Human biomonitoring of 73 elements in blood,serum, erythrocytes and urine

BackgroundHuman biomonitoring studies of trace elements in biological fluids are mostly limited to a certain number of elements or biological materials. In this study, we describe the significant extension of a biomonitoring to 73 elements being present in concentration ranges from ng/L to g/L in clinically relevant specimens such as blood, serum, erythrocytes and urine.MethodsThe samples were collected from 102 occupationally non-exposed inhabitants of northern Germany. The elements were determined either by inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) in the low concentration range or by inductively coupled plasma optical emission spectrometry (ICP-OES) for essential trace elements and electrolytes.ResultsMean values and selected percentiles of element concentrations are presented for all sample materials. From the results, we calculated the distribution of elements between plasma and blood cells. Application of ICP-MS/MS improves selectivity and accuracy in the determination of elements that are strongly spectrally interfered, such as Cr, Ge, Pd or Ti in blood samples.ConclusionsThis publication provides very valuable information for occupational or environmental hygienists, toxicologists and clinical chemists due to the particularly high number of determined elements and presented concentration ranges.     

压力罐消解ICP-MS法同时测定全血中5种微量元素

为了建立同时测定全血中微量元素铬、锰、砷、镉、铅的电感耦合等离子体质谱(ICP-MS)分析法,采用压力罐消解技术对全血样品进行消化,使用ICP-MS法对全血中的五种微量元素铬、锰、砷、镉、铅含量进行测定。结果显示,全血样品中所测定的5种微量元素浓度为0~0.20μg·mL-1时,线性关系良好,相关系数r>0.999,各元素相对标准偏差(RSD)均小于5.0%(n=6),加标回收率为88.4%~107.6%,本法对铬、锰、砷、镉、铅的最低检出浓度分别为0.60、1.4、0.70、0.048、0.18μg·L-1。表明运用ICP-MS同时检测全血中微量元素具有良好的准确度和精密度、灵敏度高、检出限低、元素之间的干扰少,方法高效可行。     

Trace element contamination in feather and tissue samples from Anna’s hummingbirds

Trace element contamination (17 elements; Be, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Ba, Hg, Tl, and Pb) of live (feather samples only) and deceased (feather and tissue samples) Anna's hummingbirds (Calypte anna) was evaluated. Samples were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS; 17 elements) and atomic absorption spectrophotometry (Hg only). Mean plus one standard deviation (SD) was considered the benchmark, and concentrations above the mean + 1 SD were considered elevated above normal. Contour feathers were sampled from live birds of varying age, sex, and California locations. In order to reduce thermal impacts, minimal feathers were taken from live birds, therefore a novel method was developed for preparation of low mass feather samples for ICP-MS analysis. The study found that the novel feather preparation method enabled small mass feather samples to be analyzed for trace elements using ICP-MS. For feather samples from live birds, all trace elements, with the exception of beryllium, had concentrations above the mean + 1 SD. Important risk factors for elevated trace element concentrations in feathers of live birds were age for iron, zinc, and arsenic, and location for iron, manganese, zinc, and selenium. For samples from deceased birds, ICP-MS results from body and tail feathers were correlated for Fe, Zn, and Pb, and feather concentrations were correlated with renal (Fe, Zn, Pb) or hepatic (Hg) tissue concentrations. Results for AA spectrophotometry analyzed samples from deceased birds further supported the ICP-MS findings where a strong correlation between mercury concentrations in feather and tissue (pectoral muscle) samples was found. These study results support that sampling feathers from live free-ranging hummingbirds might be a useful, non-lethal sampling method for evaluating trace element exposure and provides a sampling alternative since their small body size limits traditional sampling of blood and tissues. The results from this study provide a benchmark for the distribution of trace element concentrations in feather and tissue samples from hummingbirds and suggests a reference mark for exceeding normal. Lastly, pollinating avian species are minimally represented in the literature as bioindicators for environmental trace element contamination. Given that trace elements can move through food chains by a variety of routes, our study indicates that hummingbirds are possible bioindicators of environmental trace element contamination.     

