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Human Absorption of Hemoglobin-Iron
Vol. 53, No. I Printed in U.S.A.
GASTROENTEROLOGY
Copyright© 1967 by The Williams & Wilkins Co.
HUMAN ABSORPTION OF HEMOGLOBIN -IRON MARCEL E. CoNRAD, M.D., BuRTON I. BENJAMIN, M.D., H . moLD L. WILLIAMs, M.S. AND ARTHUR L. FoY, B.S. Department of Hematology, Walter Reed Army I nsti lute of Research, W asking ton, D.C.
deficient by phlebotomy of 500 ml of blood, 5 days before study .10 Absorption of iron•• from test doses of labeled hemoglobin or heme was measured in a whole-body liquid scintillation detector. The subjects were fasted 16 hr before ingestion of the test dose. Whole-body radioactivity (0.8 Mev-oo) was measured before, 4 hr, and 14 days after each test dose. Standards were prepared for each experiment by measuring a portion of the test dose into a 2.5-liter waterfilled plastic bottle. Net counts were calculated by subtracting background radioactivity. The ratio of net whole-body radioactivity 4 hr after the test dose to that of the reference standard was used as the 100% value. Similar ratios at 14 days were compared to the reference standard to determine the quantity of radionuclide retained by each subject. The percentage retained is equivalent to the quantity absorbed from test doses. 11 Oral doses of labeled hemoglobin and heme were prepared from washed red blood cells of guinea pigs that had received intraperitoneal doses of ferrous"' citrate 7 to 14 days previously. Hemolysates were made by lysis of erythrocytes in distilled water. Heme was prepared by the method of Labbe and Nishida and by dialysis of incubated mixtures of crude trypsin (N.F.) and labeled hemoglobin."'· 13 The small quantities of iron split from heme in dialysates were removed by the addition of 8-hydroxyquinoline to solutions and extracting the nonheme iron with chloroform.13 Oral doses of hemoglobin (0.30 g) and heme (11.5 mg) were made to contain 0.5 ftC of iron•• and approximately 1 mg of iron. Oral doses were administered in 200 ml of distilled water brought to pH 8.0 with N aOH. Solutions in certain studies contained niacin (0.5 M) and 5% ascorbic acid . Three normal subjects and the 3 cirrhotics ingested test doses of hemoglobin hemolysate (0.7 g) labeled with 10 ftC of iron"" and 40 p.c of chromium"'. The iron6'-labeled red blood cells had been incubated with sodium chromate6' before they were washed in saline and lysed
Populations of countries where meat is an important constituent of the diet are usually replete with iron and much of their dietary iron is bound in the porphyrin ring of myoglobin or hemoglobin. Nevertheless, it vvas widely accepted that only inorganic iron was absorbed by the gut and most investigation was limited to study of the absorption of various iron salts. 1 In 1955, ·walsh showed that man absorbed significant quantities of iron from test doses of hemoglobin. 2 Subsequent observations demonstrated that hemoglobin-iron was absorbed more efficiently from food than inorganic iron and that ascorbic acid and iron-binding chelaters failed to affect the absorption of iron from hemoglobin. 3 - 6 Recently, animal experiments supported the hypothesis that hemoglobin-iron was absorbed as an intact iron-porphyrin/· 8 but showed less selective absorption of hemoglobin-iron than was observed in man. 4 • 9 The purpose of our studies was to provide a better understanding of the mechanisms involved in the absorption of hemoglobin-iron by man.
Subjects and Methods Human subjects of this study were normal adult male volunteers and 3 patients with Laennec's cirrhosis. The cirrhotics were selected to obtain subjects with an intestinal venous drainage that bypassed the liver; each patient had undergone portacaval shunt surgery 2 to 5 years before study and recent endoscopic examination showed no recurrence of esophageal varices. Normal subjects were made iron Received January 9, 1967. Accepted January 19, 1967. Address requests for reprints to: Lt. Col. Marcel E. Conrad, MC, Hematology Department, Walter Reed Army Institute of Research, Washington, D. C. 20012. 5
6
CONRAD ET AL.
