- Email: [email protected]
Milestones in biological trace element research
The Science of the Total Environment, 100 (1991) 1-15
1
F,lsevier Science Publishers B.V., Amsterdam
M I L E S T O N E S IN BIOLOGICAL TRACE E L E M E N T RESEARCH
G.V. IYENGAR
Building 235, B125, National Institute of Standards and Technology, US Department of Commerce, Gaithersburg, MD 20899, USA
ABSTRACT Over 100 years ago, macro.scale analytical techniques were used to discover the roles of special compounds (especially of metallic elements) in living organisms, and investigations were focussed on selected proteins and pigments suspected of containing percentage quantities of metals. In contrast, present-day analytical techniques are capable of detecting extremely small quantities and have become routine ultra-trace measurement tools to probe elemental interactions at cellular levels. The scientific achievements connecting these two boundaries are punctuated with an array of analytical developments; some highlighting the phenomenal advances in the measurement ~echnology and others reflecting the exceptional bioanalytical perception and the multidisciplinary outlook of trace element investigators. An account of the events that contributed to the overall progress in biological trace element research is the essence of this communication.
INTRODUCTION
Trace elements and myth! Almost 2300 years ago Hippocrates advocated the notion of "you are what you eat". Then there are stories of prescribing rusty water to restore vigor to pale looking persons, embedding iron nails in apples for several days before recommending those apples for consumption by pregnant women and persons who were weak, and using burned sponges (later proven to contain iodine) to cure goiter. Thus, the ancient Greeks were unwittingly exploring a whole new area of science. They have also confirmed that the healing power of trace quantities was an antique phenomenon!
Nature's signatures Tracking down historical notes reveals that nature's signatures, which were sometimes subtle and often poignant, were waiting to be linked to what are now termed as trace element disorders. These hints were specific in certain geographic locations. The use of seaweeds as a source of iodine, and linking iodine to the incidence of goitre during the 18th century and consolidating these findings in the 19th century, is an outstanding example [2]. Similarly, selenium toxicity linked to livestock through a condition known as alkali0048-9697/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
disease or blind stagger was recorded in certain parts of the United States [3]. Other examples are the bush-sickness of sheep and cattle in N e w Zealand and Australia leading to the discovery of the essentiality of cobalt [4]. These events were followed by more recent findings linking selenium deficiency to the endemic Keshan disease in China [5], and the growthretardation syndromes linked to zinc deficiency in the Middle East [6].Smith has recently reviewed the historical aspects of health effects of trace elements [7]. ESSENTIALITY OF T R A C E E L E M E N T S
"Essential chemical elements whether required in major, minor or minute quantities, have the same mission -- to sustain life" [1].
Documented evidence reveals, already in the 17th century using then-available analytical expertise, the determination of percentage quantities of metals such as copper (e.g., turacin and hemocyanin, copper-containing compounds in the feathers of certain birds and in the blood of snails, respectively), zinc (sycotypin, a zinc-containing pigment in Mollusca) and vanadium (a respiratory compound in sea squirts) to investigate the roles played by these elements in sustaining essential lifeprocesses [8,9].It has been known since the 17th century (with the identification of iron in erythrocytes) that all human beings need iron if they are to survive. Similarly, iodine has also been recognized as an essential trace element since 1850. Most of our knowledge of trace elements in human and animal health, however, was acquired in this century, particularly in the last 30 years. The availability of analtyical methods refined over time has provided undisputed TABLE I
Discovery of trace element requirements Iron Iodine Copper Manganese Zinc Cobalt Molybdenum Selenium Chromium Tin Vanadium Fluorine Silicon Nickel Arsenic
17th century 1850 1928 1931 1934 1935 1953 1957 1959 1970 1971 1971 1972 1976 1977
3
evidence recognizing the essentiality of several trace elements for living organisms following various investigations. Currently no fewer than 15 such elements are thought to be essential for humans, though for some of them (e.g., arsenic, vanadium and tin) the evidence is not direct but comes from animal experiments (Table 1). In this context, the role of analytical chemistry in sustaining the progress and promoting this branch of research cannot be emphasized sufficiently. This era is also remembered for the development of criterion for defining essentiality by Cotzias [10] and the perception of a working model for dose-response relationships, which have been elegantly summarized by Mertz [9]. These and other pioneering developments have culminated in several milestones in characterizing the growth of this dynamic branch of science, namely Biological Trace Element Research (BTER), as shown in Table 2. T H E CART.BEFORE-THE-HORSE PERIOD
"The ultimate purpose of an analytical result is to address the problem, not merely generating numbers for intercomparisons" [I]. The "cure.all" situation
In earlier investigations, especially during the fifties, sixties and early seventies, a great proportion of the analytical results obtained for various TABLE 2 Milestones in biological trace element research Essentiality of trace elements Cart before the horse period! The "cure-all" situation The "artifact" variations Developments in metrology Historical perspective Trace element determinations Multielement methods Analytical quality assurance Developments in bioanalytical concepts "Metal.free environment" concept for metabolic studies Recognition of biological trace element research as a multidisciplinary science Speciation and bioavailability concept Selected applications Reference elemental concentration values for tissues and body fluids Recommended dietary allowances Elemental composition of Reference Man
biological materials were mainly intended to demonstrate the powerful capabilities of the newly emerging methodologies, e.g. multielement techniques. Unfortunately, in the wake of analysts' enthusiasm to prove the effectiveness of these extraordinary technical achievements, multielement analyses assumed a sort of "cure all" posture and little or no consideration was given to incorporate the biologic basis for the investigations. To give an example, practically countless numbers of studies have been performed on hair, due to the fact that this specimen was the easiest to procure. One wonders if there was any recognized analytical methodology that had remained un-used in the determination of some element or other in this hirsute growth. As a natural consequence of this bandwagon research, a great majority of the findings have turned out to be of no use. Their only contribution has been to leave behind an e,xasperating trail of pointless publications. The position is no better for a number of other tissues and body fluids since, in most cases, the medical and other health sciences professionals who were involved in supplying the samples for analysis were unaware of the spurious analytical implications of uncol~trolled specimen collection, and the analysts who found access to the samples on their own did not have either the insight to assess the biological integrity or possess the exacting training required to deal with real-world biological collections.
The "artifact" variations The net result of all the factors alluded to in the preceding paragraph led to ascribing part or most of the changes observed with respect to whatever was considered as "control" to so-called "biological variations" arising from a host of factors such as age, sex, environment and diet.This is of course true fvr ~ome elements that did not pose insuperable analytical difficultiesand the results were reliable. Similarly, in some cases, the conclusions drawn were based on relative measurements to estimate the differences, and there were gounds to accept these findings as valid. In addition to these, there were instances where an investigation was carried out with meticulous care to understand the problems confronted. Hc,,wever, it was not always easy to identify such cases, therefore the handful of good results was drowned in the ocean of confusion. It is now generally concedsd that a great majority of the earlier experiments suffered from methodological inadequacies. It therefore follows that part of the blame for the current state of the scientificliteratureon elemental composition of biological systems rests on the practice of demonstrating the potentials of newly developed techniques to generate numbers in unplanned investigations, rather than seeking the solution to a credible problem and using the available expertise to solve it.Or simply another case of aligning the cart before the horse! In words not devoid of irony, Pardue and W o o [11]have stated this almost as an aphorism -- tc solve the problem, analysis must start with the problem.
5 D E V E L O P M E N T S IN M E T R O L O G Y
"The analyst is the most important component of any analytical system" [1]. Metrology is the science of measurements, devoted to all aspects of perfecting an analytical measurement to generate as accurate a result as possible. This branch of science has undergone tremendous changes since the days when laboratories contained simple equipment that was conveniently termed "apparatus". The progress in analytical instrumentation and the concept of a modern analytical laboratory has brought about a bewildering array of analytical instruments whose performance is not optimally explored without the use of yet another piece of laboratory equipment, namely the computer!
Historical perspective "A false balance is an abomination to the Lord, but a just weight is his delight" [12].
