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Quantitative analysis of trace elements in human blood and plasma by energy dispersive X-ray fluorescence
Clin. Biochem. 10, (3) 127-132 (1977)
Quantitative Analysis of Trace Elements in Human Blood and Plasma by Energy Dispersive X-ray Fluorescence I. G. S T U M P ,
J. CARRUTHERS,
a n d $. M. D ' A U R I A
Department of Chemistry, Simon Fraser University, Burnaby, B.C., C a n a d a and D. A . A P P L E G A R T H
a n d A . G. F . D A V I D S O N
Department of Paediatrics, University of British Columbia, Vancouver, B.C., C a n a d a (Accepted M a r c h 1, 1 9 7 7 )
CLBIA, 10, (3), 127-132 (1977)
Clin. Biochem. Stump, I. G. ~, Carruthers, J2, D'Auria, J. M. 1, Applegarth, D. A. 2, and Davidson, A. G. F3
~Department of Chemistry, Simon Fraser University, Burnaby, B.C., and ~Department of Paediatrics, University of British Columbia, Vancouver, B.C., Canada QUANTITATIVE ANALYSIS OF TRACE ELEM E N T S IN H U M A N BLOOD A N D P L A S M A BY ENERGY DISPERSIVE X-RAY FLUORESCENCE A photon excitation, secondary t a r g e t x - r a y fluorescence system was used to measure concentrations of Cu, Zn, Br and Rb in whole blood and plasma of healthy adults. The sample p r e p a r a t i o n method and calculated limits of detection of the technique are reported. Correlation statistics for elemental concentrations have been determined. Verification of the results for copper was performed using atomic absorption spectrophotometry.
WHILE THE STUDY Of t r a c e e l e m e n t s in h u m a n biological s y s t e m s h a s been p u r s u e d f o r m a n y y e a r s ~',~', t h e i n c r e a s e d a w a r e n e s s of t h e i r role in m a n y t y p e s of d i s e a s e TM, s u g g e s t s t h e need f o r a m u l t i - e l e m e n t a l m e t h o d of a n a ~ s i s w h i c h can p r o v i d e d a t a quickly a n d e f f i c i e n t l y f o r s e v e r a l e l e m e n t s in t h e s a m e blood sample. T h e p u r p o s e of t h i s s t u d y is to r e p o r t on t h e a p p l i c a t i o n of a p h o t o n e x c i t a t i o n , s e c o n d a r y t a r g e t e n e r g y d i s p e r s i v e x - r a y f l u o r e s c e n c e ( E D X R F ) spect r o m e t e r s y s t e m , to d e t e r m i n e t h e c o n c e n t r a t i o n levels of Cu, Zn, Rb, a n d B r in p l a s m a a n d blood s a m p l e s obtained from healthy adult subjects. T h e use of an E D X R F s y s t e m can p r o v i d e m u l t i e l e m e n t a l i n f o r m a t i o n r a p i d l y in a s i n g l e m e a s u r e m e n t w i t h o u t d e s t r o y i n g a p r e p a r e d sample. S e v e r a l s t u d i e s '4"6' h a v e r e p o r t e d t h e a p p l i c a t i o n of E D X R F to a v a r i e t y of h u m a n biological samples, w h i l e a r e v i e w o f its b a s i c p r i n c i p l e s h a s been p r o v i d e d b y G o u l d i n g a n d J a k l e v i c " . D e s p i t e t h i s b o d y of work, Correspondence: Dr. J. M. D ' A u r i a , Dept. of Chemistry, Simon F r a s e r University, Burnaby, B.C., Canada
l i t t l e i n f o r m a t i o n of a q u a n t i t a t i v e n a t u r e h a s been r e p o r t e d . A r e c e n t s t u d y by F l i n t et al '8), does p r e s e n t d a t a on c e r t a i n e l e m e n t a l d i s t r i b u t i o n s in blood s e r u m s a m p l e s of c a n c e r p a t i e n t s u s i n g a p h o t o n e x c i t a t i o n , p r i m a r y Me t a r g e t E D X R F s y s t e m . F u r t h e r u t i l i z a tion of t h i s t e c h n i q u e to p r o v i d e q u a n t i t a t i v e i n f o r m a tion would be of value, however. D e t a i l s of t h e e x p e r i m e n t a l c o n d i t i o n s i n c l u d i n g s a m p l e p r e p a r a t i o n will be p r e s e n t e d as well as a c r o s s - c o m p a r i s o n of copper levels b y a t o m i c a b s o r p tion s p e c t r o p h o t o m e t r y . L i m i t s of d e t e c t i o n have been s t u d i e d to e n s u r e t h a t a full r a n g e of n o r m a l d i s t r i b u t i o n s f o r t h e e l e m e n t s could be observed. MATERIALS AND METHODS
I. Apparatus A schematic representation of the photon excitation, secondary t a r g e t E D X R F system is given in Fig. 1. The beam of photons t h a t fluoresced the sample consisted of essentially pure dichromatic (Ka and K~) characteristic X-radiation from a selectable secondary target. In this study a Me t a r g e t was used. This characteristic radiation was produced by fluorescing a secondary t a r g e t with bremsstrahlung radiation from an electron tube with a gold anode. A p p r o p r i a t e filters were used to reduce scattered bremsstrahlung radiation. The characteristic X-rays emitted by elements in the sample were detected using a S i ( L i ) spectrometer. The entire system including detector, excitation source, and pulse processing electronics were obtained from the Kevex Corporation, (Burlingame, California). Pulses from the detector were processed and accumulated in 400 channel groups in a pulse height analyzer. The d a t a were then t r a n s f e r r e d via magnetic tape to an IMB 370-165 computer and the peak energies and intensities extracted using the ' S A M P O ' spectrum-stripping computer code cs'. Shapes of s t a n d a r d x - r a y peaks were obtained by fluorescing pure samples and specimens. I t was found t h a t a Me secondary t a r g e t proved the most useful for exciting the l a r g e s t number of elements of biological interest. Elements from P ( Z = 15 to Z r ( Z = 40) could be excited and observed provided they were present in the sample in sufficient concentration. The effect of the bulk of the sample m a t r i x (carbon) is ~ r i m a r i l y as a s c a t t e r e r and absorber of the incident Me radiation and as an absorber of the emitted x - r a y s of ~nterest. Atomic absorption (A.A.) was chosen to verify the E D X R F determined, copper concentrations because this technique has proven sufficiently sensitive, specific, and reliable for determination of trace metals in biological
128
