Sampling,storage, and preparation of biological material for trace-element analysis

Sampling, storage, and preparation of biological material for analysis are some of the most important factors in the study of trace elements in biomedical fields. If due care is not given to these factors, the resulting data may often be meaningless, regardless of the analytical technique chosen for analysis. A survey of the data in the literature would support this view, in which wide differences in the trace element composition of biological tissues have been observed, and not all these differences are attributed to variations in the biological systems. In this paper, the following points are discussed in detail:

1. Sampling and Storage Sampling methods and program. Contamination of the sample during sampling and storage. Changes in the composition during storage. Various storage methods for solid and liquid samples.
2. Sample Preparation Drying and ashing. Wet digestion. Different methods for preparing samples of hard, semihard, and soft tissues, and of biological fluids.

     

Applications of ion beam analysis to calcified tissue research

Various nondestructive ion beam analysis techniques have been developed and applied to study the concentrations of fluorine and other trace elements in calcified tissues. Fluorine has been determined by prompt gamma activation analysis through the F(?,αγ)O reaction. This method is quick, convenient, and sensitive, and can also be applied to measure fluorine depth distribution nondestructively in teeth and bone samples. By the application of this technique, fluorine concentrations have been determined in a number of teeth with known histories and bones of experimental animals. Moreover, F-depth-distributions to 10-μm depths have also been obtained in a number of selected human and animal teeth. Carbon was measured by activation analysis with He-3 ions, and the technique of Proton-Induced X-ray Emission (PIXE) has been developed to simultaneously determine Ca, P, and trace elements in well-documented teeth. Dental hard tissues, i.e., enamel, dentine, cementum, and their junctions, as well as different parts of the same tissue, have been examined separately. A number of elements, Na, Mg, Al, P, Cl, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Sr, Br, Rb and Pb, have been determined in these dental tissues. The concentrations of some of these elements vary considerably in different teeth and in various parts of the same tooth. The special advantages of these nuclear techniques for studying metabolic bone diseases, renal diseases, and the effect of Pb on childrens' intelligence are discussed.     

Studies on some trace and minor elements in blood

Blood is one of the widely used specimens for biological trace element research because of its biological significance and ease of sampling. We have conducted a study of the blood of the Kalpakkam township population for trace and minor elements. For this purpose, analytical methods have been developed and standardized in our laboratory for the elemental analysis of blood plasma and red cells. Inductively coupled plasma-mass spectrometry (ICP-MS), a relatively new technique, has been applied for the analysis of trace elements. Details regarding spectral interference and matrix interference encountered in the analysis of blood and the methods of correcting them have been discussed. Flame atomic absorption spectrometry (AAS)/atomic emission spectrometry (AES) has been applied for the determination of minor elements. Precision and accuracy of these methods have also been discussed.     

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography-mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17beta]-2H(3)-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C(18) with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l (r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 microg/l (r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 microg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.     

Quantitative trace element analysis of human nails with external beam PIXE

External beam PIXE (Particle Induced X-ray Emission) analysis with a proton beam of 2.4 MeV was used to study trace element concentrations in human nails. The suitability of PIXE analysis regarding nail samples without any pretreatment besides washing was investigated. The main emphasis has been on the ability to obtain absolute concentration values and a new accurate method for nail sample standardization has been developed. Concentration values for the elements Ca, Cr, Mn, Fe, Ni, Cu, Zn, Se, Br, and Pb were determined from human nail samples. A comparison was made with nail samples taken from different fingers and toes to monitor intraindividual variation, and nails of different healthy individuals to get a view of the interindividual differences. The concentrations were also measured in relation to time in order to observe any possible short-term changes. The results are compared with the previous studies reported in the literature. The nail analysis is also compared to hair analysis in terms of detection limits, number of elements determinable, and standardization of the results.     

