Clinical Science (1991)81,113-121
113
Hepatic computed tomography for monitoring the iron status of haemodialysis patients with haemosiderosis treated with recombinant human erythropoietin SERGIO DE MARCHI
AND
EMANUELA CECCHIN
Department of Internal Medicine, University of Udine Medical School, Udine, Italy
(Received 17 December 1990/11 February 1991; accepted 26 February 1991)
SUMMARY
1. A randomized, partial-crossover study was conducted in uraemic patients with dialysis-associated anaemia and transfusional iron overload to evaluate the effects of desferrioxamine chelation therapy and of recombinant human erythropoietin treatment on hepatic iron storage determined by computed tomography, as well as by serum ferritin concentration and transferrin saturation. 2. Twenty-one haemodialysis patients with moderate iron overload, confirmed by values of serum ferritin concentration, transferrin saturation and hepatic computed tomography density exceeding 1000 pg/l, 45% and 68 Hounsfield units respectively, were randomly allocated to three groups and were followed for 12 months. 3. During the first 6 months group 1 ( n = 7) received desferrioxamine chelation therapy (30 mg/kg intravenously three times a week) and group 2 ( n= 7) underwent recombinant human erythropoietin treatment (36 units/ kg intravenously three times a week). Thereafter, in the second 6 months of observation patients in group 1 were switched to receive recombinant human erythropoietin. Because of a poor response in the desferrioxaminetreated group in the initial 6 months, patients in group 2 continued on the maintenance dose of recombinant human erythropoietin (18 units/kg three times a week) until the end of the trial. Patients in group 3 ( n = 7) were maintained on placebo throughout the study. 4. In comparison with placebo, recombinant human erythropoietin treatment, but not desferrioxamine chelation therapy, reduced serum ferritin concentration, transferrin saturation and hepatic computed tomography density, and was associated with a rise in haemoglobin and packed cell volume. Hepatic computed tomography density, serum ferritin concentration and transferrin saturation decreased in 1 3 out of 14 patients (93%) during treatment with recombinant human erythropoietin. Correspondence: Dr Sergio De Marchi, Via Tartagna, 39, 33100 Udine, Italy.
However, when the changes in hepatic computed tomography density were compared with those in the biochemical indices, we observed that the decreases in serum ferritin concentration and transferrin saturation were much slower and delayed. More specifically, within 6 months of starting recombinant human erythropoietin treatment, hepatic computed tomography density was normalized in 13 out of 1 4 patients (93%),whereas serum ferritin concentration and transferrin saturation were within the normal limits in only two (14%)and six patients (43%),respectively. 5. In conclusion, the strategies for monitoring the iron status of haemodialysis patients with transfusional haemosiderosis may evolve to a new level of sophistication with the introduction of computed tomography scanning. This technique has the advantage of estimating directly the effect of recombinant human erythropoietin treatment on hepatic iron storage. Hepatic computed tomography density is complementary to serum ferritin concentration and transferrin saturation in monitoring the iron status of haemodialysis patients treated with recombinant human erythropoietin. Key words: desferrioxamine, erythropoietin, haemodialysis, haemosiderosis, iron overload, uraemia. Abbreviations: CT, computed tomography; DFO, desferrioxamine; ESRD, end-stage renal disease; rHuEPO, recombinant human erythropoietin.
INTRODUCTION Anaemia, one of the most common complications of endstage renal disease (ESRD), renders nearly 25% of dialysis patients dependent on erythrocyte transfusions to sustain life [l].Transfusional iron overload is a serious problem for a substantial minority of the dialysis population [2]. Recombinant human erythropoietin (rHuEPO) was demonstrated to be remarkably effective in correcting dialysis-associated anaemia [l, 3-10], as well as trans-
S. De Marchi and E. Cecchin
114
Association and the European Renal Association, Vienna, Austria, 5-9 September 1990, and were published in abstract form [16a].
