PEDIATRICS Vol. 105 No. 2 February 2000, pp. 398-401

Exogenous Apotransferrin and Exchange Transfusions in Hereditary Iron Overload Disease

Vineta Fellman, MD, PhD^*, Leni von Bonsdorff, and LicTech; and Jaakko Parkkinen, MD, PhD

From the ^* Hospital for Children and Adolescents, University of Helsinki, and the  Finnish Red Cross, Blood Transfusion Service, Helsinki, Finland.

	ABSTRACT

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Objective.  To investigate whether apotransferrin administration and exchange transfusion can improve outcome in patients with the recently described recessive congenital iron overload disease, presenting with intrauterine growth retardation, severe lactic acidosis, aminoaciduria, and hemosiderosis of the liver that so far has been treatment-resistant and lethal.

Methodology.  Because the patients have hypotransferrinemia, hyperferritinemia, increased transferrin saturation, and bleomycin detectable iron in plasma, we designed a treatment regime aiming at decreasing free iron and iron overload. The serum transferrrin concentration was increased to adult level (2-5 g/L) by intravenous apotransferrin administrations and thereafter exchange transfusion was performed.

Results.  Two patients were treated. In patient 1, the transferrin saturation decreased from a baseline value of 100% and remained normal after the third exchange transfusion, and in patient 2, a reversible beneficial effect was seen on transferrin saturation and bleomycin-detectable iron. However, both infants died later of the disease, at 10 and 8 weeks of age, respectively.

Conclusions.  Exogenous apotransferrin administration proved to be safe and might deserve evaluation in other neonatal diseases with presence of free iron in plasma.hemosiderosis, metabolic acidosis, newborn infant, transferrin.

We recently described the clinical picture of a new genetic disease in a patient series including 17 newborn infants of 12 Finnish families.¹ The diagnostic criteria of the disease are severe fetal growth retardation (mean ± standard deviation [SD] birth weight SD score for gestational age: 3.8 ± .6), profound lactic acidosis (arterial pH 7.00 ± .12 and blood lactate 12.2 ± 7.5 mmol/L by 24 hours of age), Fanconi type aminoaciduria and iron overload with hemosiderosis of the liver, increased serum ferritin concentration, hypotransferrinemia, and increased transferrin iron saturation. All infants failed to thrive, had treatment-resistant acidosis, and died either early at 2 to 12 days of age (n = 9) or in infancy at 1 to 4 months of age (n = 8). Genealogical studies indicated an autosomal recessive mode of inheritance for this disease, which is clinically distinct from other lactic acidoses, neonatal hemochromatosis, and hepatitis.¹ Ongoing genome scan has recently revealed that the disease gene mutation in the Finnish patients is located to chromosome 2.² The metabolic disturbance responsible for the disease remains unidentified. We hypothesized that the observed organ dysfunction may be partly attributable to the toxic effects of free iron. Here, we aimed to investigate whether reducing the iron overload with administration of apotransferrin could improve the outcome.

	METHODS

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Patients born in or after 1995 admitted to the Hospital for Children and Adolescents, University of Helsinki, were included in the treatment study. The apotransferrin treatment was approved by the National Agency for Medicine in Finland, and informed consent was obtained from both parents in accordance with recommendations of the ethical committee of the hospital.

The treatment protocol aiming at decreasing the total body and plasma free iron content included an apotransferrin infusion, calculated to increase the serum transferrin concentration to a high normal adult level of 3 to 5 g/L, followed by an exchange transfusion (200 mL blood per kg weight) the next day. This procedure was repeated when the transferrin saturation exceeded 60% despite maintaining the transferrin serum level at 2 to 3 g/L with repeated apotransferrin infusions. Iron-free virus-inactivated apotransferrin, purified from human plasma, was provided by the Finnish Red Cross Blood Transfusion Service (Helsinki, Finland). The reconstituted solution contained 50 mg/mL apotransferrin and 250 to 400 mg/kg were given as a slow intravenous infusion over 2 to 12 hours, with continuous arterial blood pressure, heart rate, and skin temperature monitoring.

