11
Volume 69 July 1976
471
Section of Measurement in Medicine President Percy Cliffe MB
Meeting 12 January 1976
The Measurement of Trace Elements Dr H T Delves (Department of Chemical Pathology, Hospitalfor Sick Children, and Institute of Child Health, 30 Guilford Street,
London WCJNIEH) The Measurement of Trace Metals in Man The study of trace metals in certain human diseases has become increasingly important over the past ten years. In addition to the role of trace metals in acquired disorders at least two inherited disorders, Menke's syndrome and acrodermatitis enteropathica, have been shown to be associated with a deficiency of copper and of zinc respectively. The determination of these metals and of other specific single trace metals in body tissues and fluids is an important aid in the diagnosis and treatment of the diseases with which they are associated. Multi-element analyses of biological samples are important in studies of trace elements in nutrition (Wester 1971, Alexander et al. 1974) and in research into cardiovascular disorders (Masironi 1969, Sharrett & Feinleib 1975). This increase in the knowledge of the role of trace metals in human diseases has resulted from significant developments of new analytical techniques and their application in clinical chemistry. The most important and successful of these new techniques is unquestionably that of atomic absorption spectroscopy (AAS) which was first proposed as an analytical technique by Walsh (1955). The success of AAS in clinical chemistry stimulated the re-examination of many other techniques for this purpose so that the clinical chemist can now choose from at least six fundamentally different analytical techniques each capable of providing accurate analysis for up to 50 elements in biological fluids. These various techniques, shown in Fig 1, are arranged into two groups: (1) Those capable of providing
multi-element analyses from a single sample. (2) Those capable of providing analyses for a limited number of elements from a-single sample. There is some overlap between these groups with the various branches of atomic spectroscopy. The choice between the multi-element and limited-element single-sample techniques will depend mainly upon the particular clinical requirement. It is possible to determine many elements sequentially with the limited-element single-sample techniques by analysing many different portions of the same (large) sample or, if the sample size is limited, by employing a selective sequential separation procedure (Delves et al. 1971). Both approaches require a great deal of operator time and are therefore not ideal solutions for multi-element analysis problems. However, it may not always be appreciated that the results that can be obtained with multielement techniques are not always sufficiently accurate and/or precise for clinical purposes. It can be seen from the representative examples given in Table 1 that only emission spectroscopy with an inductively-coupled plasma source (ICP-ES) and neutron activation analysis (NAA) consistently produced acceptable precision data. SINGLE SAMPLE MULTI -ELEMENT
TECHNIQUES
SPARK SOURCE MASS SPECTROSCOPY X-RAY EMISSION NEUTRON ACTIVATION o EMI SSI ON - ARCSI PLASMAS
-ATOMIC SPECTROSCOPY-MAAS, AES. AFS-FLAMES SINGLE SAMPLE
LIMITEDl ELEMENTI TECHNIQUES.
NELECTROTHERMAL
ANODIC STRIPPING, POLAROGRAPHY MOL ABSIFWORIMETRY
Fig 1 Analytical techniques for trace element analysis of biological samples. AAS, atomic absorption spectroscopy. AES atomic emission spectorscopy. AFS, atomic fluorescence spectroscopy. mol. abs/fluorimetry, molecular absorption spectrophotometry/fluorimetry
472
Proc. roy. Soc. Med. Volume 69 July 1976
12
Table I Representative examples of the application of multi-element techniques to the analysis of biological tissues Technique Spark source mass spectrometry X-ray emission spectroscopy Neutron activation
analysis D.c arc emission spectroscopy Inductively coupled plasma emission spectroscopy
Sample Tissues
No.* Concentration 50 ng/g to pg/g
Relative standard. deviation 0.10-0.25
5
gg/g
0.07-0.50
Diet, fxces, 19 urine, serum Tissues 26 7 Serum Serum, 7 blood
.Lg/g
20 urine, tissues Blood, > 20 urine, tissues Blood , 5
Blood,
-
10
Relative standard Concentration deviation 0.05
«g/g to ng/g
pg/g to ng/g
0.05
ng/g to 'lg/g
0.05
±g/g
0.05-0.10
ng/g to ,ug/g
0.05-0.10
urine
Molecular absorption spectrophotometry/ fluorimetry
Blood, urine, tissues
> 20
For these reasons and those of high cost (£20 000 to £100 000), and instrumental complexity the multi-element techniques are likely to remain restricted to specialized research laboratories. It is probable that the ICP-ES procedure which is still in the early stages of development will prove to be the most useful of these techniques in clinical chemistry. The precision attainable with the limited-
