- METABOLISM OF IRON IN HEMOCHROMATOSIS*

Transcription

1 - METABOLISM OF IRON IN HEMOCHROMATOSIS* MILTON R. BEYERS, M.D., AND STANLEY E. GITLOW, M.D.t From the Department of Internal Medicine, Veterans Administration Kingsbridge Road, Bronx, New York Hospital, Hemochromatosis is characterized by extensive deposition of iron throughout the body, particularly in the liver, pancreas and skin, and by the clinical triad of hepatic cirrhosis, diabetes and pigmentation of the skin. Normally, the total amount of iron in the body, including the blood, varies from.3 to 5 grams, whereas in hemochromatosis the liver alone may contain more than 20 grams. The total iron in the tissues, exclusive of blood, has been found to be over 50 grams in some instances. 2, 6 Sheldon has reported that the iron content of the liver in "hemochromatosis varies from 2.1 to 3.6 Gm. per 100 Gm. of liver tissue as compared with the^normal average of 0.05 Gm. per 100 Gm. 16 Most authors agree and the majority of clinical and experimental studies point to the fact that the deposition of iron antecedes the characteristic widespread fibrosis seen in hemochromatosis. 1 ' 5> 6 ' 10 12, l6, 16T I8, l9 " This does not, however, signify that the pigment per se is the sole cause of the ensuing fibrosis of the organ. It is quite conceivable that excessive deposition of hemosiderin acts as an initial insult allowing other toxic factors, such as vitamin deficiencies, viral hepatitis, and generalized infections to produce the fibrosis. This initial insult may be caused by an inhibition of an essential enzyme system. 13 Assuming that deposition of iron is the factor that induces fibrosis which in turn results in the cirrhosis and diabetes in hemochromatosis, removal of the excess of stored iron shauld be of therapeutic value. Inasmuch as the diet is the " ultimate source of the excess of iron, and because it is generally accepted at the present thj^that the bodily excretion of iron is minimal, it becomes apparent that,there are two major methods by which the iron content of the body may be'.'decreased: one is control of the source of iron in the diet, and the other is withdrawal of blood, the tissue that is rich in iron. Both of these methods have been suggested in the treatment of hemochromatosis. The primary assumption for advocating phlebotomy is that the patient with hemochromatosis is able under an appropriate stimulus to utilize his excessive iron stores for the production of hemoglobin. To the best of our knowledge, this assumption has never been conclusively proven. Finch 4 and Rundles 14 failed to observe the development of anemia following repeated phlebotomies in patients with hemochromatosis who were on normal diets. This would seem to indicate that such * Reviewed in the Veterans Administration and published with the approval of the Chief Medical Director. The statements and conclusions published by the authors are a result of their own studies and do not necessarily reflect the opinion or policy of the Veterans Administration. Received for publication, October 30, f Present address, Department of Pharmacology-Physiology, University of the State of New York, College of Medicine, Brooklyn, New York. 349

