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UPTAKE OF IRON BY DUODENAL BIOPSY SPECIMENS FROM PATIENTS WITH IRON-DEFICIENCY ANÆMIA AND PRIMARY HÆMOCHROMATOSIS
123 This
study was supported by the U.S. Public Health
absorption.’ Duodenal iron absorption involves uptake by the brush border, membrane transport, intracellular handling, and transfer to the portal blood; in tinal
Service and the
Leukemia Task Force.
Requests for reprints should be addressed to J. M. G., Box 446, Mayo Bldg., Department of Pediatrics, University of Minnesota, 420 Delaware St. S.E., Minneapolis, MN 55455, U.S.A. REFERENCES 1. Krivit, W., Good, R. A. A.M.A.J. Dis. Child. 1959, 97, 137. 2. St. Geme, J. V., Prince, J. T., Burke, B. A., Good, R. A., Krivit, W. New
Engl. J. Med. 1965, 272, 229. 3. Blaese, R. M., Strober, W., Brown, R. S., Waldmann, T. A. Lancet, 1968,
i,1056. 4. Cooper, M.
D., Chase, H. P., Lowman, J. T., Krivit, W., Good, R. A. Am.
J. Med. 1968, 44, 499. 5. Kuramoto, A., Steiner, M., Baldini, M. G. New Engl. J. Med. 1970, 282, 475. 6. Krivit, W., Yunis, E., White, J. G. Pediatrics, Springfield, 1966, 37, 339. 7 Grottum, K. A., Hovig, T., Holmsen, H., Abrahamsen, A. F., Jeremic, M.,
Seys, M. Br. J. Hœmat. 1969, 17, 373 8. Baldini, M.G. Ann. N.Y. Acad. Sci. 1972, 201, 437. 9. Webb, J. L. Enzyme and Metabolic Inhibitors; vol. II, p. 386. New 1966. 10. Pumphrez, A. M. J. biol. Chem. 1962, 237, 2384. 11. Holmsen, H., Setkowsky, C. A., Day, H. J. Biochem. J. 1974, 144, 385. 12. Murer, E. H., Hellan, A. J., Rosenberg, M. C. Scand. J. clin. Invest.
York,
1967,
19, 280. 13. Rozenberg, M. C., Holmsen, H. Biochim. biophys. Acta, 1967, 155, 342. 14. Gerrard, J. M., White, J. G., Rao, G. H. R. Am. J. Path. 1974, 77, 151. 15. MacDonald, H. R. J. exp. Med. 1977, 146, 710. 16. Detwiler, T. C. Biochim. biophys. Acta, 1971, 244, 303. 17. Polmar, S. H. Clin. Hœmat. 1977, 6, 423. 18. Hirschhorn, R. in Progress in Clinical Immunology (edited by R. Schwartz); p. 67. New York, 1977. 19. Osborne, W. R. A., Chen, S. H., Giblett, E. R., Biggar, W. D., Ammann, A. A., Scott, C. R. J. clin. Invest. 1977, 60, 741.
UPTAKE OF IRON BY DUODENAL BIOPSY SPECIMENS FROM PATIENTS WITH IRON-DEFICIENCY ANÆMIA AND PRIMARY
HÆMOCHROMATOSIS T.
T. M. Cox
J. PETERS
Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 0HS
Iron uptake by human duodenal mucosa in vitro was considerably higher in irondeficient patients than in controls. In biopsy specimens taken after treatment with iron, uptake fell to normal levels, although total iron in the mucosa remained low. In iron-overloaded patients with primary hæmochromatosis, there was inappropriately high uptake of iron by the biopsy specimens. Kinetic analysis suggested that this was the result of affinity for iron being increased in a carrier in the enterocyte.