The words cited above go beyond science! At the center of the classical period is the analytical balance. The art and science of weighing was known in Egypt as early as 3000 B.C. The earliest written reference to an equal arm balance dates back to 1300 B.C. [12]. In his eloquent recollections of the history of analytical chemistry, and trace analysis in particular, Laitinen [13] presents five periods: (a) antiquity to the beginning of modern chemistry late in the 18th century; (b) late 18th century through the 19th century; (c) the period from 1900 to 1939; (d) the decade of the 1940s; and (e) the period from 1950 to the present. Laitinen characterizes each period by the following highlights: fire assay or cupellation as an earliest example of trace analysis; development of colorimetry, atomic emission spectroscopy, electroanalytical chemistry and the use of fluorescence; era of application of physical chemistry to analytical problems followed by discovery of pH, polarography and potentiometric titrations; the impact of the Second World War reflected in the development of nuclear chemistry, mass spectrometry and UV spectroscopy; and an array of modern methods as discussed below.
Trace element determinations Several analytical techniques, namely atomic absorption (flame and flameless), atomic emission (direct current and inductively coupled plasma), chemical and electroanalytical methods, gas and liquid chromatography, mass spectrometry in different modes, nuclear activation techniques and X-ray fluorescence, which offer sufficiently low detection limits for a variety of biomatrices, are now available. Yet, very few laboratories in the world carry out reliable trace element determinations, while a Idrge proportion of the laboratories working with biomaterials find it difficultto achieve a consistent
6
capability to maintain even 10-20% accuracy and precision. This is an indication that high sensitivity alone is not the solution to the problem of accuracy and precisiom and that unawareness of various interferences (e.g., matrix-related problems), flaws in sample and standard preparation and inadequate calibration procedures are evident.
Multianalyte determinations Multielement techniques are useful in obtaining simultaneous (even if partly qualitative at times) elemental composition profiles of a given specimen and have provided a big boost to our quest of understanding the elemental concentration profiles of biological systems [1,14]. Many interesting findings have surfaced unexpectedly as a result of these studies. From an analytical point of view, the non-destructive modes offer possibilities for generating simultaneous data for several elements for comparison, thus acting as internal quality control agents so that unusual situations involving any specific element can be evaluated. Moreover, in a carefully designed study, multielement assays can provide very useful information on a large number of elements at relatively low costs [15]. On the other hand, single element assays have their own role to play. In some cases, some elements have to be determined alone due to severe analytical problems. Clinical, environmental and nutritional laboratories involved with specific elements frequently need single element assays. Therefore, in a comprehensive laboratory, a combination of both single and multielement capability is essential for effective functioning.
Analytical quality assurance "Frequent analysis of appropriate certified reference materials is a key component of any well planned quality control program" [1]. Frequent analysis of reference standards is the key for overcoming procedural inconsistencies in establishing quality control in any analytical laboratory. In this context, it should be recognized that the standard set for the quality of an analytical result is an important factor and is of course dependent upon the end use of the results. Thus, for example, if the aim is restricted merely to scan different biological matrices as a provisional step to establish the relative levels of elemental concentration profiles in them, it is obvious that a reasonable degree of quality standard is sufficient; whereas for meeting the requirements of a typical biomedical trace element research laboratory aiming to use the results for medical diagnostic purposes and legal regulatory processes, depending upon the problem, results with 5-10% total analytical uncertainty may be acceptable. On the other hand, an exceptionally high quality standard (< 1% total analytical uncertainty) is mandatory in some cases, e.g. certification of reference materials. If the tolerance limits are set narrower than the investigation really requires or are not feasible under
practical laboratory conditions, it can cause unnecessary expense and loss of time [16].
Reference materials (RMs): Although there are quite a few biological RMs presently available [1,17,18] they do not fulfill all the current analytical requirements. For a group of elements (e.g., A1, F, Sb, Si, S:n and V) not many RMs are available, and in some cases the number is very small or zero. A major problem is that trace element concentrations are generally subject to large errors, and it is imperative to match the sample matrix with an appropriate RM, so that the level of a particular analyte retains its proportion in relation to the overall matrix composition of the sample. Only then can the sources of systematic errors arising from matrix effects be identified To quote one example, using instrumental neutron activation analysis, Hg and Se could be determined in human milk even in small size samples, but not in cow milk, although the levels of Hg and Se were comparable in these two matrices; this was due to matrix effects from the predominant presence of P (ratio 1:10) in cow milk [19]. D E V E L O P M E N T S IN BIOANALYTICAL CONCEPTS
"A prudent combination of analytical awareness and biological insight is crucial for success in biomedical trace element research studies" [1].
Controlled-environment concept for metabolic studies The realization that several trace elements are required at very minute concentrations and that under routine laboratory conditions the ambient levels of the metals could mask the effects of an investigation, led to the development of the controlled-environment approach or the trace element-freeisolater system. This is basically an all-plastic assembly, with filtered air, free of dust particles down to a fraction of a micrometer. Further, continuous monitoring of all the items in the system is maintained for elements of interest. Under these conditions, the diet is the main source of trace element supply, and by using a chemically well-defined diet, reliable BTER investigations can be carried out. Thus, the introduction of high purity diets on one hand, and the ability to exercise strict analytical quality control on the other, have led to rapid advances in BTER investigations [20].
B T E R - - a multidisciplinary science "The lack of a multidisciplinary approach has been the Achilles heel of biological trace element research" [1]. Biological trace element research (BTER) is a multidisciplinary science. Therefore, it is necessary for researchers in this field to strive for a reasonable degree of biological insight and analytical awareness of the problems involved.
8
This in turn will enable them to design meaningful B T E R investigations. Quite frequently, the complexities involved necessitate the use of a variety of scientific talents and a combination of several analytical techniques. Some of the special difficultiesare seen, for example, in providing a "total" quality control in the overall context of an investigation. These include experimental design, collecti:,nof biologically and analytically "valid" samples, understanding the implications of presampling factors [21,22] in the context of overall accuracy of an analytical result as reflected in Fig. 1, and finally the ability to carry out accurate analytical measurements on those specimens, data evaluation and meaningful interpretation of the findings [23]. Therefore, it follows that team work is an important component in any B T E R investigation. A RELIABLE CONCLUSION DEPENDS ON TH]RQUALITY OF THE ANALYTICAL RESULT
DataHandlingand ~ Inteipmtalion ~ Errors ~ ~
-
-
~
m --mIned~luetely e '
mllChed
conlrols
InePl~ropr*lte Stilif*shcsl treetment
t
,
o
n
K e y p u n c ~ r r o r ~ n 0 clertcel mlstlkes -- Recordmg end ¢ilculehon
Analytical
Melsuremen! Of the lnllylicII el|inllS CholcO of imDrol~lr lnilyhcII Ilchnlque
[l'lrors
-- Preplrefory fKhmques -- Specimen colllchon
Preumplln9 Factors
-........ .¢,,-,,onW"-4FJFX~ILI su=,,.,., Co.,,,on. 4 F H A g E I Cr0hcslly Smell Semples r j r l.lemolys~s
~
~ . , c . . . o . aIll'q,%,°,%"~ ~
lira ~
~
-
I" ~ " "
Long.term Phystolo~tcel Influences Short,term PhystoloQtcil Influences
Fig, 1. Elemental analysis of biological systems [22]. [Reprinted with permission from Analytical Chemistry, 54 (1982) 555A. Copyright American Chemical Society.]