element single-sample analytical techniques (Table 2) is generally much better than with the multielement methods. Of the methods indicated, only atomic absorption (AAS) with discrete sampling or electrothermal atomization, and anodic stripping voltammetry (ASV) can be regarded as microtechniques. For example, both kinds of AAS, and ASV, allow accurate blood-lead analyses to be carried out with 10-100 I-l volumes of blood and require less than 5 minutes analysis time. The older techniques of spectrophotometry and polarography would require 5-10 ml of whole blood and up to 24 hours. It is therefore apparent why these latter techniques have now been superseded by those of AAS and ASV. At present ASV is limited to the analysis of fewer than ten elements, whereas AAS can be used for more than twenty. It is probable that ASV will be
* Number of elements that have been determined in the sample indicated
most successful for the analysis of lead in blood, will be complementary to AAS for this purpose and thus allow the unequivocal establishment of accurate as well as precise results. The principal advantage of atomic spectroscopy in future studies will not simply be the ability to determine, say, twenty elements each requiring only 2-50 ,tl of a sample of blood or serum or urine, but in the ability to determine individual metalloproteins in those small samples. Electrothermal atomization AAS has been used to determine copper in protein fractions separated by electrophoresis from 2 pi volumes of serum and this has been applied to a study of Menke's syndrome (Delves 1973). More recently Kawa guchi & Auld (1975) have determined zinc in individual metalloproteins with only micro samples using a microwave plasma emission spectroscopic method. For a given metal, there are many different metal-containing species present in whole blood and since not all of these may change with a particular disease state, it will be essential to determine the concentrations of the individual metal-containing species and not simply the gross concentration of the metal in order to understand the biochemistry of the disease process. Such determinations are now
Section of Measurement in Medicine
473
cave in which are housed three plastic scintillation detectors; a separate control room contains the electronic equipment for measurement and analysis of the radioactivity. The cave was constructed by excavating an REFERENCES area 6 m deep by 5 m wide into an existing chalk Alexander F W, Clayton B E & Delves H T bank. The excavation was lined and roofed with (1974) Quarterly Journal ofMedicine 43, 89 Delves H T corrugated steel sheets on a steel joist framework, (1976) Clinica ch,imica acta (in press) and then further protected with chalk. The roof Delves H T, Shepherd G & Vinter P (1971) Analyst 96, 260 was weather proofed with 15 cm of sealed Kawaguchi H & Auld D S (1975) Clinical Chlemistry 21, 591 Masironi S concrete. The interior of the cave was lined with (1969) Bulletin ofthe World Health Organization 40, 305 3 mm steel sheets covered with 3 mm lead. The Sharrett A R & Feinleib,M (1 975) Preventive Medicine 4, 20 floor and the walls were then coated with polyWalsh A (1955) Spectrochimica Acta 7, 108 urethane to provide an impervious and easily Wester P O (1971) Acta medica Scandinavica 190, 155 cleaned surface. The detection system was designed for high sensitivity rather than high resolution and therefore has three large plastic scintillation heads, each of which is viewed by two 17.8 cm diameter photomultiplier tubes. To obtain a good approximation to whole-body geometry and to reduce variations due to the Mr M J Vagg redistribution of isotope within the body after (Agricultural Research Council, administration, the heads are arranged symInstitute for Research on Animal Diseases, metrically in a vertical plane around the animal. Compton, Newbury, Berkshire, RG16 ONN) The complete detector assembly is mounted on a rectangular frame carried on tracks in the roof Assessment of Trace Element of the cave. The frame is motor driven and is Metabolism in Farm Animals designed to traverse the length of a cow at a preset speed. In farm animals the estimation of the absorption Signals from the photomultiplier tubes pass to a of a mineral or trace element from the diet is signal mixing unit, then to an amplifier and finally important in the context of 'production disease' to a single channel pulse height analyser and as discussed by Payne (1970). scaler. The absorption of an element can be measured A 144-channel pulse height analyser (Nuclear provided the net retention of a radioisotope of the Enterprises, NE 6900) is used in the initial setting element is known, that is the difference between up of the detector for each isotope to be counted. the input, the quantity administered, and the Method of operation: The cow is restrained in a output or quantity excreted in urine, faces and mobile steel crush which is then drawn into the milk in a period of 7-10 days. However, if the cave of the whole-body counter by means of an element is poorly absorbed this difference is small electric winch. Both forward and reverse scans and cannot be accurately determined because of of the animal are made