Keywords

1 Introduction

This chapter presents different analytical procedures, which can be used to quantify the concentration of cadmium and its metabolites in biological material as well as biomarkers of effect after cadmium exposure.

Preferably, well-established standard methods of analysis are presented. In Germany, standard procedures for biological monitoring are published by the Working Group “Analyses of Hazardous Substances in Biological Materials” of the “Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area”. Additionally, analytical methods are included, that modify former methods to improve accuracy and precision or obtain lower limits of detection.

2 Biomarkers of Exposure

2.1 Overview

For human biomonitoring, the following parameters are preferably used to analyze cadmium in biological material: cadmium in blood and cadmium in urine.

Additionally, analysis of cadmium can be performed in other matrices such as saliva, hair, nail, teeth and tissues. Overviews of analytical methods for determining cadmium in biological materials are given in Tables 1 to 3 (see below).

Table 1 Analytical procedures using ICP for cadmium analysis in biological materials.
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Cadmium in blood and in urine are used as parameters for assessing occupational and environmental exposure to cadmium. Cadmium in blood is a short term parameter, reflecting recent exposure to cadmium. Short time elevations of cadmium in blood may be caused by excessive exposure situations. After the end of exposure, there is a rapid decrease in the first stage, followed by a decrease that levels off, depending on accumulated body burden. After long term exposure, cadmium concentration in blood becomes more complex to interpret, as it reflects both the present and the long term exposure [1,2].

Cadmium in urine reflects the body burden of cadmium, especially the cadmium concentration in the main accumulation organ, the kidney (organ-specific accumulation). Therefore, it can be regarded as indicator of the cumulative long term exposure. As long as the renal function remains normal, the concentration of cadmium in urine is well correlated with the total cadmium body burden. After cadmium-induced irreversible tubular renal dysfunction with microproteinuria, the cadmium excretion in urine tends to increase, as cadmium is released from renal depots [2,3].

2.2 Pre-analytic Phase

Due to the ubiquitous presence of cadmium, there is a risk for contamination during the whole process of sampling and analysis, which has to be minimized by strict laboratory procedures.

As in any trace element analysis, reagents of the highest purity and contaminant-free tips of automatic pipettes, tubes, and glassware must be used (colored plastic tubes may contain cadmium). Plastic tubes used for specimen collection and treatment must be individually cleaned with 1 M nitric acid to avoid exogenous contamination. In practice, the tubes are filled with acid and allowed to stand for at least 2 hours. Afterwards, they are rinsed two to three times first with deionized water and then with ultrapure water before drying them. The cleaning can be made more effective by warming the nitric acid [4].

2.3 Analytical Methods for the Determination of Cadmium

The most common procedures for analyzing cadmium concentrations in blood and urine are inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS). Furthermore, electrochemical methods, neutron activation analysis (NAA) and X-ray fluorescence spectrometry (XRF) can be applied. Several factors influence the choice of the analytical method, e.g. the matrix and the detection limit required.

2.3.1 Inductively Coupled Plasma Mass Spectrometry

In inductively coupled plasma mass spectrometry analysis, the sample is heated in an argon-plasma activated by a high-voltage field. Thereby, atoms are ionized. Using an electric field, the generated ions are accelerated to the analyser of the mass spectrometer, where they are separated according to the mass of the specific isotopes. In inductively coupled plasma optical emission spectroscopy (ICP-OES), also referred to as inductively coupled plasma atomic emission spectroscopy (ICP-AES), the sample is atomized in argon plasma and the excitation of an optical emission of cadmium is measured.

Using the ICP-MS and ICP-OES methods, cadmium present in urine and blood due to occupational or environmental exposure can be determined sensitively, specifically, and with little effort. Samples are usually prepared by digestion with acid [5–9] or dilution with acid [10–12]. An additional enrichment is achieved by extraction with organic solvents or by capillary micro-extraction [7,9,13]. Detection limits for ICP-MS analysis in blood or urine are predominantly reported in the lower range from 0.007 μg/L to 0.1 μg/L (for details see Table 1).