with distilled water. One and 2 hr after ingestion of the test dose, each volunteer was bled 500 mi. The plasma was separated by centrifugation and treated by a modification of the nonheme iron method of Briickmann and Zondek." Radioactivity in the supernatant fluid and precipitate was measured in a small animal liquid scintillation detector (Packard ARMAC) and compared to standards to which ferrous'" citrate or labeled heme (iron'") was added to nonradioactive plasma. Specimens of duodenum were obtained from each volunteer with small intestinal biopsy capsules.15 The specimens were washed in saline until they contained insignificant quantities of chromium51. Then they were homogenized, t reated with trichloroacetic acid and sodium pyrophosphate; the radioiron in the supernatant fluid and precipitate was measured and the values compared to standards similarly prepared. Jejunal aspirates were obtained through the polyethylene tubing of the biopsy capsules. The percentage of heme split from hemoglobulin was measured by dialysis of intestinal aspirates in a 0.2 M Tris buffer (pH 8.0) containing 2 mg of 8-hydroxyquinoline. N onheme iron was removed from the dialysate by the addition of chloroform. R adioactivity was measured in all fractions.' 3 The molecular size of heme compounds was measured on Sephadex columns.'" Sephadex gel TABLE 1. Percentage of heme and nonheme iron'• in specim ens obtained from man after an oral dose of labeled hemoglobina Duoden~l
Jejunal aspirate
Subjects
biopsy
specimen
Percent-
age radioiron
Heme
Nonheme
Heme
N on-
heme
dialyzed ---
- - --- ---
Normal 1 2 3
19 18 15
16 15 12
3 3 3
46 63 48
54 37 52
Cirrhotic 1 2 3
18 22 17
16 19 15
2 3 2
66 54 43
34 46 57
a Aspirates and biopsy specimens were obtained 90 min after ingesting an oral dose of labeled hemoglobin . Values are expressed as percentage of radioiron in the recovered specimen.
Vol. 53, No.1
filtration media (G-10 and G-25) were expanded in 0.9% NaCl solutions for 48 hr and placed in 10-ml pipettes containing a small piece of glass wool in the tip. The Sephadex was allowed to settle and 4 void volumes of the diluent for heme solutions were passed through the column. Then 0.25 ml of radioactive heme was added to the column and diluent was added to the column. The quantity of heme-iron" collected in the first displacement volume indicated the quantity of heme with a larger molecular size than compounds with a molecular weight of 700 (G-10) or 4000 (G-25). The radioactivity (0 .8 Mev-co) in collected fractions was measured in a well-type crystal scintillation detector (Packard Autogamma Spectrophotometer, Model 410A) and compared to a reference standard containing the quantity of heme-iron placed on the column. Heme was identified by the electrophoretic migration of a benzidine-positive, iron 59 -containing compound with albumin and ,B-globulin but not haptoglobin when added to normal human sera.'7
Results Normal volunteers and cirrhotics with patent portacaval shunts were given oral doses of hemoglobin (0.7 g) labeled with both 10 f-tC of iron 59 and 40 f-tC of chromium51. Jejunal aspirates, obtained 1 Y2 hr later, contained significant quantities of dialyzable heme and small amounts of nonheme iron (2%). Duodenal biopsy specimens obtained 90 min after ingestion of the test dose contained both heme and nonheme iron 59 . Plasma specimens contained only nonheme iron 59 . Similar observations in cirrhotics provided assurance that the absence of labeled heme in plasma specimens was not caused by rapid sequestration in the liver (Table 1). These data suggested that globin was split from hemoglobin within the duodenal lumen and heme was absorbed into the intestinal mucosal cell. However, more iron":. ~ovas absorbed from test doses of hemoglobin than from comparable doses of chemically prepared heme. To ascertain if de crea~ecl absorption of iron 59 from test doses of heme was caused by the chemical form of the porphyrin, we prepared heme by alkaline dialysis (pH 8.0) of incubated mixtures of crude trypsin and hemoglobin
HUMAN ABSORPTION OF HEMOGLOBIN-IRON
July 1967 'fAilLE
2. Percentage of iron 59 absm·bed vaTi01tS heme pTepaTati ons Hemin
chemically prepared
from
IHeme dialysate IHeme dialysate and degraded purified globm
Percentage of radioiron absorbed
1 2 3 4 5
1.7 1.9 2.8 2.9 3.3
24.2 19 .6 18.4 24.7 29.6
2.4 2.0 3.7 4.5 3.5
iVlean
2.5 0 .68 0.30
23 .3 4.47 2.00
3.2 1.01 0.45
SD SE
7
Discussion Previous investigation showed that human subj ects selectively absorbed significant quantities of iron from dietary hemoglobin and suggested that the metalloporphyrin was absorbed into intestinal cells where iron was split from heme and transferred into the plasma (Table 4) .2 - 5 In the duodenal lumen, hemoglobin was degraded to heme, globin degradation products, and small quantities of inorganic iron. The amount of iron split from heme was insufficient to account for the quantity of iron that was absorbed. The capability 3. P eTcentage of iTon 59 absorbed jTom test doses of heme with and without niacin
T ABLE
Percentage of radioiron in eluates from Sephadex columns
G-10 G-25
87.2 68.4
26.1 13.7
Heme and ascorbic !Heme and niacin and
84.0 70.8
acid
ascorbic acid
Percentage of radioiron absorbed
and observed a tenfold increase in absorption of radioiron. Subsequently, from these heme dialysates, hemolysates were purified by dialyzing the incubated mixtures of labeled hemoglobin and trypsin at pH 4.0; this precipitated the heme within the dialysis bag but permitted passage of many of t he globin degradation products. Then the dialysis tubing was immersed in an alkaline solution (pH 8.0) and the heme dialysate was collected. Poor absorption of this purified heme compound suggested that globin degradation products enhanced the absorption of heme-iron (Table
1 2 3 4 5
1.8 1.9 2.6 3.2 4.5
18.4 15.0 29.2 23.8 48.5
Mean
2.8 1.10 0.49
27.0 13.15 5.89
SD SE
Percentage of radioiron in eluates
from Sephadex columns
G-10 G-25
86.4 53.7
I
9.1 3.0
2).
