American Journal of Hematology 7:69-76 (1979)
Iron Chelation Therapy in Sickle Cell Anemia Alan Cohen and Elias Schwartz Department of Pediatrics, University of Pennsylvania School of Medicine and The Children’s Hospital of Philadelphia
Iron stores, ascorbic acid levels, and urinary excretion of iron in response t o deferoxamine (DFO) were studied in nine patients with sickle cell anemia who had received 8-55 liters of red blood cells. Ferritin levels ranged from 2,168 to 6,300 ng/ml and were elevated even in patients who had normal serum iron concentrations. Leukocyte ascorbic acid levels were low in three patients. Urinary iron excretion in response t o 2.0 gm DFO administered as a 12-hour subcutaneous infusion after ascorbic acid supplementation was 13-39 mg/24 hours. With this method of DFO therapy daily, urinary iron excretion would exceed transfusional iron accumulation in eight of nine patients. Following the intramuscular injection of 0.75 gm DFO, urinary iron excretion was 5-16 mg/24 hours. Iron excretion would exceed iron accumulation in only one of nine patients with this method of DFO therapy. Urinary iron excretion in response t o 1.5 gm DFO infused intravenously in 18 hours after ascorbic acid supplementation was l 1-38 mg/24 hours. In six regularly transfused but nonchelated patients iron excretion in response to intravenous DFO had increased 46-107%, in comparison with previous studies 15-22 months before. These data indicate that negative iron balance can be obtained in regularly transfused patients with sickle cell anemia by the use of overnight subcutaneous infusions of DFO. Urinary iron excretion in response t o DFO should be evaluated periodically in appropriate patients with sickle cell anemia t o determine the proper time for the institution of chelation therapy. Key words: deferoxamine, iron chelation, sickle cell
I NT RO DUCT1ON
Iron stores may be markedly increased in patients with sickle cell anemia who are frequently transfused [ 1-31. Although reports of organ dysfunction due t o iron overload in these patients have been infrequent, cardiac abnormalities due t o iron toxicity have been described [4]. In the absence of any major physiologic means of eliminating excessive iron, one can anticipate progressive hemochromatosis and early death in those patients with sickle cell anemia who continue t o receive red cell transfusions. Received for publication February 28, 1979; accepted May 23. 1979. Address reprint requests to Dr. Alan Cohen, Division of Hematology, The Children’s Hospital of Philadelphia, 34th Street and Civic Center Blvd., Philadelphia, PA 19104.
0361-8609/79/0701-0069$01.700 1979 Alan R. Liss, Inc.
70
Cohen and Schwartz
Recent clinical trials of the iron chelating agent deferoxamine (DFO) have demonstrated that negative iron balance (urinary iron excretion in excess of iron loading by transfusion) can be achieved in patients with transfusion-dependent anemia [5-lo]. Long-term studies are underway to evaluate the effects of chelation therapy on organ function and patient survival. If the accumulation of iron could be retarded or prevented in patients with sickle cell anemia, red cell transfusions would be a practical method of managing many of the serious complications of t h s disease. In a previous study we demonstrated increased excretion of iron in the urine in response to intravenous or intramuscular DFO in a group of transfused patients with sickle cell disease. In the present study we have re-evaluated urinary iron excretion in six of the previously studied patients after 15-22 months of additional transfusional therapy and in three additional patients, in order to determine whether a practical program of iron chelation could be planned to prevent further accumulation of excess iron. PATIENTS AND METHODS
Nine patients with homozygous sickle cell anemia, who range in age from six to 16 years, were studied. The diagnosis was established by a combination of methods, including cellulose acetate and agar gel electrophoresis, genetic studies, hematologic studies, globin synthesis measurement, and confirmatory tests for hemoglobin S. All patients had cerebral vascular accidents and are regularly transfused with packed red cells in a continuing study of the effect of suppression of hemoglobin S levels below 30% on the recurrence of stroke [ l l , 121. Several of the patients were also transfused in the mid-1960s as part of a study of growth and cardiac function in sickle cell anemia (Irving J. Wohnan, unpublished observations). Present blood requirements range from one to two units of sediniented red cells every three weeks, contributing 4.2 to 8.5 gm of iron yearly to body iron stores. Patients were studied in the Clinical Research