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tissue "°). A Perkin-Elmer, Model 305, A.A. spectrophotometer, equipped with a single slot burner, was used in these verification studies. 2. Sample Preparation Several different p r e p a r a t o r y techniques were explored. The most useful method from the standpoint of ease of handling, uniformity of sample produced, reproducibility and low detection limits of the elements of interest was found to be the freeze d r y i n g of one ml of sample (blood or p l a s m a ) , followed by the formation of exactly 50 mg of this dried m a t e r i a l into self-supporting pellets. Dried m a t e r i a l was mixed to provide homogeneous samples and a pressure of 3600 kg was applied to the dried powder to p r e p a r e pellets, 13 mm in diameter. The other techniques examined, such as d r y and wet ashing, were ruled out because of the large amount of m a t e r i a l required (1012 mls) to produce a sample of optimum thickness and because of possible contamination or loss of some elements during an ashing process. To minimize errors due to instrumental fluctuations, geometry effects, and v a r i a tions in the p r e p a r a t o r y steps, a measured amount (1 ml) of an aqueous y t t r i u m solution (Y(NO3)3) was added to each sample, p r i o r to the d r y i n g step. This element was used as the internal reference as its emitted x - r a y s did not interfere with determinations of other elements observed. An optimum sample thickness was determined empirically to maximize the observed fluorescence to scatter ratio. The selected thickness of 37.7 m g / c m 2 (50 mg/1.33 cm 2) lies between the thickness defined as 'thin' and 'infinitely thick' specimens ("). Linear response functions for the elements of interest were generated by s t a n d a r d additions of each element to blood or plasma, and the subsequent application of a method of least squares analysis to the normalized results (Element K a / Y Ka peak a r e a s ) . The concentration of unknown samples could be obtained directly from these f i t t e d functions, provided contributions from background had been taken into account appropriately. P r i o r to analysis of the a r e a of each peak and quantitative determination of the elements present, the effects of background were subtracted directly from the data. The magnitude of the background subtracted was proportional to the ratio of the Mo Compton peaks in a spectrum from a blank sample pellet (pure polyethylene powder) to the Mo Compton peak in spectra from the blood/plasma sample pellets. Blood from female subjects was collected using heparinized 'Lead F r e e ' Vaeutainers@ (Becton Dickinson, @4610). One ml of whole blood was removed from each tube to form the blood pellets. The remainder of the blood was centrifuged for about 10 rain and the supern a t a n t p l a s m a drawn off to p r e p a r e pellets and for analysis of copper by flame A.A. Disposable plastic syringes (Plastipak, Becton Dickin-
scattered Mo x.rays
/L~( u
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Fig. 1. - - A schematic representation of the photon excitation, secondary target x-ray fluorescence system.
L.
=
son) were used to collect blood from male subjects. The blood was t r a n s f e r r e d to polystyrene tubes from which blood and plasma pellets were p r e p a r e d as described. The technique used for the analysis of p l a s m a samples by A.A. spectrophotometry followed t h a t of Fernandez and Kahn "~). They demonstrated a 4% precision and recovery of 98-99% b y using the method of p r e p a r i n g copper s t a n d a r d s in 10% aqueous glycerol. Aliquots of the plasma specimens, simultaneously analyzed by E D X R F , were diluted 1:1 with deionized distilled w a t e r and a s p i r a t e d directly into an air-acetylene flame. The most sensitive resonant line for copper (324.7 nm) was used with a lamp c u r r e n t of 35 mA. Absorbance of these samples was recorded and compared with the results from the s t a n d a r d solutions. RESULTS T y p i c a l 15 m i n x - r a y s p e c t r a o b s e r v e d f o r whole blood a n d blood p l a s m a a r e d i s p l a y e d in f i g u r e s 2 a n d 3, r e s p e c t i v e l y . T h e s p e c t r o p h o t o m e t e r w a s o p e r a t e d w i t h an a n o d e v o l t a g e of 40 kV a n d a c u r r e n t o f 50 m A . S c a t t e r i n g o f t h e Mo K a x - r a y s f r o m t h e seconda r y t a r g e t r e s u l t s in t h e i n t e n s e l y b a c k s c a t t e r e d C o m p t o n a n d R a y l e i g h peaks. S t a n d a r d curves, p r e p a r e d f o r Cu, Zn, B r a n d Rb, a n d a n a l y z e d b y a l e a s t - s q u a r e s fitting routine, were linear over the concentration range of interest. To report Rb concentrations, the r e s p o n s e o f t h e B r Kfl p h o t o p e a k h a d to be s u b t r a c t e d f r o m t h e a p p a r e n t R b peak. ( A t low c o n c e n t r a t i o n s , and while employing an internal standard, inter-elem e n t e f f e c t s , a b s o r p t i o n a n d e n h a n c e m e n t m a y be neglected.) All Rb and Br concentrations are quoted b o t h in m i c r o g r a m s / 1 0 0 ml a n d m i c r o e q u i v a l e n t s / l i t r e as t h e s e e l e m e n t s a r e p r o b a b l y e l e c t r o l y t e s . N e v e r theless, t o t a l e l e m e n t a l c o n t e n t s w e r e m e a s u r e d a n d n o t m e r e l y ionized e l e m e n t s . T h e e l e m e n t a l c o n c e n t r a t i o n s in whole blood a n d p l a s m a f o r a g r o u p of 38 n o r m a l , h e a l t h y , a d u l t f e m a l e s b e t w e e n t h e a g e s o f 18 to 22 w e r e m e a s u r e d a n d t y p i c a l d i s t r i b u t i o n s a r e d i s p l a y e d in f i g u r e s 4 a n d 5. M e a n v a l u e s a n d t h e r a n g e of t h e d i s t r i b u t i o n s a r e p r e s e n t e d in T a b l e 1. F o r levels of copper, s e p a r a t e mean values are given for individuals taking oral c o n t r a c e p t i v e s . T h e a v e r a g e c o n t a m i n a t i o n of Zn cont r i b u t e d b y t h e c o n t a i n e r s u s e d to collect t h e f e m a l e s a m p l e s h a s been s u b t r a c t e d f r o m t h e s e values, following separate determinations of this contribution.