High-performance liquid chromatographic determination of the magnetic resonance imaging contrast agent Gadocoletate ion in human plasma, urine and faeces

Gadocoletate ion is a new paramagnetic intravascular contrast agent for magnetic resonance imaging (MRI). An high-performance liquid chromatographic method for assaying Gadocoletate ion in human plasma, urine and faecal samples is described. The analysis is based on the reversed-phase chromatographic separation of Gadocoletate ion from the endogenous components of the biological matrices and its detection during elution by ultraviolet light absorption at 200 nm. The selectivity of the method was satisfactory. The mean absolute recovery during the analytical sample preparation was greater than 87%. The precision, expressed as coefficient of variation (CV%) ranged from 0.29 to 5.90% and the accuracy, expressed as mean relative error (R.E.%) of the analytical method ranged from -3.7 to +7.1%. The detection limit in plasma and urine was 2.01 and 10.0 microg/mL (0.00203 and 0.0101 micromol/mL), respectively. The detection limit in homogenized faecal samples was 17.7 microg/g (0.0179 micromol/g). Stability studies were performed in human plasma and urine samples during the analytical cycle. Gadocoletate ion was shown to be stable in human plasma and in human urine when stored at about +4 degrees C for up 24 h, and after three freeze-thaw cycles. In addition, it was shown to be stable in samples of processed plasma and in diluted urine at about +4 degrees C for 48 h, and at room temperature for at least 24 h. As regards the long-term stability of Gadocoletate ion, the results of dedicated studies showed that Gadocoletate ion is stable in human plasma samples when stored at +4 degrees C for up to 30 days and at -80 degrees C for up to 90 days. Gadocoletate ion is stable in samples of human urine when stored at +4 degrees C for up to 30 days, and when stored at -20 degrees C and at -80 degrees C for up to 90 days. The method has been successfully validated in human plasma, urine and faeces and it has been shown to be precise, accurate and reliable.     

Compounds of Mo, V and W in biochemistry and their biomedical activity.

Molybdenum, vanadium and tungsten compounds are widely applied as analytical reagents for determination of numerous pharmacologically active substances and different biochemical parameters. Recent data from the available literature pointed to a very potent biomedical activity of compounds containing these trace elements. The present paper represents a survey on the structure and chemical properties of these compounds, as well as on their biological activity, mostly based on their interaction with cations of biomolecules, such as phospholipids and proteins. Besides, their potent inhibitory effects on cellular targets, bacterial and viral DNA and RNA polymerases will be discussed, as well. Numerous authors clearly demonstrated the antiviral (especially anti-HIV), anticoagulant and antineoplastic properties of the compounds containing the above trace elements. It has been also shown that these compounds act on some cellular enzymatic systems leading to the normalisation of blood pressure, blood glucose and serum lipid levels. Also, compounds of these trace elements represent potent antiobesity agents and express hepatoprotective and antioxidative stress activity.     

Application of a proton microprobe to biomedical research

The capability of PIXE analysis to simultaneously detect trace elements with Z≥14, with a high power of detection, can be exploited in biomedical research if the diameter of the proton beam is reduced to micrometer dimensions. In this case, trace analyses of small particles or small parts of a larger specimen are rendered possible without deteriorating the detection limits of PIXE. The measurements yield a completely new type of information on the biological microstructure. In order to fully utilize the abilities of the combined method, however, sample preparation techniques, and irradiation procedures have to be adapted to each analysis problem. Examples of application of the Bochum Proton Microprobe will be used to demonstrate how and to what extent this can be achieved for different types of biomedical problems.     

A highly sensitive ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) technique for quantitation of protein free and bound efavirenz (EFV) in human seminal and blood plasma

A combined UPLC-tandem mass spectrometric (UPLC-MS/MS) technique has been validated for quantitation of protein free efavirenz (EFV) as well as total concentrations of EFV in human blood and seminal plasma. The analytical method possesses capabilities for concentration measurements of EFV ranging from 0.5 to 10,000ng/ml with an accuracy (%dev) of -5.2-8.0% and precision (%CV) of <8%. Standard curves were linear with coefficients of variation (r(2)) >0.98. The method employs a racemic fluorinated analog of EFV (F-EFV) as the internal standard. EFV and F-EFV were eluted from a reverse-phase UPLC column via gradient elution with detection via negative ion multiple reaction monitoring (MRM). EFV and F-EFV, respectively, were detected via the following MRM transitions: m/z 314.0>244.1 and m/z 298.0>227.9. The time required for the analysis of each sample was 8.0min. The analytical technique is capable of a reliable detection limit of ～15-20fmol of EFV injected on column.     

On-line dialysis and weak cation-exchange enrichment of dialysate : Automated high-performance liquid chromatography of pholcodine in human plasma and whole blood

An automated method for the determination of pholcodine in plasma and whole blood is described. The technique combines dialysis and trace enrichment prior to high-performance liquid chromatography. Dialysis, trace enrichment on a weak cation-exchange column, separation on a cyano column and fluorescence detection was shown to be an extremely selective and sensitive method. The method has been used successfully in the analysis of real samples after administration of pholcodine. The automated method can be used, after minor modification, to determine other basic drugs in whole blood and plasma.     

