Excessive fluoride consumption increases haematological alteration in subjects with iron deficiency, thalassaemia, and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency.

Excessive fluoride consumption leads to accelerated red blood cell death and anaemia. Whether that increases the haematological alteration in subjects with haematological disorders (iron deficiency, thalassaemia, and G-6-PD deficiency) is still unclear. The fluoride in serum and urine and haematological parameters of students at Mae Tuen School (fluoride endemic area) were analysed and compared to those of students at Baan Yang Poa and Baan Mai Schools (control areas). Iron deficiency, thalassaemia, and G-6-PD deficiency were also diagnosed in these students. The students at Mae Tuen School had significantly (P < 0.001) higher levels of mean fluoride in the serum and urine than those in control areas. In both control and fluoride endemic areas, students with haematological disorders had significantly lower levels of Hb, Hct, MCV, MCH, and MCHC than those without haematological disorders. Moreover, the lowest levels of Hb, MCH, and MCHC were observed in the students with haematological disorders who live in the fluoride endemic area. Thus, the excessive fluoride consumption increased haematological alteration in subjects with iron deficiency, thalassaemia, and G-6-PD deficiency and that may increase the risk of anaemia in these subjects.

Tandem measurements of iron and creatinine by cross injection analysis with application to urine from thalassemic patients.

This work presents development of a method for the dual determination of Fe(III) and creatinine using cross injection analysis (CIA). Two CIA platforms connected in series accommodated sample and reagents plugs aspirated via y-direction channels while water was pumped through the x-direction channel toward a flow-through cell of a diode array UV-vis. detector. Iron was detected from the colorimetric reaction between Fe(II) and 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-(3-sulfopropyl)amino) aniline (5-Br-PSAA), with prior reduction of Fe(III) to Fe(II) by ascorbic acid. The Jaffe's reaction was employed for the detection of creatinine. Under the optimal conditions, good linearity ranges were achieved for iron in the range 0.5 to 7 mg L(-1) and creatinine in the range 50 to 800 mg L(-1). The CIA system was applied to spot urine samples from thalassemic patients undergoing iron chelation therapy, and was successfully validated with ICP-OES and batchwise Jaffe's method. Normalization of urinary iron excretion with creatinine is useful for correcting the iron concentration between urine samples due to variation of the collected urine volume.

Increased urinary 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine excretion in thalassemia patients: markers for lipid peroxidation-induced DNA damage.

Thalassemic diseases including homozygous beta-thalassemia and beta-thalassemia/Hb E (beta-Thal/Hb E) are prevalent in Southeast Asia. Iron overload is a common complication in beta-thalassemia patients which induces intracellular oxidative stress and lipid peroxidation (LPO). LPO end products generate miscoding etheno adducts in DNA which after their repair are excreted in urine. We investigated whether urinary levels of 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) can serve as putative cancer risk markers in beta-Thal/Hb E patients. epsilondA and epsilondC levels were assayed in collected urine samples by immunoprecipitation-HPLC-fluorescence and 32P-postlabeling TLC, respectively. Mean epsilondA (fmol/micromol creatinine) levels in urine of beta-Thal/Hb E patients ranged from 4.8 to 120.4 (33.8+/-3.9; n=37) and were 8.7 times higher compared to asymptomatic controls (1.4-13.8; 3.9+/-0.8; n=20). The respective epsilondC levels ranged from 0.15 to 32.5 (5.2+/-1.3; n=37) and were increased some 13 times over controls (0.04-1.2; 0.4+/-0.7; n=20). epsilondC levels were correlated positively with NTBI (r=0.517; P=0.002), whereas epsilondA showed only a trend (r=0.257; P=0.124). We conclude that the strongly increased urinary excretion of etheno adducts indicates elevated LPO-induced DNA damage in internal organs such as the liver. These highly promutagenic lesions may contribute to the increased risk of thalassemia patients to develop hepatocellular carcinoma.

HPLC determination of 1,2-diethyl-3-hydroxypyridin-4-one (CP94), its iron complex [Fe(III) (CP94)3] and glucuronide conjugate [CP94-GLUC] in serum and urine of thalassaemic patients.