9
Although a large body of data exists for most trace elements in biological media, in m a n y cases they are of limited use because cf inherent inaccuracies [1].This has generated a lot of skepticism among trace element researchers. Primarily, the variations in analytical findings that are stillprevalent can be linked to several pitfallsthat have failedto receive adequate attention by trace element researchers. Therefore, first of all, there is a need to reestablish credibility in trace element analytical research by generating well controlled quality data to dispel fears in the minds of many users. In this context, it may be emphasized that development of a reliable data base for the elemental composition of biological systems is a multidisciplinary task involving preparation of well defined protocols, adequately tested methods for sampling and handling biomaterials, and use of appropriate analytical procedures. Above all, itis a team effortrequiring a prudent combination of analytical awareness and biological insight, which are crucial parameters for any biomedical trace element research study. The significance of the above comments are reflected in Table 3, which illustratesdifferent perspectives of lead determinations in tissu,.~:and fluidsas seen in the scientific literature. Case I illustrates the worst situation of choosing a questionable or unsuitable biological specimen which, produces unreliable results. Case 2 is the use of a sound specimen yet it is prone to analytical pitfalls,especially at very low concentrations. Case 3 is a typical situation where extreme care is exerted to solve a problem of wrong biological perception. Smokers are usually consumers of alcohol too, and the elevated blood lead was traceable to wine consumption [24].Obviously, based on these findings ifsmoking was banned to control blood lead levels,itwould have been a futile move. Case 4 is an ideal combination of biological relevance and analytical expertise, since significant differences have been established in several studies [1]. TABLE 3 Analytical scientists versus biosciences researchers. Some examples of different perspectives! Example: Determination of lead in tissues and body-fluids Case 1: Analytically questionable and biologically unsound; Earlier studies on hair as a monitoring specimen, and Pb in blood plasma Case 2: Analytically questionable and biologically sound; Determination of Pb in liver Case 3: Analytically sound and biologically questionable; Increase of Pb in whole blood related to smoking Case 4: Analytically reliable and biologically sound; Pd in whole blood (and also Cd in blood of smokers and non-smokers)
10
Trace element specification and bioavailability "Human milk is for the human infant; cow's milk is for the calf" [25]. The late Paul Georgy stated this point almost derisively 60 years ago and persisted in his opinion when others chuckled! In my opinion, this sentence powerfully symbolizes the concept of relevance of an elemental species and its bioavailability. Conventionally conducted BTER studies generate data on the total amount of trace elements. For example, in dietary intake studies it is common to carry out elemental analysis and use the results to draw comparisons and conclusions in the context of recommended dietary allowances. It is now being recognized that the biologically available fraction of a trace element differs among different foods and, therefore: the bioavailability of various elements from single or mixed foods should be determined for accurate estimation of the intake from diets. However, metal speciation in complex biological and dietary media is a difficult task due to detection limit considerations and the danger of inducing changes in the speciation states during analysis. Many biochemical methods for the isolation of metallocomplexes such as ion-exchange chromatography, affinity chromatography, immunoprecipitation, electrophoresis and isoelectricfocussing are in use, but are faced with problems of loss and contamination of the metal and its species. Radioactive labeling of organo-arsenic compounds has been described for animal studies [26]. A two-stage in vitro system to simulate gastric and intestinal digestion of food in combination with inductively coupled plasma-mass spectrometry has been successfully used in recent investigations. Several techniques based on gas chromatography and high pressure liquid chromatography and anodic stripping voltametry have been applied to speciation chemistry. Many analytical methods are useful for speciation chemistry studies provided that their limitations are recognized [27]. Concerning bioavailability, the use of stable isotopes in mineral nutrition research is well established. Stable isotopes as isotopic tracers have no radiation risk and are therefore widely accepted. The use of various versions of mass spectrometry and neutron activation analysis for in vitro specimens following stable isotope application has resulted in considerable progress in mineral metabolism research. SELECTED APPLICATIONS
Reference elemental concentrations for tissues and body fluids "Establishment of "normal values" for trace elements in human tissues and body fluids is a desirable task with an undesirable feature: what is normal may not be easy to answer!" [1]. In dealing with human tissues and body fluids for defining baseline elemental concentrations, it is practical to consider the usage of "reference ranges" of
11 TABLE 4
Concentrations of selected trace elements in some clinical samples from adult human subjects Element Liver
Whole blood
Blood serum Urine
Milk
Hair
~g kg- ~ ~
(/~g I - i)
(ug I - I)
~g/24 h)
(~g I - ~)
(/~g kg- ~)
As Cd
5- 15 < 1000-2 000
1-10? 0.17
10-30? 1-5?
0.25-3 1?
Co Cr Cu
30-150? 5 000-7 000 100-300? 100-200? 150 000-250 000 350-550 1 100-2 100 30 150 500 800 10 50? 250-400 400C0 60000
0.1-0.3? 0.1-0.2? 800-1100 c 1100-1.400d 20-50? ,Jf~70? 8 0 0 1260 < < I? 0.5-1.0 <1 < 0.5? 1 2? 75 120 800 1 100
0.5-2? 0.5-2? 10-60
F I Fe Pb Mn Hg Mo Ni Se Zn
2-20? 0.31-1.2 a 1-4h 5-10 < 5? 800-1 I00c 1000-1400 d 200-500 ¢ 40 60 425 0 0 0 500 000 90-150 8 12 2 20? 1 3? 5? 90. 130 6000 7000
500-1500? 1 0 0 200? 100 200? 10-20? 0.6-2? 5-20? 20 30? 2 8? 25 -50 400600
150-300 400-1000
0.2-0.7 1.0-15 250400
50-300? 300-800 15 000-25 000
10-26 40-80 350-600 15 3-6 1 3 1 4 10 20 15-25 1500 2000
4 0 0 1000 30 000-60 000 2000-20000 500--1500 5002000 50-200? 20-200? 500-1000 150000-250000
"Non-smokers. bSmokers. " Males. dFemales. ¢'Value uncertain.
values thus accounting for factors such as dietary habits and geochemical and other environmental influences. For biologically controlled elements, such ranges are likely to be narrow for subjects with no known health abnormalities. For non-essential elements, such a range can be broad, depending upon the level of exposure of the subjects in question. Taking the example of AI, it is not an esseatial element and, therefore, it is not subject to homeostatic control. Its entry into human systems is highly variable with atakes fluctuating from low to very high amounts, depending upon the types of food consumed and certain other factors. Indeed, under these conditions, one should be surprised to find a "normal" value of this element in blood serum. This is also true for a number of other trace elements that are not essential for biological processes. Results of a global literature survey evaluating results for 15 trace elements in selected samples of clinical significance are presented in Table 4, and data on scarcely determined elements such as B, Li, Sn and V may also be found in the literature iZ8,291.
Recommended dietary allowances (RDA) Recommended dietary allowances are defined as the levels of intake of essential nutrients that, on the basis of scientific knowledge, are judged by the
12
Food and Nutrition Board (ofthe United States National Academy of Sciences) to be adequate to meet the known nutrient needs of practically all healthy persons [30].A similar recommendation has also been published by the World Health Organization [31] for nutrients and by the Food and Agricultural Organization for permissible exposure levels for a number of trace elements among a variety of chemicals [32,33].These developments span several decades and represent the improvements in and contributions of analytical chemistry in the context of public health problems. The recommendations for selected constituents are presented as examples in Table 5.
TABLE
5
Dietary intakes of essential and toxic elements by adult human subjects. Data source references 30-33
Recommended dietary allowance m g day- ~
/~gday- ~
Ca, Mg, P, Fe, Se, Zn, I,
80(I 30(~ (females), 350 (males) 80(] 10--18 (age/sex) 0.055 (females), 0.070 (males) 15 150
Estimated safe and adequate intake m g day- ~
~gday -t
CI, K, Na, Cu, F, Mn, Co, Cr, Mo,
1700-5100 1875-5625 1100-3300 1.5-3 1.5-~4 2.0-5 0.12 (--3/~g Vitamin B12) 50-200 75-250
Maximum acceptpble body burden ~g kg- ~ body wt day- ~
As, Cu, Fe, Sn, Zn,
2 50-500 800 20000 300-1000
Provisional tolerable intake /~gkg- l body wt day- ~
Cd, Hg, Pb,
I-1.2 0.7 (total Hg) 0.5 (methyl Hg) 7.1
13 TABLE 6 Body pools and total body burden of chromium Tissue/organ
Approx. wt (kg)
Skeletal tissues Major organs (except lungs) Blood Muscle Skin (epidermis) Lung Hair Others adipose, dermis, hypodermis, etc.