Summary
Introduction IN the absence of a major excretory pathway, iron balance is presumably controlled by regulation of intes-
studied, and the conunknown. In is particular, the mechanism trolling step which is in iron deficiency iron enhanced by absorption and the defect of control in primary haemochromatosis have not yet been established.2,3 To investigate the control of iron absorption, initial rates of iron uptake by duodenal mucosa were determined in biopsy specimens from control subjects, patients with iron-deficiency anaemia, and patients with primary haemochromatosis. man, this process has been little
Methods of ferric iron-59 (59Fe3+) into duodenal biopsy specidetermined as described elsewhere.’ Iron was presented as a chelate of nitrilo-triacetic acid in an oxygenated buffer containing 10 mmol/1 glucose. 57Co-Iabelled cyanocobalamin was used as a non-absorbed marker for extracellular fluid space.’ Uptake of Fe3+ was linear for up to 20 min, whereas the extracellular marker equilibrated within 5 min. To measure initial rates of unidirectional influx, iron uptake from a range of concentrations (18-450 mol/1) was determined after incubation for 5 min. Total iron in the mucosa was measured by atomic absorption spectrophotometry and protein content was determined fluorimetrically.6 Hxmoglobin concentration, mean corpuscular volume (M.c.v.), and serum-iron were measured by routine methods and transferrin was measured immunologically.7 These studies were approved by the local ethical committee.
Uptake
mens was
Results indices and tissue iron concentrations shown in table I. The iron content patients with iron-deficiency ansemia was significantly lower than in that obtained from control subjects. Mucosal concentrations of iron in the 2 patients with severe iron overload were greatly increased, but in the partially treated group they were low, although liver-iron concentrations were still raised. When the Fe3+ concentration of the incubation medium was 450 mol/1, there was a 3-fold increase in uptake of iron by biopsy specimens from patients with iron-deficiency anaemia (p<0.01) (fig. 1). Uptake at 180 mol/1 Fe3+ was also increased. At the higher concentration of 450 mol/1 Fe3+ the uptake of iron by biopsy specimens from patients with haemochromatosis was no different from normal. In control subjects and patients with hxmochromatosis, unidirectional uptake of iron reached saturation point over the concentration range 18-450 mol/1 Fe3+ in the incubation medium (fig. 2). Although uptake at the higher iron concentrations is similar in the 2 groups,
Haematological
for the 3 groups of mucosa from
are
TABLE I—HÆMATOLOGICAL DATA AND TISSUE-IRON CONCENTRATIONS IN PATIENT GROUPS
*Last venesections 34±6 months previously. tp<0.05,
compared with controls.
124 TABLE II—KINETIC CONSTANTS OF
59Fe
UPTAKE BY DUODENAL
BIOPSIES
fig. 1-Iron uptake at Fe3+ concentration of 450 µmol/l by duodenal biopsy specimens from 3 groups.
2-Relation between iron concentration in incubation medium and uptake of S9Fe3+ by duodenal biopsy specimens from controls and patients with primary haemochromatosis.
Fig.
the lower concentrations uptake in patients with hæmochromatosis was significantly greater. The EadieHofstee plotS and linear regression analysis by the method of least squares were used to derive values for the apparent Kt and V max from data obtained by concentration-velocity studies (table n). This kinetic analysis shows a significantly lower apparent Kt for uptake of iron by the mucosa from patients with hæmochromatosis compared with controls, although the Vmax is not different. In iron deficiency there is an increase in V max’ but in this case there is an associated rise in apparent Kt. This may be related to the limiting effect of diffusion across the unstirred layer of medium when high V max conditions are operating.9 at
Discussion An in vitro method was used to determine initial rates of iron influx in human duodenal mucosa. Uptake was demonstrated to be an energy-dependent process4 which reaches saturation point at physiological concentrations
*P<0.05 (Wilcoxon rank sum test) compared with controls. †P<0.01 (Wilcoxon rank sum test) compared with controls. of iron. There was a pronounced increase of uptake in iron-deficiency anaemia, and preliminary studies have shown that this returns to normal after treatment with iron, but not after transfusion with packed cells alone.to Treatment did not alter iron concentrations in the intestinal mucosa, at least in the short-term. In iron deficiency the enhanced uptake of iron is evidently due to an increase in Vmax for influx into the mucosa, suggesting either increased numbers or increased flux of iron-carrier molecules. Thus there is important control of iron absorption at the uptake step across the intestinal brush border. This conclusion was also reached by Acheson and Schultz" who incubated segments of rabbit intestine from normal and iron-deficient animals in a similar in-vitro system to determine initial rates of unidirectional influx of labelled iron. Intestinal absorption of iron is inappropriately high in patients with primary hæmochromatosis.12, 13 Preliminary studies10 have indicated an inverse logarithmic relation between percentage saturation of plasma transferrin and in-vitro iron uptake by duodenal mucosa from controls and patients with iron deficiency. This relation does not hold for patients with primary hsemochromatosis, who showed an inappropriately high uptake of iron by the biopsy specimens despite their high level of transferrin saturation. In addition biopsy specimens from these patients show a strikingly increased avidity for iron at low concentrations. The present experiments suggest that iron overload in primary hæmochromatosis is due to enhanced intestinal uptake. Kinetic analysis indicates an increased affinity of the carrier for inorganic iron, which may reflect a primary structural abnormality in this component. Further studies should elucidate the precise molecular defect. We thank Ms Clare Selden for the atomic absorption analyses and Ms Jean de Luca for typing the manuscript. The support of the Grocers’s Trust and the Medical Research Council is gratefully
acknowledged. Requests for reprints should be addressed to T.J.P. REFERENCES 1. 2.
