Bjørn J. Bolann

Multi-element analysis of trace element levels in human autopsy tissues by using inductively coupled atomic emission spectrometry technique (ICP-AES)

Autopsy tissue samples from the brain front lobe, cerebellum, heart, kidney (cortex and medulla), liver, pancreas, spleen and ovary were analysed for AL, B, Ba, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, Sr and Zn in 30 (17 women and 13 men) subjects ranging in age from 17 to 96 years at Haukeland University Hospital in Norway. The tissues were selected from macroscopically normal organs and samples were handled according to guidelines recommended to avoid contamination in the pre-analytical phase. Concentration of the trace elements were determined by the inductively coupled plasma atomic emission spectrometry technique (ICP-AES). In most tissues the concentrations of the essential trace elements followed the order Fe> Zn> Cu> Mn> Se> Cr> Co except in the ovary where Se was higher than Mn. The liver was the major site of deposition for Co, Cu and Mn as well as the spleen for Co, brain front lobe for Cu and pancreas for Mn. Ba, Sr and Ni built up in the ovary foLLowed by the kidney. Older subjects accumulated Ba and Sr in most tissues, whereas Al accumulated in the kidney cortex and Cd in the brain cerebellum. Generally males had higher concentrations of trace elements in the different tissue sampLes than females with the exception of Mn in the brain front lobe and heart and Sr in the liver. ICP-AES is a useful method to assess the concentration and the profiLe of trace elements in human autopsy tissues.

Evaluation of methods for trace‐element determination with emphasis on their usability in the clinical routine laboratory

Commonly used techniques for trace‐element analysis in human biological material are flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption spectrometry (GFAAS), inductively coupled plasma atomic emission spectrometry (ICP‐AES) and inductively coupled plasma mass spectrometry (ICP‐MS). Elements that form volatile hydrides, first of all mercury, are analysed by hydride generation techniques. In the absorption techniques the samples are vaporized into free, neutral atoms and illuminated by a light source that emits the atomic spectrum of the element under analysis. The absorbance gives a quantitative measure of the concentration of the element. ICP‐AES and ICP‐MS are multi‐element techniques. In ICP‐AES the atoms of the sample are excited by, for example, argon plasma at very high temperatures. The emitted light is directed to a detector, and the optical signals are processed to values for the concentrations of the elements. In ICP‐MS a mass spectrometer separates and detects ions produced by the ICP, according to their mass‐to‐charge ratio. Dilution of biological fluids is commonly needed to reduce the effect of the matrix. Digestion using acids and microwave energy in closed vessels at elevated pressure is often used. Matrix and spectral interferences may cause problems. Precautions should be taken against trace‐element contamination during collection, storage and processing of samples. For clinical problems requiring the analysis of only one or a few elements, the use of FAAS may be sufficient, unless the higher sensitivity of GFAAS is required. For screening of multiple elements, however, the ICP techniques are preferable.

Correlations of trace element levels within and between different normal autopsy tissues analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES)

Imbalance in trace metal metabolism may lead to metal interactions that may be of patho-physiological importance. Knowledge of the relation between trace metals in normal tissues is needed to assess abnormal deviations associated with disease. In this study correlations between Cu, Co, Cr, Fe, Mn, Ni, Se, Zn, Al, Ba, Cd, Pb and Sr within the same and between 6 different, normal autopsy tissues were determined using Spearman rank correlation analysis based on analytical data obtained by inductively coupled plasma atomic emission spectrometry (ICP-AES). Fe-Co were correlated in most tissues. Cu-Mn, Zn-Cu, Zn-Mn and Zn-Cd were highly correlated in the kidney medulla. Ni-Ni, Sr-Sr and Cd-Cd were correlated between several tissues, while Fe-Fe, Zn-Zn and Cu-Cu were correlated between kidney cortex and medulla. Mn-Mn was highly correlated between the liver and brain front lobe, cerebellum and heart. High correlations were found for Ni-Co and for Se-Mn between the kidney cortex and brain front lobe and pancreas respectively. Inverse correlations were found for Se-Cd between kidney cortex and cerebellum, for Se-Cd and Cd-Zn between kidney medulla and heart, for Co-Sr and Fe-Sr between the liver and kidney cortex and heart respectively, and for Sr-Mn between kidney medulla and pancreas. A large number of trace elements are statistically correlated within and between different, normal tissues. Knowledge of these correlations may contribute to increase the understanding of kinetic interactions of trace metals in the body and the role of such interactions in normal and disturbed trace metal metabolism.