Measurements of the molecular size of heme in chemical preparations and acid purified dialysates showed that most of these compounds had a molecular weight greater than 4000. On the other hand, most of heme dialysates containing globin degradation products were retained on G-10 gel filtration columns, indicating they contained large quantities of monomeric heme. Additional evidence that molecular aggregation of heme affected absorption was obtained by adding niacin to oral test doses of reduced heme. Niacin decreased polymerization and markedly increased the absorption of heme-iron (Table 3).
4. Percentage of iron 59 absoTbed jTom test doses of hemoglobin by fasting human volunteers before and afteT phlebotomy
TABLE
Subject
Normal
Postphlebotomy
1 2 3 4 5 6
15.4 18 .3 21.8 24.7 26.2 26.5
32.1 24.2 41.8 38.7 34.6 39.2
Me an
22.1 4.52 1.88
35.1 6.37 2.65
SD SE
8
Vol. 53, No.1
CONRAD ET AL.
FIG. 1. Schematic diagram of the absorption of hemoglobin-iron: In the duodenum hemoglobin is split into heme and small quantities of iron. Heme enters the intestinal cell as an intact metalloporphyrin. Iron is split from heme within the intestinal cell and absorbed selectively depending upon body requirements for iron.
of intestinal enzymes to split heme from hemoglobin and the isolation of porphyriniron59 but not chromium 51 from duodenal mucosa following oral doses of doubly labeled hemoglobin suggested that hemoglobin-iron enters intestinal cells as heme (fig. I) . Paradoxically, more iron was absorbed from test doses of hemoglobin than from comparable quantities of chemically prepared heme. To ascertain if this was caused by t he chemical form of this heme, we prepared heme dialysates from incubated mixtures of labeled hemoglobin and crude trypsin. Radioiron was poorly absorbed from purified acid-washed dialysates but was absorbed in large quantities from alkaline dialysates contaminated with globin degradation products. We believe that the degraded globin increased absorption by binding the coordinating bonds of heme and preventing polymerization. 1s, 19 Measurements of the molecular size of the heme in each of the test solutions support this hypothesis. Likewise, the addition of niacin to test doses of heme decreased polymeric aggregation of heme and increased the absorption of heme-iron. This
amide prevented polymerization by binding the coordinating bonds of heme to form a monomeric ferrohemochrome (fig. 2) _18 Thus polymerization provided an explanation for the effects of many dietary constituents and intestinal secretions upon the absorption of heme-iron. 13 · 20 • 21 Our data indicate that iron was split from heme within intestinal cells before it was transferred into the plasma. In contrast, laboratory animals absorb heme into their plasma. 7 - 9· 13 This species difference suggested an explanation for the selective absorption of hemoglobin-iron by man and the relatively unselective absorption by experimental animals. Heme was not recognized as iron and the quantity absorbed by animals was independent of body requirements for iron. 9 Conversely, the iron split from heme in human intestinal cells was recognized by the body as iron and was accepted or rejected depending upon need. Summary
Hemoglobin can be an important source of dietary iron. Hemoglobin-iron is absorbed by human subjects selectively depending upon body requirements for iron.
H UMAN ABSORPTION OF HEMOGLOBIN -IRON
July 1967
v
M
HC
CH
\i
M
_)====qM
N- - F e - - N
!\
HC
'?=====l v
CH
M
FIG . 2. The chemical structure of heme shows that iron has a coordinating valence of six. Of these, four bonds lie in one plane and link iron to the nitrogens of the pyrole rings. The remaining two bonds lie on either side of the heme molecul e and bind to oxygen or histidine when the metalloporphyrin is incorporated into hemoglobin.'' In heme solutions, the two bonds are free to coordinate with other heme molecules through oxide or water bridges to form large molecular weight polymers. The addition of many substances, such as amides, amines, cyanide or carbon monoxide, to heme solutions disrupts the polymeric bridges and binds the coordinating bonds to form hemochromes with a smaller molecula r size .' 8 • 19 " 23 " 24
Heme is split from globin within the intestinal lumen and absorbed into the duodenal mucosa as an intact metalloporphyrin. Iron is split from heme within intestinal cells and transferred into the plasma . Substances which decrease the polymeriz ation of heme increase the absorption of heme-iron. A comparison of human and animal studies suggests that the selective absorption of heme-iron is regulated either by controlled release of iron from heme within intestinal cells or by regulation of the quantity of iron transferred from intestinal cells into the plasma or both. REFERENCES 1. Granick, S. 1954. Iron metabolism. Bull. N . Y. Acad . M ed. 30: 81-105.
