The role of iron and haemochromatosis gene mutations in the progression of liver disease in chronic hepatitis C.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is frequently associated with elevated markers of iron stores. Recessively inherited mutations in the HFE gene are responsible for iron accumulation in most cases of hereditary haemochromatosis and may have a role in HCV infection. They may also be associated with progressive liver fibrosis although this remains controversial. AIMS: To assess the prevalence of HFE mutations in Scottish HCV infected patients and to explore the effect of the carrier state on serum and liver iron stores, and the severity of liver disease. PATIENTS: A total of 164 patients with antibodies to HCV who underwent liver biopsy were assessed prospectively. METHODS: Each patient was screened for HFE mutations (Cys282Tyr and His63Asp). Iron markers were assessed in serum (ferritin, transferrin saturation) and on liver biopsy (stainable iron, liver iron concentration (LIC) and hepatic iron index). RESULTS: There were 67 (41%, 26 Cys282Tyr, 33 His63Asp, eight compound) heterozygotes. Forty four (28%) patients had elevated serum iron markers, 24 (15%) had stainable liver iron, and five (3%) had elevated LICs. Carriage of HFE mutations was not associated with any clinical, biochemical, virological, or pathological features, including accumulation of liver iron. Elevated serum iron markers were associated with male sex, increased alcohol consumption, and increased liver inflammation and fibrosis. Patients with elevated LICs were older, acquired HCV infection earlier, and had more liver inflammation. CONCLUSIONS: Patients with chronic HCV infection frequently have elevated serum iron markers although elevated LICs are uncommon. Elevated serum iron studies and LICs occur in patients with more severe liver disease. Carriage of HFE mutations, although frequently observed in these HCV infected patients, does not have a role in the accumulation of iron or the progression of liver disease in HCV infection.

Direct determination of selenium in human blood serum and plasma by electrothermal atomic absorption spectrometry.

A method is described for the direct determination of selenium in serum or plasma by electrothermal atomic absorption spectrometry with deuterium-arc background correction. Samples are diluted (1 + 2) with a modifier containing palladium nitrate and Triton X-100. Samples are atomised from a L'vov platform in a pyrolytically-coated electrographite tube and peak area signals are measured. Direct determination is possible by using selenium standards matched to the physiological concentrations of sodium chloride, calcium and phosphate. The detection limit is 6 micrograms/L in the original sample. Precision at a selenium concentration of 97 micrograms/L was 2.2% RSD within batch and 3.0% RSD between batch. Accuracy is shown by (i) analysis of a Seronorm reference serum (value obtained 97 +/- 3 micrograms/L; recommended value 96 micrograms/L); (ii) recovery of added selenium (93.3 +/- 6.7% and 98.2 +/- 3.3% at additions of 30 and 60 micrograms/L, respectively) and (iii) comparison of results with mean of all laboratories in an external quality assessment scheme.

Determination of aluminium in serum, dialysate fluid and water by inductively coupled plasma optical emission spectrometry.

Methods for the determination of aluminium in serum, dialysate fluid and water by inductively coupled plasma optical emission spectrometry are described and validated. Aluminium was measured at 167 nm using an argon purged monochromator. Matrix effects in serum and dialysate fluid were overcome by using an yttrium internal standard. Serum was found to have a complicated background in the region of 167 nm: careful selection of the wavelength used for background correction is therefore a pre-requisite for accurate analysis. The method for serum was validated by comparison with electrothermal atomization atomic spectrometry and the limit of agreement determined to be +/- 0.3 mumol/L. Routine performance in a quality assessment scheme has been highly satisfactory for a period of 1 year. The method is ideal for fast and accurate monitoring of patients potentially at risk from aluminium toxicity.

Rapid partial digestion of biological tissues with nitric acid for the determination of trace elements by atomic spectrometry.

Determination of Cu, Fe, Mn and Zn in fresh bovine liver and BCR bovine liver and pig kidney reference materials, after digestion at 105 degrees C with nitric acid for various times, showed that the trace elements were completely released after heating for only 20 min. Centrifugation of the samples after digestion improved the separation from undigested fat. This partial-digestion method, based on heating about 0.2 g of sample with 2 ml of nitric acid for 20 min, was tested on a range of biological reference materials and on fresh bovine liver for the four elements. Comparison was made with results obtained after digestion by a recommended hydrogen peroxide--sulfuric acid digestion procedure. Results obtained by inductively coupled plasma atomic emission spectrometry or by flame and graphite-furnace atomic absorption spectrometry showed good agreement between the two digestion procedures, and results for the reference materials agreed well with the certified values. Between-batch precision was better than 4% for Cu, Fe and Zn at levels of 30 to 190, 210 to 320 and 100 to 140 micrograms g-1, respectively. For Mn, the precision varied between 7 and 14% for measured concentrations of 8 to 12 micrograms g-1. The partial-digestion procedure offers simplicity, speed, low cost and the ability to handle a large number of samples at the same time.