The Working Group “Analyses of Hazardous Substances in Biological Materials” of the “Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area” has published a standard procedure for the determination of cadmium in urine. Briefly, after UV digestion of the urine samples, an internal standard is added and the samples are introduced into the ICP-MS by means of a pneumatic nebulizer. Evaluation is carried out using the standard addition procedure. The limit of detection is specified with 0.02 μg/L urine. The method can be applied for samples from environmental as well as occupational-medical studies [5].

In addition to analysis in blood and urine, methods for other biological materials such as tissue of lung, liver, and kidney cortex as well as hair and nails have been published [11,14,15].

2.3.2 Atomic Absorption Spectrometry

In atomic absorption spectrometry the sample is heated by a flame or in a furnace, until the element atomizes. The atoms absorb light at the resonance line. The attenuation of intensity of the light beam can be measured.

Cadmium in blood, urine, hair, saliva, and human milk is predominantly analyzed with graphite furnace atomic absorption spectrometry (GF-AAS), also known as electrothermal AAS (ET-AAS). Samples are usually prepared by digestion with nitric acid [16–20]. Solubilizers (Triton® X-100) or matrix modifiers (e.g., diammonium hydrogen phosphate, Pd-components) are added [17,21–23]. At very low cadmium concentration, pre-concentration can be achieved by chelation and extraction with a mixture of organic solvents [4].

The limits of detection of AAS methods are with 0.02 μg/L to 0.5 μg/L higher than those of ICP-MS methods (see Tables 1 and 2).

Table 2 Analytical procedures using AAS for cadmium analysis in biological materials.
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2.3.3 Electrochemical Methods

In differential pulse anodic stripping voltammetry (DPASV), cadmium ions are first reduced and amalgamated at the working electrode (a hanging mercury drop electrode or a mercury film electrode) during pre-electrolysis at a suitable applied potential. In the second step, the reduced amalgamated cadmium is re-oxidized by means of a potential ramp imposed between the working electrode and a platinum rod electrode [24]. The resulting peak is proportional to the cadmium concentration of the solution.

DPASV procedures have been applied to analyze cadmium in urine [24–26] and in human hair [27]. The samples were prepared by digestion with acids.

DPASV can be applied as an independent reference procedure for AAS and ICP-MS. It is suitable for analyzing cadmium concentrations in environmental as well as occupational-medical samples [25] (Table 3).

Table 3 Analytical procedures using electrochemical methods for cadmium analysis in biological materials.
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During potentiometric stripping analysis (PSA), trace elements or ions are pre-concentrated by potentiostatic deposition on an electrode (e.g., mercury film on a glassy-carbon electrode). In contrast to DPASV, PSA is not subject to background interferences from organic electroactive constituents in the sample or to the presence of dissolved oxygen [28]. It is used to analyze cadmium in whole blood [29–31] and urine [32] (Table 3).

2.3.4 Further Methods

Cadmium concentrations in biological materials can also be measured with neutron activation analysis (NAA) and X-ray fluorescence spectroscopy (XRF). Both techniques depend on the detection of photons generated in cadmium by an externally incident beam of radiation. In NAA, the cadmium concentration in the sample is determined by studying the emission of γ-rays after the irradiation of the sample with neutrons [33,34]. In contrast, photon emission in XRF is produced by an incident beam of X-rays or γ-rays interacting with the atomic electrons of cadmium, resulting in the emission of characteristic X-rays [34].

The analyses of cadmium concentrations in human kidney and liver with NAA can be performed with direct in vivo [35–37] or in vitro measurements [38]. Additionally, NAA procedures for the quantification of cadmium in biological materials such as bovine liver and food samples [39], human hair [40], serum [41], and human central nervous system issue samples [42] have been described.