9
2. Walsh, R. J., I. K aldor, I. Brading, and E. P. George. 1955. Th e availabili ty of iron in meat: Some experiments with radioactive iron. Aust. Ann . Med . 4: 272-276. 3. Callender, S. T ., B. J . Mallett, and M. D . Smith. 1957. Abso rpLion of haemoglobin iron. Brit. J. H aemat. 3: 186-192 . 4. Turnbull, A., F. Cleton, and C. A. Finch. 1962. Iron absorption. IV. The absorp tion of hemoglobin iron. J . Clin. Invest. 41: 1897- 1907. 5. H allberg, L ., and L. Solve!!. 1964. Absorption of hemoglobin iron in man. Proceedings of the Xth Congress of the International Society of Haematology. Stockholm , F5. 6. Hwang, Y ., and E . Brown. 1963. Studies of the effect of desferrioxamine on human iron absorption and excretion. J. Lab. Clin. Med . 62: 885 . 7. Brown, E. B., Y-F. Hwang, and S. Nicol. 1966. Absorption of hemoglobin iron. Clin. Res. 24 : 312. 8. Conrad, M. E., S. Cortell, and L. R. Weintraub . 1966. Absorption of hemoglobin-iron: In traluminal factors and the effect of polymerization. Clin . Res. 24 : 294. 9. Conrad, M. E., L . R. Weintraub, D . A. Sears, and W. H . Crosby. 1966. Absorption of hemoglobin iron. Amer. J. Physiol. 211: 1123-1130. 10. Weintraub, L. R., M . E. Conrad, and W. H. Crosby. 1964. The significance of iron turnover in th e control of iron absorption . Blood 24 : 19-24. 11. van Hoek, R., and M. E. Conrad. 1961. Iron absorption . Measurement of ingested iron'" by a human whole-body liquid scintillation counter. J. Clin. Invest. 40: 1153-1159. 12. Labbe, R . D ., and G. Nishida. 1957. A new method of hemin isolation. Biochim. Biophys. Acta 26 : 437. 13. Conrad, M. E ., S. Cortell, H. L. Williams, and A. L. Foy. 1966. Polymerization and in traluminal factors in the absorption of hemoglobin-iron. J. Lab. Clin. Med. 68 : 659-668. 14. Briickmann, G., and S. G. Zondek. 1940. An improved method for the determination of nonh emin iron. J. Bioi. Chem. 135 : 23-30. 15. Crosby, W. H ., and H . W. Kugler. 1957. Intraluminal biopsy of the small intestine Amer. J. Digest. Dis. 2: 236-241. 16. Gelotte, B. 1960. Studies on gel filtration on the sorption properties of the bed material sephadex. J . Chromatogr. 3 : 330-342. 17. Nyman, M . 1960. On plasma proteins with heme or hemoglobin binding capacity. Scand. J. Clin. Lab. Invest. 12 : 121-130.
10
CONRAD ET AL.
18. Akoyunoglou, J . H., H . S. Olcott, and W. D . Brown. 1963. Ferrihemochrome and ferro-
hemochrome formation with amino acids, amino acid esters, pyridine derivatives and related compounds. Biochemistry (Wash .) 2 : 1033-1041. 19. Shack, J., and W . M. Clark. 1947. Metalloporphyrins. VI. Cycles of changes in systems containing heme. J. Bioi. Chern. 171: 143-187. 20. Davis, P. S., D. J. D eller, and C . G. Luke. 1966. Iron chelating ability of human gastric juice, p. 164. Abstract XIth Congress
of the In ternational Society of H aematology. Sydney. 21. Waxman, S., P. Pratt, J. Cuttner, and V. Herbert. 1966. Evidence suggesting facilitated absorption in man of organic (and inorganic) iron by a substance present in
Vol . 53 , No. 1
depepsinized neutralized norma l human gastric juice and in hog intrinsic facto r concentrates, p. 53 . Grune and Stratton, Inc., New York. Abstract IXth Annual M eeting of the American Society of H ematology . New Orleans. 22. Wyman, J ., Jr. 1948. Heme proteins, p . 407-531. In M. L . Anson, and J . T . Edsall [eels.], Advances in protein chemistry, IV. Academic Press, I nc., New York . 23. Lemberg, R., and J. vV. Legge. 1949. Hematin compounds and bile pigments, p. 177-201. Interscienc e Publishers, Inc., New York. 24. Kaziro, K., and K. T sushima. 1961. Modification of t he secondary structure of haemprotein molecules, p. 80-97. In J. E. Falk, R. Lemberg, and R. K. Morton, [eels.], Haematin enzymes, P art I. Oxford University Press, London.