McCance, R. A., Widdowson, E. M. J. Physiol. 1938, 94, 148. Turnbull, A. Iron in Biochemistry and Medicine (edited by A. Jacobs and M. Worwood); p. 369. London, 1974. 3. Crosby, W. H. Semin. Hœmat. 1977, 14, 135. 4. Cox, T. M., Peters, T. J. Gut 1977, 18, 961A. 5. Schütz, H. B., Reizenstein, P. Am. J. dig. Dis. 1963, 8, 904. 6. Peters, T. J., Batt, R. M., Heath, J. R., Tilleray, J. Biochem. Med. 1976, 15, 145. 7. Van Der Heul, C., van Eijk, H. G., Wiltink, W. F., Leijnse, B. Clinica chim. Acta 1972, 38, 347. 8. Hofstee, B. H. J. Science, 1952, 116, 329. 9. Wilson, F. A., Dietschy, J. M. Biochim. biophys. Acta 1974, 363, 112. 10. Cox, T. M., Peters, T. J. Unpublished. 11. Acheson, L. S., Schultz, S. G. Biochim. biophys. Acta 1972, 255, 479. 12. Alper, T. A., Savage, D. V., Bothwell, T. H. J. Lab. clin. Med. 1951, 37, 665. 13. Williams, R., Manenti, F.,
ii, 78.
Williams, H. S., Pitcher, C. S. Br. med. J. 1966,
study was supported by the U.S. Public Health
absorption.’ Duodenal iron absorption involves uptake by the brush border, membrane transport, intracellular handling, and transfer to the portal blood; in tinal
Service and the
Leukemia Task Force.
Requests for reprints should be addressed to J. M. G., Box 446, Mayo Bldg., Department of Pediatrics, University of Minnesota, 420 Delaware St. S.E., Minneapolis, MN 55455, U.S.A. REFERENCES 1. Krivit, W., Good, R. A. A.M.A.J. Dis. Child. 1959, 97, 137. 2. St. Geme, J. V., Prince, J. T., Burke, B. A., Good, R. A., Krivit, W. New
Engl. J. Med. 1965, 272, 229. 3. Blaese, R. M., Strober, W., Brown, R. S., Waldmann, T. A. Lancet, 1968,
i,1056. 4. Cooper, M.
D., Chase, H. P., Lowman, J. T., Krivit, W., Good, R. A. Am.
J. Med. 1968, 44, 499. 5. Kuramoto, A., Steiner, M., Baldini, M. G. New Engl. J. Med. 1970, 282, 475. 6. Krivit, W., Yunis, E., White, J. G. Pediatrics, Springfield, 1966, 37, 339. 7 Grottum, K. A., Hovig, T., Holmsen, H., Abrahamsen, A. F., Jeremic, M.,
Seys, M. Br. J. Hœmat. 1969, 17, 373 8. Baldini, M.G. Ann. N.Y. Acad. Sci. 1972, 201, 437. 9. Webb, J. L. Enzyme and Metabolic Inhibitors; vol. II, p. 386. New 1966. 10. Pumphrez, A. M. J. biol. Chem. 1962, 237, 2384. 11. Holmsen, H., Setkowsky, C. A., Day, H. J. Biochem. J. 1974, 144, 385. 12. Murer, E. H., Hellan, A. J., Rosenberg, M. C. Scand. J. clin. Invest.