Interaction of aluminium and gallium with human lymphocytes: the role of transferrin.

Aluminium-transferrin (Al-Tf) and gallium-transferrin caused a dose-dependent decrease in proliferation of human peripheral blood lymphocytes cultured for 3 days with phytohaemagglutinin (PHA). Addition of apotransferrin reduced the inhibitory effect. Al added as AlCl3 or aluminium citrate had no effect, and there was no significant difference in the response of cells from renal failure patients with or without high serum Al levels or controls. Lymphocytes cultured in the presence of Al-Tf showed a dose-dependent uptake of Al, whereas uptake from aluminium citrate was low and not dose-dependent. Uptake from AlCl3 was very high but probably involved a nonspecific uptake mechanism. Levels of Al in freshly isolated lymphocytes were approximately 1.6 ng/10(6) cells, there being no difference between cells from patients and controls. It is concluded that Al, when bound to transferrin, may have a detrimental effect on lymphocyte function and might contribute to the decreased immune responsiveness of renal failure patients on haemodialysis. However, lymphocyte Al levels are probably not useful as a marker of Al overload in such patients.

Ranitidine suppresses aluminium absorption in man.

1. Intragastric pH monitoring was performed before and after the single-blind administration of ranitidine or placebo (saline) in eight healthy subjects and four patients with end-stage renal disease who were on regular haemodialysis. 2. The subjects were studied on two occasions and were given aluminum hydroxide (1185 mg) orally 90 min after the administration of ranitidine or saline. 3. Plasma aluminum concentrations and, in normal men, urinary excretion of aluminum were monitored before and after the oral aluminum load. 4. Intragastric pH increased significantly with ranitidine but not with placebo (P less than 0.001). Urinary aluminum excretion increased significantly after the administration of aluminum hydroxide during the placebo phase (P less than 0.001) but not during the ranitidine phase. Plasma aluminium concentrations were higher in the patients with renal failure than in the normal subjects (P less than 0.05), but were unchanged in both groups after the oral aluminium load. 5. This study shows that gastric pH is an important factor in the modulation of aluminum absorption in man, and indicates that reducing gastric acid secretion with ranitidine may reduce the toxicity of orally administered aluminium compounds.

Role of iron metabolism in absorption and cellular uptake of aluminum.

The effect of iron status on aluminum (Al) absorption was investigated in this study in vivo using an animal model and in vitro using an intestinal mucosal cell line. In the in vivo model rats were rendered iron overloaded by intraperitoneal injection of iron dextran (5 mg/48 hr) or iron deficient by phlebotomy (2.5 to 3 ml blood/week). These rats, and normal controls, were then dosed with Al(OH)3 (40 mg/day) for 30 days. Urinary excretion of Al was significantly greater in the iron deficient group than in the other two groups throughout the study period, and brain Al at the end of the experiment was significantly increased in the iron depleted group (1.93 micrograms/g) and decreased in the iron overloaded group (0.73 microgram/g) compared with controls (1.42 micrograms/g). The brain Al levels in iron overloaded rats were no higher than those in normal rats that had not been dosed with Al(OH)3 (0.61 microgram/g). No significant differences were found in serum Al levels. In the in vitro experiments cultures of the rat intestinal cell line RIE1 were iron overloaded by addition of iron nitrilotriacetate (0.1 mM) or iron depleted with desferrioxamine (5 micrograms/ml) for 20 days prior to pulsing with Al transferrin (0.5 mg/ml) for 24 hours. Uptake of Al was significantly greater in the iron depleted cells (2.3 ng/micrograms cell DNA) than in iron overloaded (0.81 ng) or untreated (0.83 microgram) cells. These studies show that iron depletion markedly increases absorption and cellular uptake and suggest that susceptible individuals, such as renal failure patients, run an increased risk of toxicity if they are iron deficient.

The role of transferrin and citrate in cellular uptake of aluminium.

The ability of human erythroleukaemia K562 cells to take up aluminium from Al-transferrin and Al-citrate has been examined. Uptake from Al-transferrin was dose-dependent over the range 68-544 ng/ml of aluminium, and increased over a 12-day period. In contrast, uptake from Al-citrate was low even at an aluminium concentration of 6800 ng/ml and did not increase over time. Neither form of aluminium greatly affected cell growth. It is concluded that Al-transferrin, rather than Al-citrate, is the physiologically relevant form of this metal with respect to cellular uptake, but that any metabolic abnormalities induced by aluminium do not affect proliferation of this cell line.