Center of The Children’s Hospital of Philadelphia. Hemoglobin concentration was greater than 10 gm/dl at the time of evaluation. Serum iron concentration and iron binding capacity were measured colorimetrically. Serum ferritin levels were measured by a double-site immunoradiometric assay [13, 1 4 ) . Leukocyte vitamin C concentrations were measured according to the method of Denson and Bowers [ 151 . Liver function studies that were performed included SCOT, SGPT, prothrombin time, and total protein concentration. Following these initial studies, 24-hour urinary excretion of iron was measured in response to 1) 1.5 gm DFO administered intravenously in 18 hours, 2) 2.0 gm DFO administered subcutaneously into the abdominal wall in 12 hours using a portable, batterypowered pump (Auto-Syringe, Hookset, NH), and 3) 750 mg DFO administered intramuscularly. The intravenous dose was selected in order to compare iron excretion with previous measurements made in several of these patients as well as in patients with thalassemia major [8] . The intramuscular and subcutaneous doses, while not equivalent, represent amounts commonly used on the basis of volume limitations and dose-response relationslups. At least six hours elapsed between the completion of a 24-hour urine collection and the beginning of the next DFO dose. Urinary iron excretion returned to baseline values during this interval. After three weeks of oral vitamin C supplementation, 250 mg daily, leukocyte vitamin C levels were again measured, and the urinary iron excretion studies were repeated. Urinary iron concentration was measured by atomic absorption spectro-
Iron Chelation Therapy
71
photometry [16] . Adequacy of each urine collection was assessed by creatinine content, which was greater than 15 mg/kg of body weight in all studies. Informed consent was obtained from all patients or parents of patients. The parents were aware that administration of DFO by subcutaneous and intravenous routes is experimental. The studies were approved by the Committee on Protection of Human Subjects of The Children’s Hospital of Philadelphia. RESULTS
Iron stores as measured by serum ferritin levels were markedly increased in each of the nine patients (2,168-6,300 ng/ml) (Table I). Serum iron was elevated in four (195-240 pg/lOO ml), and saturation of iron-binding capacity was high in eight patients (70-100%). Abnormalities of liver function were detected in all patients prior to DFO administration. The SGOT was elevated in nine (44-108 milliunits/ml), the SGPT was elevated in seven (50-1 52 milliunits/ml), and the prothrombin time was prolonged in three patients (1 3.3-1 4.6 seconds). The results of studies of iron excretion are shown in Table 11. Twenty-four-hour urinary iron excretion in response to an 18-hour intravenous infusion of 1.5 gm DFO before vitamin C supplementation was 13-41 mg. Following three weeks of oral vitamin C, 250 mg daily, 24-hour urinary excretion of iron in response to the same dose of DFO was 11-38 mg. When 2.0 gm of DFO were administered subcutaneously in 12 hours, urinary iron excretion was 9-47 mg/24 hours before vitamin C supplementation and 13-39 mg/24 hours afterward. If this method of DFO administration were used daily with vitamin C supplementation, eight o f the nine patients would achieve negative iron balance (Fig. 1). Without vitamin C supplementation, six of nine patients would be in negative iron balance. Studies of iron excretion in response to subcutaneous DFO have been repeated in five patients prior to beginning regular chelation therapy. Although urinary iron excretion varies by 3-19%, negative iron balance is still achieved in these patients. Following the intramuscular injection of 750 mg of DFO, urinary iron excretion was 5-14 mg/24 hours before vitamin C supplementation and 5-16 mg/24 hours after administration of the vitamin. Only one of the patients would be in negative iron balance using this method of chelation therapy daily.
TABLE I. Iron Status Prior to Chelation Therapy in Patients With Sickle Cell Anemia ~~
Patient
Age (years)
1 6 2 8 3 9 4 14 5 12 6 12 7 15 8 15 9 15 Normal range
Transfused Serum iron packed cells concentration (liters) (pg/dl)
11 8 18 24 18 20 55 47 31
195 152 174 180 140 210 120 240 220 50-180
Total iron binding capacity (4dl)
Transferrin saturation (%)
Serum ferritin concentration (ng/ml)
240 180 210 258 180 228 222 240 282 250-420
81 84 83 70 78 92 54 100 78 15-60
3,850 2,168 6,300 2,542 3,906 3,980 4,028 2,872 4,080 20-300
11.2 13.9 31.2 12.9 18.8 18.0 23.0 41.0 32.0 23.1
Intravenous DFOa
21.8 9 .0 30.4 15.3 12.1 23.5 25.3 28.6 41.0 23.1
DFO~
Subcutaneous
al.5 gm intravenously in 18 hours. b2.0 gm subcutaneously in 12 hours. ‘0.75 gm intramuscularly.
