Arch Dis Child: first published as 10.1136/adc.63.3.250 on 1 March 1988. Downloaded from
Archives of Disease in Childhood, 1988, 63, 250-255
Depletion of trace elements and acute ocular toxicity induced by desferrioxamine in patients with thalassaemia
S DE VIRGILIIS, M CONGIA, M P TURCO, F FRAU, C DESSI, F ARGIOLU, R SORCINELLI,* A SITZIA,* AND A CAO Istituto di Clinica e Biologia dell Eta' Evolutiva, Universita' Studi Cagliari, and *Istituto di Clinica Oculistica, Universita' Studi Cagliari, Cagliari, Sardinia, Italy
SUMMARY High doses of intravenous desferrioxamine infused over a short period of time induce a large faecal and urinary iron excretion but also produce retinal abnormalities that are characterised by decreased amplitude on electroretinography and defective dark adaptation. This regimen also results in high faecal iron, zinc, and copper excretion, and reduced granulocyte zinc concentrations and alkaline phosphatase activity. The retinal abnormalities may be related to the zinc and copper deficiency and/or iron depletion 'per se' which interferes negatively with critical iron dependent enzymes.
Subcutaneous infusion of desferrioxamine is the comply with the regimen of subcutaneous adminis- most efficient method for reducing the iron burden tration. in patients with thalassaemia major who are trans- fusion dependent.1 The daily infusion of 40- Patients and methods 60 mg/kg over 12 hours for six days a week is usually sufficient to obtain iron balance.'v Because some Fifteen children, aged from 9 to 16 years, with http://adc.bmj.com/ patients fail to comply with this regimen the addi- transfusion dependent thalassaemia major and tional administration of large doses by intravenous moderate iron overload (serum ferritin concentra- infusion has been proposed.3 High doses of desferri- tions from 1100 to 2000 [tg/l), because of low oxamine, either by intravenous or subcutaneous compliance with daily infusion of subcutaneous administration, however, may result in optic neuro- desferrioxamine, were treated by a combination of pathy or high frequency sensorineural hearing loss, traditional daily subcutaneous infusion and monthly or both.7 8 Likewise, in rabbits anaesthetised with intravenous administration of very large doses on October 1, 2021 by guest. Protected copyright. urethane, the intravenous infusion of large doses of according to a previously published scheme.11 Each desferrioxamine produced a reversible reduction in patient was treated for 10 consecutive months but the amplitude of the electroretinogram.9 Toxic the balance study (see later) was carried out only concentrations are probably reached when the dose once. of desferrioxamine administered is disproportion- The dose of desferrioxamine for subcutaneous ately high in relation to the amount of iron available infusion was 40-60 mg/kg over a period of 12 hours for chelation. In these conditions desferrioxamine each night for six days a week. The intravenous may chelate critical trace elements such as copper infusion was given over a period of 24 hours at the and zinc,10 the depletion of these elements may be dosage of 10-12 g (200-450 mg/kg) dissolved in responsible for the neurotoxic effect of the drug. 750 ml of 5% glucose solution. Urinary and faecal In this study we monitored the development of iron concentrations, zinc and copper excretion, ocular abnormalities and possible zinc or copper serum zinc pattern, leukocyte zinc concentrations, depletion, or both, in a group of patients with and alkaline phosphatase activities were analysed thalassaemia major who were chelated with des- only once, as detailed later, before and after ferrioxamine by a combination of traditional daily the infusion of intravenous desferrioxamine when subcutaneous infusion and monthly intravenous we introduced the combination of both intravenous administration of large doses because of failure to and subcutaneous administration. 250 Arch Dis Child: first published as 10.1136/adc.63.3.250 on 1 March 1988. Downloaded from