Role of nuclear analytical probe techniques in biological trace-element research

Many biomedical experiments require the qualitative and quantitative localization of trace elements with high sensitivity and good spatial resolution. The feasibility of measuring the chemical form of the elements, the time course of trace element metabolism, and conducting experiments in living biological systems are also important requirements for biological trace element research. Nuclear analytical techniques that employ ion or photon beams have grown in importance in the past decade and have led to several new experimental approaches. Some of the important features of these methods are reviewed here along with their role in trace element research. Examples of their use are given to illustrate potential for new research directions. It is emphasized that the effective application of these methods necessitates a closely integrated multidisciplinary scientific team.     

Use of human teardrop fluid for the determination of trace elements in healthy individuals and diabetic patients

BackgroundThe use of unconventional biological materials in human trace element studies has increased in terms of published research studies. The aim of present study was to develop and validate the use of teardrop fluid for determining trace element levels in the human body. No study has been published in this area yet. This is a new non-invasive approach in the possible early diagnosis of the pathogenesis of type 2 diabetes.Materials and methodsHuman teardrop fluid samples were obtained from Karbala (Iraq) (n = 111) healthy individuals and with type 2 diabetes (n = 44); and London (UK) healthy individuals (n = 18). The levels of V, Cr, Mn, Fe, Cu, Zn, As, Sr and Cd were determined using an inductively coupled plasma mass equipped with collision cell technology for polyatomic ion correction (ICP-MS).Statistical analysisDiscriminate function analysis (DFA) was carried out to determine the set of variables that discriminated between the trace elements in teardrop fluid samples from healthy individuals and diabetic patients.ResultsThe trace element levels of human teardrop fluid are similar for many elements to that reported for human blood serum in the literature. This is interesting since they have different physiological functions, although overall they are mainly water containing electrolytes (∼ 90 %) and solids (antibodies, hormones, etc). In general, for the study groups in Karbala, Iraq, significantly higher teardrop fluid levels of Mn and Sr were found in type 2 diabetic patients when compared with healthy individuals (evaluated using an F-test and a two-tailed t-test). The levels of V, Cu and As were found to be significantly higher (P < 0.05) in healthy individuals than type 2 diabetic patients. Although the levels of Fe and Zn were slightly higher in type 2 diabetic patients than healthy cases, the differences were not statistically significant (P > 0.05). Cr and Cd were found to have similar levels for both study groups. Significantly higher teardrop fluid levels of V, Cr, Mn, Fe, Zn, As and Sr were found in healthy individuals from Karbala (Iraq) when compared with those from London (UK). In contrast, the levels of Cd observed to be significantly higher in London (UK) than Karbala. No statistical difference was found for Cu between the two healthy groups.Statistical analysisDiscriminate analysis showed that human teardrop fluid V, Mn, Zn, As, Sr and Cd levels could be used to discriminate between healthy and type 2 diabetes study groups in Karbala, Iraq (83 % of cases correctly classified).ConclusionThe use of human teardrop fluid for determining the trace element levels of human health conditions has been evaluated. Trace elemental levels are like that for blood serum which is widely used as an invasive method for assessing human health conditions. Sample collection for teardrop fluid is non-invasive and the application has potential for determining the trace element levels in healthy individuals and disorder conditions (like type 2 diabetes) in countries where cultural and gender sensitivity are issues with respect to the collection methods used for other body fluid samples.     

Recent advances in biomedical applications of accelerator mass spectrometry

The use of radioisotopes has a long history in biomedical science, and the technique of accelerator mass spectrometry (AMS), an extremely sensitive nuclear physics technique for detection of very low-abundant, stable and long-lived isotopes, has now revolutionized high-sensitivity isotope detection in biomedical research, because it allows the direct determination of the amount of isotope in a sample rather than measuring its decay, and thus the quantitative analysis of the fate of the radiolabeled probes under the given conditions. Since AMS was first used in the early 90's for the analysis of biological samples containing enriched ¹⁴C for toxicology and cancer research, the biomedical applications of AMS to date range from in vitro to in vivo studies, including the studies of 1) toxicant and drug metabolism, 2) neuroscience, 3) pharmacokinetics, and 4) nutrition and metabolism of endogenous molecules such as vitamins. In addition, a new drug development concept that relies on the ultrasensitivity of AMS, known as human microdosing, is being used to obtain early human metabolism information of candidate drugs. These various aspects of AMS are reviewed and a perspective on future applications of AMS to biomedical research is provided.     