10
Total body burden
Average conc. 0~g kg- ~)
Total content (/jg)
5-15
50-150
5-15 <1 5-10 50-200 10-200 250-1000
20--60 < 5.5 140-280 125-500 10-200 5- 20
19
2-5
38- 95
70
(approximate)
400-1300
4 5.5 28 2.5 1.0 9,02
Elemental composition of Reference Man A reference human body, namely The Standard Man based on a body weight of 70 kg, was formulated for use in radiological exposure and health studies by the International Commission on Radiological Protection [34]. Since its conception, several improvements have been introduced that have resulted in a revision of Standard Man to The Reference Man [35], and further improvements in this model are currently underway. These stages in the development of a reference individual are a tribute to the role of bioanalytical chemistry over a period of 40 years that has culminated in our ability to perform accurate determinations at very low concentrations. An example of this progress is reflected when data for difficult to determine elements such as Cr are reviewed. The recent estimation of total Cr indicates a range of 400-1300/~g, which is orders of magnitude lower than earlier estimations (Table 6). This is true for several other trace elements such as Li, Mo, Sn, V and W, among others. CONCLUSIONS
Obtaining analytically meaningful and biologically interpretable data for trace elements in biomedical investigations is a tedious task and requires dedicated efforts through a multidisciplinary approach by analytical chemists and health sciences investigators. The problems delineated in the preceding sections reemphasize the fact that good analytical measurements are not only crucial for success in future biological trace element research studies, but are mandatory when considering the analytical fimiings for diagnostic and related evaluations. In this context Mertz's statement "that av analytical chemist should be more than procurer of data and a life scientist more than their interpreter" [36] cannot be emphasized sufficiently.
14 THE FUTURE
Biochemical investigations involving chemical speciation to understand the mode of action of trace elements will undoubtedly dominate future BTER investigations. Concerted efforts are needed to exploit the prophylactic benefits of trace elements, and the relevance of this statement to health and nutritional problems of the developing countries cannot be emphasized sufficiently. Developing countries provide the best opportunity for carrying out biomineral-related investigations in the context of geochemical factors and availability of unusual clinical material needed to advance the clinical diagnostic implications of BTER. These investigations requir~ indepti~ vision, a high degree of scientificintegrity and genuine motivation to take ~he health benefits of science to a large cross-section of the global population, a reflection of the thoughts shared by some recent reports [37-44]. Biological trace element research is presented with a unique opportunity for interdisciplinary investigations on a massive scale, but I cannot imagine substantial progress in endeavours of this magnitude and importance without the increased participation of international scientificorganizations. REFERENCES I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
G.V. lyengar, Elemental Analysis of Biological Systems, Vol. I, C R C Press, Boca Raton, FL, 1989. A. Chatin, C.R. Acad. Sci.,(1950) 30-39; cited by E.J. Underwood, in Trace Elements in H u m a n and Animal Nutrition, 4th edn, Academic Press, N e w York, 1977, p. 1. E.J.Underwood, in W. Mertz and W.E. Cornatzer (Eds), Newer Trace Elements in Nutrition, Marcel Dekker, N e w York, 1971, p. 9. E.J. Underwood and J.F. Filmer, Aust. Vet. J., 11 (1935) 94. Keshan Disease Research Group, Chin. Med. J., 92 (1979) 477. A.S. Prasad, Zinc in Human Nutrition, C R C Press, Boca Raton, FL, 1979. J.C. Smith, in W. Mertz (Ed.), Trace Elements in H u m a n and Animal Nutrition, Academic Press, N e w York, 1987, p. 21. E.J. Underwood, Trace kLements in H u m a n and Animal Nutrition, 4th edn, Academic Press, New York, 1977. W. Mertz, Trace Elements in H u m a n and Animal Nutrition, Vols 1 and 2, Academic Press, New York, 1987. G.C. Cotzias, in D.D. Hemphill (Ed.), Proc. First Annu. Conf. Trace Substances in Environmental Health, University of Missouri, Columbia, MO, 1967, p. 5. H.L. Pardue and J. Woo, Chemtech, March, 1985, p. 183. American Chemical Society, A History of Analytical Chemistry, ACS, Washington DC, 1977. H.A. Laitinen, J. Res. Natl Bur. Stand., 93 (1988) 175. E.I.Hamilton, The Chemical Elements and Man, Charles Thomas, Springfield, IL. B. Sansoni, Instrumentelle Multielementanalyse, Verlag Chemie, Weinheim, 1985. R.M. Parr, Nutr. Res., Suppl. I (1985) 5. Y. Muramatsu and R.M. Parr, Report IAEA/RL/128, International Atomic Energy Agency, Vienna, 1985. M. lhnat, in H.A. Mckenzie and L.E. Smythe (Eds J,Quantitative Trace Analysis of Biological Materials, Elsevier, Amsterdam, 1988, p. 331. G.V. lyengar, K. Kasperek, L.E. Feinendegen, Y.X. W a n g and H. Weese, Sci. Total Environ., 24 (1982) 267.
15 20
K. Schwarz, in Proc. Nuclear Activation Techniques in the Life Sciences, IAEA, Vienna, 1972,
~I 22 23 24
t~.V.lyengar, J. Pathol., 134 (1983) 173. G.V. lyengar, Anal. Chem., 54 (1982) 554A. G.V. lyengar and D. Behne, J. Res. Natl Bur. Stand., 93 (1988) 326. P. Grandjean, in S.S. Brown and J. Savory (Eds), Chemical Toxicology and Clinical Chemistry of Metals, Academic Press, N e w York, 1983, p. 99. Editorial,N. Engl. J. Med., October 27, 1977. E. Sabb~f,~i. J. Edel and L. Goetz, Nutr. Res., Suppl. I (1985) 32. G. Toe!~ !n P. Braetter and P. Schramel (Eds), Trace Element Analytical Chemistry in Medicine and Biology, de Gruyter, Berlin, 1988, p. 1. G.V. lyengar and J.R.W. Woittiez, Clin. Chem., 34 (1988) 474. G.V. lyengar, B. Clarke, R.G. Downing and J.T. Tanner, in P. Braetter and P. Schramel (Eds), Trace Element Analytical Chemistry in Medicine and Biology, de Gruyter, Berlin, 1988, p. 267. National Research Council, Recommended Dietary Allowances, 10th edn, National Research Council, Washington, DC, 1989. W H O , Trace Elements in H u m a n Nutrition, Tech. Rep. Set. 230, World Health Organization, Geneva, 1973. F A O / W H O , Joint F A O / W H O Expert Committee on Food Additives 1972-1987, Reports 505, 631,683, 696 and 751, World Health Organization, Geneva. F A O / W H O , Joint F A O / W H O Food Standards Programme, Codex Alimentarius Commission 1984, C A C / V O L XVII, Contaminants, FAO, Rome, and W H O , Geneva. International Commission on Radiological Protection (ICRP), Report of Committee II on Permissible Dose For Internal Radiation, 1959, Health Piiys.(Suppl.),3 (!960),and ICRP Publ. 2, Pergamon Press, Oxford, 1960. International Commission on Radiological Protection (ICRP), Report of the Task Group on Reference Man, ICRP Publ. 23, Pergamon Press, Oxford. W. Mertz, Clin. Chem., 21 (1975) 468. R.M. Parr, IAEA, Bull., 25 (1983) 7. M.H.N. Goldon and B.E. Goldon, in C.F. Mills, I. Bremmer and J.K. Chesters (Eds), Trace Elements in M a n and Animals, TEMA-5, Commonwealth Agricultural Bureau, London, 1985, p. 933. G.V. lyengar, Sci. Today, India, February (1986) p. 32. C.F. Mills, M.H.N. Golden and G.V. lyengar, in M. Said (Ed.), Second Int. Conf. Elempnts in Health and Disease, Hamdard Foundation, Hamdard Press, Karachi, Pakistan, 1986, p. 7. B.S. Hetzel, J.T. Dunn and J.B. Stanbury, The Prevention and Control of Iodine Deficiency Disorders, Elsevier, Amsterdam, 1987. F A O / W H O Expert Consultation, Requirements of Vitamin A, Iron, Folate and :itamin B12, FAO, Rome, 1988. A.S. Prasad, Essential and Toxic Trace Elements in Human Health and Disease, Alan R. Liss, Inc., N e w York, 1988. M. Abdulla, H. Dashti, B. Sarkar, H. AI-Sayer and N. AI-Naqeeb, Metabolism of Minerals and Trace Elements in H u m a n Disease, Smith-Gordon Publishers, London. 1989.