TRACE E L E M E N T S IN BLOOD AND PLASMA SCATTER PEAKS
BLOOD (SPECIMEN 1536)
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ELEMENT
Rb Br
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Blood Plasma
Whole Blood Mean
Mean
Range
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Range
100 ml)
100 ml)
100 ml)
100 ml)
102 -4- 21 149 -4- 19
62 - - 147 126 - 179
113 -4- 19 186 -4- 34
85 -- 152 132 -- 246
38
707 ± 73
541 -- 840
142 ~ 39
85 -- 234
- - 42 - - 4 . 9 b)
38
287 ~ 45 206 - 386 33 -4-5.2 b) 2 4 - - 4 6 b )
28 ± 8.6 3.2 4- 1.0b)
13 - - 42 1.5 - 4.9 b)
508 ± 94 395 -- 725 64 ± 12b) 49 -- 91b)
38
404 :E 77 263 -- 590 514 -I- 46 342 -- 745 51 4-10 b) 3 7 - - 7 5 b) 64 -4- 5 . 8 b) 4 3 - - 9 3 b)
120 -4- 22
90 -- 158
28 10,)
35
776 =1=88
480 -- 1023
104 ± 17
63 - 140
35
365 -4- 45 222 -- 396 36 -4- 5.2 b) 26 -- 46b)
31 -4- 5.7 3.6 ± 0 . 7 b)
20
346 =t= 69 264 -- 537 43 =1=8.6b) 33 -- 67b)
35
Range
(~g/
Blood Plasma Mean
(-4- S)
78 -- 189
Einc
T
FEMALES
107 4- 25
32
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CONCENTRATIONS
100 ml)
7,opper
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100 ml)
t
The observed total e r r o r on a s i n g l e m e a s u r e m e n t
( ± S) (tLg/ 100 ml)
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t
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MALES
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positive c o r r e l a t i o n at the 1% s i g n i f i c a n c e level ( P - - 0 . 0 1 ) . T h i s c o r r e l a t i o n was f o u n d to be b e t w e e n B r a n d Rb levels i n the males' samples. A t t h e 5% s i g n i f i c a n c e level (P----0.05) c o r r e l a t i o n s were f o u n d b e t w e e n female Cu-Zn levels, male Cu-Zn levels, a n d B r - R b c o n c e n t r a t i o n s f o r samples f r o m f e m a l e s t a k i n g oral contraceptives. As blood a n d p l a s m a specimens f r o m the same i n d i v i d u a l were s t u d i e d e s s e n t i a l l y at t h e same time, i n t e r r e l a t i o n s h i p s in the c o n c e n t r a t i o n of the same t r a c e e l e m e n t in both were searched for. T a b l e 2 displays the slopes of the r e g r e s s i o n line a n d the correl a t i o n c o e f f i c i e n t s o b t a i n e d u s i n g the r e s u l t s f r o m both males a n d females. T h r e e positive c o r r e l a t i o n s were observed f o r the e l e m e n t a l c o n c e n t r a t i o n s bet w e e n blood a n d plasma. Specifically, the copper, b r o m i n e a n d r u b i d i u m c o n c e n t r a t i o n s in blood were r e l a t e d to the c o n c e n t r a t i o n s of these e l e m e n t s in plasma.
TABLE
(~g/
r
Fig. 3 - - S i m i l a r to f i g u r e 2 f o r a pellet o f blood p l a s m a .
The c o n c e n t r a t i o n s of Cu, Zn, Br, a n d R b i n the blood a n d p l a s m a f r o m 35 n o r m a l , healthy, a d u l t males b e t w e e n the ages of 18 a n d 33 were also d e t e r m i n e d a n d t y p i c a l d i s t r i b u t i o n s are displayed in f i g u r e s 6 a n d 7. The m e a n s a n d r a n g e s of t h e d i s t r i b u t i o n s a r e displayed i n Table 1. A c o m p a r i s o n w i t h available p u b lished r e s u l t s is i n d i c a t e d in the f i g u r e s a n d T a b l e 1 f o r both males a n d females. T h e symbols are d e f i n e d in the f i g u r e captions. T h r e e male specimens cont a i n e d copper levels up to t h r e e t i m e s the n o r m a l conc e n t r a t i o n s a n d were, t h e r e f o r e , n o t included in calc u l a t i o n of the m e a n values of the d i s t r i b u t i o n s . W h i l e these h i g h e r Cu values could r e f l e c t some u n d e r l y i n g medical condition, t h i s p o s s i b i l i t y was n o t reflected in i n f o r m a t i o n provided by the subjects. F u r t h e r s t u d i e s of t h i s were n o t p u r s u e d . C o e f f i c i e n t s of c o r r e l a t i o n (13), 'r', calculated f o r a n y two v a r i e t i e s of t r a c e - e l e m e n t c o n c e n t r a t i o n f o r males' and f e m a l e s ' p l a s m a samples, showed only one
(-4- S)
t
ENERGY (keY)
Fig. :2 - - A t y p i c a l x - r a y s p e c t r u m o b t a i n e d f r o m w h o l e blood p e l l e t w i t h the a d d i t i o n o f Y t t r i u m . T h e c o u n t i n g t i m e w a s 15 m i n u t e s f o r a p r i m a r y anode voltage o f ~0 k V a n d a c u r r e n t o f 50 m A .
No
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aThese samples were for individualson bkth control therapy. bThe values here are expressed in microequivalents/litre (~Eq/litre) also.
No
of samples
(~g/
(ug/
(~g/
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130
STUMP, CARRUTHERS, D'AURIA, APPLEGARTH AND DAVIDSON I
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/.~g/100 ml Fig. ~ - - The o b s e r v e d c o n c e n t r a t i o n d i s t r i b u t i o n f o r Cu a n d Z n in w h o l e blood specimens, o b t a i n e d f r o m a g r o u p o f a d u l t f e m a l e s ( a g e s 18-29). T h e legend f o r s y m b o l s u s e d in this a n d / o r s u b s e q u e n t f i g u r e s is g i v e n below. Legend: m = arithmetic mean for samples from normal, healthy individuals. b = a r i t h m e t i c m e a n o f o b s e r v e d Cu levels f o r s a m p l e s f o r f e m a l e s on oral c o n t r a c e p t i v e therapy. a = m e a n f r o m c o m p i l a t i o n o f A n s p a u g h "4j f o r h e a l t h y m a l e s or f e m a l e s as indicateS. u = m e a n f r o m r e p o r t o f U n d e r w o o d "~ f o r h e a l t h y m a l e s or f e m a l e s as indicated. w = m e a n f r o m r e p o r t o f W o o d "r~ f o r h e a l t h y male plasma concentration. a, = m e a n f r o m c o m p i l a t i o n o f A n s p a u g h "~J f o r healthy adult serum concentrations. uo = m e a n f r o m r e p o r t o f U n d e r w o o d "~ f o r healthy adult serum concentrations. T h e s u p e r s c r i p t (*) applies w h e n the r e p o r t e d m e a n s have been c o m b i n e d f o r m a l e s a n d f e m a l e s .
is a sum of the errors introduced by each p a r t of the preparation and analysis. These include pipetting errors, losses during preparation, nonhomogenety of pellets, instrumental variations, positioning in the E D X R F sample holder and, in particular, determination of areas of observed photopeaks. These errors were determined by analyzing the same pellet on diff e r e n t occasions over a long period of time, by study-
200
300
400
Fg/loo ml F i g . 5 - - S i m i l a r to f i g u r e $ e x c e p t f o r R b a n d B r levels in w h o l e blood s a m p l e s .
ing different pellets f r o m the same dried powder, and by comparing pellets prepared f r o m the same, original blood specimen. The total precision e r r o r in the reported concentrations, taken to two standard deviations, varied f r o m 3% for an intense peak (e.g., Br, K s ) to 13% for a weaker peak (e.g., Cu, K s ) .