GASTROENTEROLOGY
Copyright© 1967 by The Williams & Wilkins Co.
HUMAN ABSORPTION OF HEMOGLOBIN -IRON MARCEL E. CoNRAD, M.D., BuRTON I. BENJAMIN, M.D., H . moLD L. WILLIAMs, M.S. AND ARTHUR L. FoY, B.S. Department of Hematology, Walter Reed Army I nsti lute of Research, W asking ton, D.C.
deficient by phlebotomy of 500 ml of blood, 5 days before study .10 Absorption of iron•• from test doses of labeled hemoglobin or heme was measured in a whole-body liquid scintillation detector. The subjects were fasted 16 hr before ingestion of the test dose. Whole-body radioactivity (0.8 Mev-oo) was measured before, 4 hr, and 14 days after each test dose. Standards were prepared for each experiment by measuring a portion of the test dose into a 2.5-liter waterfilled plastic bottle. Net counts were calculated by subtracting background radioactivity. The ratio of net whole-body radioactivity 4 hr after the test dose to that of the reference standard was used as the 100% value. Similar ratios at 14 days were compared to the reference standard to determine the quantity of radionuclide retained by each subject. The percentage retained is equivalent to the quantity absorbed from test doses. 11 Oral doses of labeled hemoglobin and heme were prepared from washed red blood cells of guinea pigs that had received intraperitoneal doses of ferrous"' citrate 7 to 14 days previously. Hemolysates were made by lysis of erythrocytes in distilled water. Heme was prepared by the method of Labbe and Nishida and by dialysis of incubated mixtures of crude trypsin (N.F.) and labeled hemoglobin."'· 13 The small quantities of iron split from heme in dialysates were removed by the addition of 8-hydroxyquinoline to solutions and extracting the nonheme iron with chloroform.13 Oral doses of hemoglobin (0.30 g) and heme (11.5 mg) were made to contain 0.5 ftC of iron•• and approximately 1 mg of iron. Oral doses were administered in 200 ml of distilled water brought to pH 8.0 with N aOH. Solutions in certain studies contained niacin (0.5 M) and 5% ascorbic acid . Three normal subjects and the 3 cirrhotics ingested test doses of hemoglobin hemolysate (0.7 g) labeled with 10 ftC of iron"" and 40 p.c of chromium"'. The iron6'-labeled red blood cells had been incubated with sodium chromate6' before they were washed in saline and lysed
Populations of countries where meat is an important constituent of the diet are usually replete with iron and much of their dietary iron is bound in the porphyrin ring of myoglobin or hemoglobin. Nevertheless, it vvas widely accepted that only inorganic iron was absorbed by the gut and most investigation was limited to study of the absorption of various iron salts. 1 In 1955, ·walsh showed that man absorbed significant quantities of iron from test doses of hemoglobin. 2 Subsequent observations demonstrated that hemoglobin-iron was absorbed more efficiently from food than inorganic iron and that ascorbic acid and iron-binding chelaters failed to affect the absorption of iron from hemoglobin. 3 - 6 Recently, animal experiments supported the hypothesis that hemoglobin-iron was absorbed as an intact iron-porphyrin/· 8 but showed less selective absorption of hemoglobin-iron than was observed in man. 4 • 9 The purpose of our studies was to provide a better understanding of the mechanisms involved in the absorption of hemoglobin-iron by man.
Subjects and Methods Human subjects of this study were normal adult male volunteers and 3 patients with Laennec's cirrhosis. The cirrhotics were selected to obtain subjects with an intestinal venous drainage that bypassed the liver; each patient had undergone portacaval shunt surgery 2 to 5 years before study and recent endoscopic examination showed no recurrence of esophageal varices. Normal subjects were made iron Received January 9, 1967. Accepted January 19, 1967. Address requests for reprints to: Lt. Col. Marcel E. Conrad, MC, Hematology Department, Walter Reed Army Institute of Research, Washington, D. C. 20012. 5
6
CONRAD ET AL.