Depletion of trace elements and acute ocular toxicity in patients with thalassaemia 251 Ophthalmic evaluation was also carried out use, all glassware and plastic containers were acid before and after the termination of the infusion, but washed in 1-0 N hydrochloric acid for 30 minutes this assessment was repeated from two to three and rinsed six times in deionised water. times at times of successive intravenous infusion and Blood samples from peripheral veins were col- again one, two, and three months after we inter- lected with plastic syringes and stainless steel rupted the intravenous infusions while continuing needles and put into plastic tubes. Serum and the subcutaneous administration. granulocyte zinc concentrations and granulocyte alkaline phosphatase activity were measured in OPHTHALMOLOGIC ASSESSMENT samples taken before and at the end of the The ophthalmic assessment included determina- intravenous infusion as well as one month later. tion of the best corrected visual acuity by standard Basal iron, zinc, and copper excretion were methods, pupillary reaction, ocular motility, slit evaluated in urine and faeces collected for three lamp examination for changes in the anterior days before the infusion of desferrioxamine. The segment, and visual field measurement on a cali- effect of this infusion was determined by measuring brated Goldman kinetic perimeter with light inten- these elements in the urine and faeces collected on sity regulated at 31-5 apostilb. A decrease above the day of the infusion as well as three days after. 20% in the average peripheral extent of the field in During this study period the subcutaneous infusion at least two quadrants was considered abnormal. was interrupted. Adapted electroretinograms were obtained accord- An aliquot of the preinfusion and postinfusion ing to Babel12 with flashes of white light of constant urinary pools was prepared and frozen for further colour range and intensity of 2 joule, 1 hertz/second analysis. The faeces were homogenised with a at 30 cm from ocular plane (Pantops 200-Ferlux known amount of deionised water. An aliquot was Instruments). The amplitudes of the response were removed and dried at 100°C overnight and then considered abnormal when the value was decreased ashed in a muffle furnace at 550°C and left over- more than 20% from normal values (190 (SD15) ,uV). night. The resultant ash was dissolved with 10% During dark adaptation, after a preliminary light hydrochloric acid, transferred to a volumetric flask, adaptation, we noted the stage at which the ampli- and made up to volume with demineralised water. tude of the slow scotopic positive wave (the scotopic Serum, urinary, and faecal iron, zinc, and copper components of the electroretinogram are mainly due were determined by atomic absorption (IL 551 to the activity of the rods in dark adaptation) Spectrophotometer). Granulocyte alkaline phos- reached that of the photopic component. This is phatase activity was assayed with p-nitrophenyl- referred to as the 'transition phenomenon,' which is phosphate as substrate according to It-Koon and http://adc.bmj.com/ a valuable sign for the judgment of the scotopic Moss.14 Granulocytes were separated from the function. 13 other blood cells according to Boyum.'5 The speci- mens used for the analysis contained a percentage of METABOLIC STUDIES granulocytes varying from 90 to 100%. Statistical In order to carry out copper, zinc, and iron balance analysis was carried out by Student's paired t test. studies each patient was admitted to the hospital for
a week. With the aim of eliminating any residual Results on October 1, 2021 by guest. Protected copyright. effect of the subcutaneous infusion the day before admission the daily infusion of subcutaneous des- OPHTHALMOLOGIC EVALUATION ferrioxamine was interrupted. While in the hospital Visual acuity and average field size were normal in the patients were on a strict controlled diet con- all subjects. No isolated field defect (scotomata) or taining roughly 8-10 mg/day iron, 0-1 mg copper/kg enlargement of the blind spot were detected. body weight, and 0-3-0-4 mg zinc/kg body weight. Fundoscopy showed no pigment abnormalities, Diet and collection of the urine and stool were focal lesions, macular oedema, or unusual vascular carefully controlled by nurses who were specifically patterns. In three patients the electroretinograms instructed to avoid any faecal contamination with showed a decreased amplitude before and at the urine. Collection periods of stools were defined by. end of the desferrioxamine infusions as well as at administering radio-opaque markers. follow up examinations carried out at one, two, and Precautions against environmental contamination three months later. Seven patients, who had a were taken for all diet, blood, urine, and stool normal electroretinogram before the infusion, collections and analyses. Specimens were stored showed a decreased amplitude at the evaluation only in plastic containers and only low mineral carried out when the infusion was terminated. The content water and ultra high purity reagents were same pattern was recorded at least twice at each used in sample and standards preparation; before successive infusion. In these subjects a normal Arch Dis Child: first published as 10.1136/adc.63.3.250 on 1 March 1988. Downloaded from