8 9 Mean
I
1 2 3 4 5 6
Patient
Before vitamin C
5.2 6.4 10.0 11.5 9.1 10.0 5 .I 13.2 14.2 9.5
Intramuscular DFO~
16.0 18.0 32.0 11.0 18.6 19.0 31.0 31.6 30.3 23.7
Intravenous DFOa
18.0 13.0 39 .O 18.3 18.1 18.5 36.0 24.9 32.1 24.3
Subcutaneous DFO~
After vitamin C
6 .O 4.1 15.7 I .4 1.3 I .9 15.3 11.5 9.6 9.5
Intramuscular DFO~
TABLE 11. 24-Hour Urinary Iron Excretion (mg/Total Volume) in Response to Deferoxamine (DFO) Before and After Vitamin C Supplementation
4 N
Iron Chelation Therapy
73
0 TRANSFUSIONAL IRON URINARY IRON EXCRETION
1614
-
12
,"
10
L
-
0
2
0
8
E 6 4
2 0
S2
SI
S3 S 4
Sb
S6
S7
SB
S9
PATlENTS
Fig. 1. Comparison of yearly transfusional iron accumulation and urinary iron excretion in response to 2.0 gm DFO administered as a daily 12-hour subcutaneous infusion following vitamin C supplementation.
: - 1 = o
0 2 4 6 8 10 12 14 16 18 20 22
TIME BETWEEN EVALUATIONS(monthd
Fig. 2. Follow-up measurements of urinary iron excretion in response t o 1.5 gm DFO administered as an 18-hour intravenous infusion in six patients who were continuing t o receive regular red cell transfusions without chelation therapy.
Twenty-four-hour urinary iron excretion in response to the intravenous infusion of 1.5 gm DFO was compared in six patients with similar studies performed 15-22 months previously (Fig. 2). These patients had continued to receive transfusions but were not treated with DFO in the interim. Iron excretion increased 46-107%. The rates of increase in DFO-induced urinary iron excretion were remarkably similar, ranging from 0.50 to 0.63 mg/month. Initial leukocyte vitamin C levels were low in three patients (Patients 3, 4 , 6 = 3.87.8 pg/108 white cells) and normal in the remaining six patients (22.4-46.0 pg/108 white cells). No significant increase in urinary iron excretion in response to the three routes of DFO administration was found in these patients after vitamin C supplementation (Table
74
Cohen and Schwartz
11). When evaluated by Student’s t-test for paired samples, P values for iron excretion before and after vitamin C supplementation were 0.31 for intravenous, 0.42 for subcutaneous, and 0.49 for intramuscular DFO. DISCUSSION
Transfusions of red blood cells have been successfully used in the treatment of specific complications of sickle cell anemia [ l l , 12, 17, 181. The freedom from painful crises and repeated hospitalizations enjoyed by patients receiving regular transfusions emphasizes the expanded role such therapy might play in this disorder. However, we have previously shown that iron stores are markedly increased in regularly transfused patients with sickle cell anemia [ I ] , and in the present study we have found, as expected, that urinary iron excretion in response to DFO steadily increases when transfusion therapy is continued. Severe organ dysfunction due to iron overload is a common complication of chronic transfusion therapy in patients with hematologic disorders characterized by either hypoplastic bone marrow, as in Diamond-Blackfan syndrome, or hyperplastic bone marrow, as in thalassemia major. The prevention of further iron accumulation may thus be essential to protect patients with sickle cell anemia from the devastating consequences of transfusional hemochromatosis. Using an overnight subcutaneous infusion of 2.0 gm of DFO, negative iron balance can be achieved in patients with sickle cell anemia who have received as few as 32 units of packed red cells and are continuing to be transfused. In patients with larger transfusional iron stores, urinary iron excretion in response to this method of DFO administration is sufficient to prevent further iron accumulation and to deplete excessive iron stores. When DFO is administered as a daily intramuscular injection, negative iron balance was attained only in a single severely iron-overloaded patient. Urinary iron excretion in response to DFO increases in vitamin C-deficient patients with thalassemia major after vitamin C supplementation. Similar changes in DFO-induced iron excretion were not found in the present study of patients with sickle cell anemia. However, only three patients had low leukocyte vitamin C concentrations. The vitamin C concentration was unrelated to the degree of iron overload, suggesting that additional factors, perhaps including diet, may influence the vitamin C deficiency found in these patients. Mild abnormalities of liver function in these patients are consistent with hepatic hemosiderosis, repeated hepatic infarction, or viral hepatitis. Further studies of organ function, including extensive cardiac investigations, are now being performed. In addition, serial evaluations during chronic chelation therapy may distinguish those abnormalities related to iron overload from those due to the sickling disorder or infection. The retardation of hepatic fibrosis and absence of major cardiac arrhythmias in patients with thalassemia major receiving long-term DFO suggest that iron chelation should be an integral part of the treatment of that disorder [21,22]. Because excessive iron accumulation can be prevented in patients with sickle cell anemia who require chronic transfusion therapy, the deleterious effects of iron overload should similarly be prevented or delayed. The successful use of chelation therapy in these patients may ultimately enable physicians to prevent many of the severe and disabling complications of sickle cell anemia by the relatively safe use of repeated red cell transfusions.
