Biological Trace Element Research - ACS Publications - American

Chapter 1

The Need for Multidisciplinary Approaches in Biological Trace Element Research G. V. Iyengar

Downloaded by 80.82.77.83 on May 30, 2018 | https://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0445.ch001

National Institute of Standards and Technology, Reactor Building 235, B125, Gaithersburg, MD 20899

Biological Trace Element Research (BTER) is a multidisciplinary science and directing research in this field requires a combination of both biological insight and analytical awareness. Quite frequently, the complexities involved here necessitate the use of a variety of scientific talents and a combination of several analytical techniques. For example, some of the special d i f f i c u l t i e s can be visualized in meeting the requirements of a "total" quality control in the overall context of a BTER investigation. These include experimental design, collection of b i o l o g i c a l l y and a n a l y t i c a l l y "valid" samples, the a b i l i t y to carry out accurate analytical measurements on those specimens, and a proper evaluation of the analytical data (including data interpretation) . Therefore, i t follows that a team approach ensuring the required m u l t i d i s c i p l i n a r y interactions is a crucial component in any BTER endeavor. "The lack of a multidisciplinary approach has been the Achilles heel of biological trace element research" (1). Although a large body of data exists for most trace elements in biological media, in many cases they are of limited use because of inherent inaccuracies (1,2). This has generated a lot of skepticism among biological trace element researchers. Primarily, the v a r i a t i o n s in analytical findings that s t i l l are prevalent can be linked to several p i t f a l l s that have failed to receive adequate attention by trace element investigators. Therefore, f i r s t O097-6156/91/0445-O001$06.00/0 © 1991 American Chemical Society Subramanian et al.; Biological Trace Element Research ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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BIOLOGICAL TRACE ELEMENT RESEARCH

of a l l , t h e r e i s a need t o r e e s t a b l i s h c r e d i b i l i t y i n t r a c e element a n a l y t i c a l r e s e a r c h by g e n e r a t i n g w e l l c o n t r o l l e d q u a l i t y d a t a t o d i s p e l f e a r s i n t h e m i n d s o f many u s e r s . I n t h i s c o n t e x t , i t may b e e m p h a s i z e d t h a t d e v e l o p m e n t o f a reliable data base f o r the elemental composition of b i o l o g i c a l systems i s a m u l t i d i s c i p l i n a r y t a s k i n v o l v i n g an understanding of the biological basis o f t h e problem, preparation of w e l l defined protocols, adequately tested methods f o r sampling and h a n d l i n g b i o m a t e r i a l s and, use o f appropriate a n a l y t i c a l procedures. I n o t h e r words, i t i s a team e f f o r t r e q u i r i n g cumulative knowledge from s e v e r a l d i s c i p l i n e s (3) . In e a r l i e r i n v e s t i g a t i o n s , e s p e c i a l l y u n t i l about t h e early seventies, a great proportion of the analytical results obtained f o r various biological materials were mainly intended t o demonstrate t h e powerful c a p a b i l i t i e s o f the newly emerging methodologies, e.g., multielement techniques. Unfortunately, i n t h e wake o f analyst's enthusiasm to prove the effectiveness of these extraordinary t e c h n i c a l achievements, t h i s "black box" a t t i t u d e of using multielement a n a l y s e s assumed a s o r t o f " c u r e a l l " p o s t u r e a n d l i t t l e o r n o c o n s i d e r a t i o n was g i v e n to incorporate the biologic basis f o r the investigations. To g i v e a n e x a m p l e , p r a c t i c a l l y c o u n t l e s s n u m b e r o f s t u d i e s have been p e r f o r m e d on h a i r due t o t h e f a c t t h a t this s p e c i m e n was t h e e a s i e s t t o p r o c u r e , a n d a g r e a t m a j o r i t y of these i n v e s t i g a t i o n s has merely r e s u l t e d i n a t r a i l o f p o i n t l e s s p u b l i c a t i o n s . The p o s i t i o n i s no b e t t e r f o r a number o f o t h e r t i s s u e s and body f l u i d s , s i n c e i n most cases, t h e medical and other h e a l t h sciences p r o f e s s i o n a l s who w e r e i n v o l v e d i n s u p p l y i n g t h e s a m p l e s f o r a n a l y s i s , were unaware o f t h e s p u r i o u s a n a l y t i c a l i m p l i c a t i o n s o f u n c o n t r o l l e d specimen c o l l e c t i o n and, t h e a n a l y s t s f o r t h e i r p a r t who f o u n d a c c e s s t o t h e s a m p l e s o n t h e i r o w n , d i d n o t have e i t h e r t h e i n s i g h t t o assess t h e b i o l o g i c a l i n t e g r i t y or possess the exacting t r a i n i n g required t o deal with r e a l world b i o l o g i c a l c o l l e c t i o n s . I t i s n o t my i n t e n t i o n t o s u m m a r i l y d i s q u a l i f y a l l t h e e a r l i e r BTER i n v e s t i g a t i o n s . F o r e x a m p l e , r e l i a b l e r e s u l t s have been reported f o r elements that d i d not pose insuperable analytical d i f f i c u l t i e s . Similarly, i n some cases, the conclusions drawn were based on relative measurements t o e s t i m a t e t h e d i f f e r e n c e s , and t h e r e were grounds t o accept these f i n d i n g s as v a l i d . I n a d d i t i o n t o these, t h e r e w e r e i n s t a n c e s w h e r e a n i n v e s t i g a t i o n was c a r r i e d out with meticulous care t o understand t h e problems confronted. H o w e v e r , i t was n o t a l w a y s e a s y t o i d e n t i f y s u c h c a s e s so t h a t t h e h a n d f u l o f good r e s u l t s were a l s o drowned i n t h e ocean o f c o n f u s i o n . I t i s now g e n e r a l l y conceded that a great majority of the earlier BTER experiments s u f f e r e d from methodological inadequacies.