York,
1967,
19, 280. 13. Rozenberg, M. C., Holmsen, H. Biochim. biophys. Acta, 1967, 155, 342. 14. Gerrard, J. M., White, J. G., Rao, G. H. R. Am. J. Path. 1974, 77, 151. 15. MacDonald, H. R. J. exp. Med. 1977, 146, 710. 16. Detwiler, T. C. Biochim. biophys. Acta, 1971, 244, 303. 17. Polmar, S. H. Clin. Hœmat. 1977, 6, 423. 18. Hirschhorn, R. in Progress in Clinical Immunology (edited by R. Schwartz); p. 67. New York, 1977. 19. Osborne, W. R. A., Chen, S. H., Giblett, E. R., Biggar, W. D., Ammann, A. A., Scott, C. R. J. clin. Invest. 1977, 60, 741.
UPTAKE OF IRON BY DUODENAL BIOPSY SPECIMENS FROM PATIENTS WITH IRON-DEFICIENCY ANÆMIA AND PRIMARY
HÆMOCHROMATOSIS T.
T. M. Cox
J. PETERS
Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 0HS
Iron uptake by human duodenal mucosa in vitro was considerably higher in irondeficient patients than in controls. In biopsy specimens taken after treatment with iron, uptake fell to normal levels, although total iron in the mucosa remained low. In iron-overloaded patients with primary hæmochromatosis, there was inappropriately high uptake of iron by the biopsy specimens. Kinetic analysis suggested that this was the result of affinity for iron being increased in a carrier in the enterocyte.
Summary
Introduction IN the absence of a major excretory pathway, iron balance is presumably controlled by regulation of intes-
studied, and the conunknown. In is particular, the mechanism trolling step which is in iron deficiency iron enhanced by absorption and the defect of control in primary haemochromatosis have not yet been established.2,3 To investigate the control of iron absorption, initial rates of iron uptake by duodenal mucosa were determined in biopsy specimens from control subjects, patients with iron-deficiency anaemia, and patients with primary haemochromatosis. man, this process has been little
Methods of ferric iron-59 (59Fe3+) into duodenal biopsy specidetermined as described elsewhere.’ Iron was presented as a chelate of nitrilo-triacetic acid in an oxygenated buffer containing 10 mmol/1 glucose. 57Co-Iabelled cyanocobalamin was used as a non-absorbed marker for extracellular fluid space.’ Uptake of Fe3+ was linear for up to 20 min, whereas the extracellular marker equilibrated within 5 min. To measure initial rates of unidirectional influx, iron uptake from a range of concentrations (18-450 mol/1) was determined after incubation for 5 min. Total iron in the mucosa was measured by atomic absorption spectrophotometry and protein content was determined fluorimetrically.6 Hxmoglobin concentration, mean corpuscular volume (M.c.v.), and serum-iron were measured by routine methods and transferrin was measured immunologically.7 These studies were approved by the local ethical committee.
Uptake
mens was
Results indices and tissue iron concentrations shown in table I. The iron content patients with iron-deficiency ansemia was significantly lower than in that obtained from control subjects. Mucosal concentrations of iron in the 2 patients with severe iron overload were greatly increased, but in the partially treated group they were low, although liver-iron concentrations were still raised. When the Fe3+ concentration of the incubation medium was 450 mol/1, there was a 3-fold increase in uptake of iron by biopsy specimens from patients with iron-deficiency anaemia (p<0.01) (fig. 1). Uptake at 180 mol/1 Fe3+ was also increased. At the higher concentration of 450 mol/1 Fe3+ the uptake of iron by biopsy specimens from patients with haemochromatosis was no different from normal. In control subjects and patients with hxmochromatosis, unidirectional uptake of iron reached saturation point over the concentration range 18-450 mol/1 Fe3+ in the incubation medium (fig. 2). Although uptake at the higher iron concentrations is similar in the 2 groups,
Haematological
for the 3 groups of mucosa from
are
TABLE I—HÆMATOLOGICAL DATA AND TISSUE-IRON CONCENTRATIONS IN PATIENT GROUPS
*Last venesections 34±6 months previously. tp<0.05,
compared with controls.
124 TABLE II—KINETIC CONSTANTS OF
59Fe
UPTAKE BY DUODENAL
BIOPSIES
fig. 1-Iron uptake at Fe3+ concentration of 450 µmol/l by duodenal biopsy specimens from 3 groups.