and a printout obtained the considerable errors involved in the measure- of the total count and the duration of each scan. ment of both input and output of radioisotope. Application of a whole-body counter to the estimaA whole-body counter can measure directly and tion of the absorption coefficient for manganese-54 accurately the retention of a radioisotope of even in the dairy cow: A whole-body counter can a poorly absorbed element and thus give a accurately measure the retention of an oral dose measure of its absorption. The absorption of a radioisotope at various times after its coefficient of the element, defined as the fraction administration but this retention is not equal to of the dietary content of the element absorbed the absorbed fraction of that isotope for two through the gastrointestinal tract, may then be reasons: (1) Because of the unabsorbed isotope calculated from the whole-body retention values still present in the gastrointestinal tract. (2) as described below. Because of the isotope which has been absorbed This paper describes the construction of a but subsequently resecreted. into the gastrowhole-body counter suitable for cattle, and its intestinal tract and excreted in fices. application to the study of the trace element These two factors were taken into account in metabolism of farm animals. this work by oral administration of 54MnCI2 Construction: The Compton whole-body counter followed by an intravenous injection of the same consists principally of a shielded low background radioisotope about one month later, at which
possible with the new developments in atomic spectroscopy, and will no doubt be used more frequently in future studies of trace metals in human diseases.
Volume 69 July 1976
471
Section of Measurement in Medicine President Percy Cliffe MB
Meeting 12 January 1976
The Measurement of Trace Elements Dr H T Delves (Department of Chemical Pathology, Hospitalfor Sick Children, and Institute of Child Health, 30 Guilford Street,
London WCJNIEH) The Measurement of Trace Metals in Man The study of trace metals in certain human diseases has become increasingly important over the past ten years. In addition to the role of trace metals in acquired disorders at least two inherited disorders, Menke's syndrome and acrodermatitis enteropathica, have been shown to be associated with a deficiency of copper and of zinc respectively. The determination of these metals and of other specific single trace metals in body tissues and fluids is an important aid in the diagnosis and treatment of the diseases with which they are associated. Multi-element analyses of biological samples are important in studies of trace elements in nutrition (Wester 1971, Alexander et al. 1974) and in research into cardiovascular disorders (Masironi 1969, Sharrett & Feinleib 1975). This increase in the knowledge of the role of trace metals in human diseases has resulted from significant developments of new analytical techniques and their application in clinical chemistry. The most important and successful of these new techniques is unquestionably that of atomic absorption spectroscopy (AAS) which was first proposed as an analytical technique by Walsh (1955). The success of AAS in clinical chemistry stimulated the re-examination of many other techniques for this purpose so that the clinical chemist can now choose from at least six fundamentally different analytical techniques each capable of providing accurate analysis for up to 50 elements in biological fluids. These various techniques, shown in Fig 1, are arranged into two groups: (1) Those capable of providing
multi-element analyses from a single sample. (2) Those capable of providing analyses for a limited number of elements from a-single sample. There is some overlap between these groups with the various branches of atomic spectroscopy. The choice between the multi-element and limited-element single-sample techniques will depend mainly upon the particular clinical requirement. It is possible to determine many elements sequentially with the limited-element single-sample techniques by analysing many different portions of the same (large) sample or, if the sample size is limited, by employing a selective sequential separation procedure (Delves et al. 1971). Both approaches require a great deal of operator time and are therefore not ideal solutions for multi-element analysis problems. However, it may not always be appreciated that the results that can be obtained with multielement techniques are not always sufficiently accurate and/or precise for clinical purposes. It can be seen from the representative examples given in Table 1 that only emission spectroscopy with an inductively-coupled plasma source (ICP-ES) and neutron activation analysis (NAA) consistently produced acceptable precision data. SINGLE SAMPLE MULTI -ELEMENT
TECHNIQUES
SPARK SOURCE MASS SPECTROSCOPY X-RAY EMISSION NEUTRON ACTIVATION o EMI SSI ON - ARCSI PLASMAS
-ATOMIC SPECTROSCOPY-MAAS, AES. AFS-FLAMES SINGLE SAMPLE
LIMITEDl ELEMENTI TECHNIQUES.