Aluminium content of water for injection used with recombinant human erythropoietin.

Human recombinant erythropoietin is of proven value in the treatment of the anaemia of renal failure. The aluminium content of 36 ampoules of water for injection supplied for use with recombinant erythropoietin has been measured and ranged from 24 to 450 micrograms/l, with a median of 251 micrograms/l. In three samples, which may have been contaminated on opening, the range was from 1770 to 6160 micrograms/l. In Water for Injection BP, values ranged from 66 to 140 micrograms/l with a median of 99 micrograms/l. Reconstituted erythropoietin did not contain any more aluminium than could be accounted for by the water. Ampoules of a second brand of erythropoietin, supplied already in solution, contained from 506 to 837 micrograms/l aluminium (median 682 micrograms/l). In view of the lifelong duration of erythropoietin therapy clinicians and pharmaceutical companies should be aware of this potential problem. Although the amount of aluminium delivered with each injection is usually less than 4 micrograms, it is suggested that active steps are taken to establish a British Pharmacopoeia limit on the aluminium content of injections.

Hot-injection procedures for the rapid analysis of biological samples by electrothermal atomic-absorption spectrometry.

The combination of palladium/hydrogen matrix-modification and injection of samples into a graphite tube at 120 degrees has allowed the accurate determination of copper, iron, lead and nickel in biological reference materials (urine, milk powder and bovine liver). Palladium modification allowed the use of a standard ashing temperature of 1000 degrees for all four elements. Direct aqueous calibration was applied without the need for standard additions. The total heating cycle, from the start of sample injection, took 45 sec.

Determination of nine inorganic elements in human autopsy tissue.

A survey of nine elements of accepted clinical relevance has been performed on tissue sets comprising of kidney, liver, heart and muscle taken at autopsy from healthy individuals killed as the result of an accident. The concentrations are reported using tissue wet weight, dry weight and nitrogen content as frames of reference. This has allowed direct comparisons to be made with other studies without resort to the use of conversions, using average factors, between one frame of reference and another. Such conversions are shown to be unreliable and may generate spurious differences. The results obtained are largely consistent with those reported from studies in other parts of the world.

Factors influencing lead concentrations in shed deciduous teeth.

Data collected for the Edinburgh Lead Study have been used to investigate lead concentrations in children's naturally shed deciduous teeth. A within-child multiple-regression analysis has shown that the upper jaw has a higher concentration of lead than the lower, and that there is a gradient of decreasing concentration from the front to the back of the mouth. Even after the effects of jaw and tooth type have been allowed for, the concentration is still found to be negatively correlated with the weight of the tooth and with the age at which the tooth was shed. No statistically significant effects could be attributed to caries, fillings, or the incomplete resorption of roots. A single-valued index of tooth lead has been derived for each child, taking into account the fact that children gave different types of teeth.

Essential trace element provision to patients receiving home intravenous nutrition in the United Kingdom.

The methods of provision of essential trace elements to patients receiving long-term home intravenous nutrition in the U.K. have been reviewed and their trace element status has been assessed. Over a 2 year period, 57 patients based on 15 hospitals throughout the country were studied. Although biochemical abnormalities of trace element status were frequent, related clinical complications were apparently rare. Zinc provision from commercial preparations generally required further supplementation. Copper requirements were variable. Manganese provision from trace element mixtures and chromium provision from contaminants of other parenteral nutrient solutions were excessive in many cases. Aluminium contamination is not significant in the nutrient solutions currently used in the U.K. The most common depletion state is for selenium which should probably be provided on a routine basis. The clinical consequences of long-term under- or over-provision of trace elements by the intravenous route requires further study.

Does osteomalacia contribute to development of oral complications of oxalosis?

Two renal dialysis patients with oral manifestations of oxalosis had undecalcified sections of iliac and alveolar bone and teeth examined histologically in an attempt to explain the development of tooth mobility and tooth loss. Osteomalacia was detected in all bone specimens and attributed to aluminum toxicity after the histochemical localization of aluminum at the calcification front between osteoid and calcified matrix. Aluminum was also detected histochemically in the cementum of teeth. Calcium oxalate crystals were present in bone marrow, teeth, and gingiva. It is proposed that tooth mobility and tooth loss in oxalosis result from the combined effects of osteomalacia and oxalate crystal deposition within the periodontium. To prevent avoidable tooth loss it is suggested that patients with oxalosis who develop tooth mobility should have aluminum toxicity and osteomalacia excluded as causal factors.