Tea is the most consumed drink in the world after water. Green tea is a 'non-fermented' tea, and contains more catechins, than black tea or oolong tea. Catechins are in vitro and in vivo strong antioxidants. In addition, its content of certain minerals and vitamins increases the antioxidant potential of this type of tea. Since ancient times, green tea has been considered by the traditional Chinese medicine as a healthful beverage. Recent human studies suggest that green tea may contribute to a reduction in the risk of cardiovascular disease and some forms of cancer, as well as to the promotion of oral health and other physiological functions such as anti-hypertensive effect, body weight control, antibacterial and antivirasic activity, solar ultraviolet protection, bone mineral density increase, anti-fibrotic properties, and neuroprotective power. Increasing interest in its health benefits has led to the inclusion of green tea in the group of beverages with functional properties. However, although all the evidence from research on green tea is very promising, future studies are necessary to fully understand its contributions to human health, and advise its regular consumption in Western diets, in which green tea consumption is nowadays limited and sporadic.

Beneficial Effects of Green Tea—A Review

https://uhra.herts.ac.uk/bitstream/handle/2299/2030/901263.pdf;sequence=1

Platinum group elements in the environment and their health risk.

Approximately a third of the human proteome contains metal cations, either in form of cofactors with catalytic functions, or as structural support elements.1,2 To guarantee a proper maintenance of this metal ion pool, both at the cellular and whole organism levels, nature has evolved a highly sophisticated machinery comprised of a complex interplay between DNA, proteins, and biomolecules.3 Over the past decades, a steadily growing number of diseases have been identified, which are characterized by metal imbalance in cells and tissues. Among the most prominent examples rank Alzheimer’s disease and Parkinson’s disease, two neurodegenerative disorders that involve abnormal accumulation of transition metals in brain tissue.4 While some progress has been made at understanding the molecular basis of these disorders, many important questions remain unanswered. For example, little is known about the cellular structures that are involved in transiently storing metal ions prior to their incorporation into metalloproteins or the fate of metal ions upon protein degradation. An important first step towards unraveling the regulatory mechanisms involved in trace metal transport, storage, and distribution represents the identification and quantification of the metals, ideally in context of their native physiological environment in tissues, cells, or even at the level of individual organelles and subcellular compartments.

Since the inception of the first histochemical methods for the microscopic demonstration of transition metals in tissues more than 140 years ago,5 many highly sensitive microanalytical techniques and instruments have been developed for the in situ analysis of trace metals. The aim of this review is to provide an overview of the most recent achievements in trace metal imaging while at the same time also offering a historical perspective of this rapidly evolving research field. Although this survey has been structured according to the various analytical techniques, particular emphasis is given to the biological background for a better understanding of the context and importance of each discussed study.

An overview of the most important microanalytical techniques currently available for the in situ detection of trace metals in cells and tissues is compiled in Table 1. Depending on the task, each technique may offer specific advantages and, of course, also disadvantages. Currently, synchrotron- and focused ion-beam microprobes presumably offer the best combination of sensitivity and spatial resolution; however, the ionizing high-energy excitation beam is not compatible with studying live organisms. Conversely, techniques that have been specifically developed for physiological imaging in clinical medicine, notably magnetic resonance imaging and positron emission tomography, inherently offer only a low spatial resolution and are merely suitable for obtaining information at the organ or tissue level. Although fluorescence microscopy based methods provide very high sensitivity down to the single molecule level while being at the same time compatible with live cell and tissue studies, scattering and limited penetration depth renders these techniques unsuitable for imaging opaque specimens. There are also important differences regarding the type of quantitative information that can be gained by each of these analytical techniques. For example, the histochemical detection with chromogenic and fluorogenic dyes relies on a competitive exchange of the metal ion within its native environment, most likely coordinated to endogenous ligands. Depending on the exchange kinetics and thermodynamic affinity of the histochemical indicator, only a fraction of the total metal ion contents in a cell or tissue can be probed. Nevertheless, this kinetically labile pool is particularly of interest in context of understanding the uptake, distribution, and regulation of trace elements at the cellular level, and in this regard, these methods offer unique opportunities to dynamically image metal ion fluxes in live cells with high sensitivity and spatial resolution. At the same time, organelles and proteins of interest can be readily labeled with genetically encoded green fluorescent protein tags,12 thus providing direct insights into dynamic processes within a larger cellular and biochemical context. In contrast, similar correlative information is difficult to gain with the fully quantitative micro beam methods, which require xenobiotic elemental tags for identifying subcellular structures. Autoradiographic tracer experiments offer much improved resolution over PET; however, the technique is only applicable to fixed or frozen tissues and cells. Furthermore, tracer studies cannot provide direct information regarding the endogenous metal composition of cells or tissues, and are therefore primarily limited to metal uptake, distribution, and release studies. Finally, mass spectrometric analyses are surface-based methods that destroy the sample while measuring its elemental composition. Clearly, only the combination of several analytical techniques and specific biochemical studies may lead to a fully comprehensive analysis of a biological system.