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

1.

IYENGAR

The Need for Multidisciplinary Approaches

Developments

in Bioanalytical

3

Concepts


"A prudent combination of analytical awareness and b i o l o g i c a l insight i s c r u c i a l f o r success i n biomedical t r a c e element research s t u d i e s " (1). The d e v e l o p m e n t s w h i c h c o n t r i b u t e d t o t h e e m e r g e n c e o f a viable bioanalytical c o n c e p t c a p a b l e o f a d d r e s s i n g BTER p r o b l e m s a r e t h e r e s u l t o f s e v e r a l a s p e c t s such as a) t e c h n o l o g i c a l developments i n metrology, b) analytical q u a l i t y assurance and q u a l i t y s t a n d a r d , c) controlledenvironment c o n c e p t f o r m e t a b o l i c s t u d i e s , d) recognition of BTER a s a multidisciplinary s c i e n c e , and e) trace element s p e c i a t i o n and b i o a v a i l a b i l i t y . These a s p e c t s w i l l be discussed b r i e f l y i n the f o l l o w i n g paragraphs. T e c h n o l o g i c a l Developments i n Metrology. Metrology i s the science of measurements, devoted to a l l aspects of perfecting an analytical measurement to generate as a c c u r a t e a r e s u l t as p o s s i b l e . T h i s b r a n c h o f s c i e n c e has undergone tremendous changes since the d a y s when the laboratories contained simple equipment that were c o n v e n i e n t l y t e r m e d " a p p a r a t u s " . The p r o g r e s s i n a n a l y t i c a l i n s t r u m e n t a t i o n and t h e c o n c e p t of a modern analytical laboratory has brought about a bewildering array of a n a l y t i c a l i n s t r u m e n t s whose p e r f o r m a n c e i s not o p t i m a l l y e x p l o r e d w i t h o u t the use o f y e t another l a b o r a t o r y t o o l namely, computer in analytical chemistry! Several a n a l y t i c a l t e c h n i q u e s namely a t o m i c a b s o r p t i o n ( f l a m e and f l a m e l e s s ) , atomic e m i s s i o n ( d i r e c t c u r r e n t and i n d u c t i v e l y c o u p l e d p l a s m a ) , c h e m i c a l and e l e c t r o a n a l y t i c a l methods, gas and liquid chromatography, mass spectrometry in d i f f e r e n t modes, n u c l e a r a c t i v a t i o n t e c h n i q u e s and X-ray f l u o r e s c e n c e , which o f f e r s u f f i c i e n t l y low d e t e c t i o n l i m i t s applicable to a wide v a r i e t y of b i o m a t r i c e s . Furthermore, many of these methodologies possess simultaneous m u l t i e l e m e n t c a p a b i l i t i e s and a g r a d u a l u n d e r s t a n d i n g o f t h e a n a l y t i c a l p r o b l e m s w i t h b i o m a t r i c e s has significantly revolutionized BTER efforts. Thus, many interesting f i n d i n g s have s u r f a c e d u n e x p e c t e d l y as a r e s u l t o f t h e s e studies. F r o m an analytical point of view, the nondestructive modes o f f e r possibilities for generating simultaneous data f o r s e v e r a l elements f o r comparison, thus a c t i n g as i n t e r n a l q u a l i t y c o n t r o l a g e n t s so t h a t u n u s u a l s i t u a t i o n s i n v o l v i n g any s p e c i f i c e l e m e n t c a n be e v a l u a t e d . Moreover, in a carefully designed study, multielement assays can p r o v i d e v e r y u s e f u l i n f o r m a t i o n on a large number o f e l e m e n t s a t r e l a t i v e l y low c o s t s . H o w e v e r , e v e n a s we h a v e e n t e r e d t h e e r a o f t h e 9 0 s , I must s a d l y p o i n t o u t t h a t v e r y few l a b o r a t o r i e s i n t h e w o r l d c a r r y out r e l i a b l e t r a c e element d e t e r m i n a t i o n s , w h i l e a large p r o p o r t i o n of the laboratories working with biomaterials find i t difficult to achieve a consistent