25 26 27 28 29 30 31 32 33 34
35 36 37 38
39 40 41 42 43 44
1
F,lsevier Science Publishers B.V., Amsterdam
M I L E S T O N E S IN BIOLOGICAL TRACE E L E M E N T RESEARCH
G.V. IYENGAR
Building 235, B125, National Institute of Standards and Technology, US Department of Commerce, Gaithersburg, MD 20899, USA
ABSTRACT Over 100 years ago, macro.scale analytical techniques were used to discover the roles of special compounds (especially of metallic elements) in living organisms, and investigations were focussed on selected proteins and pigments suspected of containing percentage quantities of metals. In contrast, present-day analytical techniques are capable of detecting extremely small quantities and have become routine ultra-trace measurement tools to probe elemental interactions at cellular levels. The scientific achievements connecting these two boundaries are punctuated with an array of analytical developments; some highlighting the phenomenal advances in the measurement ~echnology and others reflecting the exceptional bioanalytical perception and the multidisciplinary outlook of trace element investigators. An account of the events that contributed to the overall progress in biological trace element research is the essence of this communication.
INTRODUCTION
Trace elements and myth! Almost 2300 years ago Hippocrates advocated the notion of "you are what you eat". Then there are stories of prescribing rusty water to restore vigor to pale looking persons, embedding iron nails in apples for several days before recommending those apples for consumption by pregnant women and persons who were weak, and using burned sponges (later proven to contain iodine) to cure goiter. Thus, the ancient Greeks were unwittingly exploring a whole new area of science. They have also confirmed that the healing power of trace quantities was an antique phenomenon!
Nature's signatures Tracking down historical notes reveals that nature's signatures, which were sometimes subtle and often poignant, were waiting to be linked to what are now termed as trace element disorders. These hints were specific in certain geographic locations. The use of seaweeds as a source of iodine, and linking iodine to the incidence of goitre during the 18th century and consolidating these findings in the 19th century, is an outstanding example [2]. Similarly, selenium toxicity linked to livestock through a condition known as alkali0048-9697/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
disease or blind stagger was recorded in certain parts of the United States [3]. Other examples are the bush-sickness of sheep and cattle in N e w Zealand and Australia leading to the discovery of the essentiality of cobalt [4]. These events were followed by more recent findings linking selenium deficiency to the endemic Keshan disease in China [5], and the growthretardation syndromes linked to zinc deficiency in the Middle East [6].Smith has recently reviewed the historical aspects of health effects of trace elements [7]. ESSENTIALITY OF T R A C E E L E M E N T S
"Essential chemical elements whether required in major, minor or minute quantities, have the same mission -- to sustain life" [1].
Documented evidence reveals, already in the 17th century using then-available analytical expertise, the determination of percentage quantities of metals such as copper (e.g., turacin and hemocyanin, copper-containing compounds in the feathers of certain birds and in the blood of snails, respectively), zinc (sycotypin, a zinc-containing pigment in Mollusca) and vanadium (a respiratory compound in sea squirts) to investigate the roles played by these elements in sustaining essential lifeprocesses [8,9].It has been known since the 17th century (with the identification of iron in erythrocytes) that all human beings need iron if they are to survive. Similarly, iodine has also been recognized as an essential trace element since 1850. Most of our knowledge of trace elements in human and animal health, however, was acquired in this century, particularly in the last 30 years. The availability of analtyical methods refined over time has provided undisputed TABLE I
Discovery of trace element requirements Iron Iodine Copper Manganese Zinc Cobalt Molybdenum Selenium Chromium Tin Vanadium Fluorine Silicon Nickel Arsenic
17th century 1850 1928 1931 1934 1935 1953 1957 1959 1970 1971 1971 1972 1976 1977
3
evidence recognizing the essentiality of several trace elements for living organisms following various investigations. Currently no fewer than 15 such elements are thought to be essential for humans, though for some of them (e.g., arsenic, vanadium and tin) the evidence is not direct but comes from animal experiments (Table 1). In this context, the role of analytical chemistry in sustaining the progress and promoting this branch of research cannot be emphasized sufficiently. This era is also remembered for the development of criterion for defining essentiality by Cotzias [10] and the perception of a working model for dose-response relationships, which have been elegantly summarized by Mertz [9]. These and other pioneering developments have culminated in several milestones in characterizing the growth of this dynamic branch of science, namely Biological Trace Element Research (BTER), as shown in Table 2. T H E CART.BEFORE-THE-HORSE PERIOD
"The ultimate purpose of an analytical result is to address the problem, not merely generating numbers for intercomparisons" [I]. The "cure.all" situation
In earlier investigations, especially during the fifties, sixties and early seventies, a great proportion of the analytical results obtained for various TABLE 2 Milestones in biological trace element research Essentiality of trace elements Cart before the horse period! The "cure-all" situation The "artifact" variations Developments in metrology Historical perspective Trace element determinations Multielement methods Analytical quality assurance Developments in bioanalytical concepts "Metal.free environment" concept for metabolic studies Recognition of biological trace element research as a multidisciplinary science Speciation and bioavailability concept Selected applications Reference elemental concentration values for tissues and body fluids Recommended dietary allowances Elemental composition of Reference Man
biological materials were mainly intended to demonstrate the powerful capabilities of the newly emerging methodologies, e.g. multielement techniques. Unfortunately, in the wake of analysts' enthusiasm to prove the effectiveness of these extraordinary technical achievements, multielement analyses assumed a sort of "cure all" posture and little or no consideration was given to incorporate the biologic basis for the investigations. To give an example, practically countless numbers of studies have been performed on hair, due to the fact that this specimen was the easiest to procure. One wonders if there was any recognized analytical methodology that had remained un-used in the determination of some element or other in this hirsute growth. As a natural consequence of this bandwagon research, a great majority of the findings have turned out to be of no use. Their only contribution has been to leave behind an e,xasperating trail of pointless publications. The position is no better for a number of other tissues and body fluids since, in most cases, the medical and other health sciences professionals who were involved in supplying the samples for analysis were unaware of the spurious analytical implications of uncol~trolled specimen collection, and the analysts who found access to the samples on their own did not have either the insight to assess the biological integrity or possess the exacting training required to deal with real-world biological collections.
The "artifact" variations The net result of all the factors alluded to in the preceding paragraph led to ascribing part or most of the changes observed with respect to whatever was considered as "control" to so-called "biological variations" arising from a host of factors such as age, sex, environment and diet.This is of course true fvr ~ome elements that did not pose insuperable analytical difficultiesand the results were reliable. Similarly, in some cases, the conclusions drawn were based on relative measurements to estimate the differences, and there were gounds to accept these findings as valid. In addition to these, there were instances where an investigation was carried out with meticulous care to understand the problems confronted. Hc,,wever, it was not always easy to identify such cases, therefore the handful of good results was drowned in the ocean of confusion. It is now generally concedsd that a great majority of the earlier experiments suffered from methodological inadequacies. It therefore follows that part of the blame for the current state of the scientificliteratureon elemental composition of biological systems rests on the practice of demonstrating the potentials of newly developed techniques to generate numbers in unplanned investigations, rather than seeking the solution to a credible problem and using the available expertise to solve it.Or simply another case of aligning the cart before the horse! In words not devoid of irony, Pardue and W o o [11]have stated this almost as an aphorism -- tc solve the problem, analysis must start with the problem.
5 D E V E L O P M E N T S IN M E T R O L O G Y
"The analyst is the most important component of any analytical system" [1]. Metrology is the science of measurements, devoted to all aspects of perfecting an analytical measurement to generate as accurate a result as possible. This branch of science has undergone tremendous changes since the days when laboratories contained simple equipment that was conveniently termed "apparatus". The progress in analytical instrumentation and the concept of a modern analytical laboratory has brought about a bewildering array of analytical instruments whose performance is not optimally explored without the use of yet another piece of laboratory equipment, namely the computer!
Historical perspective "A false balance is an abomination to the Lord, but a just weight is his delight" [12].