Cross-sectional analysis of trace element status in thyroid disease

BackgroundThe synthesis of thyroid hormone depends on a set of trace elements, most importantly selenium and iodine. The dietary supply with certain micronutrients is limited in many areas of the world, including central Europe and large parts of Asia and Africa. Moreover, both thyroid disease risk and therapy effects are modulated by trace element supply and status.ObjectiveAssessment of trace element status in thyroid patients in a European metropolis.Material and MethodsAdult patients visiting a medical praxis in Berlin, Germany, were enrolled into a cross-sectional analysis, and serum samples were obtained from thyroid patients (n = 323) with different conditions including goitre, hypothyroidism, malignancy or autoimmune thyroid disease. Trace elements (iodine, selenium, copper and zinc) were assessed by ICP-MS/MS or total reflection X-ray analysis, along with two protein biomarkers of selenium status (selenoprotein P, glutathione peroxidase), and compared to the clinical phenotype.ResultsThe patients displayed relatively low serum zinc and selenium concentrations as compared to a set (n = 200) of healthy subjects (zinc; 1025+/-233 vs. 1068+/−230 μg/L, p < 0.01, selenium; 76.9+/18.8 vs. 85.1+/−17.4 μg/L, p < 0.0001). A high fraction of patients (37.5%) was classified as selenium-deficient (serum selenium concentrations <70 μg/L), in particular the patients with thyroid malignancy (59%). Serum copper was not different between the groups, and total serum iodine concentrations were unrelated to thyroid disease. Explorative statistical analyses yielded no significant interactions between the trace elements and disease parameters, except for free thyroxine inversely correlating to the copper/selenium ratio.ConclusionsIn adult thyroid patients, there is no relation of circulating copper, iodine, selenium or zinc concentrations to thyroid hormone. However, a large fraction of German thyroid patients displays a considerable selenium deficit, known to constitute a disease risk potentially impairing convalescence and aggravating autoimmune disease processes. It appears advisable to testing thyroid patients for selenium deficiency, and once diagnosed, an increased supply via dietary counselling or active supplementation should be considered.     

Effects of storage conditions on the stability and distribution of clinical trace elements in whole blood and plasma: Application of ICP-MS

BackgroundKnowledge of trace element stability during sample handling and preservation is a prerequisite to produce reliable test results in clinical trace element analysis.MethodAn alkaline dissolution method has been developed using inductively coupled plasma mass spectrometry to quantify eighteen trace element concentrations: vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, bromine, molybdenum, cadmium, antimony, iodine, mercury, thallium, lead, and bismuth in human blood, using a small sample volume of 0.1 mL. The study evaluated the comparative effects of storage conditions on the stability of nutritionally essential and non-essential elements in human blood and plasma samples stored at three different temperatures (4 °C, −20 °C and −80 °C) over a one-year period, and analysed at multiple time points. The distribution of these elements between whole blood and plasma and their distribution relationships are illustrated using blood samples from 66 adult donors in Queensland.ResultsThe refrigeration and freezing of blood and plasma specimens proved to be suitable storage conditions for many of the trace elements for periods up to six months, with essentially unchanged concentrations. Substantially consistent recoveries were obtained by preserving specimens at −20 °C for up to one year. Ultra-freezing of the specimens at −80 °C did not improve stability; but appeared to result in adsorption and/or precipitation of some elements, accompanied by a longer sample thawing time. A population sample study revealed significant differences between the blood and plasma concentrations of six essential elements and their relationships also varied significantly for different elements.ConclusionBlood and plasma specimens can be reliably stored at 4 °C for six months or kept frozen at −20 °C up to one year to obtain high quality test results of trace elements.     

Concentration of 17 trace elements in serum and whole blood of plains viscachas (Lagostomus maximus) by ICP-MS, their reference ranges, and their relation to cataract

The reference ranges of the trace elements Al, As, Be, B, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, Li, Rb, Se, Sr, and Zn were determined by inductively coupled plasma-mass spectrometry (ICP-MS) in sera of a group of free-ranging plains viscachas of the pampa grasslands of Argentina. The values were compared with those of a small group of captive plains viscachas of the Zurich Zoo with diabetes and bilateral cataracts. In addition, a method for digestion of whole-blood samples is described for the trace element determination. Significant differences in the concentration of trace elements in the two groups of animals are discussed. No correlation was found between the levels of selenium and of other trace elements compared to the formation of cataracts.     

