ACP Best Practice No 163. Wilson's disease: acute and presymptomatic laboratory diagnosis and monitoring.

Wilson's disease, the most common inherited disorder of copper metabolism, is a recessive genetic condition. The clinical presentation of Wilson's disease is very variable. It is characterised by low serum copper and caeruloplasmin concentrations coupled with the pathological accumulation of copper in the tissues. However, there are diagnostic difficulties and these are discussed. The current value of DNA diagnosis, both in gene tracking in families or as applied to de novo cases, is examined. Wilson's disease can be treated successfully but treatment must be life long. Patients are best treated by specialist centres with experience and expertise in the condition.

Cadmium concentrations in human kidney in the UK: 1978-1993.

Almost 2700 samples of human renal cortex have been collected from throughout the UK over a 16 year period from 1978 to 1993. The mean Cd concentration was 19 micrograms g-1 and the median 16 micrograms g-1. Smokers were, on average, about 5 micrograms g-1 higher than non-smokers. Cd increased from low concentration in the young to a maximum of 23 micrograms g-1 in middle age followed by a decrease in old age. Subjects who had died of renal disease had lower Cd concentrations. Geographical variations in the UK are small and the concentrations appear to be static over the 16 year period. Some 3.9% of the population had Cd concentrations > 50 micrograms g-1, the critical level at which beta 2-microglobulin appears in urine.

Determination of low concentrations of potentially toxic elements in human liver from newborns and infants.

One hundred and fifty-seven liver samples from newborns and infants who had died from sudden infant death syndrome (SIDS) or other known causes have been analysed by ICP-MS for Ag, Cd, Co, Pb and Sb. The median concentrations found were: 15.4 (Ag), 2.9 (Cd), 15.9 (Co), 65.2 (Pb) and 1.8 (Sb) ng g-1 wet mass. There was no measurable difference in the concentrations of any of these elements between the SIDS and non-SIDS groups. The validity of the results was assessed by analysis of appropriate reference materials, interlaboratory comparison and isotope dilution analysis. The instrumental limits of detection were 0.25 (Ag), 0.14 (Cd), 0.21 (Co), 3.8 (Pb) and 0.38 (Sb) ng g-1 wet mass. The limits of detection of the method depend on the reagent blank and the extent of background contamination.

Assessment of status.

Laboratory methods for the measurement of micronutrients are usually based upon determinations in the peripheral blood. Most are relatively insensitive, but can screen for impending acute deficiency or toxicity. The metabolic responses to disease, injury and infection may alter the laboratory findings independently of dietary supply. Methods based upon the intra-cellular biochemical function of individual micronutrients are becoming available and can detect more marginal deficiencies. The investigation of general populations for micronutrient depletions largely relies upon clinical and epidemiological evaluation of responses to controlled trials of supplementation.

Effect of inflammation on measures of antioxidant status in patients with non-small cell lung cancer.

This study examined the effect of an inflammatory response on measures of antioxidant status in patients with non-small cell lung cancer (NSCLC). In healthy, control subjects (n = 13) and NSCLC patients (n = 22) fasting concentrations of albumin, C-reactive protein, cholesterol, and the antioxidants alpha-tocopherol, retinol, lutein, lycopene, and alpha- and beta-carotene were measured. The two groups were similar in terms of age, sex, and body mass index. However, the cancer group had an inflammatory response as evidenced by significantly increased C-reactive protein concentrations. Concentrations of all the measured antioxidants of the NSCLC group were significantly lower than those of the control group (P < 0.01). The lowest concentrations were those of the carotenoids lycopene and alpha- and beta-carotene. In the cancer group there were significant negative correlations between concentrations of C-reactive protein and retinol (r = -0.682, P < 0.01), alpha-tocopherol (r = -0.464, P < 0.05), and lutein (r = -0.599, P < 0.01). The results of this study have implications for the interpretation of circulating antioxidant concentrations in patients with NSCLC.

Nickel metabolism in humans investigated with an oral stable isotope.

