Biological Trace Element Research - ACS Publications - American

Chapter 4

In Vitro Investigations in Biological Trace Element Analysis Positive and Negative Aspects

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0445.ch004

Nicholas M . Spyrou Department of Physics, University of Surrey, Guildford, Surrey, GU2 5XH, England

'In vitro' and 'in vivo' analytical methods for trace element studies in biological systems play an increasingly important role in assessing environmental impact on health, in diagnosis of disease and in monitoring treatment and it is not possible to identify the merits and limitations of 'in vitro' methods without discussion of the capabilities of the 'in vivo' techniques. Problems in the selection of representative samples and in sample inhomogeneity are common to both. However, neither makes use of computerised tomography and of advances in tomographic techniques to help in resolving these, despite claims that the field is multidisciplinary. Difficulties in 'in vitro' investigations are illustrated by examples whose errors occur in the analytical technique, in the choice of representative sample and in sample inhomogeneity. In the last twenty years, trace element studies related to human health have continued to grow in a variety of fields, be it to assess environmental impact, to determine daily intake, to diagnose disease or to monitor the course of treatment. However, in order to carry out useful investigations in determining elemental concentrations, it is important to select samples of tissues and body fluids for analysis which will provide meaningful results. Selection should, therefore, generally be focussed on biological specimens which reflect elemental status and can be obtained from subjects preferably with ease and without trauma. The problems then arise in establishing whether these samples are representative and how elemental levels are being influenced by, for example, temporal changes due to metabolism and physiological processes, diet, medication or in general, the lifestyle of the subjects. Otherwise erroneous interpretation may result however good the analytical precision and accuracy. In other situations, when a pathological specimen is excised from the body, interpretation of results may depend on the comparison of the elemental concentrations found in this sample with those found in normal tissue adjacent to it, or in the absence of the latter, with baseline values or ranges reported in the literature for the particular type of tissue, if they exist. It is therefore highly desirable to establish for populations normal levels in tissues and body fluids so that changes during disease conditions can be detected (]_). Biopsy tissue samples, i.e. those that require surgical intervention, would require to be representative of the O097-6156/91/0445-O040$06.00/0 © 1991 American Chemical Society In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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In Vitro Investigations in Biological Trace Element Analysis

tissue or organ from which the specimen has been removed and present the additional analytical challenges of being unique and of small mass, typically less than a few mg. Autopsy samples have been useful i n determining elemental distributions i n whole organs e.g. i n liver (2), kidney and lung (3), despite the problems associated with postmortal changes i n elemental concentration and content, as a function of time after death, which can be severe (4). These elemental distribution studies are useful in indicating the extent of variation w i t h i n an organ and that found between the same organ from different individuals. They may also suggest that sampling and analysing a specific region of an organ (eg kidney cortex or medulla) may be more f r u i t f u l than determining elemental levels of the entire organ. However, there are medical, ethical and legal restrictions to contend with when acquiring postmortem specimens. Acquistion of suitable material w i l l become even more d i f f i c u l t (and more expensive) as the considerable progress made i n organ transplantation makes ever increasing demands i n obtaining healthy organs. 'IN V I V O ' M E T H O D S In order to discuss the advantages and disadvantages of 'in vitro' measurements w i t h regards to elemental analysis of biological systems it is important to assess what 'in vivo' methods have to o f f e r in this respect. A t present, there are twenty-three elements for which nuclear-based techniques exist or are being developed for determination of their concentration 'in vivo' i n the human body (5). The concentration of elements 'routinely' determined are Ca, P, Na, CI, K, N, O and Cd, whereas those of H,C,I and Pb can also be measured. There is an almost equal number of elements, L i , Be, Mg, A l , S, S i , Fe, Cu, Se, A g and Hg f o r which techniques have been developed and detection capabilities demonstrated. A l l are based on the detection of products resulting from nuclear interactions, except f o r Pb which is measured by X-ray fluorescence and K which depends on the detection of the characteristic gamma-rays emitted by the naturally occuring radionuclide, K-40. In general the methods are specific to a single element, although a group of elements may be detected simultaneously e.g. Ca, P, Na, CI and K in bone studies. Whole body or partial body 'in vivo* neutron activation analysis provides information about elemental composition f o r diagnosis and f o r monitoring therapy, which in many instances would otherwise not be available. Other 'in vivo* methods are also being explored and developed for human compositon studies to provide information, not necessarily i n the form of elemental concentrations upon which medical diagnosis can be based (6). Techniques like magnetic resonance imaging (MRI) which can define the relative amounts of bound and free water i n a biological system or total body electrical conductivity measurements which determine total lean mass or total body fat (7) are proving useful i n such body composition studies and are not only non-invasive but also ionising radiation free. In addition other methods, f o r example, employing computerised X-ray transmission tomography and photon densitometry can provide specific information about bone mineral content, i n terms of a weighted (or average) atomic number and the density of the bone matrix, which may be sufficient to serve as an index for diagnosis of osteoporosis (or other bone disease) and for monitoring its treatment (8,9). This is achieved for about the same radiation dose as (or less than) neutron activation analysis which determines Ca and P bone concentrations in the same region of interest.