fusional iron overload [ 11-14]. Recently, we demonstrated that in haemodialysis patients the assessment of hepatic density by computed tomography ( C T ) is an accurate and non-invasive alternative to liver biopsy that permits estimation of iron storage directly [15, 161. Lazarus et al. [12] observed a fall in hepatic C T density and serum ferritin level in five iron-overloaded haemodialysis patients during 9-14 months of rHuEPO treatment in combination with phlebotomy. This finding was confirmed by Nomura ef al. [13] in a haemodialysis patient with severe iron overload and ascites. However, to date the usefulness of CT scanning in monitoring the iron status of haemodialysis patients receiving rHuEPO treatment or desferrioxamine (DFO) chelation therapy for transfusional iron overload has not been established. The present study was undertaken in a group of uraemic patients with dialysis-associated anaemia and moderate iron overload to evaluate the effects of rHuEPO treatment and of DFO chelation therapy on hepatic iron storage measured by CT scanning, as well as by serum ferritin levels and transferrin saturation. The purpose of the study was to investigate the usefulness of hepatic CT density in monitoring the iron status of haemodialysis patients with transfusional haemosiderosis treated with rHuEPO or DFO. Parts of this study were presented at the 27th Annual Congress of the European Dialysis and Transplant
METHODS Study protocol
A randomized, partial-crossover study was conducted in uraemic patients with dialysis-associated anaemia and transfusional haemosiderosis to compare the effects on tissue iron storage of rHuEPO treatment and DFO chelation therapy. The experimental design is shown in Fig. 1. Twenty-one patients with moderate iron overload were randomly allocated to three groups by a table of random numbers. After a 2-week run-in period for baseline measurements, the three groups of patients of equal number ( n= 7) were followed for 12 months. During the first 6 months (phase 1)group 1 received DFO chelation therapy, group 2 underwent rHuEPO treatment, and group 3 was on a placebo. Thereafter, in the second 6 months of observation (phase 2) the patients in group 1 were switched over to the rHuEPO treatment protocol. Because of a poor response in the group receiving DFO chelation therapy in the initial 6 months, patients on rHuEPO treatment were not switched over to the DFO chelation therapy protocol, but continued on the maintePhase 2
Phase 1
Haemodialysis patients ( n= 21):
Group 1 ( n= 7)
rHuEPO
DFO
Group 2 ( n= 7)
rHuEPO
rHuEPO
Group 3 ( n= 7 )
Placebo
Placebo
Investigations: Hepatic CT density Serum ferritin concn. Transferrin saturation Haematological indices
* * * *
* * * *
* * * *
I
l
l
l
l
l
l
l
l
l
0
1
2
3
4
5
6
7
8
9
l
l
l
1 0 1 1 1 2
Time (months)
Fig. 1. Study design. During the first 6 months group 1 received DFO chelation therapy (30 mg/ kg intravenously at the end of each dialysis three times a week), group 2 underwent rHuEPO treatment (rHuEPO was administered intravenously three times a week at the end of each dialysis starting with 36 units/kg, patients were continued on this dose until the packed cell volume reached 30%, and thereafter rHuEPO treatment was reduced to the maintenance dose of 18 units/kg), and group 3 was on a placebo. In the second 6 months group 1 was switched over to the rHuEPO treatment protocol, group 2 continued on the maintenance dose of rHuEPO, and group 3 was maintained on placebo.
Hepatic computed tomography during recombinant human erythropoietin treatment nance dose of rHuEPO until the end of the trial. Patients in group 3 were maintained on placebo even in phase 2 of the study. Hepatic CT density, haemoglobin, packed cell volume, transferrin saturation and serum ferritin concentration were measured in each of the patients before and after each 6-month course. All blood samples were obtained immediately before dialysis. In the DFO chelation therapy protocol patients received intravenous DFO at a dose of 30 mg/kg over 30-40 min after the termination of each dialysis three times weekly for 6 months. In the rHuEPO treatment protocol patients received intravenous rHuEPO (Boehringer Mannheim, Italia SPA, Milan, Italy) three times weekly at the end of each dialysis starting with a dose of 36 units/kg. Patients were continued on this dose until the packed cell volume reached or exceeded 30%. Thereafter, rHuEPO treatment was reduced to a maintenance dose of 18 units/kg in all patients, except one, until the end of the 6-month course of therapy. In one patient from group 1, who did not have a positive response, as indicated by an increase in haemoglobin of less than 2 g/dl, the dose of rHuEPO was doubled every second week until a dose of 144 units/kg was reached. This study was conducted in accordance with the principles of the Declaration of Helsinki of the World Medical Association.
115
flow was set at 500 ml/min. The ultrafiltration rate was standardized at 4 ml/min. None of the patients had evidence of aluminium-related toxicity or of positive DFO aluminium challenge. Serum aluminium concentrations before DFO challenge ranged from 10 to 45 pg/l (meanIf:sD, 31 f 13 pgll). The values of hepatic CT density ranged from 69 to 81 Hounsfield units ( m e a n k s ~ ,76.3k 3.7 Hounsfield units). In a control group of 40 healthy subjects, well matched for age and sex, the values of hepatic CT density ranged from 53 to 65 Hounsfield units (mean fSD, 60.2 f 5.6 Hounsfield units). In these subjects the mean ( fSD) values of serum ferritin concentration and transferrin saturation were 100 (If:59) pg/l and 28 ( k 9)%. Estimation of hepatic CT density The C T scan of each patient was obtained by a GE CT/T 8000 scanner with a 4.5 s scan time according to the usual protocols using 8 mm thick slices. No intravenous contrast material was given. Five 1cm2 areas were selected at the periphery of the liver slice, approximately 1 cm from the liver edge, avoiding obvious bile ducts, vascular structures, fissures and infiltrations. The CT densities of these areas were averaged to obtain the hepatic CT density.