2 350 BEYERS AND GITLOW patients are able to utilize their excess of stored iron for the production of hemoglobin. However, without control of the dietary iron, such a conclusion is only presumptive. The utilization of stored iron in hemochromatosis may be quantitatively evaluated by repeatedly removing large quantities of hemoglobin in such a patient, maintaining an iron-free diet and observing the degree of regeneration of hemoglobin. If, on such a regimen, regeneration of hemoglobin occurs, it is a priori evidence that the patient has utilized his iron stores. We were afforded the opportunity of conducting such a study in a young patient with proven hemochromatosis, whose physical condition permitted the rigors of this regimen. METHOD A 27-year-old white man, weighing GO Kg., with hemochromatosis proven by biopsy of liver and skin, was maintained throughout the study, except for an initial seven-day control period, on a diet calculated to provide: (1) a total of 2.5 mg. of iron or less daily; (2) 1400 to 1600 calories a day; and (3) 50 to 60 Gm. of protein daily. This diet was supplemented by the daily oral administration of 10 mg. of thiamine, 25 mg. of nicotinic acid, 5 mg. of riboflavin, and 100 mg. of ascorbic acid, and weekly intramuscular injections of 15 /ig. of crystalline vitamin B 12 (Cobione, Merck and Co., Rahway, N. J.). It was felt that this diet provided all the necessary hemoglobin precursors except adequate amounts of iron. The total iron content of one day's sample diet was found to be 2.7 mg. The patient drank only iron-free distilled water (less than one part iron in a million) and all cooking was done in aluminum utensils using iron-free distilled water. Control values for hemoglobin, erythrocyte count, hematocrit reading and plasma volume were determined during the initial seven-day period. In the next 36 days of the study, 13 phlebotomies were performed, the amount of blood withdrawn varying from 175 to 490 ml. Following an interval of 30 days, an additional 1500 ml. of blood Avas removed from the patient and he was again observed for 18 days. Serial determinations of hemoglobin, erythrocyte count, hematocrit reading, reticulocyte count and plasma A'olume were performed throughout the study. The hemoglobin determinations were performed four to five times a week by the same technician, using a Klett-Summerson photoelectric colorimeter. Two simultaneous determinations on two different fingers for 25 successive days established the average error for this method as ±0.22 grams per 100 ml. The hematocrit reading was obtained simultaneously with the hemoglobin determinations using the technic outlined by Wintrobe. 17 The accepted error for this method is ±0.5 per cent. The Evans blue dye (T1824) method of determining plasma volume was used at intervals of seven to twelve days. Although the dye method for plasma volume determination is not reliable for the calculation of an absolute value, it is, nevertheless, quite accurate in performing serial determinations for comparison in the same person. In this laboratory, consecutive determinations in a large series of

3 IRON METABOLISM IN HEMOCHROMATOSIS 351 patients have shown a maximum error of ±200 ml. The variation between two control values for this patient was 180 ml. The total circulating hemoglobin was calculated by the formula: Plasma volume X hemoglobin (Gm. per 100 ml.) 100 hematocrit reading = total circulating hemoglobin (Gm.) Using this formula and noting the previously listed errors, the maximum total circulating hemoglobin error was calculated as ±70 Gm. One clay's sample diet, which we calculated to contain 2.5 mg. of iron, was analyzed by the method of Farrar 3 and found to contain 2.7 mg. of iron. We consider the difference of 0.2 mg. to be Avithin our range of error since the diet Avas undoubtedly contaminated to a slight degree during the long process of desiccation. RESULTS All pertinent experimental data are presented in graphic form in Figures 1 and 2. For purpose of discussion, the total study is dianded into five periods: Control period (clays 1 through 7), first phlebotomy period (days 8 through 43), first observation period (days 44 through 73), second phlebotomy period (days 74 and 75), and second observation period (clays 76 through 93). /. Control period (days 1 through 7). Successive hemoglobin determinations were 14.8, 14.6, and 14.7 grams per 100 ml.; repeated hematocrit readings gaa r e a value of 42 per cent; and the reticulocyte count was 1.2 per cent. Plasma volume determinations at a two-day interval were 3240 ml. and 3060 ml. Avith a resultant total circulating hemoglobin of 827 Gm. and 786 Gm., averaging 807 Gm. During this period, the patient Avas on a normal diet and his body weight Avas 132 pounds (60 Kg.). Liver function studies including cephalin flocculation, total protein, albuminglobulin ratio, alkaline phosphatase, serum bilirubin, thymol turbidity, and bromsulfalein excretion were within normal limits. Urinary urobilinogen excretion was consistently elevated. The glucose tolerance Avas slightly diminished but the initial fasting blood sugar level was 109 mg. per 100 ml. II. First phlebotomy period (days 8 through 48). At the beginning of this period, the patient was placed on the special iron-deficient diet and this was continued for the remainder of the study. A total of 4190 ml. of blood was withdrawn. This contained a calculated 514 Gm. of hemoglobin, equivalent to 1721 mg. of iron (hemoglobin in Gm. X 3.35 = mg. of iron). 2 The hemoglobin and hematocrit A'alues diminished progressia'ely. The former reached its lowest A'alue of 9.1 Gm. per 100 ml. on the thirty-eighth day and the latter fell to 27 per cent at the end of the period. It may be noted that the hemoglobin A'alue increased from 9.1 to 11.4 Gm. per 100 ml. between the thirtyeighth and forty-third days, during which time the phlebotomies were less frequent. An increased reticulocyte count, 2.3 per cent, was first noted on the eighteenth clay and it steadily rose to 6.2 per cent on the thirty-ninth day.