with distilled water. One and 2 hr after ingestion of the test dose, each volunteer was bled 500 mi. The plasma was separated by centrifugation and treated by a modification of the nonheme iron method of Briickmann and Zondek." Radioactivity in the supernatant fluid and precipitate was measured in a small animal liquid scintillation detector (Packard ARMAC) and compared to standards to which ferrous'" citrate or labeled heme (iron'") was added to nonradioactive plasma. Specimens of duodenum were obtained from each volunteer with small intestinal biopsy capsules.15 The specimens were washed in saline until they contained insignificant quantities of chromium51. Then they were homogenized, t reated with trichloroacetic acid and sodium pyrophosphate; the radioiron in the supernatant fluid and precipitate was measured and the values compared to standards similarly prepared. Jejunal aspirates were obtained through the polyethylene tubing of the biopsy capsules. The percentage of heme split from hemoglobulin was measured by dialysis of intestinal aspirates in a 0.2 M Tris buffer (pH 8.0) containing 2 mg of 8-hydroxyquinoline. N onheme iron was removed from the dialysate by the addition of chloroform. R adioactivity was measured in all fractions.' 3 The molecular size of heme compounds was measured on Sephadex columns.'" Sephadex gel TABLE 1. Percentage of heme and nonheme iron'• in specim ens obtained from man after an oral dose of labeled hemoglobina Duoden~l
Jejunal aspirate
Subjects
biopsy
specimen
Percent-
age radioiron
Heme
Nonheme
Heme
N on-
heme
dialyzed ---
- - --- ---
Normal 1 2 3
19 18 15
16 15 12
3 3 3
46 63 48
54 37 52
Cirrhotic 1 2 3
18 22 17
16 19 15
2 3 2
66 54 43
34 46 57
a Aspirates and biopsy specimens were obtained 90 min after ingesting an oral dose of labeled hemoglobin . Values are expressed as percentage of radioiron in the recovered specimen.
Vol. 53, No.1
filtration media (G-10 and G-25) were expanded in 0.9% NaCl solutions for 48 hr and placed in 10-ml pipettes containing a small piece of glass wool in the tip. The Sephadex was allowed to settle and 4 void volumes of the diluent for heme solutions were passed through the column. Then 0.25 ml of radioactive heme was added to the column and diluent was added to the column. The quantity of heme-iron" collected in the first displacement volume indicated the quantity of heme with a larger molecular size than compounds with a molecular weight of 700 (G-10) or 4000 (G-25). The radioactivity (0 .8 Mev-co) in collected fractions was measured in a well-type crystal scintillation detector (Packard Autogamma Spectrophotometer, Model 410A) and compared to a reference standard containing the quantity of heme-iron placed on the column. Heme was identified by the electrophoretic migration of a benzidine-positive, iron 59 -containing compound with albumin and ,B-globulin but not haptoglobin when added to normal human sera.'7
Results Normal volunteers and cirrhotics with patent portacaval shunts were given oral doses of hemoglobin (0.7 g) labeled with both 10 f-tC of iron 59 and 40 f-tC of chromium51. Jejunal aspirates, obtained 1 Y2 hr later, contained significant quantities of dialyzable heme and small amounts of nonheme iron (2%). Duodenal biopsy specimens obtained 90 min after ingestion of the test dose contained both heme and nonheme iron 59 . Plasma specimens contained only nonheme iron 59 . Similar observations in cirrhotics provided assurance that the absence of labeled heme in plasma specimens was not caused by rapid sequestration in the liver (Table 1). These data suggested that globin was split from hemoglobin within the duodenal lumen and heme was absorbed into the intestinal mucosal cell. However, more iron":. ~ovas absorbed from test doses of hemoglobin than from comparable doses of chemically prepared heme. To ascertain if de crea~ecl absorption of iron 59 from test doses of heme was caused by the chemical form of the porphyrin, we prepared heme by alkaline dialysis (pH 8.0) of incubated mixtures of crude trypsin and hemoglobin
HUMAN ABSORPTION OF HEMOGLOBIN-IRON
July 1967 'fAilLE
2. Percentage of iron 59 absm·bed vaTi01tS heme pTepaTati ons Hemin
chemically prepared
from
IHeme dialysate IHeme dialysate and degraded purified globm
Percentage of radioiron absorbed
1 2 3 4 5
1.7 1.9 2.8 2.9 3.3
24.2 19 .6 18.4 24.7 29.6
2.4 2.0 3.7 4.5 3.5
iVlean
2.5 0 .68 0.30
23 .3 4.47 2.00
3.2 1.01 0.45
SD SE
7
Discussion Previous investigation showed that human subj ects selectively absorbed significant quantities of iron from dietary hemoglobin and suggested that the metalloporphyrin was absorbed into intestinal cells where iron was split from heme and transferred into the plasma (Table 4) .2 - 5 In the duodenal lumen, hemoglobin was degraded to heme, globin degradation products, and small quantities of inorganic iron. The amount of iron split from heme was insufficient to account for the quantity of iron that was absorbed. The capability 3. P eTcentage of iTon 59 absorbed jTom test doses of heme with and without niacin
T ABLE
Percentage of radioiron in eluates from Sephadex columns
G-10 G-25
87.2 68.4
26.1 13.7
Heme and ascorbic !Heme and niacin and
84.0 70.8
acid
ascorbic acid
Percentage of radioiron absorbed
and observed a tenfold increase in absorption of radioiron. Subsequently, from these heme dialysates, hemolysates were purified by dialyzing the incubated mixtures of labeled hemoglobin and trypsin at pH 4.0; this precipitated the heme within the dialysis bag but permitted passage of many of t he globin degradation products. Then the dialysis tubing was immersed in an alkaline solution (pH 8.0) and the heme dialysate was collected. Poor absorption of this purified heme compound suggested that globin degradation products enhanced the absorption of heme-iron (Table
1 2 3 4 5
1.8 1.9 2.6 3.2 4.5
18.4 15.0 29.2 23.8 48.5
Mean
2.8 1.10 0.49
27.0 13.15 5.89
SD SE
Percentage of radioiron in eluates
from Sephadex columns
G-10 G-25
86.4 53.7
I
9.1 3.0
2).