In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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TOMOGRAPHY

The advantages of 'in vivo' methods of analysis are that measurements are carried out at the site of interest and therefore no variations in elemental concentrations arise from removing the tissue from the body and the possibility of contaminating the sample is avoided. But, unless the 'in vivo' measurements are carried out for total body analysis or total organ analysis (the degree of localisation may be d i f f i c u l t to achieve anyway), the problem of how representative is the region selected for the investigation of the total organ or tissue, as in 'in vitro' analysis, still remains. However, i f the technique has tomographic capabilities or can be combined with tomography i.e it can represent the distribution of a physical property in a plane or slice through the region of interest, without interference from overlying and underlying planes or slices (10), then variations w i t h i n the 'volume sampled* may be detected and taken into account. When photon transmission tomography is used, for example, for determination of bone mineral content, the distribution of the photon linear attenuation coefficient (a function of the atomic number and the physical density) in a selected slice through the body, be it the wrist, the ankle or the spinal column, can represent the amounts of cortical and trabecular bone, as well as soft tissues, marrow, fat blood, muscle etc., that may be present. This can be achieved with a spatial resolution (expressed in terms of a pixel or picture element) as good as 1mm by 1mm and slice thickness of 10mm (or less, i f desired). It depends on the photon energy, photon source intensity and time of irradiation i.e the radiation dose. X-ray transmission computerised tomography is now established worldwide as a method of diagnosis, made possible with the introduction of the first commercial scanner by Hounsfield in 1972. There has been a demand over the years for more accurate, quantitative data regarding the photon linear attenuation coefficients of tissues and organs (and hence their composition) in the body, so that not only accurate determinations of the size of organs and tissues can be made but also for normal variations in the values can be established and compared with pathological states. Some of the techniques developed, especially those using discrete photon energies, can be sensitive to changes in the attenuation coefficients of soft tissue (water is usually used as the comparator) of 1 in 1000. This represents a detectable mass fraction (\\) for higher atomic number elements (above, say, atomic number 30) of about 10 Mg/g, with a 5 percent precision, in water. If an organ or a region of the body is to be analysed by an 'in vivo' method, in order to determine its elemental concentrations, or a tissue sample is to be removed from the body for 'in vitro* trace element analysis, selection of the appropriate site in terms of its homogeneity or specific heterogeneity can be made more accurate by obtaining tomographic information. Radiation dose must be taken into consideration for *in vivo' analysis and sampling but i f an organ or tissue is removed from the body, for example, for specimen banking, then tomography can be performed and variations measured without restriction. The measurements can also be repeated, in order to monitor changes in the stored tissue or organ with time. We first used tomography in conjunction with neutron activation analysis i n an environmental study where the elemental composition of tree rings was determined and ring to ring variations found (JJ2). Use of