Patient selectior. Twenty-one patients with ESRD on maintenance haemodialysis, who had evidence of iron overload, confirmed by values of serum ferritin concentration, transferrin saturation and hepatic C T density exceeding 1000 pg/l, 45% and 68 Hounsfield units, respectively, were selected for the study. All patients showed moderate levels of iron excess, manifested by mean values of serum ferritin concentration and transferrin saturation of 1391 pg/l (range 1004-3000 pg/l) and 62% (range 48-95%), respectively. None had evidence of symptomatic haemosiderosis. More specifically, they had no clinical evidence of myopathy, arthropathy, cardiomyopathy or pancreatic insufficiency related to iron deposition. They were the only patients in our unit with elevated values of serum ferritin concentration, transferrin saturation and hepatic CT density that fulfilled the following criteria: no history of alcohol abuse, absence of obesity ( > 20% increase in relative weight), and no evidence of erythrocyte microcytosis or hypochromia, diabetes mellitus, liver disease, malignancy or chronic inflammatory disease. They ranged in age from 25 to 76 years ( m e a n f s ~51.6 , k 10.7 years). Nine of the patients were male and 12 were female. One patient was anephric. They required an average transfusion of 1.5 ( If:0.3) units of concentrated erythrocytes over the 6 months before the study to maintain their packed cell volume at an acceptable level. The lifetime estimated transfusional iron load was 2952k983 mg. No iron supplementation was administered during the course of the study. Patients were given our standard 4 h haemodialysis treatment three times weekly with an 8 pm, 1.0 m2 hollow fibre cuprophane dialyser throughout the trial. The blood flow was set at 300 ml/min and the dialysate
Laboratory investigations Serum iron concentration, total iron-binding capacity and transferrin saturation were determined by the methods of the International Committee for Standardization in Hematology [17]. Serum ferritin concentration was measured by an r i a . method using a commercial kit (Becton Dickinson, Milan, Italy). The 95% confidence limits for normality in both sexes were 18-185 pg/l. Serum aluminium concentration was determined by atomic spectrophotometry with a graphite furnace. Blood cell counts were performed by a Coulter counter. Statistical analysis Results are expressed as mean sf^^^. Paired and unpaired two-tailed Student’s t-tests were used to compare group means and to assess the statistical significance of changes. In the analysis of the effects of rHuEPO treatment, and DFO chelation therapy, we compared changes in the treated groups with changes in the placebo group. In addition, when appropriate, tests were done by comparing groups 1 and 2, and changes in each group, separately. A P value of less than 0.05 was considered to indicate a significant difference. RESULTS Changes in hepatic C T density, serum ferritin concentration, transferrin saturation, haemoglobin and packed cell volume during the course of DFO chelation therapy and rHuEPO treatment are shown in Table 1 and in Figs. 2 and 3.
SEM
Group 3 1 2 3 4 5 6 7 Mean
SEM
Group 2 1 2 3 4 5 6 7 Mean
SEM
Group 1 1 2 3 4 5 6 7 Mean
Patient no.