Sensitive and selective high performance liquid chromatographic (HPLC) methods for the quantification of 1,2-diethyl-3-hydroxypyridin-4-one (CP94), its iron complex [Fe(III) (CP94)3] and glucuronide metabolite (CP94-GLUC) in urine and serum of thalassaemic patients are described. Three separate analyses are involved. The first assay quantifies both CP94 and its iron complex. This procedure requires the conversion of the iron complex to the free ligand and is carried out using diethylenetriaminepentaacetic acid (DTPA). CP94 and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95) present in either serum or urine are then extracted at pH 7.0 with dichloromethane. Extraction efficiency is 96.0 +/- 5.6% and 100 +/- 7.1% for CP94 and CP95, respectively, and 31.2 +/- 2.1% at 30 microM and 53.2 +/- 4.2% at 300 microM for the corresponding iron complex. In the second assay, samples are incubated (16 h) with beta-glucuronidase and processed as before. In this assay, the drug, its iron complex and glucuronide conjugate are measured. In the third assay the iron complex of CP94, [Fe(III) (CP94)3] is quantified. From the three separate analyses it is possible to calculate the individual concentrations of the three separate components present in serum and urine of thalassaemic patients. Calibration for both components, i.e. CP94 (assays 1 and 2) and its iron complex (assay 3) are linear with correlation coefficients > 0.99 and are reproducible over the required concentration range of 0-500 microM for the free ligand and 0-100 microM for the iron complex. The minimum quantifiable level is 0.5 microM for the free ligand and 1.0 microM for the iron complex.

Efficacy and possible adverse effects of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in thalassemia major.

Eleven patients with beta thalassemia major were entered into the trial of the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1). Their ages ranged from 17 to 26 years (mean +/- SD, 22.3 +/- 2.7). Six were male and five were female. L1 was administered at an initial daily dose of 42.5 to 60 mg/kg as a single dose. After 4 weeks, the dose was increased to 85 to 119 (102 +/- 10.7) mg/kg for 191 to 352 days divided into either two or four doses daily, except for one patient who developed agranulocytosis after 11 weeks and was taken off the trial. Initial serum ferritin values in the remaining 10 patients ranged between 1,000 and 9,580 (5,549 +/- 3,333) micrograms/L and at end of the trial their mean serum ferritin was significantly lower (4,126 +/- 2,278; P less than .05 using the paired t-test). Urinary iron excretion at a daily dose of 85 to 119 mg/kg administered as two divided doses ranged between 0.14 and 0.82 (0.44 +/- 0.26) mg/kg/24 h. In three patients, the four doses per day schedule caused substantially more iron excretion than the same total dose divided into two. During the course of the trial, several possible adverse effects have been encountered. One patient (female, aged 20) developed agranulocytosis 11 weeks after starting treatment and 6 weeks after beginning treatment with a daily dose of 105 mg/kg. This patient's neutrophil count recovered spontaneously 7 weeks after the discontinuation of L1. A decrease in serum zinc levels to subnormal levels was observed in four patients with symptoms of dry skin, with an itchy scaly rash in two that was associated with low serum zinc levels that responded to zinc therapy. Urinary zinc levels ranged from 4.7 to 23.4 (13 +/- 5.5) mumol/24 h and were above 9 mumol/24 h (upper limit of normal) in eight patients. Mild nausea occurred in three patients and transient diarrhea in a fourth. Mild musculoskeletal symptoms occurred in three patients but settled without discontinuation of L1 therapy in two and with temporary discontinuation of L1 in the third. A transient increase in serum aspartate transaminase was also noted in five patients, but serum aspartate transaminase levels subsequently decreased in all of them. No cardiovascular, neurologic, renal, or retinal toxicities were demonstrable. These results confirm that L1 is an effective oral iron chelator. Further clinical trials are needed to determine the incidence and severity of adverse effects.

L1 (1,2-dimethyl-3-hydroxypyrid-4-one) for oral iron chelation in patients with beta-thalassaemia major.