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capability t o maintain even a 10-20 % a c c u r a c y and precision. This i s an i n d i c a t i o n t h a t high detection c a p a b i l i t y and s e n s i t i v i t y alone a r e not t h e s o l u t i o n t o the problem o f accuracy and p r e c i s i o n , and t h a t unawareness of v a r i o u s i n t e r f e r e n c e s (e.g., m a t r i x r e l a t e d problems), flaws i n sample and standard preparation and inadequate c a l i b r a t i o n procedures are evident.


Analytical

Quality

Assurance

and

Quality

Standard.

Frequent analysis of reference standards i s the key f o r overcoming procedural inconsistencies i n establishing quality control i n any a n a l y t i c a l laboratory. Major a d v a n c e s h a v e b e e n made i n t h e a r e a o f d e v e l o p m e n t o f reference materials with diverse matrix properties (1,48) . I n t h i s c o n t e x t , i t should be r e c o g n i z e d that the s t a n d a r d s s e t f o r t h e q u a l i t y o f an a n a l y t i c a l r e s u l t i s an i m p o r t a n t f a c t o r and i s o f c o u r s e dependent upon t h e end use o f t h e r e s u l t s . Thus, f o r example, i f t h e a i m i s r e s t r i c t e d merely t o scan d i f f e r e n t b i o l o g i c a l matrices as a p r o v i s i o n a l step t o e s t a b l i s h the r e l a t i v e l e v e l s of elemental concentration p r o f i l e s i n them, i t i s o b v i o u s that a reasonable degree o f q u a l i t y standard i s s u f f i c i e n t ; w h e r e a s , f o r m e e t i n g t h e r e q u i r e m e n t s o f a t y p i c a l BTER l a b o r a t o r y aiming t o use the r e s u l t s f o r medical d i a g n o s t i c purposes and l e g a l r e g u l a t o r y processes, depending upon t h e p r o b l e m , r e s u l t s w i t h 5 t o 10 % t o t a l u n c e r t a i n t y may b e acceptable. On t h e o t h e r hand, an e x c e p t i o n a l l y high q u a l i t y standard (< 1% t o t a l u n c e r t a i n t y ) i s mandatory i n some c a s e s , e . g . , c e r t i f i c a t i o n o f r e f e r e n c e m a t e r i a l s . I f the tolerance limits are set narrower than the investigation really requires or not feasible under p r a c t i c a l l a b o r a t o r y c o n d i t i o n s , i t can cause unnecessary e x p e n s e a n d l o s s o f t i m e (9) . C o n t r o l l e d - E n v i r o n m e n t Concept f o r M e t a b o l i c S t u d i e s . The r e a l i z a t i o n that several trace elements a r e required at very minute concentrations and that under routine l a b o r a t o r y c o n d i t i o n s t h e ambient l e v e l s of the metals c o u l d mask t h e e f f e c t s o f an i n v e s t i g a t i o n l e d t o t h e development o f controlled-environment approach o r the trace element-free-isolater system. This i s b a s i c a l l y an a l l plastic assembly, with filtered a i r , free of dust p a r t i c l e s d o w n t o f r a c t i o n o f a \im. Further, continuous monitoring o f a l l the items i n t h e system i s maintained f o r elements o f i n t e r e s t . Under these c o n d i t i o n s , t h e d i e t i s the main source o f t r a c e element supply, and by u s i n g a c h e m i c a l l y w e l l - d e f i n e d d i e t , r e l i a b l e BTER i n v e s t i g a t i o n s can be c a r r i e d o u t . Thus, t h e i n t r o d u c t i o n o f h i g h p u r i t y d i e t s on one hand, and t h e a b i l i t y t o e x e r c i s e strict a n a l y t i c a l q u a l i t y c o n t r o l on t h e o t h e r , have l e d t o r a p i d a d v a n c e s i n BTER i n v e s t i g a t i o n s ( 1 0 - 1 3 ) . BTERa M u l t i d i s c i p l i n a r y Science. Biological trace element research i s a m u l t i d i s c i p l i n a r y science. Therefore,