Iron Chelation Therapy
75
ACKNOWLEDGMENTS
The authors wish to thank Dr. Marie Russell and Marie Martin, RN, for their cooperation and assistance in the performance of these studies. We thank Carol Way for her many contributions in the preparation of tlus manuscript and Judith Ann Williams for her secretarial assistance. This work was supported in part by a grant (AM 16691) and a research training award (HL 07 150) from the National Institutes of Health and by a grant from the CibaGeigy Corporation. The studies were conducted in the Clinical Research Center, supported by grant FR-240 of the Division of Research Resources, National Institutes of Health. REFERENCES 1. Cohen A, Schwartz E : Excretion of iron in response to deferoxamine in sickle cell anemia. J Pediatr 92:659, 1978. 2. Peterson CM, Graziano JH, deCiutiis A, Grady RW, Cerami A, Worwood M, Jacobs A: Iron metabolism, sickle cell disease, and response to cyanate. Blood 46:583, 1975. 3. O’Brien R : Iron burden in sickle cell anemia. J Pediatr 92:579, 1978. 4. Buja LM, Roberts WC: Iron in the heart. Am J Med 51:209, 1971. 5. Propper RD, Shurin SB, Nathan DG: Re-assessment of the use of desferrioxamine B in iron overload. N Engl J Med 294:1421,1976. 6. Propper RD, Cooper B, Rufo R R , Nienhuis AW, Anderson WF, Bunn HF, Rosenthal A, Nathan DG: Continuous subcutaneous administration of deferoxamine in patients with iron overload. N Engl J Med 297:418,1977. 7. Modell CB, Beck J: Long-term desferrioxamine therapy in thalassemia. Ann NY Acad Sci 232:201, 1974. 8. Cohen A, Schwartz E: Iron chelation therapy with deferoxamine in Cooley anemia. J Pediatr 92: 643,1978. 9. Graziano JH, Markenson A, Miller DR, Chang H, Bestak M, Myers P, Pisciotto P, Rifkind A: Chelation therapy in P-thalassemia major. I . Intravenous and subcutaneous deferoxamine. J Pediatr 92:648,1978. 10. Weiner M, Karpatkin M, Hart D, Seaman C, Vora SK, Henry WL, Piomelli S: Cooley anemiai: High transfusion regimen and chelation therapy, results, perspective. J Pediatr 92:653, 1978. 11. Russell MO, Goldberg HI, Reis L, Friednian S , Slater R , Reivich M, Schwartz E:Transfusion therapy for cerebrovascular abnormalities in sickle cell disease. J Pediatr 88:382, 1976. 12. Russell MO, Goldberg HI, Kim HC, Halus J , Reivich M, Schwartz E: Cerebrovascular abnormalities in sickle cell patients with stroke: Initial findings and effects of prolonged transfusion therapy. Blood 5 2 3 9 , 1 9 7 8 . 13. Addison GM, Beamis MR, Hales CN, Hoskins M, Jacobs A, Llewillin P: An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J Clin Pathol25:326, 1972. 14. Miles LEM, Lipschitz DA, Bieber CP, Cook JD: Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Biochem 61:209, 1974. 15. Denson KW, Bowers EF: The determination of ascorbic acid in white blood cells. Clin Sci 21: 157, 1961. 16. Olson AD, Hamlin WB: A new method for serum iron and total iron-binding capacity by atomic absorption spectrophotometry. Clin Chem 15:438, 1974. 17. Chernoff AL, Shapleigh JB, Moore CV: Therapy of chronic ulceration of the legs associated with sickle cell anemia. JAMA 155:1487,1954. 18. Nathan DG, Pearson HA: Sickle cell syndromes and hemoglobin C disease. In Nathan DG, Oski FA (eds): “Hematology of Infancy and Childhood.” Philadelphia: WB Saunders, 1974, p 435. 19. Wapnick AA, Lynch S R , Charlton RW, Seftel HC, Bothwell TH: The effect of ascorbic acid deficiency o n desferrioxamine-induced urinary iron excretion. Br J Haematol 1 7 5 6 3 , 1969.
76
Cohen and Schwartz
20. O’Brien RT: Ascorbic acid enhancement of desferrioxamine-induced urinary iron excretion in thalassemia major. Ann NY Acad Sci 232:221, 1974. 21. Barry M, Flynn DM, Letsky EA, Risdon RA: Long-term chelation therapy in thalassemia major: Effect on liver iron concentration, liver histology, and clinical progress. Br Med J 2:16, 1974. 22. Kaye SB, Owen M: Cardiac arrhythmias in thalassemia major: Evaluation of chelation treatment using ambulatory monitoring. Br Med J 1:342, 1978.