L1 was given to eight patients with beta-thalassaemia major who had previously been treated with deferoxamine (DF) for 4-10 years. The patients' ages ranged from 11 to 27 years. Serum ferritin values ranged from 1.3 to 11.5 x 10(3) micrograms/l. L1 was given twice daily at a daily dose of 55-80 mg/kg body weight and was continued for 10 months in two patients, 9 months in three, 7 months in two patients and 4 months in one patient. As previously observed with DF, each patient's urinary iron excretion (UIE) varied greatly from day to day. The mean UIE of the eight patients ranged from 11 to 49 mg/d (0.2-0.87 mmol/d) on subcutaneous DF and from 16 to 53 mg/d (0.28-0.95 mmol/d) on L1. Two patients excreted significantly more and one patient significantly less iron while on L1. If the UIE was calculated as mmol Fe/mmol creatinine there was no statistically significant difference. Serum ferritin values fluctuated widely in all, with a consistent downward trend in three, no change in four and an increase in one of two non-splenectomized patients. This patient's splenomegaly and need for transfusions continued to increase while on L1. No toxicities attributable to the drug were detected during the period of study and tolerance of the drug was excellent.

Comparison of oral iron chelator L1 and desferrioxamine in iron-loaded patients.

The efficacy of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) was compared with that of subcutaneous desferrioxamine in 26 patients with transfusional iron overload. Immediately after red-cell transfusion, 20 patients were randomised to receive either desferrioxamine (50 mg/kg daily as a 12 h subcutaneous infusion), or L1 (50 mg/kg daily by mouth). Patients were evaluated during treatment with the other drug after transfusion the next month. Mean (SD) daily urinary iron excretion was lower during L1 than during desferrioxamine (12.3 [6.7] vs 18.2 [15.3] mg/day). In 5 patients the dose of L1 was raised from 50 to 75 mg/kg daily; mean urinary iron excretion rose from 13.8 (7.0) mg/day to 26.7 (17.8) mg/day, comparable with that during desferrioxamine (24.9 [24.3] mg/day). Faecal iron excretion rose slightly over baseline in 6 patients studied during L1 administration (from 8.5 [0.9] mg/day to 12.2 [0.9] mg/day). Pharmacokinetic studies showed an elimination half-life for L1 of 117-237 min. Studies in dogs and in volunteers showed no absorption of the L1-iron complex, excluding a contribution of absorption of intraluminal complexes of L1 and food iron to urinary iron excretion. Further animal toxicity testing is needed before L1 can be studied in a broader group of patients.

Microalbuminuria in sickle cell disease

The kidney is involved in virtually all individuals who inherit the suckle cell form of hemoglobin. though asymptomatic and relatively common, proteinuria in patients with sickle cell anemia (SS) over 40 years old is associated with reduced creatinine clearance. The subclinical incrase in urinary albumin is termed microalbuminuria and is a marker of preclinical glomerular damage. The aim of the present study was to determine the presence of microalbuminuria measured by radioimmunoassay in patients with sickle cell disease. The study inclused 41 patients with SS, 11 patients with hemoglobin SC disease, 4 subjects with Sß-thalassemia and 10 normal controls. All subjects were teenagers or adults. Sixteen SS patients (40%) and 1 SC (9%) and 1 Sß (25%) patient presented mean urinary albumin excretion (UAE) above normal values (30 mg/l). No correlation was observed between UAE and age, creatinine clearance, hemoglobin level or %HbF. These parameters, as well as the presence of ulcers, were not significantly differente between SS patients with and without UAE above 30 mg/dl. The high prevalence of microalbuminuria in patient with sickle cell anemia indicates that glomerular damage is common. The connection between microalbuminuria and clinical neplhropathy has been demonstrated in diabetes and may indicate a sign of early disease rather than a marker for susceptibility. Thus, microalbuminuria may be an early indicator of flomerular damage for patients with sickle cell disease

[Findings in relation to serum and urinary iron in heterozygotic beta-thalassemia]. / Indagine intorno al ferro sierico ed urinario nella beta-talassemia eterozigote.