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photon transmission tomography of a tree trunk section r a p i d l y identified the areas where elemental variations occured and therefore made po.ssible the selection of specific samples for detailed trace element anlaysis. In another investigation the changes in the linear photon attenuation coefficient in the cortical and trabecular compartments across and along the length of a human tibia, which had been removed from a subject, were correlated with the results of elemental levels obtained from neutron activation analysis of a number of thin transverse sections cut from the bone along its length (13). Tomographic information about the relative size of the cortical and trabecular compartments, which could not be easily separated by microtome sectioning of the samples for trace element analysis, allowed calculations of relative weighting and estimates of elemental compositon of each compartment to be carried out. In carrying out 'in vivo' neutron activation analysis of liver for determination of Cd and Se concentrations in the organ, an X-ray transmission tomographic image through the region of interest was found to be useful in calculating the volume of interaction between the neutron beam and the tissues and also in estimating the volume of irradiated tissues emitting radiations 'seen' by the gamma-ray detector (J_4). In addition, since the sizes, the distribution and the value of the photon linear attenuation coefficients of the tissues were known, the attenuation of the gamma-rays, resulting from neutron interactions, on reaching the detector could also be calculated. More recently, the techniques of neutron transmission tomography and neutron induced emission tomography have been developed (\5± \6) which depend on the neutron linear attenuation coefficient (the macroscopic neutron cross-section) and the neutron capture cross-section, respectively. The latter technique combines the principles of tomography with neutron activation analysis, thus providing a method which can determine the concentration of elements in a selected plane or section, through a sample, nondestructively. The technique, which is multielemental, has at present a spatial resolution of 0.5 mm for relatively small samples of about 50 mm diameter and because of radiation dose is only applied to 'in vitro' analysis. 'IN V I T R O ' P R O B L E M S There are many examples cited in the literature, concerning problems in the elemental analysis of biological systems and sources of error in sampling and sample preparation (e.g. J_7, |8). Here, a small number of examples from recent work using instrumental nuclear-based methods of in vitro trace element analysis are briefly discussed. First an example where the analytical technique as applied may lead to errors. In a collaborative project with the European Community Laboratories at Ispra, Italy, a preliminary investigation to determine the fluorine concentration in bone biopsy samples removed from patients with osteoporosis and on renal dialysis treatment, was carried out (Spyrou N.M., A l t a f W.J., G i l l B.S., Jeynes C, Nicolaou G.E., Pietra R., Sabbioni E and Surian M., J. Biol. Trace Analysis Research, in press). Concern has been expressed about the uptake of certain elements, particularly aluminium, in bone and other tissues of patients receiving kidney dialysis treatment. It has also been suggested that incidence of osteoporosis and bone disease where aluminium may be playing a part is reduced in the presence of fluorine. Therefore one of the objectives was to f i n d out whether a correlation between the two elements existed. Fresh biopsy samples from the iliac crest of the subjects were obtained, each had an average diameter of 3 mm with masses varying between 10 and 140 mg.


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Two methods were employed for the determination of fluorine concentrations. C y c l i c neutron activation analysis ( C N A A ) to determine f l u o r i n e and other elemental levels in the whole bone sample and proton induced gamma-ray emission (PIGE) analysis to measure variations of the element along the length and across the surface of the bone sample. Determination of the trace element concentration using P I G E depends on the range of penetration of the proton beam into the sample and thus on its major element composition, so that the mass of the sample excited by the protons can be calculated. If there are variations in the range of penetration of protons and the gross elemental composition between comparator and sample, or sample to sample or w i t h i n or along a sample, then these must be corrected, otherwise errors in interpretation occur since these are not "true variations' in the concentration of the trace element of interest. The major element composition (C, O, P and Ca) of the bone samples and bone standard was obtained using R u t h e r f o r d Backscattering (RBS), f r o m which the mass excited by the proton beam was calculated and the fluorine concentration in the bone accurately determined. If this correction was not applied, the differences in the mass excited, amounting to 30 percent, would have been attributed entirely to variations in the f l u o r i n e composition of the specimens. The second example is from an on-going Se supplementation project where elemental changes in blood components are monitored (19); it relates to which component of blood should be analysed, i f not whole blood, and also emphasises that conditions under which blood samples are collected, should be stated as f u l l y as possible. Healthy, young adults of both sexes, partaking in a gastric motility study, in which the rate of stomach empyting of a l i q u i d meal was being measured non-invasively, were given as a supplement a commerical product containing the element together with vitamins A, C and E. Blood samples were obtained on a one-day-a-weck basis from each i n d i v i d u a l over a period of about 10 weeks. The subject fasted overnight, then in the morning was given to drink 500 ml of water flavoured w i t h orange squash and stomach emptying measurements were made over an hour or so or until emptying was complete and there was a return to a baseline value. Blood was collected prior to the d r i n k and then again at the half-emptying stage, as well as at the end of the experiment. This was repeated in the afternoon, after four hours, with no food or d r i n k taken i n between. Blood samples were separated into erythrocytes and plasma and a whole blood sample was also retained for trace element analysis. Results indicated that the level of selenium in whole blood and its components, erythrocytes and plasma, increased with dose and time and concentration of the element was maintained for at least six weeks following cessation of supplementation. However, the influence of the supplement on some of the electrolytes, simultaneously determined with Se in the samples, such as Na, CI, Br and Rb could only be detected clearly in the erythrocyte and plasma and not in the whole blood. Further, when the variations of Se concentrations during the morning and afternoon sessions of the same day were examined, it was interesting to note a measureable decrease in the concentration for both plasma and erythrocytes during the day (20). One may have expected, f r o m reading the literature, that the plasma levels would vary for the element during the day but not those for erythrocytes, which we are told may reflect a longer term intake and accumulation of the element. It is obvious, as with other elements, that more work requires to be done in determining the uptake (and washout) of Se in blood, under controlled conditions of food intake.