Table 1

Spatially resolved microanalytical techniques for in situ imaging of trace metals in biology.6–11




2. Histochemical Techniques
Histology is the branch of biology dealing with the study of microscopic anatomy of cells and tissues of plants and animals. Histological studies are typically carried out on thin sections of tissue or with cultured cells. To visualize and identify particular structures, a broad spectrum of histological stains and indicators are available. Among the most widely used dyes are hematoxylin and eosin, which stain nuclei blue and the cytoplasm pink, respectively.13 The history of detecting biological trace metal by histological methods dates back more than 140 years. Although these techniques have been today mostly replaced by the much more sensitive modern analytical methods described in this review article, histochemical approaches for visualizing metals mark the very beginning in the exploration of the inorganic physiology of transition metals. Given this special place in history, we deemed it necessary to briefly review some of the early achievements in this field.

2.1. Chromogenic Detection with Chelators and Ligands
Ever since the inception of Perls Prussian blue method for staining of non-heme iron, numerous indicators have been developed for the in situ visualization of trace metals in biological tissues and cells.13 Due to their limited sensitivity; however, most of these techniques were only suitable for the diagnosis of pathological conditions, typically associated with excess metal accumulations, thus preventing their application for routine staining of normal tissue. Furthermore, because the dyes are engaged in a competitive exchange equilibrium with endogenous ligands, histological stains are not suitable for the analytical determination of the total metal contents in tissues and thus limited to the visualization of the histologically reactive fraction of loosely bound labile metal ions.



2.1.1. Histochemistry of Iron 
The histochemical demonstration of labile iron reported by Perls in 1867 is among the earliest accounts describing the in situ visualization of a trace metal in biological tissues.5 The method was originally described by Grohe, who observed the formation of a blue coloration when he treated cadaver tissues with potassium ferrocyanide in acidic solution.14 Due to its low cost and simplicity, the technique is still used today for the histological visualization of non-heme iron. Some variations focused on optimizing the concentrations and proportions of the reagents,15–17 among which Lison’s protocol17 appears to be most popular today. An intensification of Perls’ staining can be obtained by exploiting the use of ferric ferrocyanide in catalyzing the oxidation of diaminobenzidine (DAB) to polymeric benzidine black by hydrogen peroxide.18

An alternative method employs the reaction of ferricyanide with Fe(II) resulting in Turnbull blue.19 Since almost all of the Fe in tissues is in the ferric form, the staining procedure requires the in situ conversion of Fe(III) to Fe(II) with ammonium sulfide.15 Due to often incomplete reduction, the method never gained much attention. More recently, an application of Turnbull blue, named the ‘perfusion Turnbull method’ has been developed, where in vivo perfusion of acidic ferricyanide is followed by DAB intensification.20 The direct in vivo perfusion avoids artifacts associated with tissue fixation, including the loss of loosely bound iron and oxidation of Fe(II) to Fe(III). Similarly, Perls method was modified by employing in vivo perfusion with acidic ferrocyanide. Both methods are capable of identifying organs and tissues containing histochemically reactive iron over a broad pH range, including the low endosomal pH.21,22