The words cited above go beyond science! At the center of the classical period is the analytical balance. The art and science of weighing was known in Egypt as early as 3000 B.C. The earliest written reference to an equal arm balance dates back to 1300 B.C. [12]. In his eloquent recollections of the history of analytical chemistry, and trace analysis in particular, Laitinen [13] presents five periods: (a) antiquity to the beginning of modern chemistry late in the 18th century; (b) late 18th century through the 19th century; (c) the period from 1900 to 1939; (d) the decade of the 1940s; and (e) the period from 1950 to the present. Laitinen characterizes each period by the following highlights: fire assay or cupellation as an earliest example of trace analysis; development of colorimetry, atomic emission spectroscopy, electroanalytical chemistry and the use of fluorescence; era of application of physical chemistry to analytical problems followed by discovery of pH, polarography and potentiometric titrations; the impact of the Second World War reflected in the development of nuclear chemistry, mass spectrometry and UV spectroscopy; and an array of modern methods as discussed below.
Trace element determinations Several analytical techniques, namely atomic absorption (flame and flameless), atomic emission (direct current and inductively coupled plasma), chemical and electroanalytical methods, gas and liquid chromatography, mass spectrometry in different modes, nuclear activation techniques and X-ray fluorescence, which offer sufficiently low detection limits for a variety of biomatrices, are now available. Yet, very few laboratories in the world carry out reliable trace element determinations, while a Idrge proportion of the laboratories working with biomaterials find it difficultto achieve a consistent
6
capability to maintain even 10-20% accuracy and precision. This is an indication that high sensitivity alone is not the solution to the problem of accuracy and precisiom and that unawareness of various interferences (e.g., matrix-related problems), flaws in sample and standard preparation and inadequate calibration procedures are evident.
Multianalyte determinations Multielement techniques are useful in obtaining simultaneous (even if partly qualitative at times) elemental composition profiles of a given specimen and have provided a big boost to our quest of understanding the elemental concentration profiles of biological systems [1,14]. Many interesting findings have surfaced unexpectedly as a result of these studies. From an analytical point of view, the non-destructive modes offer possibilities for generating simultaneous data for several elements for comparison, thus acting as internal quality control agents so that unusual situations involving any specific element can be evaluated. Moreover, in a carefully designed study, multielement assays can provide very useful information on a large number of elements at relatively low costs [15]. On the other hand, single element assays have their own role to play. In some cases, some elements have to be determined alone due to severe analytical problems. Clinical, environmental and nutritional laboratories involved with specific elements frequently need single element assays. Therefore, in a comprehensive laboratory, a combination of both single and multielement capability is essential for effective functioning.
Analytical quality assurance "Frequent analysis of appropriate certified reference materials is a key component of any well planned quality control program" [1]. Frequent analysis of reference standards is the key for overcoming procedural inconsistencies in establishing quality control in any analytical laboratory. In this context, it should be recognized that the standard set for the quality of an analytical result is an important factor and is of course dependent upon the end use of the results. Thus, for example, if the aim is restricted merely to scan different biological matrices as a provisional step to establish the relative levels of elemental concentration profiles in them, it is obvious that a reasonable degree of quality standard is sufficient; whereas for meeting the requirements of a typical biomedical trace element research laboratory aiming to use the results for medical diagnostic purposes and legal regulatory processes, depending upon the problem, results with 5-10% total analytical uncertainty may be acceptable. On the other hand, an exceptionally high quality standard (< 1% total analytical uncertainty) is mandatory in some cases, e.g. certification of reference materials. If the tolerance limits are set narrower than the investigation really requires or are not feasible under
practical laboratory conditions, it can cause unnecessary expense and loss of time [16].
Reference materials (RMs): Although there are quite a few biological RMs presently available [1,17,18] they do not fulfill all the current analytical requirements. For a group of elements (e.g., A1, F, Sb, Si, S:n and V) not many RMs are available, and in some cases the number is very small or zero. A major problem is that trace element concentrations are generally subject to large errors, and it is imperative to match the sample matrix with an appropriate RM, so that the level of a particular analyte retains its proportion in relation to the overall matrix composition of the sample. Only then can the sources of systematic errors arising from matrix effects be identified To quote one example, using instrumental neutron activation analysis, Hg and Se could be determined in human milk even in small size samples, but not in cow milk, although the levels of Hg and Se were comparable in these two matrices; this was due to matrix effects from the predominant presence of P (ratio 1:10) in cow milk [19]. D E V E L O P M E N T S IN BIOANALYTICAL CONCEPTS
"A prudent combination of analytical awareness and biological insight is crucial for success in biomedical trace element research studies" [1].
Controlled-environment concept for metabolic studies The realization that several trace elements are required at very minute concentrations and that under routine laboratory conditions the ambient levels of the metals could mask the effects of an investigation, led to the development of the controlled-environment approach or the trace element-freeisolater system. This is basically an all-plastic assembly, with filtered air, free of dust particles down to a fraction of a micrometer. Further, continuous monitoring of all the items in the system is maintained for elements of interest. Under these conditions, the diet is the main source of trace element supply, and by using a chemically well-defined diet, reliable BTER investigations can be carried out. Thus, the introduction of high purity diets on one hand, and the ability to exercise strict analytical quality control on the other, have led to rapid advances in BTER investigations [20].
B T E R - - a multidisciplinary science "The lack of a multidisciplinary approach has been the Achilles heel of biological trace element research" [1]. Biological trace element research (BTER) is a multidisciplinary science. Therefore, it is necessary for researchers in this field to strive for a reasonable degree of biological insight and analytical awareness of the problems involved.
8
This in turn will enable them to design meaningful B T E R investigations. Quite frequently, the complexities involved necessitate the use of a variety of scientific talents and a combination of several analytical techniques. Some of the special difficultiesare seen, for example, in providing a "total" quality control in the overall context of an investigation. These include experimental design, collecti:,nof biologically and analytically "valid" samples, understanding the implications of presampling factors [21,22] in the context of overall accuracy of an analytical result as reflected in Fig. 1, and finally the ability to carry out accurate analytical measurements on those specimens, data evaluation and meaningful interpretation of the findings [23]. Therefore, it follows that team work is an important component in any B T E R investigation. A RELIABLE CONCLUSION DEPENDS ON TH]RQUALITY OF THE ANALYTICAL RESULT
DataHandlingand ~ Inteipmtalion ~ Errors ~ ~
-
-
~
m --mIned~luetely e '
mllChed
conlrols
InePl~ropr*lte Stilif*shcsl treetment
t
,
o
n
K e y p u n c ~ r r o r ~ n 0 clertcel mlstlkes -- Recordmg end ¢ilculehon
Analytical
Melsuremen! Of the lnllylicII el|inllS CholcO of imDrol~lr lnilyhcII Ilchnlque
[l'lrors
-- Preplrefory fKhmques -- Specimen colllchon
Preumplln9 Factors
-........ .¢,,-,,onW"-4FJFX~ILI su=,,.,., Co.,,,on. 4 F H A g E I Cr0hcslly Smell Semples r j r l.lemolys~s
~
~ . , c . . . o . aIll'q,%,°,%"~ ~
lira ~
~
-
I" ~ " "
Long.term Phystolo~tcel Influences Short,term PhystoloQtcil Influences
Fig, 1. Elemental analysis of biological systems [22]. [Reprinted with permission from Analytical Chemistry, 54 (1982) 555A. Copyright American Chemical Society.]