Iron Chelation Therapy in Sickle Cell Anemia Alan Cohen and Elias Schwartz Department of Pediatrics, University of Pennsylvania School of Medicine and The Children’s Hospital of Philadelphia
Iron stores, ascorbic acid levels, and urinary excretion of iron in response t o deferoxamine (DFO) were studied in nine patients with sickle cell anemia who had received 8-55 liters of red blood cells. Ferritin levels ranged from 2,168 to 6,300 ng/ml and were elevated even in patients who had normal serum iron concentrations. Leukocyte ascorbic acid levels were low in three patients. Urinary iron excretion in response t o 2.0 gm DFO administered as a 12-hour subcutaneous infusion after ascorbic acid supplementation was 13-39 mg/24 hours. With this method of DFO therapy daily, urinary iron excretion would exceed transfusional iron accumulation in eight of nine patients. Following the intramuscular injection of 0.75 gm DFO, urinary iron excretion was 5-16 mg/24 hours. Iron excretion would exceed iron accumulation in only one of nine patients with this method of DFO therapy. Urinary iron excretion in response t o 1.5 gm DFO infused intravenously in 18 hours after ascorbic acid supplementation was l 1-38 mg/24 hours. In six regularly transfused but nonchelated patients iron excretion in response to intravenous DFO had increased 46-107%, in comparison with previous studies 15-22 months before. These data indicate that negative iron balance can be obtained in regularly transfused patients with sickle cell anemia by the use of overnight subcutaneous infusions of DFO. Urinary iron excretion in response t o DFO should be evaluated periodically in appropriate patients with sickle cell anemia t o determine the proper time for the institution of chelation therapy. Key words: deferoxamine, iron chelation, sickle cell
I NT RO DUCT1ON
Iron stores may be markedly increased in patients with sickle cell anemia who are frequently transfused [ 1-31. Although reports of organ dysfunction due t o iron overload in these patients have been infrequent, cardiac abnormalities due t o iron toxicity have been described [4]. In the absence of any major physiologic means of eliminating excessive iron, one can anticipate progressive hemochromatosis and early death in those patients with sickle cell anemia who continue t o receive red cell transfusions. Received for publication February 28, 1979; accepted May 23. 1979. Address reprint requests to Dr. Alan Cohen, Division of Hematology, The Children’s Hospital of Philadelphia, 34th Street and Civic Center Blvd., Philadelphia, PA 19104.
0361-8609/79/0701-0069$01.700 1979 Alan R. Liss, Inc.
70
Cohen and Schwartz
Recent clinical trials of the iron chelating agent deferoxamine (DFO) have demonstrated that negative iron balance (urinary iron excretion in excess of iron loading by transfusion) can be achieved in patients with transfusion-dependent anemia [5-lo]. Long-term studies are underway to evaluate the effects of chelation therapy on organ function and patient survival. If the accumulation of iron could be retarded or prevented in patients with sickle cell anemia, red cell transfusions would be a practical method of managing many of the serious complications of t h s disease. In a previous study we demonstrated increased excretion of iron in the urine in response to intravenous or intramuscular DFO in a group of transfused patients with sickle cell disease. In the present study we have re-evaluated urinary iron excretion in six of the previously studied patients after 15-22 months of additional transfusional therapy and in three additional patients, in order to determine whether a practical program of iron chelation could be planned to prevent further accumulation of excess iron. PATIENTS AND METHODS
Nine patients with homozygous sickle cell anemia, who range in age from six to 16 years, were studied. The diagnosis was established by a combination of methods, including cellulose acetate and agar gel electrophoresis, genetic studies, hematologic studies, globin synthesis measurement, and confirmatory tests for hemoglobin S. All patients had cerebral vascular accidents and are regularly transfused with packed red cells in a continuing study of the effect of suppression of hemoglobin S levels below 30% on the recurrence of stroke [ l l , 121. Several of the patients were also transfused in the mid-1960s as part of a study of growth and cardiac function in sickle cell anemia (Irving J. Wohnan, unpublished observations). Present blood requirements range from one to two units of sediniented red cells every three weeks, contributing 4.2 to 8.5 gm of iron yearly to body iron stores. Patients were studied in the Clinical Research Center of The