1. IYENGAR


5

i t i s necessary f o r researchers i n t h i s f i e l d t o s t r i v e f o r a reasonable degree o f b i o l o g i c a l i n s i g h t and a n a l y t i c a l awareness t o t h e problems i n v o l v e d . This i n t u r n will e n a b l e t h e m t o d e s i g n m e a n i n g f u l BTER i n v e s t i g a t i o n s . Q u i t e frequently, the complexities i n v o l v e d here n e c e s s i t a t e the use o f a v a r i e t y o f s c i e n t i f i c t a l e n t s and a combination o f several analytical techniques. Some of the special d i f f i c u l t i e s a r e seen i n , f o r example, p r o v i d i n g a " t o t a l " q u a l i t y c o n t r o l i n t h e o v e r a l l c o n t e x t o f an i n v e s t i g a t i o n . These include: experimental design; collection of b i o l o g i c a l l y and analytically "valid" samples; understanding t h e i m p l i c a t i o n s o f presampling f a c t o r s such as biological variations, post mortem changes and i n t r i n s i c e r r o r s (14,15) i n c o n t e x t o f o v e r a l l a c c u r a c y o f a n a n a l y t i c a l r e s u l t a s r e f l e c t e d i n F i g u r e 1; a n d f i n a l l y , the a b i l i t y t o c a r r y o u t a c c u r a t e a n a l y t i c a l measurements on those specimens, data e v a l u a t i o n and a meaningful i n t e r p r e t a t i o n o f t h e f i n d i n g s ( 1 6 ) .F o r example, " s t a t u s a t s a m p l i n g " c a n be an i m p o r t a n t source o f v a r i a t i o n o f a n a l y t i c a l r e s u l t s (Table I) r e f l e c t i n g the differences i n r e s i d e n c e t i m e s o f t r a c e e l e m e n t s a n d t h e i r compounds i n t h e b l o o d s t r e a m . T o u n d e r s t a n d t h i s , some k n o w l e d g e o f g u t absorption of chemicals and n u t r i e n t s from foods i s necessary. Therefore, i t f o l l o w s t h a t team work i s an i n t e g r a l c o m p o n e n t o f a n y BTER i n v e s t i g a t i o n . I n a c a r e f u l l y p l a n n e d BTER i n v e s t i g a t i o n , s e v e r a l p a r a m e t e r s d e s e r v e e q u a l c o n s i d e r a t i o n . The a c t u a l p r o c e s s o f an a n a l y t i c a l measurement i s m e r e l y one c r i t i c a l p o i n t t h a t i s preceded by a s e r i e s o f b i o a n a l y t i c a l p r e c a u t i o n s and f o l l o w e d by a meaningful data interpretation. The parameters involved often cross boundaries of different d i s c i p l i n e s , a s i g n a l that i t i s prudent t o i n c o r p o r a t e a team approach t o s u s t a i n t h e comprehensiveness i n such i n v e s t i g a t i o n s . I t s h o u l d be r e c o g n i z e d t h a t sample q u a l i t y as a p p l i e d t o b i o m e d i c a l s p e c i m e n s demands b o t h t h e r o u t i n e a n a l y t i c a l p r e c a u t i o n s such as p r e v e n t i o n o f extraneous c o n t a m i n a t i o n o r l o s s o f an a n a l y t e , as w e l l as r e t e n t i o n of b i o l o g i c a l integrity o f t h e specimen. F o r example, a n a l y s i s o f a specimen r e p r e s e n t i n g t h e c e l l s o f an organ (e.g., blood o r l i v e r cells) implies that the results obtained were finger prints of that cellular system a t t r i b u t a b l e t o a v i a b l e s t a t u s . Any impairment t o such a s t a t u s would v i t i a t e t h e r e s u l t s o f an a n a l y s i s , depending upon t h e q u e s t i o n posed i n utilizing that analytical f i n d i n g . I f the question i s t o address t h e net elemental contents o f t h a t c e l l u l a r system, then t h e requirement i s s i m p l y no e x t r a n e o u s c o n t a m i n a t i o n a n d no l o s s o f a n y o f the c e l l u l a r fragments (and t h e r e f o r e l o s s o f t h e a n a l y t e ) d u r i n g a n a l y s i s . On t h e o t h e r h a n d , i f t h e q u e s t i o n i s t o address the biochemical moiety o f a metal species i n a specific compartment of t h e chosen cellular system, d e p e n d i n g upon t h e s i t u a t i o n , a series of biochemical measures w i l l be r e q u i r e d and f o r t h e s e r e s u l t s t o be v a l i d , a t o t a l q u a l i t y c o n t r o l concept i s o b l i g a t o r y . An



6


Sampling and Sample Preparation of Platelets

Contamination -

(trace elements)

Large volume of Blood

Differential Centrifugation Other Methods

Estimation Using

1 3

'I-HSA

Washing

Dependent on the Sampling Methodology

( e g 5 ° c Glucosei

Figure 1. Sources of errors in elemental analysis of biological systems. Reprinted from ref. 15. Copyright 1982 American Chemical Society.