252 De Virgiliis, Congia, Turco, Frau, Dessi, Argiolu, Sorcinelli, Sitzia, and Cao 240- electroretinogram was seen at follow up examina- tion after the termination of intravenous treatment (fig 1). 200- In the three patients with permanent abnormali- ties shown on their electroretinograms and in six out of the seven with the transient effects we noted 200- when control measurements were taken immedi- ately after infusion of intravenous desferrioxamine a 180- 'transition phenomenon' appeared after 14 to 18 minutes in the dark. In our age matched controls this phenomenon occurred from a minimum of 8 to a 160- maximum of 10 minutes. The remaining five patients did not show any abnormalities of their electroretinograms. 140- METABOLIC STUDIES Fig 2 shows the effeLt of the intravenous infusion of 120- desferrioxamine on serum zinc concentrations. The patients were divided into three groups: those with 100- no retinal abnormalities, those who showed a decreased amplitude on an electoretinogram after infusion of desferrioxamine, and those with per- 80 manent abnormalities shown on an electroretino- gram. During the 24 hours of the infusion all serum Before infusion After infusion One month zinc concentrations fell to within the normal ranges after infusion (84.2 (SD 12) [tg% (12-9 (1-8) ,tmol/1)). During this Fig 1 Effect ofintravenous infusion ofdesferrioxamine on period fluctuations in zinc concentrations occurred the amplitude on electroretinography. Patients with normal with peak concentrations in the morning and the amplitude (@); patients with transitory decreased amplitude lowest concentrations in the afternoon. No signifi- (A); and patients with persistent decreased amplitude (*). cant differences in the zinc concentrations or the
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0cm on October 1, 2021 by guest. Protected copyright. 2 100-
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60- I I I I I I --" 8 10 12 14 16 18 20 22 24 2 4 6 8 Hours Fig 2 Effect of intravenous injusion of desferrioxamine on serum zinc concentrations. Patients with normal amplitude on electroretinography (@); patients with transitory decreased amplitude (A); and patients with persistent decreased amplitude (*). (Results are in Mg%; to convert to ,molll multiply by 0153.) Arch Dis Child: first published as 10.1136/adc.63.3.250 on 1 March 1988. Downloaded from
Depletion of trace elements and acute ocular toxicity in patients with thalassaemia 253 zinc pattern were observed between the three and zinc; p<0.01 for copper) than in the first group groups of subjects investigated. (p<0-01). In the second group there was an inverse The effects of desferrioxamine infusion on iron, correlation between total zinc and iron elimination; zinc, and copper excretion are summarised in fig 3. this, however, did not reach significance. Those After infusion of desferrioxamine there was a subjects who had persistent abnormalities on their significant (p<0001) increase of mean urinary and electroretinograms had less iron, copper, and zinc faecal iron excretion. On average 45% of the iron excretion both in urine and faeces when compared was eliminated through the urine and 55% with the with the other two groups. stools. After the infusion, faecal and urinary zinc In all patients granulocyte zinc content and and copper excretion were also significantly in- creased (p<0-001 and <005, respectively). Con- Table Effect of intravenous infusion of desferrioxamine sidering all the patients together no significant on zinc concentrations and alkaline phosphatase activity in correlation was observed between zinc and iron granulocytes elimination both in faeces and urine. Zinc Alkaline phosphatase If the patients were divided according to the (timol/g activity abnormalities shown on their electroretino- protein) (sM/minutelg protein) grams-that is, the first group without abnormali- Patients with thalassaemia ties, the second group with transitory alterations Basal* 6-53 (1-83) 20 (8-21) after intravenous infusion, and the third group with At the end of the the infusiont -4-86 (1-8) -15-23 (9-15) permanent abnormalities- it can be seen that 1 month after second group of patients had significantly (p<005) infusion* 0-41 (2-27) 2-36 (14-5) higher mean urinary zinc elimination compared with Controls the first group; there were no differences observed 6-92 (1-75) 23 (3.8) in iron and copper elimination. Mean faecal excre- *Results are mean (SEM). tions of all the three trace elements were signifi- tResults are mean differences from the basal value (standard errors cantly higher in the second group (p<0-001 for iron of the differences).
