Atomic spectrometry and the clinical chemistry of trace elements

Andrew Taylor is a Clinical Biochemist at the Royal Surrey County Hospital and Honorary Reader in the School of Biomedical and Molecular Sciences of the University of Surrey, in Guildford, United Kingdom. He is Director of a Trace Element Reference Laboratory, which analyses clinical and environmental specimens received from hospitals and other institutions within the UK and other countries. In addition to the supervision of the analytical work of the laboratory, much time is given to consultation and provision of advice of a clinical and technical nature to other laboratories, physicians and to members of the public. He has been involved with many topics in this field including work with mercury, gold, aluminium and lead. Current research interests include the possible protective effects of magnesium and selenium in aluminium toxicity and the influence of diet on iron, zinc and copper status of infants.This issue of JAAS includes the 21st Atomic Spectrometry Update: Clinical and Biological Materials, Foods and Beverages.¹ Each year these comprehensive reviews provide a snapshot of developments in the previous 12–18 months, but rarely reflect back over longer periods. The 21st year of JAAS presents an opportunity to consider how atomic spectrometry has influenced the clinical chemistry of trace elements.

Clinical chemistry is the application of chemical science to the understanding of biochemical and physiological aspects of normal health, and of disease. The primary objectives are summarised in Table 1 and much of the work carried out in clinical chemistry departments involves measurement of chemical species in body fluids and tissues. Examples include biochemicals such as hormones, proteins, enzymes, carbohydrates and lipids. Also measured are minerals (the bulk elements Na, K, Ca, Mg, etc.) and the trace elements, i.e., elements present in the body at concentrations less than 0.01% of the dry weight.

Table 1 Objectives addressed by clinical chemists

Diagnosis of disease

Monitor the severity and progress of disease

Assess response to treatment

Assess response of the body to the presence of disease

Assess the risk of disease

Further the understanding of disease

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Early work on the clinical chemistry of trace elements employed colorimetric and polarographic techniques and was mainly directed to veterinary science, environmental iodine deficiency, occupational exposure to lead and poisonings due to arsenic. It is the development of modern analytical atomic spectrometry that is responsible for our present understanding of the importance of trace elements to human health and disease.

The marriage between atomic spectrometry and clinical chemistry took place with the introduction of the simple flame photometer for atomic emission spectrometry. Prior to this, measuring Na and K in serum involved spectrophotometric procedures that took several hours to complete and large volumes of sample. Very few assays could be processed per day and there was almost no way by which physicians could monitor the fluid balance of sick patients. These instruments revolutionised patient care and paved the way for modern clinical laboratory medicine with the concept of looking at the biochemistry of a patient almost in real-time.

When flame atomic absorption spectrometry (FAAS) was developed a few years later it was to clinical situations that the technique was first applied and clinical measurements of Ca and Mg were soon shown to be possible. Compared with the colorimetric and titimetric procedures hitherto in use, FAAS provided for rapid, accurate measurements with relatively small volumes of sample. As soon as commercial instruments became readily available the number of analyses for these elements increased enormously and the importance of Ca to bone metabolism and to neurophysiology became widely appreciated. It was not long before FAAS opened up completely new areas of clinical investigation as methods were developed for measuring some of the trace elements in clinical samples. While iron deficiency anaemia has long been recognised and the link between goitre and a lack of iodine was understood, it was generally believed that deficiencies of other essential trace elements would not occur because the amounts required were so low that it was impossible not to receive sufficient in the diet. However, with methods to measure Zn in serum by FAAS, it became evident that concentrations could be unacceptably low and dietary intakes were not always adequate. It is now realised that Zn deficiency occurs across the world with an incidence that is second only to a lack of Fe.