We report the results of the first complete study of nickel metabolism in human subjects using a stable nickel isotope (62Ni) as tracer. Four healthy adult subjects (two women and two men) fasted overnight before ingesting 10 microg 62Ni/kg body wt. Blood samples were drawn after fixed intervals of time and the total daily output of urine and feces was collected for the first 5 d after dose ingestion. 62Ni in plasma, urine, and feces was determined by isotope-dilution inductively coupled plasma-mass spectrometry with 61Ni. The direct measurement of the fecal excretion of the tracer allowed a reliable assessment of nickel absorption from the gastrointestinal tract and we found no evidence of the excretion of absorbed nickel via the gut. The percentage absorption calculated from the amount of 62Ni excreted in the feces ranged from 29% to 40%. Urinary excretion over 5 d ranged from 51% to 82% of the absorbed dose. Plasma 62Ni peaked between 1.5 and 2.5 h after ingestion and decreased by a factor of > 10 over the next few days. We observed low between-subject variability of nickel absorption and excretion. Confounding factors such as contamination and dietary intake of nickel, which hampered earlier measurements in subjects dosed with naturally abundant nickel, were eliminated by using the tracer isotope 62Ni.

Erythrocyte magnesium in elderly patients: not a reliable guide to magnesium status.

To assess body magnesium status in various illness states in older people by measurement of serum magnesium (S Mg) and erythrocyte magnesium (E Mg) and to explore the limitations of E Mg measurement. S Mg and E Mg were measured in 150 consecutive out-patients, mean age 77 years, and in 100 consecutive in-patient admissions, mean age 80 years. Results were analysed for different diagnostic groups S Mg was normally distributed for both in-patients and out-patients, mean values 0.79 mmol/l and 0.77 mmol/l respectively. In-patient E Mg concentrations were often higher but the distribution was considerably skewed, median 2.28 mmol/l, mean 2.35 mmol/l. Out-patient E Mg concentration followed a near normal distribution, median 2.32 mmol/l, mean 2.30 mmol/l. There was a significant correlation between E Mg and S Mg for out-patients, R = 0.29 (p < 0.001). In-patients with infections and pressure sores had significantly raised E Mg concentrations but normal or low S Mg. High E Mg concentrations in illness are likely to be due to alterations in characteristics of the erythrocytes themselves rather than an indication of body magnesium excess. E Mg concentrations in illness should be interpreted with caution.

Acute-phase reactants and plasma trace element concentrations in non-small cell lung cancer patients and controls.

This study examined the effect of an acute-phase response on plasma trace element concentrations of non-small cell lung cancer (NSCLC) patients. In normal subjects (n = 13) and NSCLC patients (n = 22), fasting concentrations of albumin, C-reactive protein, the trace elements iron, zinc, copper, and selenium, and their associated proteins transferrin, albumin, ceruloplasmin, and glutathione peroxidase were measured. The NSCLC patients were subdivided into two equal groups depending on whether they had a C-reactive protein concentration < 35 mg/l (Group 1) or > 35 mg/l (Group 2). Circulating zinc, iron, and transferrin concentrations were significantly lower in NSCLC Group 1 than in the control group (p < 0.05). Circulating concentrations of iron, zinc, and the binding proteins transferrin and albumin were significantly lower in NSCLC Group 2 than in the control group and NSCLC Group 1 (zinc not significantly different) (p < 0.01). In contrast circulating concentrations of copper and its binding protein ceruloplasmin were significantly increased in NSCLC Group 2 compared with NSCLC Group 1 and the control group (p < 0.01). Additionally, plasma selenium and glutathione peroxidase concentrations were significantly lower (p < 0.05) in NSCLC Group 2 than in NSCLC Group 1 and the control group. In the NSCLC patients there were significant negative correlations between concentrations of C-reactive protein and iron, transferrin, zinc, albumin, and selenium (p < 0.05). Furthermore, there were also significant positive correlations between C-reactive protein and copper (r = 0.788, p < 0.001) and ceruloplasmin (r = 0.831, p < 0.001) concentrations. The presence of an acute-phase response has implications for the interpretation of circulating trace element concentrations, the status of patients with NSCLC, and supplementation with trace elements in patients with NSCLC.

Determination of antimony in urine, blood and serum and in liver and lung tissues of infants by inductively coupled plasma mass spectrometry.

Methods are presented for the determination by ICP-MS of antimony in body fluids and tissues of infants. Urine, serum and whole blood specimens are prepared for analysis by simply diluting 200 microliters sample volumes (1 + 14) with water and adding indium as internal standard. Liver and lung tissues are digested using 16 M HNO3 either in open quartz vessels at 150 degrees C or in sealed vessels with microwave heating. The acid digests are diluted with water and indium is added as internal standard for ICP-MS measurements. All analyses were subjected to stringent internal quality control protocols. Accuracy was assessed by recoveries, repeated analyses and by analysis of NIST SRMs 1577a Bovine Liver and 1566a Oyster Tissue. Precisions of analyses were better than 5-10% in the ranges 0.1-0.3 microgram l-1 for urine, serum and blood; and at 7-25 ng g-1 in tissues. Detection limits were 0.7 ng g-1 in liver, 0.8 ng g-1 in lung, and 0.01 microgram l-1 in urine, serum and blood. The need to employ validated procedures for specimen collection and to give considerable attention to pre-analytical factors in order to avoid adventitious contamination with antimony is demonstrated.

