of Fe 59 from test doses of FeClz and FeCla in normal and iron -deficient rats Ferrous ch loride
Ligated
Patent Iron deficient
N ormal
Ferric chloride
Normal
Patent
Iron deficient
Normal
Ligated
Iron deficient
Normal
Iron defi cient
%
%
%
%
%
%
%
%
Fe 59 absorbed
4.3 5 .3 8.6 14.2 16.1 17 . 1 17. 6 19.5 20.9 23 .3
13 .4 18.1 19.5 22 .7 23.6 25.0 29 .5 30.9 31.8 37 .6
1.9 1.9 2.2 3.2 3.6 8.9 9.8 15. 9 16. 3
2.6 11.4 14.0 16 .5 17 .4 21.4 24.1 37 .0 41.6 46 .9
2.1 3 .6 3.9 5.7 6 .6 9.5 9.6 10 .8 11.2 14 .2
9 .5 14 .8 19 .8 24.4 26.0 27 . 2 38 .7 40.8 44 .9 45 .8
0.1 0 .1 0.6 0.7 1.1 1.7 2.6 2 .6 3. 9 5.4
10 .8 18 .2 19. 9 21.4 22 .5 22 .6 22.7 24.9 25.8 36 .6
Mea n
14 .6 6 .5 2.1
25.2 7.3 2.3
6 .8 6.2 2.1
23 .3 14 .2 4.5
7.7 4.0 1.3
29 .2 12.7 4.0
1.9 1.7 0.6
22.5 6.5 2.1
SD SE
I
July 1968
ACID CHELATES IN IRON ABSORPTION
chloride, most of the iron hydroxide precipitate is not brought into solution or chelated. In contrast, ferrous chloride and ascorbic acid form violet colored complexes at the alkaline pH of the duodenum. This chelate produces a modest increase in the quantity of low molecular weight iron that remains in solution between pH 7 and 8. Biochemical observations indicate that the chelates formed by adding ascorbic acid to ferrous chloride and ferric chloride are different compounds. These include differences in solubility, infrared spectroscopy, and perhaps chemical composition. The ferric iron may bind to different biochemical sites on the ascorbate in the process of undergoing reduction. The similarity between the sequential requirements for reaction of ferric chloride and ascorbate and the successive conditions encountered by food in the normal gut suggested that it might be physiologically significant. To test this hypothesis, we injected pH-adjusted doses (pH 8) of the iron chelate and precipitate into the duodenum of rats. There was poorer absorption of iron from the insoluble precipitate than from the soluble chelate. If the preparations were injected into the stomach, there was no significant difference in iron absorption because the hydrochloric acid converted the precipitate into a chelate. The biochemical reaction and physiological response of animals to chemical mixtures of ferrous chloride and ascorbic acid were less striking. The ferrous chloride-ascorbate chelate increased the solubility of iron at the slightly alkaline pH of the duodenum. Probably the chelat e increases solubility by interfering with the form ation of macromolecular iron in which one iron molecule binds to others through water bridges. 29 Likewise, it may diminish the effect of precipitating chelates in the diet (phytates, phosphates, carbonates, and oxalates ) or in pancreatic secretions (bicarbonate) upon dietary iron. 29 • 31 • 32 The capability of iron-deficient rats to absorb increased amounts of iron from various iron preparations in the absence of either bile or hydrochloric acid indicated that these intraluminal factors were less important regulators of iron absorption
43
than the body requirement for iron. This would provide an explanation for the slow development of iron deficiency in gastrectomized patients and the relatively mild state of iron deficiency observed in both achlorhydric and gastrectomized subjects. We studied iron-ascorbate complexes because vitamin C is a common ingredient in the diet, a normal constituent of bile, and an effective substance for increasing iron absorption. Similar to sugars and polyols, 33 • 34 ascorbic acid forms soluble chelates with iron which enhance iron absorption. Unlike the sugars and polyols, ascorbate complexes iron in equimolar concentrations. Both the efficient chelation of iron by ascorbic acid and its capability to reduce ferric iron may provide an explanation for its greater effect upon iron absorption. Previous observations indicated that only ferrous iron is absorbed by the intestinal epithelium of rats and man. 18 The studies reported in this article do not permit a different conclusion because ferric iron is reduced by the addition of ascorbic acid. However, our observations in combination with those of Charley et al. suggest that the primary effect of valence upon iron a bsorption is the change in solubility at the alkaline pH of the duodenum. 29 • 3 5 Rodents may absorb more iron from dietary ferric chloride than man because they are not scorbutic animals and they may secrete greater quantities of ascorbate in their bile. 3 G Summary