Measurements of the molecular size of heme in chemical preparations and acid purified dialysates showed that most of these compounds had a molecular weight greater than 4000. On the other hand, most of heme dialysates containing globin degradation products were retained on G-10 gel filtration columns, indicating they contained large quantities of monomeric heme. Additional evidence that molecular aggregation of heme affected absorption was obtained by adding niacin to oral test doses of reduced heme. Niacin decreased polymerization and markedly increased the absorption of heme-iron (Table 3).
4. Percentage of iron 59 absoTbed jTom test doses of hemoglobin by fasting human volunteers before and afteT phlebotomy
TABLE
Subject
Normal
Postphlebotomy
1 2 3 4 5 6
15.4 18 .3 21.8 24.7 26.2 26.5
32.1 24.2 41.8 38.7 34.6 39.2
Me an
22.1 4.52 1.88
35.1 6.37 2.65
SD SE
8
Vol. 53, No.1
CONRAD ET AL.
FIG. 1. Schematic diagram of the absorption of hemoglobin-iron: In the duodenum hemoglobin is split into heme and small quantities of iron. Heme enters the intestinal cell as an intact metalloporphyrin. Iron is split from heme within the intestinal cell and absorbed selectively depending upon body requirements for iron.
of intestinal enzymes to split heme from hemoglobin and the isolation of porphyriniron59 but not chromium 51 from duodenal mucosa following oral doses of doubly labeled hemoglobin suggested that hemoglobin-iron enters intestinal cells as heme (fig. I) . Paradoxically, more iron was absorbed from test doses of hemoglobin than from comparable quantities of chemically prepared heme. To ascertain if this was caused by t he chemical form of this heme, we prepared heme dialysates from incubated mixtures of labeled hemoglobin and crude trypsin. Radioiron was poorly absorbed from purified acid-washed dialysates but was absorbed in large quantities from alkaline dialysates contaminated with globin degradation products. We believe that the degraded globin increased absorption by binding the coordinating bonds of heme and preventing polymerization. 1s, 19 Measurements of the molecular size of the heme in each of the test solutions support this hypothesis. Likewise, the addition of niacin to test doses of heme decreased polymeric aggregation of heme and increased the absorption of heme-iron. This
amide prevented polymerization by binding the coordinating bonds of heme to form a monomeric ferrohemochrome (fig. 2) _18 Thus polymerization provided an explanation for the effects of many dietary constituents and intestinal secretions upon the absorption of heme-iron. 13 · 20 • 21 Our data indicate that iron was split from heme within intestinal cells before it was transferred into the plasma. In contrast, laboratory animals absorb heme into their plasma. 7 - 9· 13 This species difference suggested an explanation for the selective absorption of hemoglobin-iron by man and the relatively unselective absorption by experimental animals. Heme was not recognized as iron and the quantity absorbed by animals was independent of body requirements for iron. 9 Conversely, the iron split from heme in human intestinal cells was recognized by the body as iron and was accepted or rejected depending upon need. Summary
Hemoglobin can be an important source of dietary iron. Hemoglobin-iron is absorbed by human subjects selectively depending upon body requirements for iron.
H UMAN ABSORPTION OF HEMOGLOBIN -IRON
July 1967
v
M
HC
CH
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FIG . 2. The chemical structure of heme shows that iron has a coordinating valence of six. Of these, four bonds lie in one plane and link iron to the nitrogens of the pyrole rings. The remaining two bonds lie on either side of the heme molecul e and bind to oxygen or histidine when the metalloporphyrin is incorporated into hemoglobin.'' In heme solutions, the two bonds are free to coordinate with other heme molecules through oxide or water bridges to form large molecular weight polymers. The addition of many substances, such as amides, amines, cyanide or carbon monoxide, to heme solutions disrupts the polymeric bridges and binds the coordinating bonds to form hemochromes with a smaller molecula r size .' 8 • 19 " 23 " 24
Heme is split from globin within the intestinal lumen and absorbed into the duodenal mucosa as an intact metalloporphyrin. Iron is split from heme within intestinal cells and transferred into the plasma . Substances which decrease the polymeriz ation of heme increase the absorption of heme-iron. A comparison of human and animal studies suggests that the selective absorption of heme-iron is regulated either by controlled release of iron from heme within intestinal cells or by regulation of the quantity of iron transferred from intestinal cells into the plasma or both. REFERENCES 1. Granick, S. 1954. Iron metabolism. Bull. N . Y. Acad . M ed. 30: 81-105.