Trace-element contents of human fetal liver of 12–22 weeks gestation

There are some papers in the literature on the trace element contents of fetal livers of 20-wk gestation time and over. However, there is very little information on this subject for fetal livers of less than 20-wk gestation. We have initiated a program on the measurement of trace elements in fetal livers of 12–22-wk gestation, using thick-target X-ray fluorescence analysis. The liver samples were obtained from freshly aborted fetuses. After removing blood from the samples, they were chopped into small pieces and freeze dried. The resulting material was ground into fine powder and compressed into 3-mm thick pellets, with boric acid backing. A similar pellet was also made of NBS—Bovine Liver—which was used as the standard for calculating the absolute concentrations of different trace elements. The measurements were carried out using a commercial Wave-Length-Dispersive XRF-System. Different X-ray tubes were used for different sets of elements in order to maximize the detection sensitivity. The results are compared with those of fetal liver of longer gestation and adult livers.     

Alterations of Mg<Superscript>2+</Superscript> After Hemorrhagic Shock

The objectives of this study were to measure the concentrations of elements in raw milk by inductively coupled plasma-mass spectrometry (ICP-MS) and evaluate differences in element concentrations among animal species and regions of China. Furthermore, drinking water and feed samples were analyzed to investigate whether the element concentrations in raw milk are correlated with those in water and feed. All samples were analyzed by ICP-MS following microwave-assisted acid digestion. The mean recovery of the elements was 98.7 % from milk, 103.7 % from water, and 93.3 % from a certified reference material (cabbage). Principal component analysis results revealed that element concentrations differed among animal species and regions. Correlation analysis showed that trace elements Mn, Fe, Ni, Ga, Se, Sr, Cs, U in water and Co, Ni, Cu, Se, U in feed were significantly correlated with those in milk (p < 0.05). Toxic and potential toxic elements Cr, As, Cd, Tl, Pb in water and Al, Cr, As, Hg, Tl in feed were significantly correlated with those in milk (p < 0.05). Results of correlation analysis revealed that elements in water and feed might contribute to the elements in milk.     

Cumulative Author Index

Abstract

Bread represents an important source of trace elements in the human diet. This study is focused on home prepared bread in the Czech Republic. The amounts of Cu, Mo, Mn, Ni and Zn (total and soluble in Tris-HCl buffer, pH 7.5), Cd, Co, Fe, Pb and Tl (total only) as well as Hg (total and soluble in mercaptoethanol-HCl mixture) in raw materials and baked bread were determined using ICP-MS. Moreover, the speciation of elements was investigated using HPLC/ICP-MS. Isolated peptide ligands of the trace elements were analysed for amino acids and characterised by MALDI-MS. The concentrations of all elements were in accordance with Czech legislation. The solubility of the Ni species was not affected by the baking process, whereas the solubilities of Mo, Mn and Zn species decreased. Soluble mercury was found only in the inorganic form. The soluble species of Cu, Mo, Mn, Ni and Zn were found in two fractions with the apparent molecular weights of 1–2 kDa and 4–5 kDa. Ligands of trace metals isolated from these fractions contained appreciable amounts of Asx, Glx, Gly, Ser and Cys. No phytochelatin-like peptides were found in the MALDI-MS spectra of isolated ligands,. Using MALDI-MS/MS, the partial amino-acids sequences of peptide ligands were obtained, and the linkages of peptides and saccharides confirmed. The MS analysis of the trypsin digest of the medium molecular weight fraction revealed several proteins rich in cysteine (e.g., barwin and amylase inhibitors).     

INAA and PIXE of Atmospheric and Combustion Aerosols

Using instrumental neutron activation analyses and photoninduced x-ray emission techniques for analysis of size-fractionated atmospheric and combustion aerosols and other emission samples arising from fluidized-bed combustion of North Bohemian lignites up to 42 elements were determined in all sample types. This allowed the evaluation of element enrichment, time trends, and interelement correlations and the performance of factor analysis of various fractions of atmospheric aerosols. The data obtained on mass and element size distributions of aerosols and emission samples obtained upon lignite combustion in an experimental scale atmospheric fluidized-bed combustor without and with added hydrated lime and limestone were used to elucidate the mechanism of abatement of toxic trace and matrix elements from flue gas.     