Daily blood samples were obtained in iron-free tubes for evaluation of iron metabolism. Serum iron, transferrin, transferrin saturation, and ferritin were measured with routine laboratory methods. Free iron in serum was determined by the bleomycin-detectable iron (BDI) assay³ with the following modifications. The reaction was conducted in a total volume of 1 mL instead of 2 mL, and the absorbance of the butanol phase was measured at 350 nm using a microplate reader (Titertek Multiscan RC, Labsystems, Helsinki, Finland). The samples were measured in parallel with a corresponding sample blank without the addition of bleomycin. The absorbance value of the blank was reduced from each sample absorbance value. Reagent blank was reduced from the absorbance values of the standards (iron atomic absorption standard; Sigma Chemical Co, St Louis, MO) and a standard curve between .1 and 3 µmol/L was calculated by linear regression for each series.

Liver biopsy specimen was investigated with Perls' stain and iron content graded as described.^1,4 Production of superoxide radicals, on addition of nicotinamide adenine dinucleotide, was measured in isolated fibroblast mitochondrial membranes from the patients and 2 normal infants using the luminometric probe lucigenin;⁵ the results are expressed as counts per second per microgram mitochondria.

	RESULTS

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Two of the original 17 patients were included in the treatment study. Their clinical characteristics are shown in Table 1 in comparison with the entire published patient series.¹ They were born in 1995 and 1997; 1 patient born in 1996 died before the treatment could be started.

When hypotransferrinemia, increased transferrin iron saturation, and grade IV liver hemosiderosis⁴ in a biopsy specimen were confirmed, apotransferrin administration and subsequent exchange transfusion treatment was started, at 3 weeks of age in case 1, and 4 days in case 2. Patient 1 received 3 exchange transfusions between 3 to 6 weeks of age, and patient 2 received a total of 6 between 4 and 20 days of age. The results of iron metabolism analyses before and during the treatment are shown in Table 2. In case 1, an iron-chelating substance, deferoxamine (20 mg/kg/day for 1 week) was administered as a continuous infusion before the first apotransferrin infusion. In both infants, apotransferrin administration caused a temporary decrease in transferrrin saturation, as did exchange transfusion. In case 1, transferrin saturation remained within the normal range after the third exchange transfusion. A similar treatment response was found in case 2, but because the effect was not persistent, a total of 6 exchange transfusions were performed preceeded by apotransferrin administration with the exception of the fourth transfusion (Table 2). In both infants, a slight temperature increase (.2-1°C) was associated with 3 apotransferrin infusions. No other adverse events or deterioration of the disease were observed during the treatment. In total, case 1 received 11 and case 2 received 10 apotransferrin infusions. During the treatment period, a mean arterial pH of 7.27 (range: 7.14-7.40) was maintained in case 1 with a slower alkali infusion (5 mmol/kg/day) than before the treatment (11 mmol/kg/day). However, in case 2, the daily requirement of alkali to keep the mean pH at 7.30 (7.21-7.38) was unchanged (10-20 mmol/kg/day). Because both infants failed to thrive with no improvement of the disease, intensive care was withheld after 6 weeks of age.¹ The infants died at 10 and 8 weeks of age, respectively. The amount of iron staining was decreased in the liver autopsy specimen, compared with that obtained at a diagnostic liver biopsy at 16 days of age in case 1. However, grade IV hemosiderosis persisted in case 2, despite apotransferrin infusion and exchange transfusion. No iron deposits or histologic abnormality was found in neuropathological examination of the brain.¹

The superoxide production in fibroblast mitochondrial membranes was increased in both patients (2090 ± 360 and 4970 ± 435 counts/second/µg mitochondria, respectively), compared with controls (750 ± 160 counts/second/µg), the higher production being measured in the patient with more abundant liver iron content.

	DISCUSSION

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In our patients with no overt liver failure at birth, progressive hepatic dysfunction and significant fibrosis of the liver were found in the cases with extended survival.¹ Whether this progression is attributable to the toxic effects of iron overload is unclear. Liver injury caused by iron overload is presumed to involve lipid peroxidation and the formation of products such as 4-hydroxynonenal, which has been implicated in hepatic fibrogenesis.⁶ Whether the observed free iron in plasma^7-10 may cause toxic effects in the newborn period is unclear. Several studies have reported free iron in approximately one half of preterm infants, even when transferrin is not fully saturated.^7,8,10 In one study, increased serum ferritin levels were found in preterm infants who developed retinopathy, a multifactorial disease in which oxygen-free radicals have been implicated.¹¹ Presence of free iron in term asphyxiated infants has been associated with an adverse neurodevelopmental outcome,⁹ suggesting that free iron may play a role in reperfusion injury. We found that transferrin was fully saturated, and serum ferritin was increased approximately 10-fold in our patients.¹ For measurement of free iron, we used the BDI method. In a recent study, it was shown that the bleomycin assay is a valid biological method for measuring low molecular mass iron, which appears in plasma when transferrin becomes fully iron loaded and poses a risk for oxidative damage.¹² The increased superoxide production in fibroblasts of our patients suggests a possible connection between the iron overload and free radical production.