1.

Table I .


7


IYENGAR

I n f l u e n c e o f " s t a t u s a t sampling" on the c o n c e n t r a t i o n l e v e l s o f s e l e c t e d e l e m e n t s : some examples

Sample Element

Condition

Influence

Serum

Fasting

Elevated

Even o v e r n i g h t

A f t e r normal food

Lowered

During

A f t e r high Zn food,

Elevated

e.g., O y s t e r s , etc.

Low Zn i n t a k e

Lowered

Rather sudden e f f e c t

Stress

Elevated

e.g., S t a n d i n g

Pregnancy

Lowered

Progressive decline

Cu

Pregnancy

Elevated

Progressive

F

Fasting

Normal

Overnight

A f t e r normal food

Variable

I f sampled

immediately

Tea consumption E l e v a t e d

I f sampled

immediately

Low F i n t a k e

Over a few days

Zn

Milk

f i r s t few hours liver,

posture

increase

fasting

Uptake i s r a p i d but d e c l i n e i s slow s i n c e biological half-life i s 2.5 weeks

Low I i n t a k e

Lowered

Over a few days

AS

Fish intake

Elevated

W e l l absorbed and slowly excreted

Cd

Tobacco smoking

Elevated

Remains c h r o n i c a l l y h i g h i n smokers

Pb

Alcohol consumption

Elevated

E s p e c i a l l y wine drinkers

Mn

High i n p u t

Elevated

Mn r i c h c e r e a l s

Fe

Fore-milk Hind-milk

Elevated Elevated

V a r i a t i o n due t o high f a t content

Low I i n t a k e High I i n t a k e e.g., d i e t a r y algae

Lowered Elevated

Over a few days Extremely h i g h l e v e l s of I e x c r e t e d w i t h i n a short period

Variations

Sensitive to f l u i d intake, requiring 24-h c o l l e c t i o n s .

I

Urine

fasting

Elevated High i n t a k e e.g. sea f o o d s , iodinated salts etc.,

I

Blood

Lowered

Remarks

S e v e r a l Low or h i g h elements i n t a k e s


8



excellent example of t o t a l quality c o n t r o l concept is p r o v i d e d b y t h e s t u d y t h a t was d e s i g n e d t o o b t a i n p l a t e l e t s f r o m human b l o o d f o r t r a c e e l e m e n t i n v e s t i g a t i o n s . The steps i n v o l v e d namely, the problems of sample s i z e , cell viability, cell purity, trapped p l a s m a and extraneous contamination (Figure 2), required the e x p e r t i s e of s e v e r a l d i s c i p l i n e s t o o b t a i n v a l i d samples (17).