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The third example is related to the above but underlines the importance of selecting the appropriate tissue. P u b l i c consumption of m u l t i v i t a m i n and mineral supplements in the U K has grown significantly in the last decade, f o l l o w i n g trends set earlier in the USA. A reliable and preferably inexpensive method for monitoring the levels of these supplements in humans is sought in order to ensure that toxicity does not arise from prolonged usage. This is expecially true for trace elements where 'windows' of intake for good health can be very narrow and synergistic effects between elements may alter body burdens significantly. The possibility of using fingernails as a biomonitor for Se and other elements is being investigated (2J_), as blood samples become more d i f f i c u l t to obtain because of the rigorous 'screening' procedures required before analysis. The ease of collection, storage, preparation and handling of nail tissue has always been an attraction. The degree to which nail stores different elements normally depends on the supply of these elements during nail growth and the presence of 42 elements in the normal nail implies that the tissue may be a suitable candidate for monitoring. However, what are the elemental variations from finger to finger and from hand to hand? Does one select, as many do, the thumb nail from the right or left hand (whichever is used more) because it provides the largest mass ? Fingernail clippings were collected from two healthy male adults, aged 45 and 28 years, over a period of 6 months. The first subject (A) was on supplements; Se (lOO/zg/day) with vitamins A, C and E and Zn (5mg/day) provided by commercial products, whereas the second subject (B) was not given any supplement. Nail-to-nail variations and hand-to-hand variations were obtained for both subjects and Table I, shows the Se concentrations over the whole six month period; deviations for the same finger over this period were less than 20%. It can be seen that the highest concentrations for Se occur in both subjects in the nails of the little fingers of both hands and that variations exist between the fingers Table I: Average Se concentrations (Mg/g * 20%) over six month period of six sets of fingernail samples from both hands of subjects A (supplemented) and B (unsupplemented).

Subject

little finger

ring finger

middle

forefinger

thumb

A(left) (right)

17 17

N/A N/A

5 6

N/A N/A

10 10

B(left) (right)

37 48

13 15

16 20

17 19

19 13

N/A

a l l samples not available

of one hand. It may also be worth noting that Se levels in the fingernails of subject A did not exhibit the same degree of variation as those found in subject B (not supplemented) and that supplementation may have acted as a regulating mechanism i n the excretion of the element from the body and its distribution through the fingernails. The profile of elemental


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concentrations i n the fingernails may therefore give us additional information about the regular intake or otherwise, of trace elements by the subject. F i n a l l y , it was decided to determine the homogeneity of the nail tissue and estimate the minimum mass required f o r a representative sample to be collected f r o m the unsupplemented i n d i v i d u a l (subject B), who exhibited large variations i n the nail-to-nail and hand-to-hand elemental concentrations. This was done by employing the concept of the sampling factor (22, 23) f o r the elements detected and using a l l 140 fingernail samples collected f r o m subject B over the six month period. The values f o r the sampling factors, defined at being homogeneous to w i t h i n 1 percent, are given i n Table II. These values are very high but should not be considered unusual. Fingernails must be accepted as inhomogeneous materials for analysis, providing an integrated value of elemental concentration only f o r the specific period of growth f o r which the sample has been collected. Table II: The sampling factor, K, at one percent inhomogeneity, for 12 elements detected i n 140 f i n g e r n a i l samples f r o m both hands of subject B (unsupplemented), collected over 6 months

Element K(g)

Au 53.0

Br 5.6

CI 1.1

K 8.5

Mg 1.4

Mn 3.1

Element K(g)