The history of iron histochemistry would be incomplete without mentioning Quincke’s method, which employed ammonium sulfide for the precipitation of tissue iron as its sulfide.23 A detailed account on the various techniques, including a comprehensive historical overview of non-heme iron chemistry, has been recently published.24

In Situ Imaging of Metals in Cells and Tissues

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a highly accepted, widely used method for the determination of major, minor and trace elements in solids, as well as isotope-ratio measurements. However, it suffers from non-stoichiometric effects occurring during sampling, aerosol transport, vaporization, atomization and ionization within the ICP, described as elemental fractionation. This matrix-dependent phenomenon has been the limitation for quantitative analysis without matrix-matched calibration standards. Significant insights into the processes responsible for elemental fractionation have been obtained and, with improved understanding of LA-ICP-MS, a variety of strategies for more precise, more accurate quantitative analyses have been developed. This review aims to summarize recent developments in LA-ICP-MS based on the fundamental understanding of the LA process and particle formation but also includes the importance of the ICP and its operating conditions. We discuss figures of merit and new trends in quantification in order to demonstrate the capabilities of this direct solid-sampling technique. We present a few selected applications to underline why LA is a fast-expanding analytical technique.

Solid sample analysis using laser ablation inductively coupled plasma mass spectrometry

Levels of essential elements with antioxidant activity, as well as catechins, gallic acid, and caffeine levels, in a total of 45 samples of different teas commercialized in Spain have been evaluated. Chromium, manganese, selenium, and zinc were determined in the samples mineralized with HNO(3) and V(2)O(5), using ETAAS as the analytical technique. The reliability of the procedure was checked by analysis of a certified reference material. Large variations in the trace element composition of teas were observed. The levels ranged from 50.6 to 371.4 ng/g for Cr, from 76.1 to 987.6 microg/g for Mn, from 48.5 to 114.6 ng/g for Se, and from 56.3 to 78.6 ng/g for Zn. The four major catechins [(-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC)], gallic acid (GA), and caffeine were simultaneously determined by a simple and fast HPLC method using a photodiode array detector. In all analyzed samples, EGCG ranged from 1.4 to 103.5 mg/g, EGC from 3.9 to 45.3 mg/g, ECG from 0.2 to 45.6 mg/g, and EC ranged from 0.6 to 21.2 mg/g. These results indicated that green tea has a higher content of catechins than both oolong and fermented teas (red and black teas); the fermentation process during tea manufacturing reduces the levels of catechins significantly. Gallic acid content ranged from 0.039 to 6.7 mg/g; the fermentation process also elevated remarkably gallic acid levels in black teas (mean level of 3.9 +/- 1.5 mg/g). The amount of caffeine in the analyzed samples ranged from 7.5 to 86.6 mg/g, and the lower values were detected in green and oolong teas. This study will be useful for the appraisal of trace elements and antioxidant components in various teas, and it will also be of interest for people who like drinking this beverage.