Zinc Deficiency Is Common among Healthy Women of Reproductive Age in Bhaktapur, Nepal

Zinc deficiency is a major public health problem in many developing countries. However, its prevalence is still unknown in most populations. Women of reproductive age in developing countries are highly vulnerable to nutritional deficiencies, including that of zinc. To estimate the prevalence of zinc deficiency and to identify important dietary sources of zinc, we undertook a cross-sectional survey in 500 nonpregnant Nepalese women and measured their plasma zinc concentrations. We also examined the associations between plasma zinc and dietary intake of zinc or phytate, iron status, plasma concentrations of C-reactive protein, albumin, and hemoglobin. Food intake was estimated by 2 24-h dietary recalls and 1 FFQ for each woman. The plasma zinc concentration was (mean +/- SD) 8.5 +/- 2.4 micromol/L and more than three-quarters of the women were zinc deficient. Dietary zinc intake did not predict plasma zinc concentration, whereas phytate intake was negatively and significantly associated with plasma zinc. The other variables that were associated with plasma zinc were plasma albumin and hemoglobin concentration. Rice contributed 50% to the total estimated daily zinc intake and wheat and meat each contributed 15%. Rice also contributed 68% to the daily intake of phytate. In conclusion, we found that zinc deficiency was common in women of reproductive age and that the foods contributing substantial amounts of zinc also contributed importantly to the intake of phytate.

Validation of inductively coupled plasma atomic emission spectrometry technique (ICP-AES) for multi-element analysis of trace elements in human serum.

The use of inductively coupled plasma atomic emission spectrometry (ICP-AES) for the simultaneous determination of Al, B, Ba, Be, Cd, Co, Cr, Cu, Fe, Li, Mn, Ni, Pb, Se, Sr and Zn in human serum in a clinical laboratory was validated. Samples were digested and then analysed using yttrium as an internal standard and a serum-matched calibration standard. The criteria used to assess the analytical performance of the ICP-AES were detection and quanti® cation limits, linearity, sensitivity, recovery, interference from alkali and acid, trueness and precision. Detection limits were 0.002 ± 0.003 mmol/L for Mn, Sr, Ba, and Cd; 0.014 ± 0.07 mmol/L for Co, Zn, Fe, Be, Li, Pb, Cu, Ni, and Cr; and 0.2 ± 0.9 mmol/L for B, Se, and Al. Trueness, as controlled by analysis of bovine serum certi® ed reference material, was acceptable for Co, Cu, Se and Zn, while Fe was 5.1% and Mn 6.2% below the lowest limit of the certi® ed material interval. We conclude that ICP-AES can be used for multi-element analysis of B, Ba, Cu, Fe, Li, Se, Sr and Zn in serum. Serum levels of Al, Be and Co were below the detection limits while serum levels of Cd, Cr, Ni and Pb were below the quanti® cation limits of the ICP-AES. These trace metals cannot be analysed as routine by the ICP-AES. However, in cases of intoxication with elevated serum concentrations mean recovery of 100¡10% was obtained at an addition of 2.22 mmol/L for Al, 0.11 mmol/L for Be, 0.03 mmol/L for Co, 0.39 mmol/L for Cr, 0.14 mmol/L for Ni, and 0.12 mmol/L for Pb.The use of inductively coupled plasma atomic emission spectrometry (ICP-AES) for the simultaneous determination of Al, B, Ba, Be, Cd, Co, Cr, Cu, Fe, Li, Mn, Ni, Pb, Se, Sr and Zn in human serum in a clinical laboratory was validated. Samples were digested and then analysed using yttrium as an internal standard and a serum-matched calibration standard. The criteria used to assess the analytical performance of the ICP-AES were detection and quantification limits, linearity, sensitivity, recovery, interference from alkali and acid, trueness and precision. Detection limits were 0.002-0.003 micromol/L for Mn, Sr, Ba, and Cd; 0.014-0.07 micromol/L for Co, Zn, Fe, Be, Li, Pb, Cu, Ni, and Cr; and 0.2-0.9 micromol/L for B, Se, and Al. Trueness, as controlled by analysis of bovine serum certified reference material, was acceptable for Co, Cu, Se and Zn, while Fe was 5.1% and Mn 6.2% below the lowest limit of the certified material interval. We conclude that ICP-AES can be used for multi-element analysis of B, Ba, Cu, Fe, Li, Se, Sr and Zn in serum. Serum levels of Al, Be and Co were below the detection limits while serum levels of Cd, Cr, Ni and Pb were below the quantification limits of the ICP-AES. These trace metals cannot be analysed as routine by the ICP-AES. However, in cases of intoxication with elevated serum concentrations mean recovery of 100+/-10% was obtained at an addition of 2.22 micromol/L for Al, 0.11 micromol/L for Be, 0.03 micromol/L for Co, 0.39 micromol/L for Cr, 0.14 micromol/L for Ni, and 0.12 micromol/L for Pb.