Serum transferrin concentrations in normal term infants are of the same magnitude as in adults.¹³ In previous studies on newborn infants with BDI, transferrin levels were not reported.^7-10,14 A low transferrin level does not seem to be the fundamental disturbance in iron metabolism in our patients, because, in human atransferrinemia, no overt abnormality is found in the newborn period, the primary finding later being sideroblastic anemia.¹⁵ The low level found in our patients is probably secondary to hepatic dysfunction.¹ Plasma-soluble transferrin-receptor concentrations were normal in our patients,¹ suggesting that exogenous apotransferrin could be handled at the receptor site. Because the observed hypotransferrinemia may cause or enhance occurrence of free iron in blood, we corrected the hypotransferrinemia with the aim to decrease plasma free iron and performed exchange transfusions to reduce total body iron content. Apotransferrin infusions have previously been used to correct anemia in a few cases of congenital atransferrinemia.¹⁵ In an animal study, hyperoxic lung injury attenuated when the amount of free iron was decreased by apotransferrin administration.¹⁶ Exchange transfusions in newborn infants with rhesus hemolytic disease were associated with decreases in plasma iron and ferritin levels and variable changes in prooxidants.¹⁷

With our treatment regime, we could reduce plasma free iron. Administration of apotransferrin seemed to mobilize iron from tissues as the total serum iron content typically increased after the infusion. Repeating the apotransferrin-exchange transfusion caused a decreasing trend of transferrin saturation in both patients, with a persisting result in case 1 but a recurrence of free iron in case 2 even after 6 exchange transfusions. However, exchange transfusion without a preceding apotransferrin administration had no clear effect on free iron. The combination treatment might have had a beneficial effect on the disease, because the infants' condition during the treatment did not deteriorate. Whether the treatment had any effect on survival is unclear, although the majority of the so far diagnosed patients died early and these 2 treated patients survived clearly longer than the median age of 12 days.

Adult type hemochromatosis is a disease manageable with venesections and iron chelators,¹⁸ whereas neonatal hemochromatosis, a disease with an unknown gene locus and clearly distinguishable from the disease in our patients, presents at birth with severe progressive liver dysfunction resulting in death in early infancy.¹⁹ A few cases have survived as a result of liver transplantation.^20,21 Treatment has been attempted before liver transplantation with an iron chelator combined with antioxidants, aiming at reducing the toxic effects of oxygen radical generated by free iron but without success.²¹ The possible benefits of iron chelators in other neonatal disorders with BDI are still unclear.

The observed good acute safety of the apotransferrin administration indicated that the benefits of the treatment would overweigh the risks in our setting. Thus, we considered this approach superior to iron chelator, such as deferoxamine, which has several adverse effects, eg, neurotoxicity,²² and which has failed in neonatal hemochromatosis.²¹ Because this disease is very rare (so far not reported from other countries), the treatment cannot be evaluated in a trial. However, the possibility to reduce plasma free iron level with no significant adverse events suggests new treatment prospects for newborn infants with plasma free iron or iron overload.

	ACKNOWLEDGMENTS

This study has been supported by grants from the Finnish Pediatric Research Foundation, Ulla Hjelt Fund.

We thank Dr Sari Pitkänen for the superoxide determinations.

	FOOTNOTES

Received for publication Dec 11, 1998; accepted Apr 27, 1999.

Reprint requests to (V.F.) Hospital for Children and Adolescents, University of Helsinki, Stenbäckinkatu 11, Helsinki, PB 281, 00029 Helsinki, Finland. E-mail: vineta.fellman{at}huch.fi

	ABBREVIATIONS

SD, standard deviation; BDI, bleomycin-detectable iron.

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