Determination of Tea Components with Antioxidant Activity

Particle size distributions and compositions of primary aerosols produced by means of near-IR femtosecond laser ablation (λ
				= 775 nm) of brass in He or Ar at atmospheric pressure have been measured. Aerosols were characterized using a 13-stage low-pressure impactor covering a size range from 5 nm up to 5 μm and subsequently analyzed applying total reflection X-ray fluorescence spectrometry. The results indicate, that for femtosecond laser ablation in the low-fluence regime (<5 J cm−2) ultra-fine aerosols (mean diameter dp
				≈ 10 nm/peak width wp
				≈ 35 nm) are produced. Furthermore, the total Cu/Zn ratio of these aerosols corresponds to the composition of the bulk material. In contrast, ablation above 10 J cm−2 results in the formation of polydisperse, bimodal aerosols, which are distributed around dp1
				≈ 20 nm (wp1
				≈ 50 nm) and dp2
				≈ 1 μm (wp2
				≈ 5 µm), respectively, and whose total Cu/Zn ratio slightly deviates from the bulk composition. In order to examine the influence of pulse duration on particle size distribution and aerosol composition, comparative measurements by means of near-IR nanosecond ablation were also performed. The data show that nanosecond ablation generally leads to an intensified formation of particles in the micrometer range. Moreover, the composition of these aerosols strongly departs from the stoichiometry of the bulk. Aspects concerning the formation of particles during ablation as well as implications for the element-selective analysis by inductively coupled plasma spectrometry are discussed.

Particle size distributions and compositions of aerosols produced by near-IR femto- and nanosecond laser ablation of brass

First report on the uptake of automobile catalyst emitted palladium by European eels (Anguilla anguilla) following experimental exposure to road dust.

Determination of the critical thickness and the sensitivity for thin-film analysis by total reflection X-ray fluorescence spectrometry

A review of total reflection x-ray fluorescence (TXRF) as an effective excitation mode for energy-dispersive x-ray spectral analysis is presented. The instrumental conditions of excitation under grazing incidence (ψ < 0.1°) are emphasized and the analytical features of powerful detection and simple and reliable quantification are characterized. The applicability of TXRF to environmental analyses is illustrated by some typical examples. The analysis of pure water samples leads to detection limits at the ppt (ng/l) level. A special matrix separation is only needed for river, sea and waste waters. The analysis of air dust is directly possible with a sampling volume of 1 m 3 and a sampling time of 1 h. Organ tissue can be analysed down to the lower ppm range after freeze-cutting of μm thick sections. Plant material has to be pulverized and digested prior to analysis, e.g. with nitric acid In combination with a chromatographic separation, speciation is made possible for small 0.5 ml fractions, e.g. for vegetable foodstuffs. For all these applications a multi-element determination can be carried out, for about 20-25 elements simultaneously. Simple and reliable quantification is effected by internal standardization. The reliability of the method has been proved by intercomparison tests. Second-generation instruments that are compact and user-friendly are now commercially available.

Elemental Analysis of Environmental Samples by Total Reflection X-Ray Fluorescence: a Review

Old oil paintings and illuminated historical manuscripts are valuable objects of cultural heritage. Pigments and inks once used for these artefacts today allow insights of art historical or archaeological relevance. For their identification, a number of non-destructive spectroanalytical methods can be applied. This paper first gives a historical review and describes fundamentals of optical and x-ray fluorescence spectrometry including instrumentation and analytical characteristics. The variant of total reflection x-ray fluorescence (TXRF) is shown to be a highly convenient tool for the characterization of inorganic pigments and inks. Since only micro-samples are needed, a very gentle sampling technique can be applied. It consists of rubbing a dry cotton-wool bud (Q-tip) over the painted surface without causing any visible damage. Only a minute amount of a few micrograms is removed from the object, transferred to a glass carrier and analyzed by TXRF. Some remarkable examples of TXRF analysis of old paintings and illuminated manuscripts are given. Rather than emphasizing the analytical details, the archaeometric potential of the method is illustrated. Copyright © 2000 John Wiley & Sons, Ltd.

A. von Bohlen

Papers

Particle size distributions and compositions of aerosols produced by near-IR femto- and nanosecond laser ablation of brass

First report on the uptake of automobile catalyst emitted palladium by European eels (Anguilla anguilla) following experimental exposure to road dust.

Determination of the critical thickness and the sensitivity for thin-film analysis by total reflection X-ray fluorescence spectrometry

Elemental Analysis of Environmental Samples by Total Reflection X-Ray Fluorescence: a Review

Analysis of pigments and inks on oil paintings and historical manuscripts using total reflection x-ray fluorescence spectrometry