Thus, using FAAS, the clinical chemistry of essential trace elements was established. Moreover, the importance of this topic was soon seen to extend beyond simple dietary problems. Prasad reported on a group of young men who were very small and had failed to enter puberty. After many investigations they were found to have low serum Zn concentrations and, as soon as supplements were given, they increased in height and became sexually mature. Further work established that they failed to absorb Zn because they were eating soil, which made the dietary metal insoluble and non-bioavailable.² Malabsorption of Zn and other elements has since been observed as a consequence of several other pathological conditions affecting the gut. The most profound deficiencies occur when patients receive intra-venous nutrition without adequate supplements, and where there are genetic disorders affecting the intestinal absorptive mechanisms. Severe Zn deficiency, for example, causes diarrhoea and eruptive pustular lesions in the skin around the face and genital areas. In the genetic disorder, acrodermatitis enteropathica, these same disturbing symptoms appear when affected infants are weaned from breast milk. Although the condition had been described many years previously it was only when Zn could be measured in serum by FAAS that the disease was shown to be due to a deficiency of the metal and that symptoms were easily resolved by providing an oral supplement.³Table 2 lists a number of other genetic disorders that were identified because of atomic spectrometric measurements. Both deficiencies and excessive accumulations of elements have been identified, depending on the site of the dysfunctional metal transport system within a cell.

Table 2 Genetic disorders of trace element metabolism

Haemochromatosis	Fe accumulation
Acrodermatitis enteropathica	Zn deficiency
Wilson’s disease	Cu accumulation
Menke’s disease	Cu deficiency

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The development of analyte enrichment sample preparation procedures allows low concentrations of non-essential elements to be determined by FAAS. Chelation and solvent extraction, with reagents like ammonium pyrrolidinedithiocarbamate and methyl isobutyl ketone, provided for regular measurements of Pb in blood. While a considerable foundation for the recognition and control of occupational exposure to Pb had been established many decades earlier, using a colorimetric assay, the opportunities for more extensive work that were afforded by FAAS prompted a re-evaluation of these matters. In addition, the greater sensitivity meant that concentrations in non-occupationally exposed subjects could be determined so that environmental issues associated with leaded petrol and Pb in household paints began to assume a considerable public profile.

The impact of FAAS was further extended with devices that overcome the inefficiency of the pneumatic nebuliser. Atom trapping techniques provided for useful reduction in detection limits⁴ but the greatest success was achieved with the micro-cup introduced directly into the flame. With this Delves’ cup, Pb can be determined in only 10 μl blood and within a few minutes’ analysis time.⁵ It is no exaggeration to say that this technique made possible the epidemiological and other studies that brought about the removal of Pb from petrol in the latter part of the last century.

Other aspects of the clinical chemistry of trace elements have been addressed with the use of FAAS. To exploit the pharmacological properties of trace elements regular monitoring may be necessary to ensure that therapeutic levels are maintained in the blood. Thus, safe and effective use of lithium to treat depression is only possible when the serum concentrations can be measured. Linked to this are attempts to understand the mechanisms involved in metal pharmacology and toxicology. Metallothionein is an unusual protein involved in the normal metabolism of essential elements, in protection against toxicity, and in mechanisms by which metals counteract disease.⁶ The role of the protein became evident when levels of trace elements were measured in tissues. It is now well established that several elements rapidly move from one pool to another within the body in response to infection or other insults, as part of the body’s normal defence mechanisms (acute phase reaction). Changes in blood concentrations may be sufficiently sensitive to predict when a clinical condition is about to improve or to remit, and monitoring serum Cu has been used to help in the manipulation of treatment for cancer.⁷

In many haemodialysis centres around the world, patients experienced neurological symptoms before becoming unconscious and dying. This distressing condition, sometimes called dialysis dementia, was observed over a number of years. Several possible explanations were proposed but the one that eventually proved to be correct was Al toxicity.⁸ Understanding the aetiology, early diagnosis of affected patients, treatment and prevention was made possible entirely by the ability to measure low concentrations of Al in serum and dialysis fluids by electrothermal atomisation atomic absorption spectrometry (ETAAS). The lives of hundreds of thousands of patients have been saved following this work. As a consequence of lower detection limits and greater range of elements that can be measured in clinical samples by ETAAS, the type of investigations started with FAAS has been extended to other elements and to other clinical situations. Thus, examples of fatal cardiomyopathy in patients receiving intra-venous nutrition were linked to Se deficiency,⁹ while elsewhere toxicity was seen in those whose fluids had been erroneously over-supplemented with Mn.¹⁰ Occupational monitoring to elements such as Co and Ni are now routine, with fewer cases of toxicity. Many more examples of how ETAAS has been used to develop the objectives noted in Table 1 have been reported in the 21 years of ASU.