Verification As mentioned, determinations of Cu concentrations in blood plasma for the 38 female specimens studied were also'performed using A.A. These data are shown in fig. 8. The mean (-- S) of the A.A. results was 132 ± 40 ~g/100 ml as compared to a value of 136 ± 40 ~g/100 ml f r o m E D X R F (includes all specimens studied). The observed precision of a single A.A. determination was 6% while the large S values reflect the elemental distributions ir~ our population. The correlation coefficient, ( r ) , was calculated to be 0.92, (P=0.01) indicating excellent agreement~'3L The estimated variance of the calculated regression line ( Y = 6 . 1 + 0 . 9 3 X ) was determined as 0.004.
T R A C E E L E M E N T S IN BLOOD AND P L A S M A I
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80
1 00
120
140
~g/100
2
,IL
,I,
I
160
180
200
20
ml
Fig. 6 - - Similar to figure 4 e~ccept for Zn and Cu levels ire whole plasma samples from healthy adult modes. Detection
I
[
30
40
I
50
60
/zg/ 100 ml Fig. 7 - - Similar to figure 5 except for Br and Rb levels in blood plasma samples.
Limits
L o w e r l i m i t s of d e t e c t a b i l i t y w e r e e s t i m a t e d to ensure that meaningful trace element analyses were possible over a r e l e v a n t r a n g e of e l e m e n t a l c o n c e n t r a tions. T h e c a p a c i t y of E D X R F to m e a s u r e t r a c e quant i t i e s of e l e m e n t s is d e t e r m i n e d by t h e a r e a of t h e o b s e r v e d s p e c t r a l p e a k above t h e b r e m s s t r a h l u n g backg r o u n d . T h e m i n i m u m d e t e c t a b l e l i m i t h a s been def i n e d as t h e e l e m e n t a l c o n c e n t r a t i o n g e n e r a t i n g a peak a p p r o x i m a t e l y equal to t h r e e t i m e s t h e backg r o u n d v a r i a n c e c o n t a i n e d in t h e full w i d t h a t h a l f m a x i m u m of t h a t peakC'L T a b l e 3 p r e s e n t s d e t e c t i o n l i m i t s c a l c u l a t e d in t h i s m a n n e r f o r t h e p r e s e n t E D X R F s y s t e m . C h r o m i u m , an e s s e n t i a l e l e m e n t in i n t e r m e d i a t e c o n c e n t r a t i o n in b i o l o g i c a l m a t e r i a l , is included to show t h a t t h i s s y s t e m d e s c r i b e d h e r e i n is TABLE 2
not s u f f i c i e n t l y s e n s i t i v e to be used in t h e a n a l y s i s of e l e m e n t s at c o n c e n t r a t i o n s much lower t h a n t h e ones chosen. DISCUSSION T h e m e a n s of t h e o b s e r v e d e l e m e n t a l c o n c e n t r a t i o n s f o r h e a l t h y s u b j e c t s a r e in good a g r e e m e n t w i t h rep o r t e d values ' ' ' i ' . Small d e v i a t i o n s m a y be a t t r i b u t e d to e n v i r o n m e n t a l and g e o g r a p h i c f a c t o r s . A d d i t i o n a l discussion, h o w e v e r , is r e q u i r e d in t h e i n s t a n c e s of blood a n d p l a s m a Cu in f e m a l e s u b j e c t s a n d p l a s m a Rb results. T h e m e a n v a l u e s o b t a i n e d f o r blood a n d p l a s m a Cu levels in a d u l t f e m a l e s a r e c o n s i d e r e d s e p a r a t e l y f o r those on oral c o n t r a c e p t i v e t h e r a p y a s h i g h e r levels a r e expected ~'~. T h e n e a r l y b i m o d a l d i s t r i b u t i o n obTABLE 3
REGRESSION AND CORRELATION ANALYSIS OF BLOOD-PLASMACONCENTRATIONS FROM THE SAME SUBJECT
LIMITS OF DETECTION
Regression Equation: Y(plasma) = aX(blood) + b Element Variate
n
a
Copper Zinc Bromine Rubidium
69 69 68 70
0.56 -0.05 1.02 0.051
S2 0.0086 0.0026 0.0029 0.0004
r
P
0.59 -0.12 0.85 0.31
<0.01 >0.10 <<0.01 --~'0.01
Cr . . . . . . . . . . Cu . . . . . . . . . . Zn . . . . . . . . . . Br . . . . . . . . . . Rb . . . . . . . . . .
Blood Plasma (~g/100 ml) 25 17 16 8 (1.0 t~ Eq/litre) 15 (1.8 I~ Eq/litre)
Whole Blood (txg/100 ml) 34 26 22 10 (1.3 t~ Eq/litre) 26 (3.0 ~ Eq/litre)
132
STUMP, CARRUTHERS, D'AURIA, APPLEGARTH AND DAVIDSON CONCLUSION
--- 300 O0
000 /
E 280 Cb
o
r = 092
o~ 240 ::L. 220
u_
nx 200 >" m 180 rr 160 uJ
13_
n
. /
"
~"
.:;/+
140
0
o
/
y = 6.1 +0.93x
260
The potential value of the E D X R F technique described as a fast, efficient, relatively accurate multielemental analytic tool has been illustrated. While the present system was applied for measuring only four elements, in principle the elements P, C1, Ca, S. K and Fe could easily have been included. It is anticipated that with more sensitive E D X R F systems concentrations of such elements as Cr, V, Ge, Se and Cd could also be determined quantitatively.