Trace elements distribution and species fractionation in the wheat (Triticum aestivum) plant

Abstract

The status of 13 trace elements’ (both essential and toxic) was investigated in individual parts of the winter wheat plant(Triticum aestivum) taken during its whole cultivation period. The study includes the determination of total concentrations, portions soluble in 0.02 mol L^?1 Tris-HCI buffer solution (pH = 7.5), and the fractionation of soluble species of elements by SEC and ICP/MS. Ligands of trace elements from a low-molecular weight SEC fraction were isolated by affinity chromatography and characterised by MALDI/MS analyses and by amino acids composition. Inhomogeneous accumulation of trace elements was found in the analysed plant tissues. The concentrations of elements are also affected by the maturity of the plants. The distribution of the soluble species of the elements between chromatographic fractions exhibited some regularity in all the samples. Substantial amounts of trace elements are located in a low-molecular weight fraction (< 2 kDa). Only chromatograms of Zn (grain) and Cu (all samples) contain significant medium-molecular and high-molecular weight fractions. Compounds isolated from the low-molecular weight fractions are rich in cystein and dicarboxylic amino acids or their amides. MALDI/MS spectra of these compounds isolated from shoots, straw and grain confirmed the presence of the phytochelatin PC₅.     

Evaluation of total contents of Al,As, Ca,Cd, Fe,K, Mg,Ni, Pb,Zn and their fractions leached to the infusions of different tea samples. A multivariate study

ABSTRACT

Total aluminum, arsenic, calcium, cadmium, iron, lead, potassium, magnesium, nickel and zinc were determined in black tea by electro thermal atomic absorption spectrometry after ultrasonic assisted pseudo-digestion with mixture of acid and oxidant. All the metals were also determined in the tea infusions in order to know the percentage of each element leached into the liquor. A conventional acid digestion on electric hot plate was used to obtain total metals under study for comparative purpose. Analytical results for Al, As, Ca, Cd, Fe, K, Mg, Ni, Pb and Zn obtained by ultrasound assisted pseudodigestion, and conventional wet digestion methods showed a good agreement, thus indicating the possibility of using ultrasonic assisted digestion sample preparation instead of intensive treatments inherent with the acid digestion methods on electric hot plate. The validity and accuracy of both procedures were checked by using certified sample of NIES No. 7 (Tea Leaves). Non significant differences were observed for P>0.05 when comparing the values obtained by both methods (paired t-test).

Principal component analysis (PCA) was applied in order to determine the different amount of metals as main sources of variability in the data sets and to establish the relation between samples (branded and nonbranded tea samples) and micronutrient, trace and toxic metals (variables). Hierarchical cluster analysis (HCA) was used to explore the different branded and nonbranded tea samples grouping according to the essential and toxic metals as additional information to the out put obtained by PCA.     

Evaluation of methods for total selenium determination in yeast

The selenium determination in biological materials by the classical fluorometric method (FM) is time-consuming and also hazardous, as it requires the destruction of the organic matrix samples with hot HNO₃/HClO₄ mixtures prior to analysis. Accordingly, commercial analytical laboratories are increasingly using faster instrumental methods; for sample digestion, avoid using HClO₄. Because of these procedural changes, the results obtained by commercial laboratories may be unreliable, especially for samples containing Se in organic forms. One such “difficult” substrate is Se yeast, which contains most of its Se as selenomethionine. To establish which methods for Se analysis and sample digestion are applicable, samples of Se yeast and of selenomethionine standards were sent to laboratories employing either flame atomic absorption spectrometry (FAAS), inductively coupled plasma-mass spectrometry (ICP-MS), or hydride generation atomic absorption spectrometry (HGAAS). The result were compared with those obtained by FM and non-destructive instrumental neutron activation analysis (INAA). ICP-MS, after microwave digestion of sample with HNO₃/H₂O₂, produced results within 5% of the expected values, as did those obtained by FM and INAA. With FAAS, acceptable results were obtained after digestion with HNO₃/HCl. With HGAAS, sample digestion with HNO₃/H₂O₂ produced values that were systematically elevated by about 10% and exhibited standard deviations of ≥10%. Thus, current methods of sample digestion are applicable for Se yeast analysis by ICP-MS and FAAS, but not by HGAAS.     

Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry

Interest in the biological behavior of a growing number of elements, along with increasing recognition of the importance of interactions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improvements over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been realized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of ICP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, performance criteria, and certain problems peculiar to ICP-MS.     

Room temperature solvent extraction for simple and fast determination of total concentration of Ca,Cu, Fe,Mg, Mn,and Zn in bee pollen by FAAS along with assessment of the bioaccessible fraction of these elements using in vitro gastrointestinal digestion

Background and aimBee pollen is recognized to be a source of different nutrients, including minerals. As a food supplement, its quality and safety due to concentrations of essential macro- and microelements, and harmful trace elements has to be verified. Fast and simple element analysis of bee-collected pollen can be regarded as an important part of its quality assurance and control. The present study aimed at developping a new method for determination of selected elements (Ca, Cu, Fe, Mg, Mn, Zn) of bee pollen based on solvent extraction and completely avoiding a high temperature treatment with concentrated reagents. In addition, in vitro gastrointestinal digestion was used to assess bioavailability of elements from this food supplement.MethodsBee pollen samples were dried and pulverized. Total concentrations of Ca, Cu, Fe, Mg, Mn, and Zn were determined by flame atomic absorption spectrometry (FAAS) in sample solutions obtained by wet digestion (WD) in concentrated HNO₃ or alternatively by solvent extraction (SE) with diluted solutions of HNO₃. Gastrointestinal digestion was mimicked using simulated solutions of gastric and intestinal juices followed by determination of Ca, Cu, Fe, Mg, Mn and Zn concentrations in the bioaccessible fraction by FAAS.ResultsA new simple and fast method for determination of total concentrations of Ca, Cu, Fe, Mg, Mn, and Zn in bee pollen was developed and validated. The method combined room temperature, two-hour SE with 0.5 mol L⁻¹ HNO₃ with FAAS measurements versus simple standard solutions. It provided precision within 1–5 % and trueness better than 8%, and was shown to be suitable for fast analysis of different polyfloral bee pollens. In vitro gastrointestinal digestion revealed that elements were well (70–85 % for Ca, Mg) and fairly (27–43 % for Cu, Fe, Mn, and Zn) bioaccessible from bee pollen. By pouring with water and swelling overnight, bioaccessibility of studied elements from such prepared bee pollen was increased on average by less than 15 % (Mn), 20 % (Ca, Cu, Fe, Zn) or 30 % (Mn).ConclusionsAvoiding long-lasting, high-temperature wet digestion with concentrated reagents, the proposed sample treatment along with FAAS provided precise and true results of total concentrations of Ca, Cu, Fe, Mg, Mn, and Zn in bee pollen. The method was simple and fast, and enabled to analyze a higher number of samples. Simulated gastrointestinal digestion of bee pollen have shown for the first time that Ca and Mg are the most bioaccessible from this bee product. Bioaccessibility of Cu, Fe, Mg, and Zn from bee pollen are close to or lower than 40 %.     

The first insight into the trace element status of human adrenal gland accompanied by elemental alterations in adrenal adenomas

BackgroundThe baseline status of trace metals in adrenal tissue is unresolved, while the elemental profile for any adrenal pathology has not been examined so far. This study aimed to determine the baseline status of important toxic (Ni, As, Cd, Pb, Th, U) and essential trace elements (Mn, Co, Cu, Zn, Se) in healthy adrenal tissues (HATs) as well as to examine whether there are alterations in the elemental composition of adenomatous adrenal tissues (AATs). Furthermore, this study aimed to find potential trace metals that could play a role in the pathogenesis of adrenal adenoma (AA).MethodsThe study included 45 patients diagnosed with AA. Impacts of relevant parameters such as gender, age, smoking habits and nodular sizes were considered. All samples were subjected to microwave digestion and the trace elements were determined by inductively coupled plasma mass spectrometry (ICP-MS).ResultsThis is the first study that provided an insight into the elemental status of HATs. It was also shown that AATs had altered trace metal contents. Compared to HATs, the most significant findings were related to the high content of essential (Cu, Mn, Se, Zn) and Pb as a non-essential metal. Although gender, age and smoking habits had a modest effect on metal profiles, the most significant alterations were related to the nodular diameter above 4 cm, indicating that the growth of benign tumor could influence changes in elemental composition.ConclusionFor the first time the baseline contents of essential and toxic trace metals in HATs were determined. The results of this study may highlight the role of toxic and essential trace metals in AAs and could provide new insights into the molecular basis of pathophysiological changes caused by the hazardous effects of trace metals on adrenal structure and function.     

