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Iron Overload and Antioxidant Status inPatients with β-Thalassemia Major

K. RELLER,a B. DRESOW,a M. COLLELL,b R. FISCHER,a,e

R. ENGELHARDT,a P. NIELSEN,a M. DÜRKEN,c C. POLITIS,d

AND A. PIGAb

aMedizin. Biochemie, UKE-Universität Hamburg, Germany bCentro Microcitemie, Università di Torino, ItalycKinderklinik, Universität Hamburg, Germany dHellenic Red Cross Blood Transfusion Centre, Athens, Greece

In iron overload diseases as in thalassemia or genetic hemochromatosis, a low molecularweight iron, non-transferrin-bound iron (NTBI), is thought to cause the formation of

highly reactive oxygen radicals that generate lipid peroxidation processes with subsequentdevelopment of tissue and organ damage. The molecular mechanism leading to such organdamage are not fully clarified, but have been mostly attributed to iron-induced lipid per-oxidation (LPO), leading to a breakdown of biomembrane functions and finally to celldeath. The long-term clinical consequences of this process are heart failure, liver fibrosisor cirrhosis, and endocrinopathies. An increased consumption of antioxidants may resultfrom these disorders. In the present study we measured hematological iron parameters,NTBI and the status of endogenous antioxidants vitamin A, E and C. Its relation to thedegree of iron overload was studied in 249 patients with ß-thalassemia major (ages 4–38year) from Italy, Greece and Germany.

Patients treated with deferoxamine (DFO: n = 209) and with deferiprone (DFP: n = 40)were admitted for SQUID biomagnetic liver susceptometry (BLS) with non-invasiveassessment of liver and spleen iron concentration.1 In order to calculate total body ironstores, organ volumes were determined by sonographical scanning. Blood samples weretaken after overnight fasting and suspension of DFO treatment at least for 24 hours.Hematological iron parameters (serum iron, transferrin saturation, ferritin) were deter-mined by routine laboratory methods. Serum NTBI was measured by HPLC according toa modified method of Singh et al.2 In short, After addition of NTA and ultrafiltration of theFe-NTA complex NTBI was measured by RP-HPLC with deferiprone (L1) in the eluent.Serum lipid soluble antioxidants vitamin E (Vit E) and A (Vit A) were measured simulta-neously after lipid extraction by RP-HPLC with UV and fluorescence detection. Serumvitamin C (Vit C) was determined by RP-HPLC using UV and electrochemical detection.

LIVER IRON, NTBI AND VITAMINS

Liver iron concentration measured by biomagnetometry (bLIC) was in the range of 170to 11100 (median 1340) µg/gliver. Taking into account the enlarged organ volumes (meanliver volume: 1581 ± 583 ml), total body iron stores were calculated in the range of 0.3 to

eAuthor for correspondence: Dr. Roland Fischer, UKE-Abt. Medizin. Biochemie, Martinistr. 52,D 20246 Hamburg, Germany; Tel.: +49-40-4717-3385; Fax: +49-40-4717-4797; E-mail:fischer@uke.uni-hamburg.de

39.0 g (median 2.5 g) assuming 80% of the total iron in liver and spleen. Hematologicaliron parameters (median; range) were elevated for serum ferritin (2186; 81–17519 µg/l),serum iron (225; 31–337 µg/dl) and transferrin saturation (77 %; range 10–100%).

The serum from thalassemic patients contained NTB-iron in the range of –0.8 to 13.9µmol/L with a mean value of 5.4 ± 2.8 µmol/L. NTBI was increased significantly when thetransferrin saturation exceeded 60% (r = 0.63; p < 10–3). NTBI values correlated also sig-nificantly with liver iron concentrations and ferritin (r = 0.39 and 0.35; p < 10–3). However,NTBI is not only a function of transferrin saturation, but seems to depend simultaneouslyfrom liver iron stores (linear bivariate regression analysis: r = 0.7; p < 10– 4). NTBI values> 3 µmol/L were found only above liver iron concentrations of 2000 µg/gliver correspond-ing with specific total body iron stores of more than 120 mg Fe/kg (FIG. 1).

Plasma levels of lipid soluble antioxidants were between 0.01 and 11.3 µg/ml for VitE and 0.4 to 5.7 µg/ml for Vit A. Serum Vit C concentration of 0.1 to 25.2 µg/ml wereobserved in patients not substituted with oral vitamin preparations. Low antioxidantplasma levels were observed more often in severely iron loaded patients than in wellchelated patients. Significant exponential relationships with liver iron concentration werefound for vitamin C and E, but not for vitamin A. About 50% of patients with bLIC >4000 µg/gliver had subnormal vitamin C and E concentrations. An inverse correlationbetween the vitamin E status of the patients and serum NTBI was observed (r = -0.43,p < 10-3). Subnormal vitamin E plasma levels were observed especially for NTBI valuesbeyond 5 µmol/L.

CONCLUSION

Iron overload, determined by non-invasive biomagnetic liver susceptometry, isassociated with high transferrin saturation and high serum NTBI. This toxic iron frac-tion, not found in normal human blood is able to generate lipid peroxidation processes,with subsequent consumption of antioxidants, especially in patients beyond liver iron

464 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. Non-transferrin-bound iron (NTBI) in patients with β-thalassemia major 24 hours afterlast s. c. deferoxamine treatment. Arrows at 380 mg Fe/kg and 120 mg Fe/kg indicate liver ironregions of low (safe) and high (risky) plasma NTBI levels.

concentrations > 2000 µg/gliver. This seems to agree with recent recommendations foran “optimal” hepatic iron concentration of 1000 to 2100 µg/gw.w. in patients with tha-lassemia major.3 From this point of view, the limit of 380 mg Fe/kg4 (equivalent toabout 5000µg/gliver), below which total body iron stores are considered relatively“safe,” should be reduced to about 120 mg Fe/kg.

REFERENCES

1. FISCHER, R., A. PIGA, F. TRICTA, P. NIELSEN, R. ENGELHARDT, F. GAROFALO, A. DI PALMA & C.VULLO. 1997. The use of biomagnetic liver susceptometry in the Ferrara-Hamburg-Turin studyon thalassemia. In Proceedings of the 10th International Conference on Biomagnetism, SantaFe 96. Elsevier. In press.

2. SINGH, S., R. C. HIDER & J. B. PORTER. 1990. A direct method for quantification of non-transferrin-bound iron. Anal. Biochem. 186: 320–323.

3. OLIVIERI, N. F. & G. M. BRITTENHAM. 1997. Iron-chelating therapy and the treatment of tha-lassemia. Blood 89: 739–761.

4. MODELL, B. & V. BERDOUKAS. 1984. The Clinical Approach to Thalassaemia. Grune andStratton. London.

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