With XRF, cadmium in the kidney can be analyzed in vivo [43–45]. These procedures are used for clinical measurements in the kidney of persons occupationally exposed to cadmium. The detection limit of XRF is strongly dependent on the distance between skin and kidney, which has to be analyzed with ultrasound [45].

2.4 Quality Control

An internal quality control should be performed with each analytical run. Control materials for internal quality control of cadmium analytical procedures are commercially available, e.g., certified reference material for trace elements in urine, plasma, serum, and whole blood [46,47].

To assure the accuracy and comparability of the results with other laboratories, an appropriate external quality assessment is necessary. For external quality control, participation in a round robin test is recommended. There are various quality control programs containing cadmium in blood and urine, e.g., the international program of the German External Quality Assessment Scheme (E-QUAS), where cadmium analysis can be tested for the concentration range found in occupational and environmental medicine [48], or the external quality assessment schemes of the Centre of Toxicology in the National Institute of Public Health of Québec [49].

2.5 Body Burden after Environmental and Occupational Exposure

The German commission “Human-Biomonitoring” evaluated reference values (95th percentile of the cadmium background exposure) of 1.0 μg cadmium/L blood and 0.8 μg cadmium/L urine for non-smokers aged 18 to 69 [50,51]. Smokers showed higher 95th percentile values of 3.32 μg cadmium/L blood and 1.20 μg cadmium/L urine [52,53].

Occupational exposure to cadmium leads to higher levels in blood and urine. Workers exposed to cadmium in a non-ferrous smelter showed mean levels of 6.23 μg cadmium/g creatinine (range 0.87–165 μg/g creatinine) and 6.54 μg cadmium/L blood (range 1.6–51 μg/L) [54].

3 Biomarkers of Effect

3.1 Overview

Long term exposure to cadmium results in kidney diseases, especially tubular damage, as early and frequent health damage. The earliest sign of nephropathy induced by chronic cadmium exposure is an increased urinary excretion of low-molecular-weight proteins with a molecular weight of less than 40 kDa, such as β2-microglobulin (β2-M) and retinol-binding protein (RBP). In healthy persons, the reabsorption of those small proteins is almost complete. A decrease in the tubular reabsorption capacity causes an increase in the urinary excretion of microproteins like β2-M and RBP [55].

There are sensitive methods available for the quantification of tubular proteinuria in populations exposed to cadmium occupationally or environmentally. RBP, β2-M, α1-microglobulin (α1-M, also called protein HC), metallothionein, and enzymes such as N-acetyl- β-D-glucosaminidase (NAG) in human urine are used as biomarkers of cadmium-induced effects [56–61]. However, the effects are not specific to cadmium exposure, but may also occur due to various renal diseases or nephrotoxic agents other than cadmium.

Urinary β2-M is a highly sensitive and widely used parameter [3,55]. Because of the instability of β2-M in acidic urine (pH < 5.6), a strict pH-control of the urine is necessary. Even if patients are given bicarbonate before urine collection, a decomposition of β2-M in up to 30% of the urine samples is observed [55]. α1-M is very stable in urine, but less specific to tubular damage because of its larger size and slightly less sensitive than β2-M and RBP. RBP on the other hand is described as stable, specific, and as sensitive as β2-M [61].

3.2 Analytical Methods for β2-Microglobulin Quantification

The quantification of β2-M concentration in human urine and serum can be performed with immunoassays, e.g., enzyme-linked immunosorbent assay, radio immunoassay [62], latex immunoassay [63,64], immunoenzymatic assay with chemiluminescence detection, and immunoturbidimetric assay [65].