45/F 45/M 68/F 45/F 61/M 50/M 42/F
51/F 60/F 49/F 51/F 25/M 76/M 62/M
49/F 52/F 61/F 42/F 50/M 54/M 45/M
Age (years)/ sex
3015 826 954 2523 1100 875 807 1442 349
After
90 49 48 55 56 51 92 63 7
After
840 3000 960 1150 810 804 2000 1366 316
Before 407 1980 172 476 305 312 761 630 236
After
2950 810 805 889 954 826 2430 1380 344
Before 3180 850 865 830 920 832 2650 1446 384
After
First 6-month course of placebo
Before
After 41 66 53 52 37 41 56 49 4
96 51 52 61 59 54 95 67 8
2800 900 899 2600 1030 895 853 1425 33 1
Before 79.5 81 77 70.5 76.5 76 70.5 75.8 1.5
Before
80.5 75 69 77.5 77.5 77.5 80.5 76.8 1.5
Before
79.5 80 77.5 77 69 74 76.5 76.2 1.4
Before
81 75 70 77.5 77 77 80 76.8 1.4
After
49.5 47.5 53 53.5 54 53 56.4 52.4 1.1
After
80.5 80.5 77.5 68 76.5 74 69 75.2 2.0
After
Hepatic CT density (HU)
First 6-month course of rHuEPO treatment
92 51 50 86 68 50 51 64 7
After
59 95 66 77 55 47 95 71 7
Before
90 72 54 49 68 7
55
95 58
Before
Serum ferritin concn. (pgll)
6-month course of DFO chelation therapy
Transferrin saturation ( O h )
90 49 48 55 56 51 92 63 7
Before
41 66 53 52 37 41 56 49 4
Before
92 51 50 86 68 50 51 64 7
Before
Serum ferritin concn. ( p g l l )
3015 826 954 2523 1100 875 807 1442 349
Before 1945 400 154 2466 416 300 280 851 356
After
80.5 80.5 77.5 68 76.5 74 69 75.2 2.0
Before
407 1980 172 476 305 312 76 1 630 236
Before
120 617 65 150 93 85 180 187 73
After
49.5 47.5 53 53.5 54 53 56.4 52.4 1.1
Before
96 59 47 53 55 55 94 66 8
After
3180 850 865 830 920 832 2650 1446 384
Before
3050 830 835 875 941 830 2590 1421 365
After
81 75 70 77.5 77 77 80 76.8 1.4
Before
Second 6-month course of placebo
11 44 37 28 18 15 41 28 5
After
Second 6-month course of rHuEPO treatment
65 47 28 88 48 25 24 46 9
After
81 75 70 77 77 78 79 76.7 1.3
After
48.5 46.5 51 51.5 50 51 54 50.4 0.9
After
47.5 50.5 53.5 72.5 53 51 47.5 53.6 3.3
After
Hepatic CT density (HU)
6-month course of rHuEPO treatment
Transferrin saturation ( O h )
Table 1. Transferrin saturation, serum ferritin concentration and hepatic CT density in the three groups of haemodialysis patients before and after each phase of the study
r
5
6 g
m
6
z 55
0
;
o\
Hepatic computed tomography during recombinant human erythropoietin treatment I
1
117
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$ 35-
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30-
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20-
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*** 40
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0 1 2 3
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1
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5 6 7 8 9101112
Time (months)
Fig. 2. Changes in transferrin saturation ( a ) , serum ferritin concentration ( b )and hepatic CT density (c) in the three groups of haemodialysis patients (0, group 1; 0 , group 2; 0 , group 3) over the full experimental period. Statistical significance: * P < 0.05, **P< 0.005, ***P< 0.0001 compared with placebo.
Effects on hepatic CT density
The mean baseline levels of hepatic CT density did not differ among the three groups of haemodialysis patients as shown in Table 1. The mean ( fSEM) values were significantly higher than that observed in healthy subjects (60.2 f 1.3 Hounsfield units, P < 0.0001). In group 1 patients the 6-month course of DFO chelation therapy did not induce any change in the mean value of hepatic CT density as compared with that in the placebo group. Furthermore, in this group of patients there was no difference in the mean value of hepatic CT density between the beginning and end of phase 1. Conversely, in these patients the subsequent rHuEPO treatment induced a significant fall in the mean value of hepatic CT density as compared either with that in the placebo group (53.6 f 3.3 versus 76.7 f 1.3 Hounsfield units, P< 0.0001) or with the value previously obtained in the same group of patients at the end of the course of DFO chelation therapy (53.6 f 3.3 versus 75.2 f2 Hounsfield units, P< 0.0001). More specifically, six patients returned to low-normal levels of hepatic CT density at the end of the rHuEPO treatment, and the mean ( fSEM) value of the individual decreases was 25.8 ( f 1.8) Hounsfield units. In
1050’
1
1
0
1 2 3 4 5 6 7 89101112 Time (months)
1
1
1
1
1
1
1
1
1
1
1
Fig. 3. Changes in haemoglobin ( a ) and packed cell volume ( b )in the three groups of haemodialysis patients (0,group 1; 0 , group 2; 0 , group 3) over the full experimental period. Statistical significance: *P< 0.001, **P< 0.0001 compared with placebo.