4 352 BEYERS AND GITLOW The total circulating hemoglobin fell progressively and reached 450 Gm. by the end of this period. Although his general condition remained excellent, the patient's weight decreased to 122 pounds (55.4 Kg.). III. First observation period (days 44 through 73). The hemoglobin concentration continued to fall, reaching 10.8 Gm. per 100 ml. on the forty-sixth day; thereafter it rose progressively to 14.0 Gm. per 100 ml. by the seventy-third day. The hematocrit value increased gradually to 40 per cent by the end of D A Y S FIG. 1. Hematocrit, hemoglobin and reticulocyte values during the study this period. Reticulocyte counts remained elevated until the forty-eighth day and then rapidly fell, reaching 1 per cent on the fifty-fourth day. The total circulating hemoglobin reached the lowest value, 415 Gm. on the forty-eighth day, representing a loss of 392 Gm. when compared with the average control value. Thereafter, the total circulating hemoglobin rose steadily to reach 845 Gm. by the end of this observation period, an increase of 430 Gm. during a 26-day interval. The discrepancy between the calculated loss of 514 Gm. of hemoglobin in the blood removed during the first phlebotomy period, and the observed loss of 392 Gm. of hemoglobin, as determined by the total circulating hemoglobin values, was probably the result of continued hemoglobin production during

5 IRON METABOLISM IN HEMOCHROMATOSIS 353 the phlebotomy period. In the second phlebotomy period, limited to two days, this discrepancy was not noted. Repeated liver function studies gave results which were within normal limits; the fasting blood sugar on the sixtieth day was 123 mg. per 100 ml., but repeated urine examinations for glycosuria were negative. The patient's weight remained 122 pounds (55.4 Kg.). 4CO-l-T-T-1-TTT-r UJ WOO- D A Y S FIG. 2. Plasma volume and total circulating hemoglobin values during the study IV. Second phlebotomy period (days 7J, and 75). A total of 1500 ml. of blood was withdrawn; this contained a calculated 197 Gm. of hemoglobin, equivalent to 661 mg. of iron. V. Second observation period (days 76 through 93). Hemoglobin values fell to a low of 10.0 Gm. per 100 ml. on the eighty-first day. At this time the hematocrit reading was also at its lowest level, 29 per cent. By the end of this period, the hemoglobin had risen to 13.8 Gm. per 100 ml., and the hematocrit reading to 39.5 per cent. The reticulocytes gradually increased to 8.3 per cent on the eightyfourth day following which, they diminished to 1.0 per cent at the end of the period.