Iron status (expressed as serum ferritin and iron levels) has been compared in normal and in heterozygous beta-thalassemic subjects. A higher serum ferritin concentration has been found in beta-thalassemic males, showing, therefore, a shift towards super-normal values of the balance between tissue iron and serum ferritin levels. In beta-thalassemic subjects the serum ferritin levels have been found in the normal range and this seems to be correlated with an adequate and ready iron supply by protein transferrin to hyperplastic bone marrow. The higher urinary iron values in normal male subjects can be explained in this way: a large iron supply from the transferrin to the thalassemic erythroid cells limits the contribution from this protein to the urinary iron.

Iron excretion in thalassemia children treated with deferoxamin methansulfonat: intravenous drip compared to the subcutaneous routes.

The iron excretion in the three beta-Thal/Hb E patients were determined comparing the effect of DF given by subcutaneous push, subcutaneous drip and intravenous drip. The subcutaneous drip or intravenous drip increased urine iron excretion by 5.6-11.2 times whereas the subcutaneous push, 3.5-5.3 times only. It is recommended that for countries where the infusion machine is very expensive the DF should be given by intravenous drip or the modified, simple and inexpensive equipment for subcutaneous drip.

Effective chelation of iron in beta thalassaemia with the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one.

The main iron chelator used for transfusional iron overload is desferrioxamine, which is expensive, has toxic side effects, and has to be given subcutaneously. An orally active iron chelator is therefore required. The effects of oral 1,2-dimethyl-3-hydroxypyrid-4-one on urinary iron excretion were studied in eight patients who had received multiple transfusions: four had myelodysplasia and four beta thalassaemia major. Different daily doses of the drug up to 100 mg/kg/day, alone or in combination with ascorbic acid, were used. In three patients with thalassaemia the effect of the drug was compared with that of subcutaneous desferrioxamine at the same daily dose. In all eight patients a single dose of oral 1,2-dimethyl-3-hydroxypyrid-4-one resulted in substantial urinary iron excretion, mainly in the first 12 hours. Urinary iron excretion increased with the dose and with the degree of iron loading of the patient. Giving two or three divided doses over 24 hours resulted in higher urinary iron excretion than a single dose of the same amount over the same time. In most patients coadministration of oral ascorbic acid further increased urinary iron excretion. 1,2-Dimethyl-3-hydroxypyrid-4-one caused similar iron excretion to that achieved with subcutaneous desferrioxamine at a comparable dose. In some cases the iron excretion was sufficiently high (maximum 99 mg/day) to suggest that a negative iron balance could be easily achieved with these protocols in patients receiving regular transfusions. No evidence of toxicity was observed on thorough clinical examination or haematological and biochemical testing in any of the patients. None of the patients had any symptoms that could be ascribed to the drug. These results suggest that the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one is as effective as subcutaneous desferrioxamine in increasing urinary iron excretion in patients loaded with iron. Its cheap synthesis, oral activity, and lack of obvious toxicity at effective doses suggest that it should be developed quickly and thoroughly tested for the management of transfusional iron overload.

Host-associated iron transfer factor in normal humans and patients with transfusion siderosis.

A low molecular weight iron-binding substance that promotes bacterial growth in vitro by increasing iron availability was identified in human blood and urine. Partial purification and physical characterization indicate that this factor is similar to the host-associated iron transfer factor (HAITF) previously isolated from mammalian tissue. HAITF was found to be significantly elevated in the blood of patients with thalassemia who have transfusional siderosis. The level of HAITF in the blood of these patients was also found to correlate with that of serum iron and serum glutamic-oxaloacetic transaminase (SGOT) but not with that of serum ferritin. Thus, elevated blood levels of HAITF may explain the increased susceptibility to infection seen in patients with iron overload. Its physiologic role, however, may involve the transport of iron within cells.

Early iron overload in beta-thalassaemia major: when to start chelation therapy?

Twenty-eight children with beta-thalassaemia major aged between 11 and 48 months were given intensive transfusions. Serum iron, transferrin saturation, serum ferritin, non-transferrin iron, and subcutaneous desferrioxamine-induced urinary iron excretion were measured. The results showed that even children with a limited number of transfusions had severe iron overload as indicated, in particular, by the raised serum ferritin levels and the high excretion rates after subcutaneous infusion of desferrioxamine. The desferrioxamine test was useful, even in very young children, in assessing response to chelation therapy thus enabling such treatment to be started early to prevent harm from iron overload.