NELECTROTHERMAL
ANODIC STRIPPING, POLAROGRAPHY MOL ABSIFWORIMETRY
Fig 1 Analytical techniques for trace element analysis of biological samples. AAS, atomic absorption spectroscopy. AES atomic emission spectorscopy. AFS, atomic fluorescence spectroscopy. mol. abs/fluorimetry, molecular absorption spectrophotometry/fluorimetry
472
Proc. roy. Soc. Med. Volume 69 July 1976
12
Table I Representative examples of the application of multi-element techniques to the analysis of biological tissues Technique Spark source mass spectrometry X-ray emission spectroscopy Neutron activation
analysis D.c arc emission spectroscopy Inductively coupled plasma emission spectroscopy
Sample Tissues
No.* Concentration 50 ng/g to pg/g
Relative standard. deviation 0.10-0.25
5
gg/g
0.07-0.50
Diet, fxces, 19 urine, serum Tissues 26 7 Serum Serum, 7 blood
.Lg/g
20 urine, tissues Blood, > 20 urine, tissues Blood , 5
Blood,
-
10
Relative standard Concentration deviation 0.05
«g/g to ng/g
pg/g to ng/g
0.05
ng/g to 'lg/g
0.05
±g/g
0.05-0.10
ng/g to ,ug/g
0.05-0.10
urine
Molecular absorption spectrophotometry/ fluorimetry
Blood, urine, tissues
> 20
For these reasons and those of high cost (£20 000 to £100 000), and instrumental complexity the multi-element techniques are likely to remain restricted to specialized research laboratories. It is probable that the ICP-ES procedure which is still in the early stages of development will prove to be the most useful of these techniques in clinical chemistry. The precision attainable with the limited-
element single-sample analytical techniques (Table 2) is generally much better than with the multielement methods. Of the methods indicated, only atomic absorption (AAS) with discrete sampling or electrothermal atomization, and anodic stripping voltammetry (ASV) can be regarded as microtechniques. For example, both kinds of AAS, and ASV, allow accurate blood-lead analyses to be carried out with 10-100 I-l volumes of blood and require less than 5 minutes analysis time. The older techniques of spectrophotometry and polarography would require 5-10 ml of whole blood and up to 24 hours. It is therefore apparent why these latter techniques have now been superseded by those of AAS and ASV. At present ASV is limited to the analysis of fewer than ten elements, whereas AAS can be used for more than twenty. It is probable that ASV will be
* Number of elements that have been determined in the sample indicated
most successful for the analysis of lead in blood, will be complementary to AAS for this purpose and thus allow the unequivocal establishment of accurate as well as precise results. The principal advantage of atomic spectroscopy in future studies will not simply be the ability to determine, say, twenty elements each requiring only 2-50 ,tl of a sample of blood or serum or urine, but in the ability to determine individual metalloproteins in those small samples. Electrothermal atomization AAS has been used to determine copper in protein fractions separated by electrophoresis from 2 pi volumes of serum and this has been applied to a study of Menke's syndrome (Delves 1973). More recently Kawa guchi & Auld (1975) have determined zinc in individual metalloproteins with only micro samples using a microwave plasma emission spectroscopic method. For a given metal, there are many different metal-containing species present in whole blood and since not all of these may change with a particular disease state, it will be essential to determine the concentrations of the individual metal-containing species and not simply the gross concentration of the metal in order to understand the biochemistry of the disease process. Such determinations are now
Section of Measurement in Medicine
473