120
ACKNOWLEDGEMENTS
< o3
< _-I
100
,~,l".'i"
80
n
60
1 O0
' ,- , , , , , , 140
180
220
260
300
PLASMA COPPER BY A . A . ( p . g / 100 mls) Fig. 8 - - Displayed here are the observed conce~ztrations 'of Cu in the same set of blood plasma specimens as measured by x-ray fluorescence and by atomic absorption techniques. Also included are the observed experimental errors for each technique. A correlation factor (r) of 0.92 was calculated indicating good agreement between these sets of values. The regression equation for this set of data was found to be y = 6.1 + 0.93X (S z : 0.004). The line shown in this figure represents Y----X.
served for blood (Fig. 4) clearly reflects this expected increase. The means of the values obtained for females not using oral contraceptives is in good agreement with reported values"". The observed differences in copper concentrations could be used to assess the relative reactivity of female subjects to oral contraceptives ~,G~. Plasma and blood Rb values are difficult to measure by other analytical techniques, at least to this degree of sensitivity and convenience. Earlier studies ''~ do indicate slightly lower values for plasma Rb. The E D X R F technique is fairly sensitive to the element Rb and such data could ultimately be used for research and therapy in psychiatric disorders. This element has been found to exert positive effects in the treatment of manic-depressives ''~). Total bromine (as opposed to bromide ion) is easily determined by E D X R F and, although no clinical applications yet exist, application of E D X R F to analysis of this element may prove valuable in the future. The variation from a high detection limit for chromium to a low limit for bromine reflects the characteristic limiting feature of x-ray fluorescence analysis. Where a range of elements is to be analyzed while exciting the sample with essentially monochromatic radiation, the limit of detection invariably decreases with decreasing atomic number. The detection limit for chromium could be increased to about that of copper by utilizing a selenium secondary target. Even this, however, would not yield a limit of detection for which Cr could be determined in biological material using the present system.
The financial support for this interdisciplinary project from the Medical Research Council of Canada, the Vancouver Foundation, and the National Research Council of Canada is gratefully acknowledged. We are also greatly indebted to P. M. MacLeod, L. Wong, and A. Wu for clinical assistance without which the study could not have been completed. REFEREN C ~ S
1. Underwood, E. J., Trace Eleme)~ts in Human and Animal Nut'ritio~ (Academic Press, New York, N.Y., 1971). 2. Schroeder, H. A., and Nason, A. P., Clinical Chem., 17, 461 (1971). 3. McCall, J. T., Goldstein, N. P., & Smith, L. H., Fed. Proc. 30, lOll, 1971. 4. Agarwal, M., Bennett, R. B., Stump, I. G., and D'Auria, J. M., Anal. Cem., 47, 924 (1975). 5. Ong, P. S., Lund, P. K., Litton, C. E., and Mitchell, B. A., Adv. X - R a y Anal., ed. by L. S. Birks, 16, 124 (1972). 6. Bearse, R. C., Chase, D. A., Malanify, J. J., and Umbarger, C. J., Anal. Chem., 46, 499 (1974). 7. Goulding, F., and Jaklevic, J. M., Ann. Rev. Nuc. Sci., 23, 45 (1973). 8. Flint, R. W., Lawson, C. D., and Standel, S., J. Lab. Clin. Meth., 85, 155 (1975). 9. Routti, J. T., and Prussin, S. G., Nucl. Instr. Meth., 72, 125 (1969). 10. Sunderman, Jr., F. W., H u m a n Pathology, 4, 549 (1973). 11. Ziegler, C. A. ed., Application of Low Energy X a~d Gamma Rays, p. 317, Gordon and Breach (1971). 12. Fernandez, F. J., and Kahn, H. L., Clin. Chem. Newsletter, 3, 24 (1971). 13. Zuwaylif, F. H., General Applied Statistics, second ed., (Addison Wesley Publishing Co., Inc., Don Mills, Ontario, 1974). 14. Anspaugh, L. R., Robinson, W. L., et al, Compilations of published Information on Elemental Concentration in Human Organs in Both Normal and Diseased States., U.C.R:L. 51013 (microfiche) (1971). 15. Schenker, J. G., Jungries, E., Polishuk, W. Z., Int. J. Fertil., 17:28 (1972). 16. Horwitt, M. K., Harvey, C. C., Danum, C. H., Amer. J. of Clin. Nutr., 28; 403 (1975). 17. Wood, O. Lew, Bioehem. Med., 3, 458 (1970). 18. Fieve, R. R., Meltzer, H., et al. Amer. J. Psychiatry, 130(1): 55, (1973).
#t
#t
Quantitative Analysis of Trace Elements in Human Blood and Plasma by Energy Dispersive X-ray Fluorescence I. G. S T U M P ,
J. CARRUTHERS,
a n d $. M. D ' A U R I A
Department of Chemistry, Simon Fraser University, Burnaby, B.C., C a n a d a and D. A . A P P L E G A R T H
a n d A . G. F . D A V I D S O N
Department of Paediatrics, University of British Columbia, Vancouver, B.C., C a n a d a (Accepted M a r c h 1, 1 9 7 7 )
CLBIA, 10, (3), 127-132 (1977)
Clin. Biochem. Stump, I. G. ~, Carruthers, J2, D'Auria, J. M. 1, Applegarth, D. A. 2, and Davidson, A. G. F3
~Department of Chemistry, Simon Fraser University, Burnaby, B.C., and ~Department of Paediatrics, University of British Columbia, Vancouver, B.C., Canada QUANTITATIVE ANALYSIS OF TRACE ELEM E N T S IN H U M A N BLOOD A N D P L A S M A BY ENERGY DISPERSIVE X-RAY FLUORESCENCE A photon excitation, secondary t a r g e t x - r a y fluorescence system was used to measure concentrations of Cu, Zn, Br and Rb in whole blood and plasma of healthy adults. The sample p r e p a r a t i o n method and calculated limits of detection of the technique are reported. Correlation statistics for elemental concentrations have been determined. Verification of the results for copper was performed using atomic absorption spectrophotometry.