Trace element reference values in serum determined by inductively coupled plasma atomic emission spectrometry.

Abstract Serum reference values for Ba, B, Cd, Cu, Fe, Mn, Li, Se, Sr, and Zn in 141 healthy Norwegians were determined. The trace element concentrations were determined by the inductively coupled plasma atomic emission spectrometry technique that we have recently validated. The reference intervals were established according to the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine. Also coverage intervals with coverage uncertainties were calculated according to the International Union of Pure and Applied Chemistry. The population studied consisted of 69 men and 72 women of the ages 21–87 years. The effects of gender, age, smoking, and oral contraceptives on serum levels of trace elements were investigated. Median concentrations of the different trace elements in (μmol/l) were as follows: Ba (0.44), B (1.50), Cd (0.004), Cu (17.1), Fe (21.4), Li (0.06), Mn (0.003), Se (1.26), Sr (0.17), and Zn (13.3). An increase in serum Ba and Sr was detected with age. These metals and Se were also significantly higher in women over 50 years of age in comparison to younger women. Women had higher serum Cu than men and those on oral contraceptives had higher serum Cu and Fe. Serum B tended to increase with age, while it was significantly reduced with smoking.

Zinc status in HIV infected Ugandan children aged 1-5 years: a cross sectional baseline survey

BackgroundLow concentrations of serum zinc have been reported in HIV infected adults and are associated with disease progression and an increased risk of death. Few studies have been conducted in HIV infected children in Africa. We determined serum zinc levels and factors associated with zinc deficiency in HIV infected Ugandan children.MethodsWe measured the baseline zinc status of 247 children aged 1-5 years enrolled in a randomised trial for multiple micronutrient supplementation at paediatric HIV clinics in Uganda (http://ClinicalTrials.gov NCT00122941). Zinc status was determined using inductively coupled atomic emission spectrophotometry (ICP-AES). Clinical and laboratory characteristics were compared among zinc deficient (zinc < 10.0 μmol/L) and non deficient children. Logistic regression was used to determine predictors of low serum zinc.ResultsOf the 247 children, 134 (54.3%) had low serum zinc (< 10.0 μmol/L). Of the 44 children on highly active antiretroviral therapy (HAART), 13 (29.5%) had low zinc compared to 121/203 (59.6%) who were not on HAART. Overall, independent predictors of low zinc were fever (OR 2.2; 95%CI 1.1 - 4.6) and not taking HAART (OR 3.7; 95%CI 1.8 - 7.6).ConclusionAlmost two thirds of HAART naïve and a third of HAART treated HIV infected children were zinc deficient. Increased access to HAART among HIV infected children living in Uganda might reduce the prevalence of zinc deficiency.