one patient, hepatic CT density, serum ferritin concentration, transferrin saturation, as well as haemoglobin and packed cell volume, were not influenced by rHuEPO treatment. This patient had evidence of severe hyperparathyroidism. In group 2 patients the first 6-month course of rHuEPO treatment induced a significant decrease in the mean value of hepatic CT density as compared either with that in the placebo group (52.4k 1.1 versus 76.8 f 1.4 Hounsfield units, P
113
Hepatic computed tomography for monitoring the iron status of haemodialysis patients with haemosiderosis treated with recombinant human erythropoietin SERGIO DE MARCHI
AND
EMANUELA CECCHIN
Department of Internal Medicine, University of Udine Medical School, Udine, Italy
(Received 17 December 1990/11 February 1991; accepted 26 February 1991)
SUMMARY
1. A randomized, partial-crossover study was conducted in uraemic patients with dialysis-associated anaemia and transfusional iron overload to evaluate the effects of desferrioxamine chelation therapy and of recombinant human erythropoietin treatment on hepatic iron storage determined by computed tomography, as well as by serum ferritin concentration and transferrin saturation. 2. Twenty-one haemodialysis patients with moderate iron overload, confirmed by values of serum ferritin concentration, transferrin saturation and hepatic computed tomography density exceeding 1000 pg/l, 45% and 68 Hounsfield units respectively, were randomly allocated to three groups and were followed for 12 months. 3. During the first 6 months group 1 ( n = 7) received desferrioxamine chelation therapy (30 mg/kg intravenously three times a week) and group 2 ( n= 7) underwent recombinant human erythropoietin treatment (36 units/ kg intravenously three times a week). Thereafter, in the second 6 months of observation patients in group 1 were switched to receive recombinant human erythropoietin. Because of a poor response in the desferrioxaminetreated group in the initial 6 months, patients in group 2 continued on the maintenance dose of recombinant human erythropoietin (18 units/kg three times a week) until the end of the trial. Patients in group 3 ( n = 7) were maintained on placebo throughout the study. 4. In comparison with placebo, recombinant human erythropoietin treatment, but not desferrioxamine chelation therapy, reduced serum ferritin concentration, transferrin saturation and hepatic computed tomography density, and was associated with a rise in haemoglobin and packed cell volume. Hepatic computed tomography density, serum ferritin concentration and transferrin saturation decreased in 1 3 out of 14 patients (93%) during treatment with recombinant human erythropoietin. Correspondence: Dr Sergio De Marchi, Via Tartagna, 39, 33100 Udine, Italy.
However, when the changes in hepatic computed tomography density were compared with those in the biochemical indices, we observed that the decreases in serum ferritin concentration and transferrin saturation were much slower and delayed. More specifically, within 6 months of starting recombinant human erythropoietin treatment, hepatic computed tomography density was normalized in 13 out of 1 4 patients (93%),whereas serum ferritin concentration and transferrin saturation were within the normal limits in only two (14%)and six patients (43%),respectively. 5. In conclusion, the strategies for monitoring the iron status of haemodialysis patients with transfusional haemosiderosis may evolve to a new level of sophistication with the introduction of computed tomography scanning. This technique has the advantage of estimating directly the effect of recombinant human erythropoietin treatment on hepatic iron storage. Hepatic computed tomography density is complementary to serum ferritin concentration and transferrin saturation in monitoring the iron status of haemodialysis patients treated with recombinant human erythropoietin. Key words: desferrioxamine, erythropoietin, haemodialysis, haemosiderosis, iron overload, uraemia. Abbreviations: CT, computed tomography; DFO, desferrioxamine; ESRD, end-stage renal disease; rHuEPO, recombinant human erythropoietin.
INTRODUCTION Anaemia, one of the most common complications of endstage renal disease (ESRD), renders nearly 25% of dialysis patients dependent on erythrocyte transfusions to sustain life [l].Transfusional iron overload is a serious problem for a substantial minority of the dialysis population [2]. Recombinant human erythropoietin (rHuEPO) was demonstrated to be remarkably effective in correcting dialysis-associated anaemia [l, 3-10], as well as trans-
S. De Marchi and E. Cecchin
114
Association and the European Renal Association, Vienna, Austria, 5-9 September 1990, and were published in abstract form [16a].