A comparison of trace-elemental composition of natural color and gray hair

In order to get some information on the possible causes of graying of hair, we have used the technique of X-ray fluorescence (XRF) analysis for comparing the trace element contents of natural color and gray hair from a number of subjects. The technique of XRF was preferred to other analytical methods for this kind of comparative studies since it appeared to be simple, convenient, quick, and contamination free. Natural color and gray hair from each subject were obtained from the same scalp region. The hair samples were washed in the recommended fashion. The natural color and gray hair from different subjects were mounted separately on hollow plastic cylindrical sample holders, assuring that the hair were parallel to, and not on top of one another. The samples were analyzed in a commercial wave length dispersive XRF system, with different X-ray tubes being used for obtaining maximum sensitivity for different elements. The scattered X-ray peak from each sample was also monitored and gave a measure of the sample volume being investigated. So far, hair samples from 10 subjects have been analyzed. Their results are presented in the paper, and advantages of XRF, for trace element analysis on hair are discussed.     

Liquid-phase and dispersive liquid-liquid microextraction techniques with derivatization: recent applications in bioanalysis

Liquid phase microextraction (LPME), especially hollow fiber liquid-phase microextraction (HF-LPME), and dispersive liquid-liquid microextraction (DLLME) offer high enrichments of target analytes in a single step. The analytical usefulness of these techniques is significantly enhanced by coupling them with suitable derivatization methods. Due to their simplicity, diverse bioanalytical applications have recently been reported. This review focuses on the recent developments of the combined LPME (mainly HF-LPME and single drop microextraction (SDME)) and DLLME techniques with derivatization for the analysis of biological samples. A broad range of sample matrices such as urine, blood, plasma and human hair samples with various derivatization methods for polar or ionizable organic compounds will be considered. These techniques can also be extended to the determination of trace metal ions, such as the heavy metal ions (Hg, Pb, and Co) and Se. Future trends of the techniques will also be discussed.     

Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS

Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.     

Cell analysis with the new Leipzig high-energy ion nanoprobe

The high-energy ion nanoprobe LIPSION at the University of Leipzig has been in operation since 1998. The ultrastable, 3.5 MV SINLETRON accelerator supplies the H+ or He+ ion beam. A magnetic scanning system moves the focused beam across the sample. At present, a resolution of 41 +/- 4 nm in the low current mode and 300 nm at 5 pA can be achieved. The experimental chamber is equipped with electron-, energy dispersive X-ray-, and particle detectors. They can be used simultaneously to analyse the sample by means of PIXE (particle induced X-ray emission), RBS (Rutherford backscattering), and in the case of thin sections or monolayer samples STIM (scanning transmission ion microscopy). A goniometer allows the application of channeling measurements in single crystals in combination with these methods. In contrast to previous publication describing microbeam facility at LIPSION, the current biomedical research has concentrated on microscopy and tomography on chondrocytes in pig cartilages and fixed single endothelial cells (HUVEC). For the irradiation of single living cells, an external beam facility with irradiation platform, fast beamgate and mini-Petri dishes is under construction.     

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.     

Impact of lead pollution on the status of other trace metals in blood and alterations in hepatic functions

Lead pollution and its impact on the status of four other trace elements—Fe, Zn, Br, and Rb—have been studied in the whole blood samples of different population groups employing energy dispersive X-ray fluorescence technique. These population groups included normal, automobile workers and lead battery manufacturers. The maximum increase in the concentration of trace elements in the blood samples of automobile workers and battery manufacturers was observed for Pb, when compared with normal Pb-B levels. The effect of lead pollution had significantly reduced Zn levels in automobile workers. Fe-B levels in automobile workers had been found to be reduced significantly as compared to control, whereas in battery workers the reduction was not significant. The concentration of Br was greatly enhanced in the blood samples of automobile workers, whereas Rb-B levels were significantly higher in both the automobile and battery workers. Oral administration of lead acetate (100 mg/kg body wt) to experimental rats significantly decreased the activities of hepatic transaminases after 3 and 4 mo of treatment, whereas the activity of hepatic alkaline phosphatase decreased significantly after 4 mo of treatment. It is concluded from this study that higher Pb-B levels greatly influence the levels of other trace elements in human blood samples and also the activities of hepatic transaminases as well as alkaline phosphatase in experimental rats.     

Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility

Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31 mg/mL to 44 ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.