A. Gremio docente en Guanaquín
III. B.2 “Unos se portan bien, otros mal, con otros no sabemos qué hacer” Maestras y
6.1. INTRODUCTION
In haemochromatosis or transfusion dependent refractory anaemia, plasma transferrin is saturated leading to excessive accumulation of iron in various tissues. Moreover, some iron released from tissues to plasma may remain 'free'. The precise nature o f this non transferrin-bound iron(NTBI) is uncertain. Hershko et <3/(1978) suggested that NTBI is a low molecular weight iron complex probably loosely bound to albumin, while others postulated that NTBI exists mainly as iron citrate(Grootveld et al 1989). Whatever its form, NTBI is known to be effective in the formation of potentially toxic oxygen derivatives(Gutteridge et at 1985, Halliwell & Gutteridge 1986).
Two other factors have been suggested which may have a bearing on the mechanism of NTBI-induced tissue injury. First, plasma is deficient in antioxidant enzymes, such as superoxide dismutase(SOD), that are capable of inactivating toxic oxygen species and normally exist inside cells(Hershko 1987) and second, NTBI is more readily taken up by tissues than transferrin-bound iron(Craven et al 1987, Fawwaz et al 1967).
Several methods are currently in use for measuring NTBI(Batey et al 1980, Gower et al 1989, Gutteridge et al 1981, Gutteridge et al 1986, Hershko et al 1978, Singh et al 1989, Singh et al 1990). The most recent one has been claimed to be simple and sensitive with high reproducibility(Singh et al, 1990).
In this study, NTBI levels were measured in 52 patients with TM to determine its relation to degree o f iron overload. When NTBI was found to correlate significantly with iron overload in these patients its measurements were used to monitor the efficacy of Lj.
6.2. MATERIALS AND METHODS
The method used in this study was modified from that described by Singh et a/(1990) as follows: First, the more convenient and rapid Amicon Centrifree Micropartition system
(Amicon, Beverly, MA) was used instead of Amicon Centriflo-CF 25 cone membranes to prepare the serum ultrafiltrates. Second, 5 mmol of L i was substituted for the 3mmol/1 of LiNPr(CP-22)(l-propyl-2-methyl-3-hydroxypyrid-4-one) in the HPLC mobile phase since Lj-iron complexes elute faster than the corresponding LiNPr-iron complexes. The change in polarity caused by the use of Lj instead o f LjNPr necessitated the reduction of acetonitrile in the HPLC mobile phase from 20% to 5%.
The modified method was, therefore, as follows. To 0.9ml o f serum was added 0 .1ml 800mmol/l nitrilotriacetic acid (NTA: Aldrich, Gillingham, Dorset, UK), which had previously been adjusted to pH 7.0 with 1 mol/1 NaOH, and allowed to stand at room temperature for 30min. The solution was then ultrafiltered with an Amicon centrifree micropartition system with an applied centrifugal force of lOOOg for 30min using a fixed-angle rotor (MSB GF- 8 centrifuge, Crawley, Sussex, UK). A 20ul aliquot o f the
ultrafiltrate (approx. total volume SOOul) was injected directly onto the HPLC system using an iron-free syringe. The HPLC system comprised of an LDC constrametric III pump, LDC spectromonitor 3000 uv-visible detector, LDC model 308 integrator (Milton- Roy, Stone, Staffs, UK) and Rheodyne 7125 injector (Cotati, CA). A 100 x 3mm Chromspher-ODS (Chrompack, The Netherlands) glass column, filled with 5um material, was used with a mobile phase of 5% acetonitrile and 5mmol/l L^ in 5mmol/1 MOPS, pH 7.0, at a flow rate of 0.6ml/min. Detection was at 460nm. A standard curve was generated by injecting 0, 2, 4, 6, 8 and lOumol/1 iron prepared in 80mmol/l NTA
(adjusted to pH 7.0). All injections were performed in duplicate with a standard after each sample.