6 354 BEYERS AND GITLOW The total circulating hemoglobin diminished to 680 Gm. by the eighty-second day, representing a loss of 165 Gm. of hemoglobin when compared with the value just prior to the second phlebotomy period. It will be noted that the figure of 165 Gm. of hemoglobin, as determined from the total circulating hemoglobin values, compared favorably with the calculated loss of 197 Gm. of hemoglobin in the 1500 ml. of blood removed during the second phlebotomy period. The total circulating hemoglobin rose to reach 939 Gm. by the ninety-third day, an increase of 259 Gm. in a 12-day interval. Immediately after this period, liver function studies were again found to be normal. A second glucose tolerance curve did not differ significantly from the control study. DISCUSSION It has already been noted that in the first phlebotomy period, the calculated loss of 514 Gm. of hemoglobin in the blood withdrawn failed to correlate with the observed loss of 392 Gm. of hemoglobin as determined by comparison of the total circulating hemoglobin values. This discrepancy was obviously caused by the patient's continued synthesis of hemoglobin during the 36-day interval of blood loss. The total circulating hemoglobin value at the end of this phelbotomy period represented the amount of hemoglobin remaining after the phelbotomies plus the amount regenerated during this interval. In the second phelbotomy period, of only two days' duration, the calculated loss of 197 Gm. of hemoglobin in the withdrawn blood more closely approximated the actual 165 Gm. difference between the total circulating hemoglobin vames before and after the phlebotomies. It is, therefore, evident that comparisons of the total circulating hemoglobin values cannot be used in determining the absolute amount of hemoglobin lost or regenerated, unless the blood loss occurred over a short period of time and frequent determinations of the total circulating hemoglobin have been made. The only accurate method for determining the total amount of synthesized hemoglobin is by adding the total calculated hemoglobin in the phlebotomized blood to the difference between the initial and final total circulating hemoglobin levels [total synthesized hemoglobin = total hemoglobin in withdraavn blood + (final total circulating hemoglobin-initial total circulating hemoglobin)]. During the entire study, 711 grams of hemoglobin, equivalent to 2382 milligrams of iron, were removed by phlebotomy. The initial total circulating hemoglobin was 807 grams and the final value 939 grams, an increase of 132 grams. The total synthesized hemoglobin was, therefore, 843 grams ( ). The calculated maximum error for total circulating hemoglobin was ±70 Gm. Assuming this error in both the initial and final total circulating hemoglobin determinations, the maximum error in computing the total synthesized hemoglobin was ±140 Gm. If this error were actually present the minimum total synthesized hemoglobin would have been 703 grams ( ). This value of 703 grams of total synthesized hemoglobin can be divided into three components: (1) that derived from dietary iron; (2) that derived from the

7 IRON METABOLISM IN HEMOCHROMATOSIS 355 iron stores normally available in man; and (3) that derived from iron stores in excess of those found in normal persons. During the 86 days of the special diet, the patient received a total of 215 mg. iron from his food (86 X 2.5 mg. Fe per day). Part of this dietary iron was in an unavailable form; part of the available iron was probably not absorbed; and a portion of the diet offered was not eaten. However, assuming 100 per cent availability, absorption and ingestion, the 215 mg. of dietary iron would have enabled the patient to synthesize 64 Gm. of hemoglobin. The normal total body iron is about 45 mg. per kilogram body weight, of which 20 per cent is stored but available for hemoglobin synthesis. 2 Therefore, a normal person of the patient's weight (60 Kg.) would have 2700 mg. of total iron, of which 540 mg. Avould be available for the production of 161 Gm. of hemoglobin. The third component of the total synthesized hemoglobin, that derived from the iron stores in excess of those found in normal humans, is equal to the difference between the total synthesized hemoglobin and the sum of the first two components listed above. These first two components (dietary iron, and normally available storage iron) could account for 225 grams of the total 703 grams of synthesized hemoglobin. The remaining 478 grams of hemoglobin must have been produced from the excessive iron stores present in this patient. This represents the utilization of 1600 mg. of stored iron in excess of that amount ordinarily found in a person of the patient's weight. We feel that this-isdefinite proof of the physiologic availability of the excess iron stores in this patient with hemochromatosis. It is impossible to determine, on the basis of this study alone, the molecular structure of stored iron which was mobilized for the production of hemoglobin. The storage iron in the patient with hemochromatosis exists as hemosiderin and as a high concentration of structurally normal ferritin, which contains iron of the three unpaired electron type (three unpaired electrons in the outer electron shell per atom of iron). 6, Si 9 Granick has stated that hemosiderin does not yield utilizable iron as readily as ferritin. He implied, however, that hemosiderin iron could be given up to the body for utilization slowly over a long period of time. 7 Accepting this hypothesis, it may be assumed that the iron mobilized during this relatively short study was derived for the most part from ferritin. Apparently the stored iron in patients with hemochromatosis is normal in physiologic function as well as structure. It is the consensus that excessive iron deposition is the primary factor in causing the fibrosis found in hemochromatosis. Having now established in one such patient that this excess iron is physiologically available for hemoglobin regeneration, we suggest that an iron-deficient diet and repeated phlebotomies offer a rational means of therapy. SUMMARY Blood was repeatedly withdrawn from a patient with hemochromatosis while he was on an iron-deficient diet.