fusional iron overload [ 11-14]. Recently, we demonstrated that in haemodialysis patients the assessment of hepatic density by computed tomography ( C T ) is an accurate and non-invasive alternative to liver biopsy that permits estimation of iron storage directly [15, 161. Lazarus et al. [12] observed a fall in hepatic C T density and serum ferritin level in five iron-overloaded haemodialysis patients during 9-14 months of rHuEPO treatment in combination with phlebotomy. This finding was confirmed by Nomura ef al. [13] in a haemodialysis patient with severe iron overload and ascites. However, to date the usefulness of CT scanning in monitoring the iron status of haemodialysis patients receiving rHuEPO treatment or desferrioxamine (DFO) chelation therapy for transfusional iron overload has not been established. The present study was undertaken in a group of uraemic patients with dialysis-associated anaemia and moderate iron overload to evaluate the effects of rHuEPO treatment and of DFO chelation therapy on hepatic iron storage measured by CT scanning, as well as by serum ferritin levels and transferrin saturation. The purpose of the study was to investigate the usefulness of hepatic CT density in monitoring the iron status of haemodialysis patients with transfusional haemosiderosis treated with rHuEPO or DFO. Parts of this study were presented at the 27th Annual Congress of the European Dialysis and Transplant
METHODS Study protocol
A randomized, partial-crossover study was conducted in uraemic patients with dialysis-associated anaemia and transfusional haemosiderosis to compare the effects on tissue iron storage of rHuEPO treatment and DFO chelation therapy. The experimental design is shown in Fig. 1. Twenty-one patients with moderate iron overload were randomly allocated to three groups by a table of random numbers. After a 2-week run-in period for baseline measurements, the three groups of patients of equal number ( n= 7) were followed for 12 months. During the first 6 months (phase 1)group 1 received DFO chelation therapy, group 2 underwent rHuEPO treatment, and group 3 was on a placebo. Thereafter, in the second 6 months of observation (phase 2) the patients in group 1 were switched over to the rHuEPO treatment protocol. Because of a poor response in the group receiving DFO chelation therapy in the initial 6 months, patients on rHuEPO treatment were not switched over to the DFO chelation therapy protocol, but continued on the maintePhase 2
Phase 1
Haemodialysis patients ( n= 21):
Group 1 ( n= 7)
rHuEPO
DFO
Group 2 ( n= 7)
rHuEPO
rHuEPO
Group 3 ( n= 7 )
Placebo
Placebo
Investigations: Hepatic CT density Serum ferritin concn. Transferrin saturation Haematological indices
* * * *
* * * *
* * * *
I
l
l
l
l
l
l
l
l
l
0
1
2
3
4
5
6
7
8
9
l
l
l
1 0 1 1 1 2
Time (months)
Fig. 1. Study design. During the first 6 months group 1 received DFO chelation therapy (30 mg/ kg intravenously at the end of each dialysis three times a week), group 2 underwent rHuEPO treatment (rHuEPO was administered intravenously three times a week at the end of each dialysis starting with 36 units/kg, patients were continued on this dose until the packed cell volume reached 30%, and thereafter rHuEPO treatment was reduced to the maintenance dose of 18 units/kg), and group 3 was on a placebo. In the second 6 months group 1 was switched over to the rHuEPO treatment protocol, group 2 continued on the maintenance dose of rHuEPO, and group 3 was maintained on placebo.
Hepatic computed tomography during recombinant human erythropoietin treatment nance dose of rHuEPO until the end of the trial. Patients in group 3 were maintained on placebo even in phase 2 of the study. Hepatic CT density, haemoglobin, packed cell volume, transferrin saturation and serum ferritin concentration were measured in each of the patients before and after each 6-month course. All blood samples were obtained immediately before dialysis. In the DFO chelation therapy protocol patients received intravenous DFO at a dose of 30 mg/kg over 30-40 min after the termination of each dialysis three times weekly for 6 months. In the rHuEPO treatment protocol patients received intravenous rHuEPO (Boehringer Mannheim, Italia SPA, Milan, Italy) three times weekly at the end of each dialysis starting with a dose of 36 units/kg. Patients were continued on this dose until the packed cell volume reached or exceeded 30%. Thereafter, rHuEPO treatment was reduced to a maintenance dose of 18 units/kg in all patients, except one, until the end of the 6-month course of therapy. In one patient from group 1, who did not have a positive response, as indicated by an increase in haemoglobin of less than 2 g/dl, the dose of rHuEPO was doubled every second week until a dose of 144 units/kg was reached. This study was conducted in accordance with the principles of the Declaration of Helsinki of the World Medical Association.