Trace E l e m e n t S p e c i a t i o n a n d B i o a v a i l a b i l i t y . A great m a j o r i t y o f c o n v e n t i o n a l l y c o n d u c t e d BTER s t u d i e s e v e n now g e n e r a t e d a t a o n l y on t h e t o t a l amount o f t r a c e e l e m e n t s . F o r e x a m p l e i n d i e t a r y i n t a k e s t u d i e s i t i s common t o carryout elemental a n a l y s i s and use t h e r e s u l t s t o draw c o m p a r i s o n s and conclusions i n context of recommended dietary allowances ( R D A ) . I t i s b e i n g r e c o g n i z e d now that the b i o l o g i c a l l y a v a i l a b l e f r a c t i o n of a trace element d i f f e r s among different foods and therefore, b i o a v a i l a b i l i t y of v a r i o u s elements from s i n g l e or mixed f o o d s s h o u l d be d e t e r m i n e d f o r an a c c u r a t e e s t i m a t i o n o f t h e i n t a k e f r o m d i e t s . However, m e t a l s p e c i a t i o n i n complex b i o l o g i c a l a n d d i e t a r y m e d i a i s a d i f f i c u l t t a s k due to d e t e c t i o n l i m i t c o n s i d e r a t i o n s and t h e d a n g e r o f i n d u c i n g changes i n the s p e c i a t i o n s t a t e s d u r i n g a n a l y s i s . Many b i o c h e m i c a l methods f o r i s o l a t i o n of m e t a l l o c o m p l e x e s such as i o n - e x c h a n g e chromatography, a f f i n i t y chromatography, i m m u n o p r e - c i p i t a t i o n , e l e c t r o p h o r e s i s and i s o e l e c t r i c f ocussing are i n use, but are faced with p r o b l e m s o f l o s s and c o n t a m i n a t i o n o f t h e m e t a l and i t s species. Radioactive l a b e l i n g of organo-arsenic compounds has been d e s c r i b e d f o r a n i m a l s t u d i e s (18). A t w o - s t a g e i n v i t r o s y s t e m t o s i m u l a t e g a s t r i c and i n t e s t i n a l d i g e s t i o n of food i n combination with I n d u c t i v e l y Coupled PlasmaMass S p e c t r o m e t r y has been s u c c e s s f u l l y used i n recent investigations (19) . S e v e r a l techniques based on gas c h r o m a t o g r a p h y and h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y and anodic stripping voltametry have been applied for s p e c i a t i o n c h e m i s t r y . Many a n a l y t i c a l m e t h o d s a r e u s e f u l for speciation chemistry work provided that their l i m i t a t i o n s are recognized (20) . Concerning bioavailability, use of stable isotopes in mineral n u t r i t i o n r e s e a r c h i s w e l l e s t a b l i s h e d . S t a b l e i s o t o p e s as i s o t o p i c t r a c e r s h a v e no r a d i a t i o n r i s k a n d t h e r e f o r e , a r e widely accepted. Use of various versions of Mass S p e c t r o m e t r y and N e u t r o n a c t i v a t i o n a n a l y s i s f o r i n v i t r o specimens following stable isotope a p p l i c a t i o n have resulted i n considerable progress in mineral metabolism research. Experimental

D e s i g n and

Data

Interpretation

"The u l t i m a t e p u r p o s e o f a n a n a l y t i c a l r e s u l t the problem, not merely generating intercomparisons" (1) .

i s to address numbers for


IYENGAR



A RELIABLE CONCLUSION DEPENDS ON THE QUALITY OF THE ANALYTICAL RESULT

IBIOLOGICAU VARIATIONS \ _ Genetic Factor$|_ Seasonai Changes!. „ Long-term Physiological Influences Short-term Physiological Influences

Figure 2. Multidisciplinary aspects of sampling human blood platelets for trace element analysis. (Reprinted from ref. 17. Copyright 1979 American Chemical Society.)


10



S t u d i e s on t h e e l e m e n t a l c o m p o s i t i o n studies i n biological systems c a n be divided into 4 stages: e x p e r i m e n t a l d e s i g n , c o l l e c t i o n o f v a l i d samples, chemical analysis and data evaluation (and i n t e r p r e t a t i o n ) . Collection of v a l i d samples and c h e m i c a l a n a l y s i s have been touched upon i n p r e v i o u s sections. Experimental d e s i g n and d a t a i n t e r p r e t a t i o n w i l l be b r i e f l y commented upon h e r e . Experimental Design. The d e s i g n i n g o f a BTER e x p e r i m e n t i s possibly the most important stage at which m u l t i d i s c i p l i n a r y approach i s crucial. A lack of this approach has l e d t o e i t h e r a total failure o f an investigation o r has generated false c o n c l u s i o n s . To u n d e r s t a n d t h i s s i t u a t i o n o n e may c o n s i d e r t h e f o l l o w i n g four s i t u a t i o n s i n r e s o l v i n g a problem r e l a t e d t o l e a d i n the environment: Case 1 : A n a l y t i c a l l y q u e s t i o n a b l e and b i o l o g i c a l l y u n s o u n d - e.g., e a r l i e r s t u d i e s on h a i r as a m o n i t o r i n g s p e c i m e n , a n d Pb i n b l o o d p l a s m a . Case 2 : A n a l y t i c a l l y q u e s t i o n a b l e and b i o l o g i c a l l y sound- e.g., d e t e r m i n a t i o n o f Pb i n l i v e r . Case 3: Analytically sound and biologically q u e s t i o n a b l e - e.g., i n c r e a s e o f Pb i n whole b l o o d r e l a t e d to smoking. C a s e 4: A n a l y t i c a l l y r e l i a b l e a n d b i o l o g i c a l l y s o u n d e.g., Pb i n whole b l o o d (and a l s o Cd i n b l o o d o f smokers and non-smokers). Case 1 i l l u s t r a t e s a worst s i t u a t i o n o f c h o o s i n g a questionable o r u n s u i t a b l e b i o l o g i c a l specimen presented with unreliable results. T h e r e a r e many p r o b l e m s i n m e a s u r i n g t h e l o w P b l e v e l s i n p l a s m a a n d m o r e t h a n 95 % o f t h e w h o l e b l o o d Pb i s a s s o c i a t e d w i t h t h e e r y t h r o c y t e s a n d therefore, blood plasma i s n o t an i d e a l indicator to reflect changes i n Pb c o n c e n t r a t i o n s . Case 2 i s a r e f l e c t i o n o f a sound b i o l o g i c a l specimen t o study, y e t prone t o a n a l y t i c a l pitfalls, especially a t very low c o n c e n t r a t i o n l e v e l s . Case 3 i s a t y p i c a l s i t u a t i o n where extreme care i s exerted t o solve a problem o f wrong b i o l o g i c a l p e r c e p t i o n . Smokers a r e u s u a l l y consumers o f alcohol t o o , and i t has been demonstrated that the e l e v a t e d b l o o d l e a d was t r a c e a b l e t o w i n e c o n s u m p t i o n ( 2 1 ) . O b v i o u s l y , b a s e d on t h e s e f i n d i n g s i f s m o k i n g h a d been banned as a measure t o c o n t r o l b l o o d l e a d l e v e l s , t h e e f f o r t w o u l d h a v e b e e n a f u t i l e move! C a s e 4 i l l u s t r a t e s a n i d e a l combination o f b i o l o g i c a l relevance and a n a l y t i c a l soundness s i n c e t h e v a l i d i t y o f t h i s approach has been d o c u m e n t e d t h r o u g h s e v e r a l s t u d i e s (22) . Data P r e s e n t a t i o n and I n t e r p r e t a t i o n . I t i s important t o p r e s e n t r e s u l t s f r o m BTER i n v e s t i g a t i o n s i n a n u n a m b i g u o u s way a n d i t i s p r u d e n t t o choose s i m p l e methods o f d a t a p r e s e n t a t i o n and a n a l y s i s o v e r c o m p l i c a t e d p r o c e d u r e s . As