The chemical reaction of ferric chloride a nd ascorbic acid must be initiated at an acid pH to insure solubility of the iron at the pH of th e duodenum: (FeCI.+ ascorbic acid) +base-> soluble iron chelate (FeCla + base ) +ascorbic acid-> insoluble iron precipitate
In this reaction, iron displaces hydrogen ions from ascorbate to form purple colored complexes that can be isolated by alcoholic precipitation and which hold iron in solution over a wide range of pH (2 to 11). In contrast, FeCb combines with ascorbate at the pH of the duodenum making HCl less
44
CONRAD AND SCHADE
important for iron absorption from this compound. Biochemical differences were found which suggested that FeCb and FeCla ascorbate complexes were different compounds. The similarity between the sequence of the FeC1 3 and ascorbic acid reaction and successive conditions encountered by food in the intact gut suggested that it might be physiologically significant. To test this hypothesis, we introduced pH-adjusted doses of the iron chelate and precipitate into the duodenum of normal rats and administered oral doses to gastrectomized animals. There was poorer absorption of iron from the insoluble precipitate than from the soluble chelate. If the preparations were administered through the stomach, there was no significant difference in iron absorption because acid converted the precipitate into a chelate. Ligation of the bile duct decreased the absorption of iron from test doses of FeC1 3 . This may be caused by the ascorbate content of the bile; in vitro bile dissolves iron from FeC1 3 . These data provide information of the importance of an intact gastrointestinal tract in maintaining iron equilibrium. Gastrectomized patients might become iron deficient because of an inability to utilize ascorbate from the diet or bile to bring dietary ferric iron into solution and make it available for absorption. REFERENCES 1. Mettier, S. R., and G. R. Minot. 1931. The
effect of iron on blood formation as influ enced by changing the acidity of the gastroduod0nal contents in certain cases of anemia. Amer. J. Med . Sci. 181: 25-36. 2. MPttier, S. R., F. Kellogg, and J. F. Rinehart . 1933. Chronic idiopathic hy pochromic anemia. Etiologic relationship of achlorhydria to the anemia, with sper-ial reference to the effect of large doses of iron, organic (dietary) iron and of predigested food upon formation of erythro cytes. Amer. J . Med. Sci. 1S6 : 694-704. 3. K ellogg, F ., and S. R. Mettier. 1936. Effect of alkaline therapy for peptic ulcer on utilization of dietary iron in the regeneration of hemoglobin. Arch. Intern. Med. (Chicago) 58: 278-284. 4. Leroux, R., and E. Vermes. 1939. Anemies agas triques. Sang. 13: 241-267.
Vol. 55, No. 1
5. Lyngar, E. 1950. Blood changes after partial
gastrectomy for ulcer. Acta Med. Scand ., sup pl. 247 : 5-71. 6. Owren, P. A. 1952. The pathogenesis and treatment of iron deficiency anemia after partial gastrectomy. Acta Chir. Scand. 104: 206-214.
7. Baird, I. MeL., D. A. Podmore, and G. M. Wilson. 1957. Changes in iron metabolism following gastrectomy and other surgical operations. Clin. Sci. 16: 463-473 . 8. Stevens, A. R., Jr., G. Pirzio-Biroli, H . N. Harkins, L. M. Nyhus, and C. A. Finch. 1959. Iron metabolism in patients after partial gastrectomy. Ann. Surg. 149: 534538. 9. Choudhury, M . R., and J. Williams. 1959.
10.
11.
12.
13.