115
flow was set at 500 ml/min. The ultrafiltration rate was standardized at 4 ml/min. None of the patients had evidence of aluminium-related toxicity or of positive DFO aluminium challenge. Serum aluminium concentrations before DFO challenge ranged from 10 to 45 pg/l (meanIf:sD, 31 f 13 pgll). The values of hepatic CT density ranged from 69 to 81 Hounsfield units ( m e a n k s ~ ,76.3k 3.7 Hounsfield units). In a control group of 40 healthy subjects, well matched for age and sex, the values of hepatic CT density ranged from 53 to 65 Hounsfield units (mean fSD, 60.2 f 5.6 Hounsfield units). In these subjects the mean ( fSD) values of serum ferritin concentration and transferrin saturation were 100 (If:59) pg/l and 28 ( k 9)%. Estimation of hepatic CT density The C T scan of each patient was obtained by a GE CT/T 8000 scanner with a 4.5 s scan time according to the usual protocols using 8 mm thick slices. No intravenous contrast material was given. Five 1cm2 areas were selected at the periphery of the liver slice, approximately 1 cm from the liver edge, avoiding obvious bile ducts, vascular structures, fissures and infiltrations. The CT densities of these areas were averaged to obtain the hepatic CT density.
Patient selectior. Twenty-one patients with ESRD on maintenance haemodialysis, who had evidence of iron overload, confirmed by values of serum ferritin concentration, transferrin saturation and hepatic C T density exceeding 1000 pg/l, 45% and 68 Hounsfield units, respectively, were selected for the study. All patients showed moderate levels of iron excess, manifested by mean values of serum ferritin concentration and transferrin saturation of 1391 pg/l (range 1004-3000 pg/l) and 62% (range 48-95%), respectively. None had evidence of symptomatic haemosiderosis. More specifically, they had no clinical evidence of myopathy, arthropathy, cardiomyopathy or pancreatic insufficiency related to iron deposition. They were the only patients in our unit with elevated values of serum ferritin concentration, transferrin saturation and hepatic CT density that fulfilled the following criteria: no history of alcohol abuse, absence of obesity ( > 20% increase in relative weight), and no evidence of erythrocyte microcytosis or hypochromia, diabetes mellitus, liver disease, malignancy or chronic inflammatory disease. They ranged in age from 25 to 76 years ( m e a n f s ~51.6 , k 10.7 years). Nine of the patients were male and 12 were female. One patient was anephric. They required an average transfusion of 1.5 ( If:0.3) units of concentrated erythrocytes over the 6 months before the study to maintain their packed cell volume at an acceptable level. The lifetime estimated transfusional iron load was 2952k983 mg. No iron supplementation was administered during the course of the study. Patients were given our standard 4 h haemodialysis treatment three times weekly with an 8 pm, 1.0 m2 hollow fibre cuprophane dialyser throughout the trial. The blood flow was set at 300 ml/min and the dialysate
Laboratory investigations Serum iron concentration, total iron-binding capacity and transferrin saturation were determined by the methods of the International Committee for Standardization in Hematology [17]. Serum ferritin concentration was measured by an r i a . method using a commercial kit (Becton Dickinson, Milan, Italy). The 95% confidence limits for normality in both sexes were 18-185 pg/l. Serum aluminium concentration was determined by atomic spectrophotometry with a graphite furnace. Blood cell counts were performed by a Coulter counter. Statistical analysis Results are expressed as mean sf^^^. Paired and unpaired two-tailed Student’s t-tests were used to compare group means and to assess the statistical significance of changes. In the analysis of the effects of rHuEPO treatment, and DFO chelation therapy, we compared changes in the treated groups with changes in the placebo group. In addition, when appropriate, tests were done by comparing groups 1 and 2, and changes in each group, separately. A P value of less than 0.05 was considered to indicate a significant difference. RESULTS Changes in hepatic C T density, serum ferritin concentration, transferrin saturation, haemoglobin and packed cell volume during the course of DFO chelation therapy and rHuEPO treatment are shown in Table 1 and in Figs. 2 and 3.
SEM
Group 3 1 2 3 4 5 6 7 Mean
SEM
Group 2 1 2 3 4 5 6 7 Mean
SEM
Group 1 1 2 3 4 5 6 7 Mean
Patient no.