Na 18.1

Rb 0.08

S 4.5

Se 191

V 1.9

Zn 4.9

CONCLUSIONS The advantages and disadvantages of 'in vitro' trace element studies are better illustrated in the context of 'in vivo' methods of analysis. Both 'in vitro' and 'in vivo' methods have common problems when it comes to selecting a sample or site which is representative of the particular biological system under investigation and i n assessing sample inhomogeneity. The use of tomographic techniques and of the vast amount of data already accumulated i n hospital departments from computerised tomography scanning of body tissues, fluids and organs, should serve as aids i n resolving some of these problems and i n providing information about homogeneity and size of organs and compartments i n terms of major element composition. It is advocated that analytical techniques w i l l benefit significantly by applying whenever possible the principles of tomography in order to obtain information about elemental distribution i n a sample, non-destructively. In addition a number of non-invasive, radiation free methods are becoming available f o r composition studies. They define body compartments e.g. fat, water and lean mass, which can be useful i n normalising elemental concentrations. It should then be possible, by employing the d i f f e r e n t information supplied f r o m a combination of techniques, to reduce the variations i n composition generally attributed to 'individual physiology and anatomy' and therefore allow the more subtle differences that trace element analysis may reveal to be interpreted correctly. A v a i l a b i l i t y ,


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acquisition and interpretation of such information a m u l t i d i s c i p l i n a r y a p p r o a c h i n such studies.

is o n l y

possible through

LITERATURE CITED 1.

2.


3.

4. 5.

6.

Iyengar, G.V. Clin. Nutr. 1987, 6, 4, 154-58. Lievens, P.; Versieck, J.; Cornells, R.; Hoste, J. 1977, J. Radioanal. Chem. 1977, 7, 483-96. Vanoeteren, C.; Cornells, R.; Sabbioni, E. Critical Evaluation of Normal Levels of Major and Trace Elements in Human Lung Tissue, Report EUR 10440 En, Commission of the European Communities, Luxembourg, 1986. Iyengar, G.V. J. Path. 1981, 134, 173-80. Spyrou, N.M. J. Trace and Microprobe Tech. 1988, 6, 4, 603-20. In Vivo Body Composition Studies, Ellis K.J.; Yasumura, S.; Morgan, W. D. Eds., The Institute of Physical Sciences in Medicine: London, 1987.

7. 8. 9.

Proceedings Symposium: Body Composition Methods, Roche, A.F. Ed., 1987, Human Biology 59, 209-335. Non-Invasive Bone Measurements and Their Clinical Application, Cohn, S.H. Ed., CRC Press: Florida, 1981. Banks, L.M.; Stephenson, J.C. J. Comput. Assist. Tomogr. 1986, 10, 463-67.

10. 11.

12.

13.

Kouris, K.; Spyrou, N.M.; Jackson, D.F. Imaging with Ionising Radiations, Surrey University Press/Blackie and Son Ltd: Glasgow and London, 1982, Vol. 1. Kouris, K.; Spyrou, N.M. Nucl. Inst. & Meth. 1978, 153, 477-87. Spyrou, N.M.; Neofotistou, V.; Kouris, K. In Proceedings Third International Conference on Nuclear Methods in Environmental and Energy Research, 1977, Columbia, Missouri, Technical Information Centre, U.S. Department of Energy, CONF-771072, 126-135. Kidd, P.M.; Nicolaou, G.E.; Spyrou, N.M., J. Radioanal. Chem. 1982, 71, 489-507.

14. 15.

16.

Nicolaou, G.E.; Matthews, I.P.; Stephens-Newsham, L.G.; Othman, I.; Spyrou, N.M. J. Radioanal. Chem. 1982, 71, 519-27. Spyrou, N.M.. J. Radioanal. Nucl. Chem. 1987, 110, 641-53. Spyrou, N.M.; Kusminarto,; Nicolaou, G.E. J. Radioanal. Nucl. Chem. 1987, 112, 57-64.

17.

18. 19. 20. 21. 22.

23.

Behne, D. J. Clin. Chem. Clin. Biochem. 1981, 19, 115-120. Iyengar, G.V. In Trace Element Metabolism in Man and Animals, McHowell, J.; Gawthorn, J.M.; White, L. Eds., Australian Academy of Sciences: Canberra, 1981, 667-73. Damyanova, A.A.; Akanle, O.A.; Spyrou, N.M. J. Radioanal. and Nucl. Chem. 1987, 113, 431-36. Spyrou, N.M.; Akanle, O.A.; Damyanova, A.A. Am. Nucl. Soc. Transact. 1986, 53, 187-89. Spyrou, N.M.; Altaf, W.J. Am. Nucl. Soc. Transact. 1989, 60, 28-29. Heydorn, K.; Damsgaard, E.; Rietz, B. Anal. Chem. 1980, 52, 1045-53. Spyrou, N.M.; Al-Mugrabi, M.A. J. Trace and Microprobe Techn. 1988, 6, 425-35.

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Biological Trace Element Research - ACS Publications - American

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