9
Although a large body of data exists for most trace elements in biological media, in m a n y cases they are of limited use because cf inherent inaccuracies [1].This has generated a lot of skepticism among trace element researchers. Primarily, the variations in analytical findings that are stillprevalent can be linked to several pitfallsthat have failedto receive adequate attention by trace element researchers. Therefore, first of all, there is a need to reestablish credibility in trace element analytical research by generating well controlled quality data to dispel fears in the minds of many users. In this context, it may be emphasized that development of a reliable data base for the elemental composition of biological systems is a multidisciplinary task involving preparation of well defined protocols, adequately tested methods for sampling and handling biomaterials, and use of appropriate analytical procedures. Above all, itis a team effortrequiring a prudent combination of analytical awareness and biological insight, which are crucial parameters for any biomedical trace element research study. The significance of the above comments are reflected in Table 3, which illustratesdifferent perspectives of lead determinations in tissu,.~:and fluidsas seen in the scientific literature. Case I illustrates the worst situation of choosing a questionable or unsuitable biological specimen which, produces unreliable results. Case 2 is the use of a sound specimen yet it is prone to analytical pitfalls,especially at very low concentrations. Case 3 is a typical situation where extreme care is exerted to solve a problem of wrong biological perception. Smokers are usually consumers of alcohol too, and the elevated blood lead was traceable to wine consumption [24].Obviously, based on these findings ifsmoking was banned to control blood lead levels,itwould have been a futile move. Case 4 is an ideal combination of biological relevance and analytical expertise, since significant differences have been established in several studies [1]. TABLE 3 Analytical scientists versus biosciences researchers. Some examples of different perspectives! Example: Determination of lead in tissues and body-fluids Case 1: Analytically questionable and biologically unsound; Earlier studies on hair as a monitoring specimen, and Pb in blood plasma Case 2: Analytically questionable and biologically sound; Determination of Pb in liver Case 3: Analytically sound and biologically questionable; Increase of Pb in whole blood related to smoking Case 4: Analytically reliable and biologically sound; Pd in whole blood (and also Cd in blood of smokers and non-smokers)
10
Trace element specification and bioavailability "Human milk is for the human infant; cow's milk is for the calf" [25]. The late Paul Georgy stated this point almost derisively 60 years ago and persisted in his opinion when others chuckled! In my opinion, this sentence powerfully symbolizes the concept of relevance of an elemental species and its bioavailability. Conventionally conducted BTER studies generate data on the total amount of trace elements. For example, in dietary intake studies it is common to carry out elemental analysis and use the results to draw comparisons and conclusions in the context of recommended dietary allowances. It is now being recognized that the biologically available fraction of a trace element differs among different foods and, therefore: the bioavailability of various elements from single or mixed foods should be determined for accurate estimation of the intake from diets. However, metal speciation in complex biological and dietary media is a difficult task due to detection limit considerations and the danger of inducing changes in the speciation states during analysis. Many biochemical methods for the isolation of metallocomplexes such as ion-exchange chromatography, affinity chromatography, immunoprecipitation, electrophoresis and isoelectricfocussing are in use, but are faced with problems of loss and contamination of the metal and its species. Radioactive labeling of organo-arsenic compounds has been described for animal studies [26]. A two-stage in vitro system to simulate gastric and intestinal digestion of food in combination with inductively coupled plasma-mass spectrometry has been successfully used in recent investigations. Several techniques based on gas chromatography and high pressure liquid chromatography and anodic stripping voltametry have been applied to speciation chemistry. Many analytical methods are useful for speciation chemistry studies provided that their limitations are recognized [27]. Concerning bioavailability, the use of stable isotopes in mineral nutrition research is well established. Stable isotopes as isotopic tracers have no radiation risk and are therefore widely accepted. The use of various versions of mass spectrometry and neutron activation analysis for in vitro specimens following stable isotope application has resulted in considerable progress in mineral metabolism research. SELECTED APPLICATIONS
Reference elemental concentrations for tissues and body fluids "Establishment of "normal values" for trace elements in human tissues and body fluids is a desirable task with an undesirable feature: what is normal may not be easy to answer!" [1]. In dealing with human tissues and body fluids for defining baseline elemental concentrations, it is practical to consider the usage of "reference ranges" of
11 TABLE 4
Concentrations of selected trace elements in some clinical samples from adult human subjects Element Liver
Whole blood
Blood serum Urine
Milk
Hair
~g kg- ~ ~
(/~g I - i)
(ug I - I)
~g/24 h)
(~g I - ~)
(/~g kg- ~)
As Cd
5- 15 < 1000-2 000
1-10? 0.17
10-30? 1-5?
0.25-3 1?
Co Cr Cu
30-150? 5 000-7 000 100-300? 100-200? 150 000-250 000 350-550 1 100-2 100 30 150 500 800 10 50? 250-400 400C0 60000
0.1-0.3? 0.1-0.2? 800-1100 c 1100-1.400d 20-50? ,Jf~70? 8 0 0 1260 < < I? 0.5-1.0 <1 < 0.5? 1 2? 75 120 800 1 100
0.5-2? 0.5-2? 10-60
F I Fe Pb Mn Hg Mo Ni Se Zn
2-20? 0.31-1.2 a 1-4h 5-10 < 5? 800-1 I00c 1000-1400 d 200-500 ¢ 40 60 425 0 0 0 500 000 90-150 8 12 2 20? 1 3? 5? 90. 130 6000 7000
500-1500? 1 0 0 200? 100 200? 10-20? 0.6-2? 5-20? 20 30? 2 8? 25 -50 400600
150-300 400-1000
0.2-0.7 1.0-15 250400
50-300? 300-800 15 000-25 000
10-26 40-80 350-600 15 3-6 1 3 1 4 10 20 15-25 1500 2000
4 0 0 1000 30 000-60 000 2000-20000 500--1500 5002000 50-200? 20-200? 500-1000 150000-250000
"Non-smokers. bSmokers. " Males. dFemales. ¢'Value uncertain.
values thus accounting for factors such as dietary habits and geochemical and other environmental influences. For biologically controlled elements, such ranges are likely to be narrow for subjects with no known health abnormalities. For non-essential elements, such a range can be broad, depending upon the level of exposure of the subjects in question. Taking the example of AI, it is not an esseatial element and, therefore, it is not subject to homeostatic control. Its entry into human systems is highly variable with atakes fluctuating from low to very high amounts, depending upon the types of food consumed and certain other factors. Indeed, under these conditions, one should be surprised to find a "normal" value of this element in blood serum. This is also true for a number of other trace elements that are not essential for biological processes. Results of a global literature survey evaluating results for 15 trace elements in selected samples of clinical significance are presented in Table 4, and data on scarcely determined elements such as B, Li, Sn and V may also be found in the literature iZ8,291.
Recommended dietary allowances (RDA) Recommended dietary allowances are defined as the levels of intake of essential nutrients that, on the basis of scientific knowledge, are judged by the
12
Food and Nutrition Board (ofthe United States National Academy of Sciences) to be adequate to meet the known nutrient needs of practically all healthy persons [30].A similar recommendation has also been published by the World Health Organization [31] for nutrients and by the Food and Agricultural Organization for permissible exposure levels for a number of trace elements among a variety of chemicals [32,33].These developments span several decades and represent the improvements in and contributions of analytical chemistry in the context of public health problems. The recommendations for selected constituents are presented as examples in Table 5.
TABLE
5
Dietary intakes of essential and toxic elements by adult human subjects. Data source references 30-33
Recommended dietary allowance m g day- ~
/~gday- ~
Ca, Mg, P, Fe, Se, Zn, I,
80(I 30(~ (females), 350 (males) 80(] 10--18 (age/sex) 0.055 (females), 0.070 (males) 15 150
Estimated safe and adequate intake m g day- ~
~gday -t
CI, K, Na, Cu, F, Mn, Co, Cr, Mo,
1700-5100 1875-5625 1100-3300 1.5-3 1.5-~4 2.0-5 0.12 (--3/~g Vitamin B12) 50-200 75-250
Maximum acceptpble body burden ~g kg- ~ body wt day- ~
As, Cu, Fe, Sn, Zn,
2 50-500 800 20000 300-1000
Provisional tolerable intake /~gkg- l body wt day- ~
Cd, Hg, Pb,
I-1.2 0.7 (total Hg) 0.5 (methyl Hg) 7.1
13 TABLE 6 Body pools and total body burden of chromium Tissue/organ
Approx. wt (kg)
Skeletal tissues Major organs (except lungs) Blood Muscle Skin (epidermis) Lung Hair Others adipose, dermis, hypodermis, etc.