8 356 BEYERS AND GIT-LOW By serial studies of the total circulating hemoglobin, it was observed that this patient synthesized hemoglobin far in excess of that which would be expected from normal stores of iron. The physiologic availability of the excessive stores of iron for hemoglobin production was demonstrated in a patient with hemochromatosis. Acknowledgment. We gratefully acknowledge the guidance of Dr. Leo M. Meyer, the assistance of Miss Constance H. Shine in preparing the diet, the technical assistance of Mr. Randolph S. Douglas, Mr. Manuel J. Villazon, Dr. Bernard Klein and Mr. Milton Weissman, and the cooperation of Mrs. Clara J. Hart, librarian. Author'8 note. Since the completion of this study, Davis, W. D., and Arrovvsmith, W. R. (J. Lab. and Clin. Med., 36: , 1950) have reported the therapeutic use of repeated phlebotomies in patients with hemochromatosis and demonstrated histologic evidence of depletion of the iron deposits in the liver. REFERENCES 1. BOYD, W.: A Text-Book of Pathology. Ed. 4. Philadelphia: Lea and Febiger, 1943, p; CANTAROW, A., AND TRUMPER, M.: Clinical Biochemistry. Ed. 4. Philadelphia and London: W. B. Saunders Company, 1949, pp FARRAR, C. E., JR.: Determination of iron in biological materials. J. Biol. Chem., 110: , FINCH, C. A.: Iron metabolism in hemochromatosis. J. Clin. Investigation, 28: , GRAEF, I., NEWMAN, W., GORDON, B., AND OLIVETTI, R.: Observations on exogenous hemochromatosis apparently due to multiple transfusions. Read before the N. Y. Path. Soc, May 25, Proc N. Y. Path. Soc, in press. 6. GRANICK, S.: Iron metabolism and hemochromatosis. N. Y. Acad. Med., 25: , GRANICK, S.: Discussion at the N. Y. Path. Soc, May 25, Proc. N. Y. Path. Soc, in press. 8. GRANICK, S., AND HAHN, P. F.: Ferritin; speed of uptake of iron by the liver and its conversion to ferritin iron. J. Biol. Chem., 155: , GRANICK, S., AND MICHABLIS, L.: The presence of ferritin in the duodenal mucosa and liver in hemochromatosis. Proc. Soc. Exper. Biol, and Med., 66: , HOWARD, C. P., AND MILLS, E. S.: Oxford Medicine Vol. VIII. New York: Oxford University Press, 1949, pp KINNEY, T. D., HEGSTED, D. M., AND FINCH, C. A.: The influence of diet on iron absorption. I. The pathology of iron excess. J. Exper. Med., 90: , MUIRHEAD, E. E., CRASS, G., JONES, F., AND HILL, J. M.: Iron overload (hemosiderosis) aggravated by blood transfusions. Arch. Int. Med., 83: , RACHER, E., AND KRIMSKY, I.: Inhibition of coupled phosphorylation in brain homogenates by ferrous sulfate. J. Biol. Chem., 173: , 1948.' 14. RUNDLES, W.: Personal communication to the authors. 15. SCHWARTZ, S. O., AND BLUMENTHAL, S. A.: Exogenous hemochromatosis resulting from blood transfusions. Blood, 3: , SHELDON, J. H.: Haemochromatosis. New York and London: Oxford University Press, 1935, 382 pp. 17. WINTROBE, M. M.: Clinical Hematology. Ed. 2. Philadelphia: Lea and Febiger, 1946, pp IS. WYATT, J. B., AND GOLDENBURC, H.: Hemosiderosis in refractory anemia. Arch. Int. Med., 83: 67-76, ZELTMACHER, K., AND BEVANS, M.: Aplastic anemia and its association with hemochromatosis. Arch. Int. Med., 75: , 1945.