2-Relation between iron concentration in incubation medium and uptake of S9Fe3+ by duodenal biopsy specimens from controls and patients with primary haemochromatosis.
Fig.
the lower concentrations uptake in patients with hæmochromatosis was significantly greater. The EadieHofstee plotS and linear regression analysis by the method of least squares were used to derive values for the apparent Kt and V max from data obtained by concentration-velocity studies (table n). This kinetic analysis shows a significantly lower apparent Kt for uptake of iron by the mucosa from patients with hæmochromatosis compared with controls, although the Vmax is not different. In iron deficiency there is an increase in V max’ but in this case there is an associated rise in apparent Kt. This may be related to the limiting effect of diffusion across the unstirred layer of medium when high V max conditions are operating.9 at
Discussion An in vitro method was used to determine initial rates of iron influx in human duodenal mucosa. Uptake was demonstrated to be an energy-dependent process4 which reaches saturation point at physiological concentrations
*P<0.05 (Wilcoxon rank sum test) compared with controls. †P<0.01 (Wilcoxon rank sum test) compared with controls. of iron. There was a pronounced increase of uptake in iron-deficiency anaemia, and preliminary studies have shown that this returns to normal after treatment with iron, but not after transfusion with packed cells alone.to Treatment did not alter iron concentrations in the intestinal mucosa, at least in the short-term. In iron deficiency the enhanced uptake of iron is evidently due to an increase in Vmax for influx into the mucosa, suggesting either increased numbers or increased flux of iron-carrier molecules. Thus there is important control of iron absorption at the uptake step across the intestinal brush border. This conclusion was also reached by Acheson and Schultz" who incubated segments of rabbit intestine from normal and iron-deficient animals in a similar in-vitro system to determine initial rates of unidirectional influx of labelled iron. Intestinal absorption of iron is inappropriately high in patients with primary hæmochromatosis.12, 13 Preliminary studies10 have indicated an inverse logarithmic relation between percentage saturation of plasma transferrin and in-vitro iron uptake by duodenal mucosa from controls and patients with iron deficiency. This relation does not hold for patients with primary hsemochromatosis, who showed an inappropriately high uptake of iron by the biopsy specimens despite their high level of transferrin saturation. In addition biopsy specimens from these patients show a strikingly increased avidity for iron at low concentrations. The present experiments suggest that iron overload in primary hæmochromatosis is due to enhanced intestinal uptake. Kinetic analysis indicates an increased affinity of the carrier for inorganic iron, which may reflect a primary structural abnormality in this component. Further studies should elucidate the precise molecular defect. We thank Ms Clare Selden for the atomic absorption analyses and Ms Jean de Luca for typing the manuscript. The support of the Grocers’s Trust and the Medical Research Council is gratefully
acknowledged. Requests for reprints should be addressed to T.J.P. REFERENCES 1. 2.
McCance, R. A., Widdowson, E. M. J. Physiol. 1938, 94, 148. Turnbull, A. Iron in Biochemistry and Medicine (edited by A. Jacobs and M. Worwood); p. 369. London, 1974. 3. Crosby, W. H. Semin. Hœmat. 1977, 14, 135. 4. Cox, T. M., Peters, T. J. Gut 1977, 18, 961A. 5. Schütz, H. B., Reizenstein, P. Am. J. dig. Dis. 1963, 8, 904. 6. Peters, T. J., Batt, R. M., Heath, J. R., Tilleray, J. Biochem. Med. 1976, 15, 145. 7. Van Der Heul, C., van Eijk, H. G., Wiltink, W. F., Leijnse, B. Clinica chim. Acta 1972, 38, 347. 8. Hofstee, B. H. J. Science, 1952, 116, 329. 9. Wilson, F. A., Dietschy, J. M. Biochim. biophys. Acta 1974, 363, 112. 10. Cox, T. M., Peters, T. J. Unpublished. 11. Acheson, L. S., Schultz, S. G. Biochim. biophys. Acta 1972, 255, 479. 12. Alper, T. A., Savage, D. V., Bothwell, T. H. J. Lab. clin. Med. 1951, 37, 665. 13. Williams, R., Manenti, F.,
ii, 78.
Williams, H. S., Pitcher, C. S. Br. med. J. 1966,