WHILE THE STUDY Of t r a c e e l e m e n t s in h u m a n biological s y s t e m s h a s been p u r s u e d f o r m a n y y e a r s ~',~', t h e i n c r e a s e d a w a r e n e s s of t h e i r role in m a n y t y p e s of d i s e a s e TM, s u g g e s t s t h e need f o r a m u l t i - e l e m e n t a l m e t h o d of a n a ~ s i s w h i c h can p r o v i d e d a t a quickly a n d e f f i c i e n t l y f o r s e v e r a l e l e m e n t s in t h e s a m e blood sample. T h e p u r p o s e of t h i s s t u d y is to r e p o r t on t h e a p p l i c a t i o n of a p h o t o n e x c i t a t i o n , s e c o n d a r y t a r g e t e n e r g y d i s p e r s i v e x - r a y f l u o r e s c e n c e ( E D X R F ) spect r o m e t e r s y s t e m , to d e t e r m i n e t h e c o n c e n t r a t i o n levels of Cu, Zn, Rb, a n d B r in p l a s m a a n d blood s a m p l e s obtained from healthy adult subjects. T h e use of an E D X R F s y s t e m can p r o v i d e m u l t i e l e m e n t a l i n f o r m a t i o n r a p i d l y in a s i n g l e m e a s u r e m e n t w i t h o u t d e s t r o y i n g a p r e p a r e d sample. S e v e r a l s t u d i e s '4"6' h a v e r e p o r t e d t h e a p p l i c a t i o n of E D X R F to a v a r i e t y of h u m a n biological samples, w h i l e a r e v i e w o f its b a s i c p r i n c i p l e s h a s been p r o v i d e d b y G o u l d i n g a n d J a k l e v i c " . D e s p i t e t h i s b o d y of work, Correspondence: Dr. J. M. D ' A u r i a , Dept. of Chemistry, Simon F r a s e r University, Burnaby, B.C., Canada
l i t t l e i n f o r m a t i o n of a q u a n t i t a t i v e n a t u r e h a s been r e p o r t e d . A r e c e n t s t u d y by F l i n t et al '8), does p r e s e n t d a t a on c e r t a i n e l e m e n t a l d i s t r i b u t i o n s in blood s e r u m s a m p l e s of c a n c e r p a t i e n t s u s i n g a p h o t o n e x c i t a t i o n , p r i m a r y Me t a r g e t E D X R F s y s t e m . F u r t h e r u t i l i z a tion of t h i s t e c h n i q u e to p r o v i d e q u a n t i t a t i v e i n f o r m a tion would be of value, however. D e t a i l s of t h e e x p e r i m e n t a l c o n d i t i o n s i n c l u d i n g s a m p l e p r e p a r a t i o n will be p r e s e n t e d as well as a c r o s s - c o m p a r i s o n of copper levels b y a t o m i c a b s o r p tion s p e c t r o p h o t o m e t r y . L i m i t s of d e t e c t i o n have been s t u d i e d to e n s u r e t h a t a full r a n g e of n o r m a l d i s t r i b u t i o n s f o r t h e e l e m e n t s could be observed. MATERIALS AND METHODS
I. Apparatus A schematic representation of the photon excitation, secondary t a r g e t E D X R F system is given in Fig. 1. The beam of photons t h a t fluoresced the sample consisted of essentially pure dichromatic (Ka and K~) characteristic X-radiation from a selectable secondary target. In this study a Me t a r g e t was used. This characteristic radiation was produced by fluorescing a secondary t a r g e t with bremsstrahlung radiation from an electron tube with a gold anode. A p p r o p r i a t e filters were used to reduce scattered bremsstrahlung radiation. The characteristic X-rays emitted by elements in the sample were detected using a S i ( L i ) spectrometer. The entire system including detector, excitation source, and pulse processing electronics were obtained from the Kevex Corporation, (Burlingame, California). Pulses from the detector were processed and accumulated in 400 channel groups in a pulse height analyzer. The d a t a were then t r a n s f e r r e d via magnetic tape to an IMB 370-165 computer and the peak energies and intensities extracted using the ' S A M P O ' spectrum-stripping computer code cs'. Shapes of s t a n d a r d x - r a y peaks were obtained by fluorescing pure samples and specimens. I t was found t h a t a Me secondary t a r g e t proved the most useful for exciting the l a r g e s t number of elements of biological interest. Elements from P ( Z = 15 to Z r ( Z = 40) could be excited and observed provided they were present in the sample in sufficient concentration. The effect of the bulk of the sample m a t r i x (carbon) is ~ r i m a r i l y as a s c a t t e r e r and absorber of the incident Me radiation and as an absorber of the emitted x - r a y s of ~nterest. Atomic absorption (A.A.) was chosen to verify the E D X R F determined, copper concentrations because this technique has proven sufficiently sensitive, specific, and reliable for determination of trace metals in biological
128
STUMP, C A R R U T H E R S ,
FILTER ~ (Mo) ~
I
~41~
SELECTABLE I-I / SECONDARY [ [
•Mo AR ,,
LI,,, \ __
~' ,f'
s
D'AURIA, APPLEGARTH
AND
DAVIDSON
SAMPLE ~
n -, %t ANk ~'t.~lP" PULSES , ~ A ~,t/ ]l= ~ "u" ...... 7N/v
~
(,
MULTIPLESAMPLEHOt.DER
-
I
\ \
VACUUMCHAMBER
'1 YE
IP~LSE
I
l I
~;|
I
I
sample x-rays
....-J u~,,.~_.J
tissue "°). A Perkin-Elmer, Model 305, A.A. spectrophotometer, equipped with a single slot burner, was used in these verification studies. 2. Sample Preparation Several different p r e p a r a t o r y techniques were explored. The most useful method from the standpoint of ease of handling, uniformity of sample produced, reproducibility and low detection limits of the elements of interest was found to be the freeze d r y i n g of one ml of sample (blood or p l a s m a ) , followed by the formation of exactly 50 mg of this dried m a t e r i a l into self-supporting pellets. Dried m a t e r i a l was mixed to provide homogeneous samples and a pressure of 3600 kg was applied to the dried powder to p r e p a r e pellets, 13 mm in diameter. The other techniques examined, such as d r y and wet ashing, were ruled out because of the large amount of m a t e r i a l required (1012 mls) to produce a sample of optimum thickness and because of possible contamination or loss of some elements during an ashing process. To minimize errors due to instrumental fluctuations, geometry effects, and v a r i a tions in the p r e p a r a t o r y steps, a measured amount (1 ml) of an aqueous y t t r i u m solution (Y(NO3)3) was added to each sample, p r i o r to the d r y i n g step. This element was used as the internal reference as its emitted x - r a y s did not interfere with determinations of other elements observed. An optimum sample thickness was determined empirically to maximize the observed fluorescence to scatter ratio. The selected thickness of 37.7 m g / c m 2 (50 mg/1.33 cm 2) lies between the thickness defined as 'thin' and 'infinitely thick' specimens ("). Linear response functions for the elements of interest were generated by s t a n d a r d additions of each element to blood or plasma, and the subsequent application of a method of least squares analysis to the normalized results (Element K a / Y Ka peak a r e a s ) . The concentration of unknown samples could be obtained directly from these f i t t e d functions, provided contributions from background had been taken into account appropriately. P r i o r to analysis of the a r e a of each peak and quantitative determination of the elements present, the effects of background were subtracted directly from the data. The magnitude of the background subtracted was proportional to the ratio of the Mo Compton peaks in a spectrum from a blank sample pellet (pure polyethylene powder) to the Mo Compton peak in spectra from the blood/plasma sample pellets. Blood from female subjects was collected using heparinized 'Lead F r e e ' Vaeutainers@ (Becton Dickinson, @4610). One ml of whole blood was removed from each tube to form the blood pellets. The remainder of the blood was centrifuged for about 10 rain and the supern a t a n t p l a s m a drawn off to p r e p a r e pellets and for analysis of copper by flame A.A. Disposable plastic syringes (Plastipak, Becton Dickin-
scattered Mo x.rays
/L~( u
E
Fig. 1. - - A schematic representation of the photon excitation, secondary target x-ray fluorescence system.
L.