Tetradecylthioacetic acid and tetradecylselenoacetic acid inhibit lipid peroxidation and interact with superoxide radical.

Reactive oxygen species are thought to induce cellular damage and to play a pathological role in several human diseases. Tetradecylthioacetic acid (TTA) was previously reported to prevent the oxidative modification of low-density lipoprotein (LDL) particles and to act as an antioxidant. In this study we present a new fatty acid analogue, namely tetradecylselenoacetic acid (TSA), in which the sulfur atom of TTA is replaced by a selenium atom. TSA was more potent than TTA in increasing the lag time before the onset of LDL oxidation and this effect was dose dependent. TTA and TSA were shown to reduce the iron-ascorbate-induced microsomal lipid peroxidation, TSA being more efficient than TTA. TTA and TSA, in the presence of iron, interacted with the superoxide radical as assessed by direct and indirect testing methods. TSA like TTA failed to scavenge 1.1-diphenyl-2-picrylhydrazyl radicals. TSA bound copper ions as shown by the wavelength spectra measurement. These results suggest that TTA and TSA exert their antioxidant capacity by interaction with copper or iron ions in radical scavenging, TSA being more potent than TTA. Nevertheless, a chelating effect resulting in chemically inactive metal ions cannot be excluded.

Normothermic liver ischemia in rats: xanthine oxidase is not the main source of oxygen free radicals.

We studied the effect of allopurinol (ALL) on the activity of xanthine dehydrogenase (XDH), xanthine oxidase (XOX), superoxide dismutase (SOD), and catalase (CAT) in rat liver during ischemia followed by 60 min of reperfusion. We induced 60-min ischemia in the median and left lobes by clamping the hepatic artery and portal branches. The percentage XOX relative to total oxidase activity increased significantly in the control group, from 10% during the stabilization period to 18% after 60 min of reperfusion. The XDH activity decreased during reperfusion. Activity of both XDH and XOX was almost completely blocked by ALL. The activity of SOD and CAT did not differ significantly between the ALL group and controls after 60 min of reperfusion. ALL treatment did not affect liver injury parameters, as concentrations of lactate dehydrogenase (LDH) and alanine transferase (ALT) increased in plasma after ischemia, both in controls and in the ALL-treated group. We concluded that ischemia promotes conversion of XDH to XOX during reperfusion. XOX may not be the main source of free radical production, since intracellular scavengers (SOD and CAT) did not differ significantly between controls and the ALL-treated group, despite the fact that ALL blocked XOX activity completely.

Decay kinetics of O2.− studied by direct spectrophotometry. Interaction with catalytic and non-catalytic substances

The kinetic behaviour of O2.- during spontaneous dismutation and in the presence of Cu,Zn-superoxide dismutase and other compounds, was studied by monitoring the decrease in absorbance (A250nm-A360nm) on a time-scale of > or = 1 min, at pH 9.5. O2.- was generated from KO2, and calculations were performed between 25 and 4 microM of O2.-. An algorithm for the simultaneous calculation of the 1st and 2nd-order rate constants from the decay curve, was evolved. The respective fractions of O2.- which interacted with catalysts or disappeared spontaneously, in various experimental situations, could be estimated. Substances could be classified as inert, catalysts or scavengers. The high assay pH excluded examination of the effect of alkali sensitive substances, e.g., Mn-superoxide dismutase. However, the high pH minimized the interfering effect of trace amounts of Cu(II). Therefore a metal chelator was superfluous and even the effect of metals and metal complexes could be tested. The extremely high sensitivity of the method allowed minute concentrations of reagents to be used, including proteins absorbing in the UV-region. The rate constants found by this simple method, agreed with those obtained by more sophisticated and inaccessible techniques like pulse radiolysis and stopped-flow spectrophotometry.