1. IYENGAR


11

e x p r e s s e d b y C h e r r y , t h e numbers g e n e r a t e d b y t r a c e element studies a r e often very l a r g e ; t h e r e f o r e , an a p p r e c i a b l e f r a c t i o n o f r e s o u r c e s must be d e v o t e d f o r d a t a a c q u i s i t i o n and p r o c e s s i n g (23). Unfortunately, relating the elemental concentration data t o meaningful base has n o t received adequate a t t e n t i o n . T h i s c a n l e a d t o wrong i n t e r p r e t a t i o n of t h e r e s u l t s even though t h e a n a l y s i s has been c a r r i e d out properly. This i s due t o t h e f a c t t h a t biological m a t e r i a l s c o n s i s t o f m u l t i p l e components w i t h d i f f e r e n t element content, a n d t h e r a t i o o f t h e s e c o m p o n e n t s may d i f f e r e v e n i n t h e same t y p e o f s a m p l e s (24) . S e v e r a l examples, recommendations concerning parameters f o r evaluation o f elemental analysis data and a c h e c k l i s t o f essential features related t o elemental analysis of b i o l o g i c a l systems a r e presented ( 1 6 ) . Conclusions

" C e l l s i n t h e body the t r a c e element (1) .

do n o t f u n c t i o n i n i s o l a t i o n . Perhaps researchers may w i s h t o e m u l a t e t h e m "

Obtaining a n a l y t i c a l l y meaningful and b i o l o g i c a l l y interpretable data f o r trace elements i n biomedical i n v e s t i g a t i o n s i s a tedious task and requires dedicated e f f o r t s through a m u l t i d i s c i p l i n a r y approach by a n a l y t i c a l chemists and h e a l t h sciences i n v e s t i g a t o r s . Examples such a s t h e RDAs ( 2 5 ) , t h e p e r m i s s i b l e l i m i t s f o r e x p o s u r e t o v a r i o u s t r a c e e l e m e n t s ( 2 6 ) , c o m p o s i t i o n o f R e f e r e n c e Man (27) a n d e v a l u a t i o n o f r e f e r e n c e elemental concentration v a l u e s i n human m i l k (28) a n d i n o t h e r c l i n i c a l s p e c i m e n s (29-31) f o r m o n i t o r i n g purposes, demonstrate t h e p u b l i c h e a l t h b e n e f i t s o f 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 e s i n BTER studies. The p r o b l e m s a c c o u n t e d f o r i n the preceding sections reemphasize the fact that good analytical measurements a r e n o t o n l y c r u c i a l f o r success w i t h future biological trace element research studies, but are mandatory f o r considering the analytical findings f o r diagnostic and r e l a t e d evaluations. In t h i s context Mertz's s t a t e m e n t " t h a t an a n a l y t i c a l c h e m i s t s h o u l d be more t h a n procurer o f data and a l i f e s c i e n t i s t more t h a n their i n t e r p r e t e r " (32) c a n n o t b e e m p h a s i z e d s u f f i c i e n t l y .