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2. Walsh, R. J., I. K aldor, I. Brading, and E. P. George. 1955. Th e availabili ty of iron in meat: Some experiments with radioactive iron. Aust. Ann . Med . 4: 272-276. 3. Callender, S. T ., B. J . Mallett, and M. D . Smith. 1957. Abso rpLion of haemoglobin iron. Brit. J. H aemat. 3: 186-192 . 4. Turnbull, A., F. Cleton, and C. A. Finch. 1962. Iron absorption. IV. The absorp tion of hemoglobin iron. J . Clin. Invest. 41: 1897- 1907. 5. H allberg, L ., and L. Solve!!. 1964. Absorption of hemoglobin iron in man. Proceedings of the Xth Congress of the International Society of Haematology. Stockholm , F5. 6. Hwang, Y ., and E . Brown. 1963. Studies of the effect of desferrioxamine on human iron absorption and excretion. J. Lab. Clin. Med . 62: 885 . 7. Brown, E. B., Y-F. Hwang, and S. Nicol. 1966. Absorption of hemoglobin iron. Clin. Res. 24 : 312. 8. Conrad, M. E., S. Cortell, and L. R. Weintraub . 1966. Absorption of hemoglobin-iron: In traluminal factors and the effect of polymerization. Clin . Res. 24 : 294. 9. Conrad, M. E., L . R. Weintraub, D . A. Sears, and W. H . Crosby. 1966. Absorption of hemoglobin iron. Amer. J. Physiol. 211: 1123-1130. 10. Weintraub, L. R., M . E. Conrad, and W. H. Crosby. 1964. The significance of iron turnover in th e control of iron absorption . Blood 24 : 19-24. 11. van Hoek, R., and M. E. Conrad. 1961. Iron absorption . Measurement of ingested iron'" by a human whole-body liquid scintillation counter. J. Clin. Invest. 40: 1153-1159. 12. Labbe, R . D ., and G. Nishida. 1957. A new method of hemin isolation. Biochim. Biophys. Acta 26 : 437. 13. Conrad, M. E ., S. Cortell, H. L. Williams, and A. L. Foy. 1966. Polymerization and in traluminal factors in the absorption of hemoglobin-iron. J. Lab. Clin. Med. 68 : 659-668. 14. Briickmann, G., and S. G. Zondek. 1940. An improved method for the determination of nonh emin iron. J. Bioi. Chem. 135 : 23-30. 15. Crosby, W. H ., and H . W. Kugler. 1957. Intraluminal biopsy of the small intestine Amer. J. Digest. Dis. 2: 236-241. 16. Gelotte, B. 1960. Studies on gel filtration on the sorption properties of the bed material sephadex. J . Chromatogr. 3 : 330-342. 17. Nyman, M . 1960. On plasma proteins with heme or hemoglobin binding capacity. Scand. J. Clin. Lab. Invest. 12 : 121-130.
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CONRAD ET AL.
18. Akoyunoglou, J . H., H . S. Olcott, and W. D . Brown. 1963. Ferrihemochrome and ferro-
hemochrome formation with amino acids, amino acid esters, pyridine derivatives and related compounds. Biochemistry (Wash .) 2 : 1033-1041. 19. Shack, J., and W . M. Clark. 1947. Metalloporphyrins. VI. Cycles of changes in systems containing heme. J. Bioi. Chern. 171: 143-187. 20. Davis, P. S., D. J. D eller, and C . G. Luke. 1966. Iron chelating ability of human gastric juice, p. 164. Abstract XIth Congress
of the In ternational Society of H aematology. Sydney. 21. Waxman, S., P. Pratt, J. Cuttner, and V. Herbert. 1966. Evidence suggesting facilitated absorption in man of organic (and inorganic) iron by a substance present in
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depepsinized neutralized norma l human gastric juice and in hog intrinsic facto r concentrates, p. 53 . Grune and Stratton, Inc., New York. Abstract IXth Annual M eeting of the American Society of H ematology . New Orleans. 22. Wyman, J ., Jr. 1948. Heme proteins, p . 407-531. In M. L . Anson, and J . T . Edsall [eels.], Advances in protein chemistry, IV. Academic Press, I nc., New York . 23. Lemberg, R., and J. vV. Legge. 1949. Hematin compounds and bile pigments, p. 177-201. Interscienc e Publishers, Inc., New York. 24. Kaziro, K., and K. T sushima. 1961. Modification of t he secondary structure of haemprotein molecules, p. 80-97. In J. E. Falk, R. Lemberg, and R. K. Morton, [eels.], Haematin enzymes, P art I. Oxford University Press, London.