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5ol F I I 1 1 1 1ol12 I Ixcrvtio n Daily iron excretion Daily zinc excretion - Daily copper excretion Fig 3 Effect ofintravenous infusion ofdesferrioxamine on iron, copper, and zinc excretion. Patients without abnormalities on electroretinography (@); patients with transitory alterations (A); patients with permanent abnormalities (*). Patients had baseline concentrations higher than normal because they had stopped subcutaneous administration ofdesferrioxamine only 24 hours previously. Arch Dis Child: first published as 10.1136/adc.63.3.250 on 1 March 1988. Downloaded from
254 De Virgiliis, Congia, Turco, Frau, Dessi, Argiolu, Sorcinelli, Sitzia, and Cao alkaline phosphatase activity were within the normal organism'9 decreased significantly after the infusion ranges at the outset of the infusion and decreased of desferrioxamine. We cannot exclude, however, significantly (p
because during the balance study the patients were suggest, however, that zinc depletion induced by on October 1, 2021 by guest. Protected copyright. in the hospital and urine and faecal collections were desferrioxamine in the retinal pigment epithelium carefully controlled by a nurse who had been may have played a part in producing the transient adequately instructed. Furthermore it is already abnormalities that we have seen on electroretino- known from a study carried out by Pippard et al that graphy. large doses of desferrioxamine result in an increase Blake et al, who studied a patient with retinal of faecal iron loss but have little effect on urinary toxicity induced by desferrioxamine, also found elimination. 18 An inverse but not significant correla- abnormalities in the loosely bound iron and copper tion between iron and zinc excretion was observed content of the cerebrospinal fluid.25 Copper and solely in those subjects with transient abnormalities iron complexes may stimulate an autocatalytic on electroretinography. process of lipid peroxidation and might therefore be Increased copper and zinc excretion may be responsible for retinal abnormalities in patients explained by assuming that the rapid infusion of treated with desferrioxamine.26 large doses of desferrioxamine left a certain amount Desferrioxamine acts on a labile iron pool, which of the drug non-iron chelated and thus able to is presumably intermediate in the interchange of chelate these trace elements. Accordingly granulo- iron molecules between storage and transport cyte alkaline phosphatase activity, which is one of forms.27 Acute iron depletion of this pool may have the best indicators of the zinc content of the a negative effect on the iron dependent enzymes and Arch Dis Child: first published as 10.1136/adc.63.3.250 on 1 March 1988. Downloaded from
Depletion of trace elements and acute ocular toxicity in patients with thalassaemia 255 help cause the production of retinal anomalies. We De Virgiliis S, Frau F, Sanna G, et al. Iron elimination following have excluded the intravenous desferrioxamine infusion in patients with thalass- possibility that ocular abnormali- emia major. European Paediatric HaematolylOncology 1985;2: ties in our patients were related to a diabetic or 47-8. prediabetic condition via the disruption of the 9 Olivieri NF, Buncic JR, Chew E, et al. Visual and auditory integrity of the blood-retinal barrier, as has been neurotoxicity in patients receiving subcutaneous deferoxamine proposed,28 because none of our had a infusions. N Engl J Med 1986;314:869-73. patients 10 Arden GB. Desferrioxamine administered intravenously by reduced glucose tolerance (data not shown). infusion causes a reduction in the electroretinogram in rabbits A limited number (three out of 15) of the patients anaesthetized with urethane. Hum Toxicol 1986;5:229-36. investigated showed persistent abnormalities on " Zaino EC. Desferrioxamine and trace metal excretion in electroretinography. Because it is difficult to assess chelation therapy in chronic iron overload. In: Zaino EC, Roberts RH, eds. Chelation and chronic iron overload. New the compliance to treatment we do not know York: Ciba Medical Horizons Symposium, 1977:95-107. whether these patients differed from those with 12 Babel JM, Stangos M, Korol S, Spiritus M. Ocular electro- transient abnormalities in the amount of desferri- physiology. Stuttgart: G Thiene Publishers, 1977:34-5. oxamine they received by the daily subcutaneous 3 Auerbach E. The human electroretinogram in the light and during dark adaptation. Doc Ophthalmol 1967;22:1-4. infusion in the years preceding the trial. Those 14 It-Koon T, Moss DW. The estimation of intestinal alkaline patients with persistent