Literature Cited 1.

Iyengar G . V . Elemental Analysis of Biological Systems. Vol 1, CRC P r e s s , Boca Raton, FL, 1989; pages 1, 19, 71. 2. V e r s i e c k , J . Trace E l e . Med. 1984, 1, 2-6. 3. Iyengar, G . V . Sci. Total Environ. 1981, 19, 105-109. 4. T h i r d I n t e r n a t i o n a l Symposium on Biological Reference M a t e r i a l s , Fres. Zschr. Analyt. Chem. 1988, 332, N o . 6 .


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5.

6. 7.


8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.


Ihnat, M. In Quantitative Analysis of B i o l o g i c a l Materials, McKenzie, H . A . ; Smythe, L.E., Ed.; Elsevier, Amsterdam, New York, 1988; Chapter 19, p 331. Okamoto, K . ; Fuwa, K. Anal. Lett. 1985, 1, 206-213. Muramatsu, Y . ; Parr, R.M. Survey of currently available Reference Materials for use in connection with the determination of trace elements in biological and environmental materials. IAEA Report IAEA/RL/128, 1985. Horwitz, W. In this ACS Symposium Proceedings, 1990. Parr, R. M. Nut. Res. Suppl. 1985, I, 5-11. Schwarz, K. In Proceedings of Nuclear Activation Techniques in the Life Sciences, 1972, IAEA, Vienna, p 3. Underwood, E . J . 1977. Trace Elements in Human and Animal N u t r i t i o n . 4th Edition, Academic Press, New York. Smith, J . C . In Trace Elements in Human and Animal Nutrition, Mertz, W., E d . ; Academic Press, New York, 1988, p 21. Mertz, W. Trace Elements in Human and Animal Nutrition, Volume 1 and 2, Academic Press, New York. 1987/1988. Iyengar, G.V. J. Path. 1981, 134, 173-181. Iyengar, G.V. Anal. Chem. 1982, 54, 554A-558A. Iyengar, G.V.; Behne D. J . Res. Nat. Bur. Stds. 1988, 93, 326-327. Iyengar, G . V . ; Borberg, H . ; Kasperek, K . ; Kiem, J.; Siegers, M.; Feinendegen, L.E.; Gross, R. C l i n . Chem. 1979, 25, 699-704. Sabbioni, E . ; Edel, J.; Goetz L . Nut. Res. Suppl. 1985, I, 32-43. Crews, H.M.; Massey, R.; McWeeny, D . J . J . Res. Nat. Bur. StdS 1988, 93, 349-350. Toelg, G. In Trace Element Analytical Chemistry in Medicine and Biology, Braetter, P.; Schramel P. Eds.; de Gruyter, Berlin, 1988, p 1. Grandjean, P. 1983. In Chemical Toxicology and C l i n i c a l Chemistry of Metals, Brown, S.S.; Savory, J . Eds.; p 99. Iyengar, G.V. B i o l . Trace Ele. Res. 1987, 12, 263-295. Cherry, W.H. Sci. Total Environ. 1983, 34, 199-200. Behne, D. C l i n . Chem. C l i n . Biochem. 1981, 19, 115. Recommended Dietary Allowances. National Research Council, Washington D.C. 1989, 10th Ed. Joint FAO/WHO Expert Committee on Food Additives: Technical Report Series 751, WHO, Geneva, 1987.


1.

27.

28. 29. 30.


31. 32.

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International Commission on Radiological Protection (ICRP). Report of the Task Group on Reference Man, ICRP Publication 23, Pergamon Press, Oxford, 1975, (Revision in progress). Minor and Trace Elements in Breast Milk: World Health Organization Report of a Joint WHO/IAEA Collaborative Study, WHO, Geneva, 1989. Hamilton, E . I . The Chemical Elements and Man. Charles Thomas, Spring Fields, IL, 1979. Heydorn, K. Neutron Activation Analysis in C l i n i c a l Trace Element Research. CRC Press, Boca Raton, FL, 1984. Iyengar, G.V.; Woittiez J.R.W. C l i n . Chem. 1988, 34, 474-481. Mertz, W. C l i n . Chem. 1975, 21, 468-473.

RECEIVED July

16, 1990


Biological Trace Element Research - ACS Publications - American

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