cave in which are housed three plastic scintillation detectors; a separate control room contains the electronic equipment for measurement and analysis of the radioactivity. The cave was constructed by excavating an REFERENCES area 6 m deep by 5 m wide into an existing chalk Alexander F W, Clayton B E & Delves H T bank. The excavation was lined and roofed with (1974) Quarterly Journal ofMedicine 43, 89 Delves H T corrugated steel sheets on a steel joist framework, (1976) Clinica ch,imica acta (in press) and then further protected with chalk. The roof Delves H T, Shepherd G & Vinter P (1971) Analyst 96, 260 was weather proofed with 15 cm of sealed Kawaguchi H & Auld D S (1975) Clinical Chlemistry 21, 591 Masironi S concrete. The interior of the cave was lined with (1969) Bulletin ofthe World Health Organization 40, 305 3 mm steel sheets covered with 3 mm lead. The Sharrett A R & Feinleib,M (1 975) Preventive Medicine 4, 20 floor and the walls were then coated with polyWalsh A (1955) Spectrochimica Acta 7, 108 urethane to provide an impervious and easily Wester P O (1971) Acta medica Scandinavica 190, 155 cleaned surface. The detection system was designed for high sensitivity rather than high resolution and therefore has three large plastic scintillation heads, each of which is viewed by two 17.8 cm diameter photomultiplier tubes. To obtain a good approximation to whole-body geometry and to reduce variations due to the Mr M J Vagg redistribution of isotope within the body after (Agricultural Research Council, administration, the heads are arranged symInstitute for Research on Animal Diseases, metrically in a vertical plane around the animal. Compton, Newbury, Berkshire, RG16 ONN) The complete detector assembly is mounted on a rectangular frame carried on tracks in the roof Assessment of Trace Element of the cave. The frame is motor driven and is Metabolism in Farm Animals designed to traverse the length of a cow at a preset speed. In farm animals the estimation of the absorption Signals from the photomultiplier tubes pass to a of a mineral or trace element from the diet is signal mixing unit, then to an amplifier and finally important in the context of 'production disease' to a single channel pulse height analyser and as discussed by Payne (1970). scaler. The absorption of an element can be measured A 144-channel pulse height analyser (Nuclear provided the net retention of a radioisotope of the Enterprises, NE 6900) is used in the initial setting element is known, that is the difference between up of the detector for each isotope to be counted. the input, the quantity administered, and the Method of operation: The cow is restrained in a output or quantity excreted in urine, faces and mobile steel crush which is then drawn into the milk in a period of 7-10 days. However, if the cave of the whole-body counter by means of an element is poorly absorbed this difference is small electric winch. Both forward and reverse scans and cannot be accurately determined because of of the animal are made and a printout obtained the considerable errors involved in the measure- of the total count and the duration of each scan. ment of both input and output of radioisotope. Application of a whole-body counter to the estimaA whole-body counter can measure directly and tion of the absorption coefficient for manganese-54 accurately the retention of a radioisotope of even in the dairy cow: A whole-body counter can a poorly absorbed element and thus give a accurately measure the retention of an oral dose measure of its absorption. The absorption of a radioisotope at various times after its coefficient of the element, defined as the fraction administration but this retention is not equal to of the dietary content of the element absorbed the absorbed fraction of that isotope for two through the gastrointestinal tract, may then be reasons: (1) Because of the unabsorbed isotope calculated from the whole-body retention values still present in the gastrointestinal tract. (2) as described below. Because of the isotope which has been absorbed This paper describes the construction of a but subsequently resecreted. into the gastrowhole-body counter suitable for cattle, and its intestinal tract and excreted in fices. application to the study of the trace element These two factors were taken into account in metabolism of farm animals. this work by oral administration of 54MnCI2 Construction: The Compton whole-body counter followed by an intravenous injection of the same consists principally of a shielded low background radioisotope about one month later, at which
possible with the new developments in atomic spectroscopy, and will no doubt be used more frequently in future studies of trace metals in human diseases.