Laboratory gloves as a source of trace element contamination

Contamination in a trace element laboratory can come from a variety of sources, including laboratory gloves. Therefore, vinyl and latex gloves were obtained from as many manufacturers as would supply gloves. These gloves were either prepared for acid-washing and subsequent soaking in an acid solution, or immersed in an acid solution for a duration of either 1 min or 1 h. Incubation washes were analyzed for a variety of trace elements by flame atomic abosrption spectroscopy (AAS) or inductively coupled mass spectrometry (ICP-MS). Results indicated that only three brands of vinyl gloves were acceptable for use in a trace element laboratory, whereas others had contamination of different elements. Latex gloves contained such high levels of biologically important elements that they were not considered suitable for routine trace element work. Vinyl gloves of choice should be routinely acid-washed before use in a trace element laboratory.     

Trace elements in human milk: Correlation with blood levels, inter-element correlations and changes in concentration during the first month of lactation

Using inductively coupled plasma mass spectrometry (ICP-MS) based analytical procedures, the concentration of several trace elements (Mn, As, Pb, Co, Ni, Cu, Zn and Se) was determined in human milk samples collected from a group of healthy lactating Portuguese women (n=44), both on the 2nd day postpartum (i.e., colostrum; n=34) and at 1 month postpartum (i.e., mature milk; n=19). Blood samples (n=44), collected on the 2nd day after parturition, were also analyzed for the same trace elements. No major correlations were observed between the levels of the analyzed trace elements in blood and colostrum samples. All the studied elements, except for Co, Pb and Ni, showed a significant trend for a decrease in concentration in milk during the first month of lactation. This trend was more pronounced for Zn and Se, whose levels decreased to approximately 23% and 44% of their initial mean concentration, respectively. With the exception of Co (r=0.607) and Zn (r=0.487), no significant correlations were observed when comparing the levels of each trace element between samples of colostrum and mature milk. Several inter-element correlations were found within each type of milk sample. The most significant were: (i) Se vs Cu (r=0.828) and Se vs Co (r=0.605) in colostrum samples and (ii) Ni vs Pb (r=0.756), Ni vs Mn (r=0.743) and Se vs Co (r=0.714) in mature milk samples. An inverse correlation between Zn and Se was also found in both types of milk sample; however, it only reached statistical significance for mature milk (r=-0.624).     

The association between content of the elements S,Cl, K,Fe, Cu,Zn and Br in normal and cirrhotic liver tissue from Danes and Greenlandic Inuit examined by dual hierarchical clustering analysis

ProjectMeta-analysis of previous studies evaluating associations between content of elements sulphur (S), chlorine (Cl), potassium (K), iron (Fe), copper (Cu), zinc (Zn) and bromine (Br) in normal and cirrhotic autopsy liver tissue samples.ProcedureNormal liver samples from 45 Greenlandic Inuit, median age 60 years and from 71 Danes, median age 61 years. Cirrhotic liver samples from 27 Danes, median age 71 years. Element content was measured using X-ray fluorescence spectrometry. Statistics: Dual hierarchical clustering analysis, creating a dual dendrogram, one clustering element contents according to calculated similarities, one clustering elements according to correlation coefficients between the element contents, both using Euclidian distance and Ward Procedure.ResultsOne dendrogram separated subjects in 7 clusters showing no differences in ethnicity, gender or age. The analysis discriminated between elements in normal and cirrhotic livers. The other dendrogram clustered elements in four clusters: sulphur and chlorine; copper and bromine; potassium and zinc; iron. There were significant correlations between the elements in normal liver samples: S was associated with Cl, K, Br and Zn; Cl with S and Br; K with S, Br and Zn; Cu with Br. Zn with S and K. Br with S, Cl, K and Cu. Fe did not show significant associations with any other element.ConclusionsIn contrast to simple statistical methods, which analyses content of elements separately one by one, dual hierarchical clustering analysis incorporates all elements at the same time and can be used to examine the linkage and interplay between multiple elements in tissue samples.