Children’s Hospital of Philadelphia. Hemoglobin concentration was greater than 10 gm/dl at the time of evaluation. Serum iron concentration and iron binding capacity were measured colorimetrically. Serum ferritin levels were measured by a double-site immunoradiometric assay [13, 1 4 ) . Leukocyte vitamin C concentrations were measured according to the method of Denson and Bowers [ 151 . Liver function studies that were performed included SCOT, SGPT, prothrombin time, and total protein concentration. Following these initial studies, 24-hour urinary excretion of iron was measured in response to 1) 1.5 gm DFO administered intravenously in 18 hours, 2) 2.0 gm DFO administered subcutaneously into the abdominal wall in 12 hours using a portable, batterypowered pump (Auto-Syringe, Hookset, NH), and 3) 750 mg DFO administered intramuscularly. The intravenous dose was selected in order to compare iron excretion with previous measurements made in several of these patients as well as in patients with thalassemia major [8] . The intramuscular and subcutaneous doses, while not equivalent, represent amounts commonly used on the basis of volume limitations and dose-response relationslups. At least six hours elapsed between the completion of a 24-hour urine collection and the beginning of the next DFO dose. Urinary iron excretion returned to baseline values during this interval. After three weeks of oral vitamin C supplementation, 250 mg daily, leukocyte vitamin C levels were again measured, and the urinary iron excretion studies were repeated. Urinary iron concentration was measured by atomic absorption spectro-
Iron Chelation Therapy
71
photometry [16] . Adequacy of each urine collection was assessed by creatinine content, which was greater than 15 mg/kg of body weight in all studies. Informed consent was obtained from all patients or parents of patients. The parents were aware that administration of DFO by subcutaneous and intravenous routes is experimental. The studies were approved by the Committee on Protection of Human Subjects of The Children’s Hospital of Philadelphia. RESULTS
Iron stores as measured by serum ferritin levels were markedly increased in each of the nine patients (2,168-6,300 ng/ml) (Table I). Serum iron was elevated in four (195-240 pg/lOO ml), and saturation of iron-binding capacity was high in eight patients (70-100%). Abnormalities of liver function were detected in all patients prior to DFO administration. The SGOT was elevated in nine (44-108 milliunits/ml), the SGPT was elevated in seven (50-1 52 milliunits/ml), and the prothrombin time was prolonged in three patients (1 3.3-1 4.6 seconds). The results of studies of iron excretion are shown in Table 11. Twenty-four-hour urinary iron excretion in response to an 18-hour intravenous infusion of 1.5 gm DFO before vitamin C supplementation was 13-41 mg. Following three weeks of oral vitamin C, 250 mg daily, 24-hour urinary excretion of iron in response to the same dose of DFO was 11-38 mg. When 2.0 gm of DFO were administered subcutaneously in 12 hours, urinary iron excretion was 9-47 mg/24 hours before vitamin C supplementation and 13-39 mg/24 hours afterward. If this method of DFO administration were used daily with vitamin C supplementation, eight o f the nine patients would achieve negative iron balance (Fig. 1). Without vitamin C supplementation, six of nine patients would be in negative iron balance. Studies of iron excretion in response to subcutaneous DFO have been repeated in five patients prior to beginning regular chelation therapy. Although urinary iron excretion varies by 3-19%, negative iron balance is still achieved in these patients. Following the intramuscular injection of 750 mg of DFO, urinary iron excretion was 5-14 mg/24 hours before vitamin C supplementation and 5-16 mg/24 hours after administration of the vitamin. Only one of the patients would be in negative iron balance using this method of chelation therapy daily.
TABLE I. Iron Status Prior to Chelation Therapy in Patients With Sickle Cell Anemia ~~
Patient
Age (years)
1 6 2 8 3 9 4 14 5 12 6 12 7 15 8 15 9 15 Normal range
Transfused Serum iron packed cells concentration (liters) (pg/dl)
11 8 18 24 18 20 55 47 31
195 152 174 180 140 210 120 240 220 50-180
Total iron binding capacity (4dl)
Transferrin saturation (%)
Serum ferritin concentration (ng/ml)
240 180 210 258 180 228 222 240 282 250-420
81 84 83 70 78 92 54 100 78 15-60
3,850 2,168 6,300 2,542 3,906 3,980 4,028 2,872 4,080 20-300
11.2 13.9 31.2 12.9 18.8 18.0 23.0 41.0 32.0 23.1
Intravenous DFOa
21.8 9 .0 30.4 15.3 12.1 23.5 25.3 28.6 41.0 23.1
DFO~
Subcutaneous
al.5 gm intravenously in 18 hours. b2.0 gm subcutaneously in 12 hours. ‘0.75 gm intramuscularly.