10
Total body burden
Average conc. 0~g kg- ~)
Total content (/jg)
5-15
50-150
5-15 <1 5-10 50-200 10-200 250-1000
20--60 < 5.5 140-280 125-500 10-200 5- 20
19
2-5
38- 95
70
(approximate)
400-1300
4 5.5 28 2.5 1.0 9,02
Elemental composition of Reference Man A reference human body, namely The Standard Man based on a body weight of 70 kg, was formulated for use in radiological exposure and health studies by the International Commission on Radiological Protection [34]. Since its conception, several improvements have been introduced that have resulted in a revision of Standard Man to The Reference Man [35], and further improvements in this model are currently underway. These stages in the development of a reference individual are a tribute to the role of bioanalytical chemistry over a period of 40 years that has culminated in our ability to perform accurate determinations at very low concentrations. An example of this progress is reflected when data for difficult to determine elements such as Cr are reviewed. The recent estimation of total Cr indicates a range of 400-1300/~g, which is orders of magnitude lower than earlier estimations (Table 6). This is true for several other trace elements such as Li, Mo, Sn, V and W, among others. CONCLUSIONS
Obtaining analytically meaningful and biologically interpretable data for trace elements in biomedical investigations is a tedious task and requires dedicated efforts through a multidisciplinary approach by analytical chemists and health sciences investigators. The problems delineated in the preceding sections reemphasize the fact that good analytical measurements are not only crucial for success in future biological trace element research studies, but are mandatory when considering the analytical fimiings for diagnostic and related evaluations. In this context Mertz's statement "that av analytical chemist should be more than procurer of data and a life scientist more than their interpreter" [36] cannot be emphasized sufficiently.
14 THE FUTURE
Biochemical investigations involving chemical speciation to understand the mode of action of trace elements will undoubtedly dominate future BTER investigations. Concerted efforts are needed to exploit the prophylactic benefits of trace elements, and the relevance of this statement to health and nutritional problems of the developing countries cannot be emphasized sufficiently. Developing countries provide the best opportunity for carrying out biomineral-related investigations in the context of geochemical factors and availability of unusual clinical material needed to advance the clinical diagnostic implications of BTER. These investigations requir~ indepti~ vision, a high degree of scientificintegrity and genuine motivation to take ~he health benefits of science to a large cross-section of the global population, a reflection of the thoughts shared by some recent reports [37-44]. Biological trace element research is presented with a unique opportunity for interdisciplinary investigations on a massive scale, but I cannot imagine substantial progress in endeavours of this magnitude and importance without the increased participation of international scientificorganizations. REFERENCES I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
G.V. lyengar, Elemental Analysis of Biological Systems, Vol. I, C R C Press, Boca Raton, FL, 1989. A. Chatin, C.R. Acad. Sci.,(1950) 30-39; cited by E.J. Underwood, in Trace Elements in H u m a n and Animal Nutrition, 4th edn, Academic Press, N e w York, 1977, p. 1. E.J.Underwood, in W. Mertz and W.E. Cornatzer (Eds), Newer Trace Elements in Nutrition, Marcel Dekker, N e w York, 1971, p. 9. E.J. Underwood and J.F. Filmer, Aust. Vet. J., 11 (1935) 94. Keshan Disease Research Group, Chin. Med. J., 92 (1979) 477. A.S. Prasad, Zinc in Human Nutrition, C R C Press, Boca Raton, FL, 1979. J.C. Smith, in W. Mertz (Ed.), Trace Elements in H u m a n and Animal Nutrition, Academic Press, N e w York, 1987, p. 21. E.J. Underwood, Trace kLements in H u m a n and Animal Nutrition, 4th edn, Academic Press, New York, 1977. W. Mertz, Trace Elements in H u m a n and Animal Nutrition, Vols 1 and 2, Academic Press, New York, 1987. G.C. Cotzias, in D.D. Hemphill (Ed.), Proc. First Annu. Conf. Trace Substances in Environmental Health, University of Missouri, Columbia, MO, 1967, p. 5. H.L. Pardue and J. Woo, Chemtech, March, 1985, p. 183. American Chemical Society, A History of Analytical Chemistry, ACS, Washington DC, 1977. H.A. Laitinen, J. Res. Natl Bur. Stand., 93 (1988) 175. E.I.Hamilton, The Chemical Elements and Man, Charles Thomas, Springfield, IL. B. Sansoni, Instrumentelle Multielementanalyse, Verlag Chemie, Weinheim, 1985. R.M. Parr, Nutr. Res., Suppl. I (1985) 5. Y. Muramatsu and R.M. Parr, Report IAEA/RL/128, International Atomic Energy Agency, Vienna, 1985. M. lhnat, in H.A. Mckenzie and L.E. Smythe (Eds J,Quantitative Trace Analysis of Biological Materials, Elsevier, Amsterdam, 1988, p. 331. G.V. lyengar, K. Kasperek, L.E. Feinendegen, Y.X. W a n g and H. Weese, Sci. Total Environ., 24 (1982) 267.
15 20
K. Schwarz, in Proc. Nuclear Activation Techniques in the Life Sciences, IAEA, Vienna, 1972,
~I 22 23 24
t~.V.lyengar, J. Pathol., 134 (1983) 173. G.V. lyengar, Anal. Chem., 54 (1982) 554A. G.V. lyengar and D. Behne, J. Res. Natl Bur. Stand., 93 (1988) 326. P. Grandjean, in S.S. Brown and J. Savory (Eds), Chemical Toxicology and Clinical Chemistry of Metals, Academic Press, N e w York, 1983, p. 99. Editorial,N. Engl. J. Med., October 27, 1977. E. Sabb~f,~i. J. Edel and L. Goetz, Nutr. Res., Suppl. I (1985) 32. G. Toe!~ !n P. Braetter and P. Schramel (Eds), Trace Element Analytical Chemistry in Medicine and Biology, de Gruyter, Berlin, 1988, p. 1. G.V. lyengar and J.R.W. Woittiez, Clin. Chem., 34 (1988) 474. G.V. lyengar, B. Clarke, R.G. Downing and J.T. Tanner, in P. Braetter and P. Schramel (Eds), Trace Element Analytical Chemistry in Medicine and Biology, de Gruyter, Berlin, 1988, p. 267. National Research Council, Recommended Dietary Allowances, 10th edn, National Research Council, Washington, DC, 1989. W H O , Trace Elements in H u m a n Nutrition, Tech. Rep. Set. 230, World Health Organization, Geneva, 1973. F A O / W H O , Joint F A O / W H O Expert Committee on Food Additives 1972-1987, Reports 505, 631,683, 696 and 751, World Health Organization, Geneva. F A O / W H O , Joint F A O / W H O Food Standards Programme, Codex Alimentarius Commission 1984, C A C / V O L XVII, Contaminants, FAO, Rome, and W H O , Geneva. International Commission on Radiological Protection (ICRP), Report of Committee II on Permissible Dose For Internal Radiation, 1959, Health Piiys.(Suppl.),3 (!960),and ICRP Publ. 2, Pergamon Press, Oxford, 1960. International Commission on Radiological Protection (ICRP), Report of the Task Group on Reference Man, ICRP Publ. 23, Pergamon Press, Oxford. W. Mertz, Clin. Chem., 21 (1975) 468. R.M. Parr, IAEA, Bull., 25 (1983) 7. M.H.N. Goldon and B.E. Goldon, in C.F. Mills, I. Bremmer and J.K. Chesters (Eds), Trace Elements in M a n and Animals, TEMA-5, Commonwealth Agricultural Bureau, London, 1985, p. 933. G.V. lyengar, Sci. Today, India, February (1986) p. 32. C.F. Mills, M.H.N. Golden and G.V. lyengar, in M. Said (Ed.), Second Int. Conf. Elempnts in Health and Disease, Hamdard Foundation, Hamdard Press, Karachi, Pakistan, 1986, p. 7. B.S. Hetzel, J.T. Dunn and J.B. Stanbury, The Prevention and Control of Iodine Deficiency Disorders, Elsevier, Amsterdam, 1987. F A O / W H O Expert Consultation, Requirements of Vitamin A, Iron, Folate and :itamin B12, FAO, Rome, 1988. A.S. Prasad, Essential and Toxic Trace Elements in Human Health and Disease, Alan R. Liss, Inc., N e w York, 1988. M. Abdulla, H. Dashti, B. Sarkar, H. AI-Sayer and N. AI-Naqeeb, Metabolism of Minerals and Trace Elements in H u m a n Disease, Smith-Gordon Publishers, London. 1989.
25 26 27 28 29 30 31 32 33 34
35 36 37 38
39 40 41 42 43 44