The importance of inductively coupled plasma-mass spectrometry (ICP-MS) to the clinical chemistry of trace elements is still being realised. Apart from the obvious features of multi-element analysis and low detection limits, which simplify and extend the diagnostic and monitoring aspects of nutritional, occupational and environmental medicine, there are examples of work that are practically possible only because of this technique. Successful coupling to a separation system, such as a chromatography column, has made speciation procedures much more effective. Most applications have involved As and Se with identification of species produced as part of the metabolic processes. Differentiation of organo- and inorganic-As species is now straightforward, but the more interesting work relates to recognition of short-lived but toxic As^III intermediates during metabolism. With Se, current interest relates to the number of species that have been observed in biological samples and their place in the function and metabolism of this element. However, it appears that some species may be artefacts from the sample preparation, and issues of maintaining structural integrity of species during extraction, and possible misidentification, are discussed in the current ASU.¹

Another area where ICP-MS has a valuable clinical impact involves the measurement of stable isotopes. Using tracer doses of an isotopically-enriched element, mechanisms of absorption and distribution are safely undertaken without having to use radioisotopes as tracers, as in the past. This permits metabolic studies in infants, pregnant women and other vulnerable subjects. Clever detective work with Pb isotopic ratios allows sources of exposure to be identified. In this way, children in Saudi Arabia were shown to be suffering from Pb poisoning due to ethnic remedies rather than household paint or leaded petrol.¹¹ In the last year or so isotope ratio measurements made using multiple collector ICP-MS have revealed a previously unrecognised feature of human physiology in that, under certain circumstances, the body appears to treat one isotope of an element differently from others. In haemochromatosis, for example, there are higher ⁵⁶Fe:⁵⁴Fe ratios in blood from patients than in blood from healthy individuals, suggesting that patients absorb more of the heavier Fe isotope than do normal subjects.¹² In another study, in normal individuals, the ⁶⁶Zn:⁶⁴Zn and ⁶⁸Zn:⁶⁴Zn ratios in hair were significantly lower than in red cells, revealing another example of isotopic fractionation within the body.¹³

Vapour generation techniques have made equally important contributions to clinical chemistry with measurements of Hg and the hydride forming elements. Many examples of occupational medicine, environmental disasters, suicidal and homicidal incidents, pharmaceutical applications, etc., are regularly reported where the vapour generation is coupled to measurements using AAS, AES or ICP-MS.

The importance of X-ray fluorescence (XRF) in clinical chemistry cannot be overlooked. The technique has been applied to analysis of body fluids but it is for in vivo measurements that XRF is particularly valuable in clinical chemistry. Thus far, a limited range of applications have been reported and most work is concerned with determination of Pb in bone. Difficulties with calibration and complexities of the instrumentation have impeded the rapid development of in vivo XRF but projects involving mobilisation of Pb during pregnancy, and lifetime occupational exposures, are beginning to demonstrate the unique potential of the technique.

Historically, FAAS has had the greatest impact, permitting fundamental work for the many different aspects of the clinical chemistry of trace elements. Pioneering work established by this technique has been extended by virtue of the improved sensitivity of ETAAS and ICP-MS but exciting new topics are now being discovered thanks to the features afforded by multiple collector ICP-MS and in vivo XRF.

Andrew Taylor

References

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3. E. J. Moynahan, Acrodermatitis enteropathica: a lethal inherited human zinc deficiency disorder, Lancet, 1974, ii, 399–400.
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5. H. T. Delves, A microsampling method for the rapid determination of lead in blood by atomic absorption spectrophotometry, Analyst, 1970, 95, 431.
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7. A. Taylor, Detection and monitoring for disorders of essential trace elements, Ann. Clin. Biochem., 1996, 33, 486–510.
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13. T. Ohno, A. Shinohara, M. Chiba and T. Hirata, Precise Zn isotope ratio measurements of human red blood cells and hair samples by multiple collector ICP mass spectrometry, Anal. Sci., 2005, 21, 425–8.

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