8 9 Mean
I
1 2 3 4 5 6
Patient
Before vitamin C
5.2 6.4 10.0 11.5 9.1 10.0 5 .I 13.2 14.2 9.5
Intramuscular DFO~
16.0 18.0 32.0 11.0 18.6 19.0 31.0 31.6 30.3 23.7
Intravenous DFOa
18.0 13.0 39 .O 18.3 18.1 18.5 36.0 24.9 32.1 24.3
Subcutaneous DFO~
After vitamin C
6 .O 4.1 15.7 I .4 1.3 I .9 15.3 11.5 9.6 9.5
Intramuscular DFO~
TABLE 11. 24-Hour Urinary Iron Excretion (mg/Total Volume) in Response to Deferoxamine (DFO) Before and After Vitamin C Supplementation
4 N
Iron Chelation Therapy
73
0 TRANSFUSIONAL IRON URINARY IRON EXCRETION
1614
-
12
,"
10
L
-
0
2
0
8
E 6 4
2 0
S2
SI
S3 S 4
Sb
S6
S7
SB
S9
PATlENTS
Fig. 1. Comparison of yearly transfusional iron accumulation and urinary iron excretion in response to 2.0 gm DFO administered as a daily 12-hour subcutaneous infusion following vitamin C supplementation.
: - 1 = o
0 2 4 6 8 10 12 14 16 18 20 22
TIME BETWEEN EVALUATIONS(monthd
Fig. 2. Follow-up measurements of urinary iron excretion in response t o 1.5 gm DFO administered as an 18-hour intravenous infusion in six patients who were continuing t o receive regular red cell transfusions without chelation therapy.
Twenty-four-hour urinary iron excretion in response to the intravenous infusion of 1.5 gm DFO was compared in six patients with similar studies performed 15-22 months previously (Fig. 2). These patients had continued to receive transfusions but were not treated with DFO in the interim. Iron excretion increased 46-107%. The rates of increase in DFO-induced urinary iron excretion were remarkably similar, ranging from 0.50 to 0.63 mg/month. Initial leukocyte vitamin C levels were low in three patients (Patients 3, 4 , 6 = 3.87.8 pg/108 white cells) and normal in the remaining six patients (22.4-46.0 pg/108 white cells). No significant increase in urinary iron excretion in response to the three routes of DFO administration was found in these patients after vitamin C supplementation (Table
74
Cohen and Schwartz
11). When evaluated by Student’s t-test for paired samples, P values for iron excretion before and after vitamin C supplementation were 0.31 for intravenous, 0.42 for subcutaneous, and 0.49 for intramuscular DFO. DISCUSSION
Transfusions of red blood cells have been successfully used in the treatment of specific complications of sickle cell anemia [ l l , 12, 17, 181. The freedom from painful crises and repeated hospitalizations enjoyed by patients receiving regular transfusions emphasizes the expanded role such therapy might play in this disorder. However, we have previously shown that iron stores are markedly increased in regularly transfused patients with sickle cell anemia [ I ] , and in the present study we have found, as expected, that urinary iron excretion in response to DFO steadily increases when transfusion therapy is continued. Severe organ dysfunction due to iron overload is a common complication of chronic transfusion therapy in patients with hematologic disorders characterized by either hypoplastic bone marrow, as in Diamond-Blackfan syndrome, or hyperplastic bone marrow, as in thalassemia major. The prevention of further iron accumulation may thus be essential to protect patients with sickle cell anemia from the devastating consequences of transfusional hemochromatosis. Using an overnight subcutaneous infusion of 2.0 gm of DFO, negative iron balance can be achieved in patients with sickle cell anemia who have received as few as 32 units of packed red cells and are continuing to be transfused. In patients with larger transfusional iron stores, urinary iron excretion in response to this method of DFO administration is sufficient to prevent further iron accumulation and to deplete excessive iron stores. When DFO is administered as a daily intramuscular injection, negative iron balance was attained only in a single severely iron-overloaded patient. Urinary iron excretion in response to DFO increases in vitamin C-deficient patients with thalassemia major after vitamin C supplementation. Similar changes in DFO-induced iron excretion were not found in the present study of patients with sickle cell anemia. However, only three patients had low leukocyte vitamin C concentrations. The vitamin C concentration was unrelated to the degree of iron overload, suggesting that additional factors, perhaps including diet, may influence the vitamin C deficiency found in these patients. Mild abnormalities of liver function in these patients are consistent with hepatic hemosiderosis, repeated hepatic infarction, or viral hepatitis. Further studies of organ function, including extensive cardiac investigations, are now being performed. In addition, serial evaluations during chronic chelation therapy may distinguish those abnormalities related to iron overload from those due to the sickling disorder or infection. The retardation of hepatic fibrosis and absence of major cardiac arrhythmias in patients with thalassemia major receiving long-term DFO suggest that iron chelation should be an integral part of the treatment of that disorder [21,22]. Because excessive iron accumulation can be prevented in patients with sickle cell anemia who require chronic transfusion therapy, the deleterious effects of iron overload should similarly be prevented or delayed. The successful use of chelation therapy in these patients may ultimately enable physicians to prevent many of the severe and disabling complications of sickle cell anemia by the relatively safe use of repeated red cell transfusions.