=
son) were used to collect blood from male subjects. The blood was t r a n s f e r r e d to polystyrene tubes from which blood and plasma pellets were p r e p a r e d as described. The technique used for the analysis of p l a s m a samples by A.A. spectrophotometry followed t h a t of Fernandez and Kahn "~). They demonstrated a 4% precision and recovery of 98-99% b y using the method of p r e p a r i n g copper s t a n d a r d s in 10% aqueous glycerol. Aliquots of the plasma specimens, simultaneously analyzed by E D X R F , were diluted 1:1 with deionized distilled w a t e r and a s p i r a t e d directly into an air-acetylene flame. The most sensitive resonant line for copper (324.7 nm) was used with a lamp c u r r e n t of 35 mA. Absorbance of these samples was recorded and compared with the results from the s t a n d a r d solutions. RESULTS T y p i c a l 15 m i n x - r a y s p e c t r a o b s e r v e d f o r whole blood a n d blood p l a s m a a r e d i s p l a y e d in f i g u r e s 2 a n d 3, r e s p e c t i v e l y . T h e s p e c t r o p h o t o m e t e r w a s o p e r a t e d w i t h an a n o d e v o l t a g e of 40 kV a n d a c u r r e n t o f 50 m A . S c a t t e r i n g o f t h e Mo K a x - r a y s f r o m t h e seconda r y t a r g e t r e s u l t s in t h e i n t e n s e l y b a c k s c a t t e r e d C o m p t o n a n d R a y l e i g h peaks. S t a n d a r d curves, p r e p a r e d f o r Cu, Zn, B r a n d Rb, a n d a n a l y z e d b y a l e a s t - s q u a r e s fitting routine, were linear over the concentration range of interest. To report Rb concentrations, the r e s p o n s e o f t h e B r Kfl p h o t o p e a k h a d to be s u b t r a c t e d f r o m t h e a p p a r e n t R b peak. ( A t low c o n c e n t r a t i o n s , and while employing an internal standard, inter-elem e n t e f f e c t s , a b s o r p t i o n a n d e n h a n c e m e n t m a y be neglected.) All Rb and Br concentrations are quoted b o t h in m i c r o g r a m s / 1 0 0 ml a n d m i c r o e q u i v a l e n t s / l i t r e as t h e s e e l e m e n t s a r e p r o b a b l y e l e c t r o l y t e s . N e v e r theless, t o t a l e l e m e n t a l c o n t e n t s w e r e m e a s u r e d a n d n o t m e r e l y ionized e l e m e n t s . T h e e l e m e n t a l c o n c e n t r a t i o n s in whole blood a n d p l a s m a f o r a g r o u p of 38 n o r m a l , h e a l t h y , a d u l t f e m a l e s b e t w e e n t h e a g e s o f 18 to 22 w e r e m e a s u r e d a n d t y p i c a l d i s t r i b u t i o n s a r e d i s p l a y e d in f i g u r e s 4 a n d 5. M e a n v a l u e s a n d t h e r a n g e of t h e d i s t r i b u t i o n s a r e p r e s e n t e d in T a b l e 1. F o r levels of copper, s e p a r a t e mean values are given for individuals taking oral c o n t r a c e p t i v e s . T h e a v e r a g e c o n t a m i n a t i o n of Zn cont r i b u t e d b y t h e c o n t a i n e r s u s e d to collect t h e f e m a l e s a m p l e s h a s been s u b t r a c t e d f r o m t h e s e values, following separate determinations of this contribution.
TRACE E L E M E N T S IN BLOOD AND PLASMA SCATTER PEAKS
BLOOD (SPECIMEN 1536)
129 SCATTER ,PE~KS
1051 PLASMA (SPECIMEN 1509)
Y Ka
Fe Ka
v
'
Br Ka
/ zna KI
°1
K2P Ca Ka
)AK
l
]0 t
,
,
I
,
r
*
t
TRACE
Whole Blood Mean
ELEMENT
Rb Br
s
Blood Plasma
Whole Blood Mean
Mean
Range
(-4- S)
Range
100 ml)
100 ml)
100 ml)
100 ml)
102 -4- 21 149 -4- 19
62 - - 147 126 - 179
113 -4- 19 186 -4- 34
85 -- 152 132 -- 246
38
707 ± 73
541 -- 840
142 ~ 39
85 -- 234
- - 42 - - 4 . 9 b)
38
287 ~ 45 206 - 386 33 -4-5.2 b) 2 4 - - 4 6 b )
28 ± 8.6 3.2 4- 1.0b)
13 - - 42 1.5 - 4.9 b)
508 ± 94 395 -- 725 64 ± 12b) 49 -- 91b)
38
404 :E 77 263 -- 590 514 -I- 46 342 -- 745 51 4-10 b) 3 7 - - 7 5 b) 64 -4- 5 . 8 b) 4 3 - - 9 3 b)
120 -4- 22
90 -- 158
28 10,)
35
776 =1=88
480 -- 1023
104 ± 17
63 - 140
35
365 -4- 45 222 -- 396 36 -4- 5.2 b) 26 -- 46b)
31 -4- 5.7 3.6 ± 0 . 7 b)
20
346 =t= 69 264 -- 537 43 =1=8.6b) 33 -- 67b)
35
Range
(~g/
Blood Plasma Mean
(-4- S)
78 -- 189
Einc
T
FEMALES
107 4- 25
32
I
CONCENTRATIONS
100 ml)
7,opper
i
1
Range (~g/ 100 ml)
100 ml)
t
The observed total e r r o r on a s i n g l e m e a s u r e m e n t
( ± S) (tLg/ 100 ml)
of samples
t
Error Analysis
MALES
~d
?
positive c o r r e l a t i o n at the 1% s i g n i f i c a n c e level ( P - - 0 . 0 1 ) . T h i s c o r r e l a t i o n was f o u n d to be b e t w e e n B r a n d Rb levels i n the males' samples. A t t h e 5% s i g n i f i c a n c e level (P----0.05) c o r r e l a t i o n s were f o u n d b e t w e e n female Cu-Zn levels, male Cu-Zn levels, a n d B r - R b c o n c e n t r a t i o n s f o r samples f r o m f e m a l e s t a k i n g oral contraceptives. As blood a n d p l a s m a specimens f r o m the same i n d i v i d u a l were s t u d i e d e s s e n t i a l l y at t h e same time, i n t e r r e l a t i o n s h i p s in the c o n c e n t r a t i o n of the same t r a c e e l e m e n t in both were searched for. T a b l e 2 displays the slopes of the r e g r e s s i o n line a n d the correl a t i o n c o e f f i c i e n t s o b t a i n e d u s i n g the r e s u l t s f r o m both males a n d females. T h r e e positive c o r r e l a t i o n s were observed f o r the e l e m e n t a l c o n c e n t r a t i o n s bet w e e n blood a n d plasma. Specifically, the copper, b r o m i n e a n d r u b i d i u m c o n c e n t r a t i o n s in blood were r e l a t e d to the c o n c e n t r a t i o n s of these e l e m e n t s in plasma.