There are commercially available test kits for the quantitative determination of β2-M in plasma, serum, and urine with ELISA, e.g., from Immundiagnostik [66] or ORGENTEC Diagnostika GmbH [67]. In this method, immobilized antibodies against β2-M are fixed on the surface of a microtiter plate. During the immune reaction, the antibodies on the plate bind the β2-M in the sample. After the non-bonded components have been washed out, an enzyme (horseradish peroxidase) labeled anti-human β2-M-antibody is added, which binds to β2-M. The amount of bound enzyme is directly proportional to the β2-M-concentration of the sample. A chromogen (tetramethylbenzidine) is converted by the bound enzyme to a chromogenic compound, which is photometrically quantified [66].

Latex immunoassay was introduced by Bernard et al. [63,64]. In this method, the surface of polystyrene-latex particles is coated with specific antibodies sensitized to human β2-M. They agglutinate with β2-M molecules in the serum or urine sample. Unspecific agglutination of the particles carrying the antibodies is prevented by diluting the samples with a standardized albumin solution. The quantitative evaluation of the results is performed by counting the remaining non-agglutinated particles or by measuring the decrease of absorbance with a photometer at 360 nm. The limit of detection is 0.5 μg/L [63,64].

3.3 Analytical Methods for the Quantification of the Retinol Binding Protein

Retinol binding protein (RBP) in plasma, serum, and urine can be analyzed using different immunoassays, e.g., latex immunoassay [63], ELISA [59], monoclonal antibody-based fluorescence immunoassay [68] or immunonephelometry [69,70]. For principles of latex immunoassay and ELISA, see Section 3.2.

Immunonephelometry uses the effect of a diluted suspension of small particles to scatter a light-beam angular (Tyndall effect) to quantify aggregates formed in an antigen-antibody-reaction [71]. Test systems for RBP quantification are commercially available.

3.4 Analytical Methods for the Quantification of Further Effect Markers

The total protein concentration can be analyzed with commercially available kits based on Coomassie Brilliant Blue reaction. Albumin and α1-microglobulin (protein HC) are quantified by single radial immunodiffusion techniques [72,73] and immunonephelometry [60]. For N-acetyl-β-D-glucosaminidase determination, there are commercially available test kits based on spectrophotometric assay [74].

3.5 Effect Biomarkers after Exposure to Cadmium

Long-term or high exposure to cadmium causes tubular damage that may progress to glomerular damage with decreased glomerular filtration rate and the risk of renal failure. Taking a variety of early markers of kidney damage into account, a dose–response assessment identified early effects in the kidney at concentrations between 0.5 and 3 μg cadmium/g creatinine in the general population [75].

The reversibility of glomerular lesions induced by cadmium is still under discussion [61,76]. Tubular proteinuria (β2-M and RBP in urine) between 300 and 1000 μg/g creatinine might be reversible [61], but more severe tubular proteinuria (i.e., more than 1000 μg β2-M/g creatinine) seems to be irreversible [61,77]. A large study with 1699 subjects (aged 20–80 years) of the general population showed a 10% probability of values of urinary excretion of RBP, NAG, β2-M, amino acids, and calcium being abnormal when cadmium excretion exceeded 2–4 μg/24 h [78].

4 Conclusions

The analysis of cadmium concentrations in biological material is mainly performed with ICP-MS or AAS. ICP-MS attains lower limits of detection than AAS. In addition, electrochemical methods, NAA, and XRF can be applied. The predominant sample matrices include blood and urine, but also hair, saliva, and tissue are used. The application of neutron activation analysis and X-ray fluorescence enables in vivo measurements of cadmium.

Cadmium accumulates in the human body after chronic exposure and causes kidney diseases. A tubular proteinuria causes an increase in urinary excretion of microproteins. For routine screening, the excretion of β2-M, RBP, and α1-M have been validated as biomarkers for cadmium effects. Sensitive, particularly immunological analytical procedures have been published for these parameters. However, proteinuria is not specific to cadmium, but can also occur after exposure to other nephrotoxic agents or due to various kidney diseases. Thus, a combination of parameters of exposure (cadmium in blood, cadmium in urine) and parameters of effect (e.g., β2-M, RBP) is required to reveal cadmium-induced nephrological effects.