Iron absorption and gastric operations. Clin . Sci.18: 527-532. Hines, J. D., A. V. Hoffbrand, and D. L. Mollin. 1967. The hematologic complications following partial gastrectomy. Amer. J . Med. 43: 555-569. Barer, A. P., and W. M. Fowler. 1937. Influence of gastric acidity and degree of anemia on iron retention. Arch. Intern. Med. (Chicago) 59: 785-792. Goldberg, A., A. C. Lockhead, and J. H. Dagg. 1963. Histamine fast achlorhydria and iron absorption. Lancet 1: 848-850 . Cook, J. D., G. M. Brown, and L. S. Valberg. 1964. The effect of achylia gastrica on iron absorption. J. Clin. Invest. 43: 1185-
1191. 14. Dubach, R. S., T. E. Callender, and C. V. Moore. 1948. StudiPs in iron transporta-
tion and metabo lism. VI. Absorption of radioactive iron in patients with fever and with anemias of varied etiology. Blood 3 : 526-540. 15. Calil'nder, S. T., B. J. Mallett, and M. D. Smith. 1957. Absorption of haemoglobin iron. Brit. J. Haemat. 3: 186-192. 16. Willia ms, J. 1959. The effect of ascorbic acid
on iron absorption in postgastrectomy anemia and achlorhydria. Clin . Sci. 18: 521525 . 17. Charlton, R. W., P. J acobs, H. Stefel, and T. H. Bothwell. 1964. Effect of alcohol on iron absorption. Brit. Med. J. 2: 1427-1429. 18. Venkatachalam, P . S., I. Brading, E. P. George, and R. J. Walsh. 1956. An experi-
ment in rats to determine whether iron is absorbed only in the ferrous state. Aust. J. Exp. Biol. Med. Sci. 34: 389--393. 19. Anelli , J. 1958. Combinations of iron and ascorbic acid. Acta Cient. Venez. 9: 105114.
July 1968
ACID CHELATES IN IRON ABSORPTION
20. Forrester, R. H ., M. E . Conrad, Jr., and W. H. Crosby. 1962. Measurement of total body iron 59 in anima ls using whole-body liquid scintillation detectors. Proc . Soc. Exp. Bioi. M ed . 111 : 115-119. 21. M ahin, D. T., and R. T. Lofberg. 1966. A simplified method of sample preparation for determination of tritrium, carbon-14 or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. Biochem. 16: 500-509. 22. Gelotte, B. 1960. Studies on gel filtration sorption properties of the bed material sephadex. J . Chromatogr. 3: 330-342. 23. Stimson, M. M., and M . J. O'Donnell. 1952. The infrared and ultraviolet absorption spec tra of cytosine and isocytosine in the solid state . J. Amer. Chern . Soc. 74: 18051808. 24 . Groen, J., W. A. Van D en Broek, and H. Veldman . 1947. Absorption of iron compounds from the small intestine of the rat. Biochim. Biophys. Acta 1: 315-326. 25. Bockus, H. L. 1965. Gas troenterology, Vol. 3, p. 576-577. W. B. Saunders Company, Philadelphia. 26. Kinney, T. D., C. A. Finch, N. K aufman, D . M . H cgsted, and P. F. Part hington. 1950. The relationship of the pancreas to the absorption of iron. Amer. J. Path. 26: 746. 27. Tonz, 0., S. Weiss, H . W. Strahm, and E. Rossi. 1965. Iron absorption in cystic fibrosis. Lancet 2 : 1096-1099. 28. H awkins. W. B .. F. S. Robschertt-Robbins,
29.
30. 31.
32.
33.
34.
35.
36.
45
and H. Whipple. 1938. H emoglobin production in anem ia as influenced by bile fistula. J . Exp. Med. 67 : 89-110. Benj amin, B. I., S. Cortell, and M. E. Conrad. 1967. Bicarbonate-induced iron complexes and iron absorption : one effect of pancreatic secretions. Gastroenterology 53 : 389-396. Moore, C. V. 1961. Iron metabolism and nutrition. H arvey Lect. 55: 67-101. Chaberek, S., and A. E. M artell. 1959. Organic s2questering agents. A discussion of the chemical behavior and applications of metal chelate compounds in aqueous systems, p. 187-198. John Wiley & Sons, Inc., New York. Bailar, J. C., Jr. 1956. Chemistry of the coordination compounds, p. 114-125. Rheinhold, New York. Charley, P. J ., B. Sarkar, C. F. Stitt, and P . Saltman . 1963. Chelation of iron by sugars. Biochim . Biophy ~ . Acta 69 : 313-321. Pollack, S ., R. M . K aufman, and W . H. Crosby . 1964. Iron absorption: effects of sugars and reducing agents. Blood 24: 577581. Charley, P . J., C. Stitt, E. Shore, and P. Saltman. 1963. Studies in the regulation of intestinal iron absorption. J. Lab. Clin . Med. 61: 397-410 . Farris, :K J., and J . W. Griffith, Jr. 1949. The rat in laborato ry in vestigation , p. 137. J. B. Lippincott Company, Philadelphia.