abnormalities did not differ, phosphatase in human blood serum. Clin Chim Acta 1969;25: however, from those with transient modifications in 177-9. the serum ferritin concentrations, the zinc 15 Boyum A. A one stage procedure for isolation of granulocytes content, and lymphocytes from human blood. Scand J Clin Lab Invest and alkaline phosphatase activity of leukocytes, but 1968;97(suppl 21):51-3. showed lower zinc and iron excretion after intra- 16 Lakhanpal V, Schocket SS, Jiji R. Deferoxamine (Desferal) venous infusion of desferrioxamine. This may induced toxic retinal pigmentary degeneration and presumed indicate the presence of less iron accumulation and a optic neuropathy. Ophthalmology 1984;91:443-51. 17 Markowitz ME, Rosen JF, Mizruchi M. Circandian variations in borderline zinc reserve, which may nevertheless serum zinc (Zn) concentrations: correlation with blood ionized result in defective function of those tissues, such as calcium, serum total calcium and phosphate in humans. Am J the retinal epithelium, which are most sensitive to Clin Nutr 1985;41:689-96. zinc depletion. 18 Pippard MJ, Callender ST, Finch CA. Ferrioxamine excretion in iron-loaded man. Blood 1982;60:288-94. In conclusion, administration of large doses of 19 Prasad AS. Clinical, biochemical and nutritional spectrum of desferrioxamine by intravenous infusion are able to zinc deficiency in human subjects: an update. Nut Rev produce in iron overloaded thalassaemia patients a 1983;41:197-208. very large iron excretion but they also induce 20 Karciogluo ZA. Zinc in the eye. Surv Ophthalmol 1982;27: 114-22. excretion of trace elements such as copper and zinc 21 Leure-dePree AE, McClain CJ. The effect of severe zinc
and cause transient ocular abnormalities. For those deficiency on the morphology of the rat retinal pigment http://adc.bmj.com/ patients treated with this regimen ophthalmological epithelium. Invest Ophthalmol Vis Sci 1982;23:425-34. evaluation 22 Wong Jr EK, Leopold IH. Zinc deficiency and visual dys- is recommended for determining early function. Metabolic and Pediatric Ophthalmology 1979;3:1-4. retinal toxicity induced by desferrioxamine. Further 23 Warth JA, Prasad AS, Zwas F, Frank RN. Abnormal dark studies are needed in order to assess whether a adaptation in sickle cell anemia. J Lab Clin Med 1981;98: reduction of the desferrioxamine dosage may avoid 189-94. these toxic effects. 24 Silverstone BZ, N.Bursen D, Seelenfreund MH. Plasma zinc levels in high myopia and retinitis pigmentosa. Medical and References Pediatric Ophthalmology 1981;5:187-90. Propper RD, Cooper B, Rufo RR, et al. Continuous subcuta- 25 Blake DR, Winyard P, Lunec J, et al. Cerebral and ocular on October 1, 2021 by guest. Protected copyright. neous administration of desferrioxamine in patients with iron toxicity induced by desferrioxamine. Q J Med 1985;56:345-55. overload. N Engl J Med 1977;297:418-23. 26 Gutteridge JMC. Fluorescent products of phospholipid per- 2 Graziano JH, Markenson A, Miller DR, et al. Chelation oxidation: Formation and inhibition in model systems. In: therapy in 3-thalassemia major. I. Intravenous and subcuta- Armstrong D, Koppang N, Rider JA, eds. Ceroid lipofuscinosis neous deferoxamine. J Pediatr 1978;92:648-52. (Batten's disease). Amsterdam: Elsevier, Biomedical Press, 3 Cohen A, Martin M, Schwartz E. Response to long term 1982:351-64. desferrioxamine therapy in thalassemia. J Pediatr 1981;99: 27 Laub R, Schneider YJ, Octave JN, Trouet A, Crichton RR. 689-94. Cellular pharmacology of deferrioxamine B and derivatives in 4 Modell B, Letzky AE, Flynn DM, Peto R, Weatherall DJ. cultured rat hepatocytes in relation to iron mobilization. Survival and desferrioxamine in thalassemia major. Br Med J Biochem Pharmacol 1985;34:1175-83. 1982;284:1081. 28 Arden GB, Wonke B, Kennedy C, Huehns ER. Ocular changes 5 Fargion S, Taddei MT, Gabutti V, et al. Early iron overload in in patients undergoing long-term desferrioxamine treatment. 13-thalassaemia major: when to start chelation therapy? Arch Dis Br J Ophthalmol 1984;68:873-77. Child 1982;57:929-33. This work was partially financed by the Assessorato Igiene e De Virgiliis S, Cossu P, Toccafondi C, et al. Effect of Sanita'-Regione Sardegna. subcutaneous desferrioxamine on iron balance in young thalass- emia major patients. Am J Pediatr Hemnatol Oncol 1983;5:73-7. Correspondence to Professor A Cao, Instituto di Clinica c Biologia 7 Davies SC, Marcus RE, Hungerford JL, Miller MH, Arden GB, dell' Eta' Evolutiva, Universita' Studi Cagliari, Via Jenner s/n, Huehns ER. Ocular toxicity of high dose intravenous desferri- 1-09100 Cagliari, Sardinia, Italy. oxamine. Lancet 1983;ii:181-4. Accepted 16 September 1987