Improved molecular fluorescence method for the determination of selenium in biological samples.

A procedure for the determination of selenium in whole blood and urine has been improved by optimization of the digestion and derivatization procedures. An overnight pre-digestion step with 4 + 1 concentrated nitric-perchloric acids reduced the time of mineralization at 170 degrees C from 4 h to 30-45 min. Conversion of all the selenium to selenite (SeIV) was optimized by addition of 1 ml of concentrated hydrochloric acid, with heating to 100 degrees C for 30 min. The rate of formation of the 2,3-diaminonaphthalene (DAN) complex of selenium was improved by heating to 70-100 degrees C for a minimum of 30 min. Co-addition of hexane during derivatization simplified the extraction procedure. The modified method was applied successfully to the analysis of Seronorm quality control whole blood and urine (83 and 24 micrograms l-1 Se, respectively). Samples from 12 healthy adults, gave results in expected ranges (mean concentrations of 75 +/- 8 micrograms l-1 in blood and 25 +/- 8 micrograms l-1 in urine). The structure of the Se-DAN complex was investigated using elemental analysis, FTIR spectrometry, 1H and 13C NMR spectroscopy, and FAB MS. The information obtained indicates that the selenodiazo group does not contain an Se-O bond or protonated nitrogens, as proposed in other studies.

Determination of citrate in plasma protein solutions by UV-visible spectrophotometry and ion chromatography.

Two procedures were compared for the determination of citrate in plasma protein solution (PPS). Ion chromatography with gradient elution was preferred to an enzymatic-spectrophotometric method for citrate concentrations in the range 0.4-11 mg l-1. Better comparisons of results by both methods were obtained for citrate concentrations at the g l-1 level, as dilution of the PPS reduced interferences in the enzymatic procedure.

Distribution of selenium in human blood plasma and serum.

A method has been developed which separates the three major selenium-containing proteins found in human blood serum and plasma: selenoprotein-P, glutathione peroxidase and albumin. They were separated from plasma or serum by affinity chromatography and the Se content determined directly by ETAAS. Selenoprotein-P is retained on a heparin-Sepharose column, and subsequently eluted with an excess of heparin, while glutathione peroxidase is separated by a blue-Sepharose column. The amount of Se associated with albumin was assumed to be the Se remaining in the rest of the sample. The detection limit of the ETAAS method, when applied to the separated fractions, was 0.8 microgram l-1 (2 ng absolute) and the accuracy of the determination was confirmed by comparison with spectrofluorimetry. The distribution of Se in the serum or plasma of 21 healthy people was determined, showing that 53 +/- 6% of the total present is associated with selenoprotein-P, 39 +/- 6% as glutathione peroxidase and 9 +/- 4% as albumin.

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.

Use of a stable copper isotope (65Cu) in the differential diagnosis of Wilson's disease.

1. 65Cu/63Cu stable-isotope ratios have been measured in blood serum after oral administration of the stable isotope 65Cu. The incorporation of the isotope into the plasma protein pool was followed at various times for up to 3 days. The resulting patterns of enrichment in healthy control subjects, in Wilson's disease patients and in heterozygotes for the Wilson's disease gene, were similar in appearance to those found by others using copper radioactive isotopes. After an initially high enrichment at 2 h after dosage, the Wilson's disease cases, in contrast to the control subjects, did not show a secondary rise in isotope enrichment of the plasma pool after 72 h, demonstrating a failure to incorporate copper into caeruloplasmin. The Wilson's disease heterozygotes had variable degrees of impairment of isotope incorporation, not always distinguished from those of control subjects. 2. The stability of the isotope also permits the copper tracer to be followed for a longer period. Ten healthy subjects were studied for over 40 days, allowing the biological half-time of an oral dose of copper to be determined (median 18.5 days, 95% confidence interval 14-26 days). Known heterozygotes for the Wilson's disease gene were found to have a significantly increased biological half-time for removal of copper from the plasma pool (median 43 days, 95% confidence interval 32-77 days). 3. The incorporation of 65 Cu in patients with diseases of the liver (other than Wilson's disease) was found to be similar to that in control subjects, aiding differential diagnosis.