45/F 45/M 68/F 45/F 61/M 50/M 42/F
51/F 60/F 49/F 51/F 25/M 76/M 62/M
49/F 52/F 61/F 42/F 50/M 54/M 45/M
Age (years)/ sex
3015 826 954 2523 1100 875 807 1442 349
After
90 49 48 55 56 51 92 63 7
After
840 3000 960 1150 810 804 2000 1366 316
Before 407 1980 172 476 305 312 761 630 236
After
2950 810 805 889 954 826 2430 1380 344
Before 3180 850 865 830 920 832 2650 1446 384
After
First 6-month course of placebo
Before
After 41 66 53 52 37 41 56 49 4
96 51 52 61 59 54 95 67 8
2800 900 899 2600 1030 895 853 1425 33 1
Before 79.5 81 77 70.5 76.5 76 70.5 75.8 1.5
Before
80.5 75 69 77.5 77.5 77.5 80.5 76.8 1.5
Before
79.5 80 77.5 77 69 74 76.5 76.2 1.4
Before
81 75 70 77.5 77 77 80 76.8 1.4
After
49.5 47.5 53 53.5 54 53 56.4 52.4 1.1
After
80.5 80.5 77.5 68 76.5 74 69 75.2 2.0
After
Hepatic CT density (HU)
First 6-month course of rHuEPO treatment
92 51 50 86 68 50 51 64 7
After
59 95 66 77 55 47 95 71 7
Before
90 72 54 49 68 7
55
95 58
Before
Serum ferritin concn. (pgll)
6-month course of DFO chelation therapy
Transferrin saturation ( O h )
90 49 48 55 56 51 92 63 7
Before
41 66 53 52 37 41 56 49 4
Before
92 51 50 86 68 50 51 64 7
Before
Serum ferritin concn. ( p g l l )
3015 826 954 2523 1100 875 807 1442 349
Before 1945 400 154 2466 416 300 280 851 356
After
80.5 80.5 77.5 68 76.5 74 69 75.2 2.0
Before
407 1980 172 476 305 312 76 1 630 236
Before
120 617 65 150 93 85 180 187 73
After
49.5 47.5 53 53.5 54 53 56.4 52.4 1.1
Before
96 59 47 53 55 55 94 66 8
After
3180 850 865 830 920 832 2650 1446 384
Before
3050 830 835 875 941 830 2590 1421 365
After
81 75 70 77.5 77 77 80 76.8 1.4
Before
Second 6-month course of placebo
11 44 37 28 18 15 41 28 5
After
Second 6-month course of rHuEPO treatment
65 47 28 88 48 25 24 46 9
After
81 75 70 77 77 78 79 76.7 1.3
After
48.5 46.5 51 51.5 50 51 54 50.4 0.9
After
47.5 50.5 53.5 72.5 53 51 47.5 53.6 3.3
After
Hepatic CT density (HU)
6-month course of rHuEPO treatment
Transferrin saturation ( O h )
Table 1. Transferrin saturation, serum ferritin concentration and hepatic CT density in the three groups of haemodialysis patients before and after each phase of the study
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Time (months)
Fig. 2. Changes in transferrin saturation ( a ) , serum ferritin concentration ( b )and hepatic CT density (c) in the three groups of haemodialysis patients (0, group 1; 0 , group 2; 0 , group 3) over the full experimental period. Statistical significance: * P < 0.05, **P< 0.005, ***P< 0.0001 compared with placebo.
Effects on hepatic CT density
The mean baseline levels of hepatic CT density did not differ among the three groups of haemodialysis patients as shown in Table 1. The mean ( fSEM) values were significantly higher than that observed in healthy subjects (60.2 f 1.3 Hounsfield units, P < 0.0001). In group 1 patients the 6-month course of DFO chelation therapy did not induce any change in the mean value of hepatic CT density as compared with that in the placebo group. Furthermore, in this group of patients there was no difference in the mean value of hepatic CT density between the beginning and end of phase 1. Conversely, in these patients the subsequent rHuEPO treatment induced a significant fall in the mean value of hepatic CT density as compared either with that in the placebo group (53.6 f 3.3 versus 76.7 f 1.3 Hounsfield units, P< 0.0001) or with the value previously obtained in the same group of patients at the end of the course of DFO chelation therapy (53.6 f 3.3 versus 75.2 f2 Hounsfield units, P< 0.0001). More specifically, six patients returned to low-normal levels of hepatic CT density at the end of the rHuEPO treatment, and the mean ( fSEM) value of the individual decreases was 25.8 ( f 1.8) Hounsfield units. In
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1 2 3 4 5 6 7 89101112 Time (months)
1
1
1
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Fig. 3. Changes in haemoglobin ( a ) and packed cell volume ( b )in the three groups of haemodialysis patients (0,group 1; 0 , group 2; 0 , group 3) over the full experimental period. Statistical significance: *P< 0.001, **P< 0.0001 compared with placebo.
one patient, hepatic CT density, serum ferritin concentration, transferrin saturation, as well as haemoglobin and packed cell volume, were not influenced by rHuEPO treatment. This patient had evidence of severe hyperparathyroidism. In group 2 patients the first 6-month course of rHuEPO treatment induced a significant decrease in the mean value of hepatic CT density as compared either with that in the placebo group (52.4k 1.1 versus 76.8 f 1.4 Hounsfield units, P