NTBI results obtained by the modified and original procedures were initially compared. In 38 patients with beta thalassaemia major the correlation coefficient between the NTBI
values from the 2 methods was 0.926(y=0.823x + 0.465)(p<0.0001). The within- and between-batch coefficient of variation (CV) for the modified procedure was 7 and 11% respectively. This was determined by multiple (10) analyses o f a pooled sample. The modified HPLC conditions, as with the original conditions, allows the separation of DFX-bound iron from other NTBI in patient samples. Control serum samples were included in each batch and showed consistent values.
Lj was prepared and characterised as previously described(Kontoghiorghes & Sheppard
1987). LjNPr was generous gift of Professor RC Hider.
Serum ferritin was estimated by ELISA technique as described by Flowers et a/(1986). Serum iron and TIBC were measured by routine laboratory techniques(ICSH Expert Panel on Iron 1978 a&b). Statistical analysis was performed using Student's r-test.
6.3. PATIENTS
Serum samples were obtained from 52 patients with p thalassaemia major. Their ages ranged from 3 to 34 years (19.0±7.3), 27 males and 25 females. Serum samples were also obtained from 12 normal volunteers(34.0±3.6 years).
The patients were receiving regular blood transfusions to keep their haemoglobin above 10 g/dl. They were using subcutaneous DFX(40-50mg/kg/night) for chelation with variable degree of compliance (1-5 nights/week). Subsequently 10 of the patients were entered into a trial of the oral chelator Li(Chapter 2).
To avoid contamination o f samples with external iron, all handling was performed in disposable iron-free equipment. Blood samples were separated within one hour of collection to avoid the possible release of iron from haemolysis o f erythrocytes. The serum was stored at -20”C until analysis. Samples used in this study were obtained immediately before the start of DFX infusion or L, ingestion and approximately 12 hours
after the last administration to ensure complete clearance o f Li(Chapter 4, Kontoghiorghes et al 1990 a&b).
6.4. RESULTS
The NTBI levels obtained with the modified method in the 12 normal controls ranged from -0.7umol/l to -2.3umol/l(-1.5±0.6). In the 52 patients the NTBI values ranged from -1.5 to 9.0 umol/l(3.6±2.3). As these negative values are artifactual(see below) we have assigned them zero values when statistical analyses were performed.
The patients' serum ferritin concentrations ranged from 207 to 11400ug/l(2674±2538). In 35 of the patients the total serum iron concentrations ranged from 20 to 61 umol/1 (39.5±9.6) and transferrin saturation ranged from 44% to 110% (84.5±13.8). There was a significant correlation between serum ferritin and NTBI values (r=0.502, p<0.001. Fig 6.1). There was also a significant correlation between total serum iron and NTBI (r=0.608, p<0.001, Fig 6.2) and between transferrin saturation and NTBI (r=0.481, p=0.004, Fig 6.3).
Ten of the patients were entered into a trial of the oral iron chelator L j. They, therefore, stopped using DFX and commenced chelation with Lj, initially at a total daily dose of about 40-60mg/kg and 4 weeks later at a total daily dose of 80-120mg/kg both were divided into 2 doses/day. Their ages ranged from 16 to 26 years(22.3±2.7) and their serum ferritin concentrations, at the start o f the trial, ranged from 1000 to 9580ug/l (5549±3333). Their NTBI levels were measured at the beginning o f the trial and 3 and
6 months later (Fig 6.4). The initial NTBI ranged from 3.6 to 9.0umol/l (6.1±1.6) and the
final NTBI ranged from 3.8 to 5.4umol/l (4.5±0.5). The difference between the 2 mean values is significant (p<0.001) as was the overall fall in NTBI values when paired Mest was used(p=0.007). On the other hand when measured at 6 months serum ferritin levels
100000 10000 — DO 1000 — 100 - 11=52 r=0.467 p<0.001 0 1 2 3 4 5 6 7 8 9 10