Iron Chelation Therapy
75
ACKNOWLEDGMENTS
The authors wish to thank Dr. Marie Russell and Marie Martin, RN, for their cooperation and assistance in the performance of these studies. We thank Carol Way for her many contributions in the preparation of tlus manuscript and Judith Ann Williams for her secretarial assistance. This work was supported in part by a grant (AM 16691) and a research training award (HL 07 150) from the National Institutes of Health and by a grant from the CibaGeigy Corporation. The studies were conducted in the Clinical Research Center, supported by grant FR-240 of the Division of Research Resources, National Institutes of Health. REFERENCES 1. Cohen A, Schwartz E : Excretion of iron in response to deferoxamine in sickle cell anemia. J Pediatr 92:659, 1978. 2. Peterson CM, Graziano JH, deCiutiis A, Grady RW, Cerami A, Worwood M, Jacobs A: Iron metabolism, sickle cell disease, and response to cyanate. Blood 46:583, 1975. 3. O’Brien R : Iron burden in sickle cell anemia. J Pediatr 92:579, 1978. 4. Buja LM, Roberts WC: Iron in the heart. Am J Med 51:209, 1971. 5. Propper RD, Shurin SB, Nathan DG: Re-assessment of the use of desferrioxamine B in iron overload. N Engl J Med 294:1421,1976. 6. Propper RD, Cooper B, Rufo R R , Nienhuis AW, Anderson WF, Bunn HF, Rosenthal A, Nathan DG: Continuous subcutaneous administration of deferoxamine in patients with iron overload. N Engl J Med 297:418,1977. 7. Modell CB, Beck J: Long-term desferrioxamine therapy in thalassemia. Ann NY Acad Sci 232:201, 1974. 8. Cohen A, Schwartz E: Iron chelation therapy with deferoxamine in Cooley anemia. J Pediatr 92: 643,1978. 9. Graziano JH, Markenson A, Miller DR, Chang H, Bestak M, Myers P, Pisciotto P, Rifkind A: Chelation therapy in P-thalassemia major. I . Intravenous and subcutaneous deferoxamine. J Pediatr 92:648,1978. 10. Weiner M, Karpatkin M, Hart D, Seaman C, Vora SK, Henry WL, Piomelli S: Cooley anemiai: High transfusion regimen and chelation therapy, results, perspective. J Pediatr 92:653, 1978. 11. Russell MO, Goldberg HI, Reis L, Friednian S , Slater R , Reivich M, Schwartz E:Transfusion therapy for cerebrovascular abnormalities in sickle cell disease. J Pediatr 88:382, 1976. 12. Russell MO, Goldberg HI, Kim HC, Halus J , Reivich M, Schwartz E: Cerebrovascular abnormalities in sickle cell patients with stroke: Initial findings and effects of prolonged transfusion therapy. Blood 5 2 3 9 , 1 9 7 8 . 13. Addison GM, Beamis MR, Hales CN, Hoskins M, Jacobs A, Llewillin P: An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J Clin Pathol25:326, 1972. 14. Miles LEM, Lipschitz DA, Bieber CP, Cook JD: Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Biochem 61:209, 1974. 15. Denson KW, Bowers EF: The determination of ascorbic acid in white blood cells. Clin Sci 21: 157, 1961. 16. Olson AD, Hamlin WB: A new method for serum iron and total iron-binding capacity by atomic absorption spectrophotometry. Clin Chem 15:438, 1974. 17. Chernoff AL, Shapleigh JB, Moore CV: Therapy of chronic ulceration of the legs associated with sickle cell anemia. JAMA 155:1487,1954. 18. Nathan DG, Pearson HA: Sickle cell syndromes and hemoglobin C disease. In Nathan DG, Oski FA (eds): “Hematology of Infancy and Childhood.” Philadelphia: WB Saunders, 1974, p 435. 19. Wapnick AA, Lynch S R , Charlton RW, Seftel HC, Bothwell TH: The effect of ascorbic acid deficiency o n desferrioxamine-induced urinary iron excretion. Br J Haematol 1 7 5 6 3 , 1969.
76
Cohen and Schwartz
20. O’Brien RT: Ascorbic acid enhancement of desferrioxamine-induced urinary iron excretion in thalassemia major. Ann NY Acad Sci 232:221, 1974. 21. Barry M, Flynn DM, Letsky EA, Risdon RA: Long-term chelation therapy in thalassemia major: Effect on liver iron concentration, liver histology, and clinical progress. Br Med J 2:16, 1974. 22. Kaye SB, Owen M: Cardiac arrhythmias in thalassemia major: Evaluation of chelation treatment using ambulatory monitoring. Br Med J 1:342, 1978.