TABLE
(~g/
r
Fig. 3 - - S i m i l a r to f i g u r e 2 f o r a pellet o f blood p l a s m a .
The c o n c e n t r a t i o n s of Cu, Zn, Br, a n d R b i n the blood a n d p l a s m a f r o m 35 n o r m a l , healthy, a d u l t males b e t w e e n the ages of 18 a n d 33 were also d e t e r m i n e d a n d t y p i c a l d i s t r i b u t i o n s are displayed in f i g u r e s 6 a n d 7. The m e a n s a n d r a n g e s of t h e d i s t r i b u t i o n s a r e displayed i n Table 1. A c o m p a r i s o n w i t h available p u b lished r e s u l t s is i n d i c a t e d in the f i g u r e s a n d T a b l e 1 f o r both males a n d females. T h e symbols are d e f i n e d in the f i g u r e captions. T h r e e male specimens cont a i n e d copper levels up to t h r e e t i m e s the n o r m a l conc e n t r a t i o n s a n d were, t h e r e f o r e , n o t included in calc u l a t i o n of the m e a n values of the d i s t r i b u t i o n s . W h i l e these h i g h e r Cu values could r e f l e c t some u n d e r l y i n g medical condition, t h i s p o s s i b i l i t y was n o t reflected in i n f o r m a t i o n provided by the subjects. F u r t h e r s t u d i e s of t h i s were n o t p u r s u e d . C o e f f i c i e n t s of c o r r e l a t i o n (13), 'r', calculated f o r a n y two v a r i e t i e s of t r a c e - e l e m e n t c o n c e n t r a t i o n f o r males' and f e m a l e s ' p l a s m a samples, showed only one
(-4- S)
t
ENERGY (keY)
Fig. :2 - - A t y p i c a l x - r a y s p e c t r u m o b t a i n e d f r o m w h o l e blood p e l l e t w i t h the a d d i t i o n o f Y t t r i u m . T h e c o u n t i n g t i m e w a s 15 m i n u t e s f o r a p r i m a r y anode voltage o f ~0 k V a n d a c u r r e n t o f 50 m A .
No
//
I02
ENERGY(keY)
Z
/
II .oo ;AZoK /
I/
~Ka
A// c.Kp I
'
I
Rb Ka
~o
ZnlK~ E GDI i
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B,jp
Cu Ka
>.
z i05 z_
--
/
CI Ka
lO 4
2.3
aThese samples were for individualson bkth control therapy. bThe values here are expressed in microequivalents/litre (~Eq/litre) also.
No
of samples
(~g/
(ug/
(~g/
(~/
130
STUMP, CARRUTHERS, D'AURIA, APPLEGARTH AND DAVIDSON I
I
I
I
ma*
12
I
I
~.LEq/lit re
2'5'3'1
11 10
'sr.' ' 6 b '
"/5
12
BLOOD ZINC
9 10
8
BLOOD BROMINE
7 8
6 03 UJ d
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5
A O3 LIJ .,_.1
4
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3
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2-
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1
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400
500
600
700
800
900
1000
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9
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8
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7
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6
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BLOOD COPPER
£3 <
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200
5
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I
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400
I
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600
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IJ_
b
2
v O3 .g
-
6
4
10
2'3 m
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5 4
BLOOD RUBIDIUM
8
Z
3
6
2-
,I
4 60
I
l
I
I
I
[
80
100
120
140
160
180
2
q
/.~g/100 ml Fig. ~ - - The o b s e r v e d c o n c e n t r a t i o n d i s t r i b u t i o n f o r Cu a n d Z n in w h o l e blood specimens, o b t a i n e d f r o m a g r o u p o f a d u l t f e m a l e s ( a g e s 18-29). T h e legend f o r s y m b o l s u s e d in this a n d / o r s u b s e q u e n t f i g u r e s is g i v e n below. Legend: m = arithmetic mean for samples from normal, healthy individuals. b = a r i t h m e t i c m e a n o f o b s e r v e d Cu levels f o r s a m p l e s f o r f e m a l e s on oral c o n t r a c e p t i v e therapy. a = m e a n f r o m c o m p i l a t i o n o f A n s p a u g h "4j f o r h e a l t h y m a l e s or f e m a l e s as indicateS. u = m e a n f r o m r e p o r t o f U n d e r w o o d "~ f o r h e a l t h y m a l e s or f e m a l e s as indicated. w = m e a n f r o m r e p o r t o f W o o d "r~ f o r h e a l t h y male plasma concentration. a, = m e a n f r o m c o m p i l a t i o n o f A n s p a u g h "~J f o r healthy adult serum concentrations. uo = m e a n f r o m r e p o r t o f U n d e r w o o d "~ f o r healthy adult serum concentrations. T h e s u p e r s c r i p t (*) applies w h e n the r e p o r t e d m e a n s have been c o m b i n e d f o r m a l e s a n d f e m a l e s .
is a sum of the errors introduced by each p a r t of the preparation and analysis. These include pipetting errors, losses during preparation, nonhomogenety of pellets, instrumental variations, positioning in the E D X R F sample holder and, in particular, determination of areas of observed photopeaks. These errors were determined by analyzing the same pellet on diff e r e n t occasions over a long period of time, by study-
200
300
400
Fg/loo ml F i g . 5 - - S i m i l a r to f i g u r e $ e x c e p t f o r R b a n d B r levels in w h o l e blood s a m p l e s .
ing different pellets f r o m the same dried powder, and by comparing pellets prepared f r o m the same, original blood specimen. The total precision e r r o r in the reported concentrations, taken to two standard deviations, varied f r o m 3% for an intense peak (e.g., Br, K s ) to 13% for a weaker peak (e.g., Cu, K s ) .
Verification As mentioned, determinations of Cu concentrations in blood plasma for the 38 female specimens studied were also'performed using A.A. These data are shown in fig. 8. The mean (-- S) of the A.A. results was 132 ± 40 ~g/100 ml as compared to a value of 136 ± 40 ~g/100 ml f r o m E D X R F (includes all specimens studied). The observed precision of a single A.A. determination was 6% while the large S values reflect the elemental distributions ir~ our population. The correlation coefficient, ( r ) , was calculated to be 0.92, (P=0.01) indicating excellent agreement~'3L The estimated variance of the calculated regression line ( Y = 6 . 1 + 0 . 9 3 X ) was determined as 0.004.
T R A C E E L E M E N T S IN BLOOD AND P L A S M A I
12
I
m
/ . L E q / l i t re
u
9 8
8
7
6-
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PLASMA ZINC
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140
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PLASMA COPPER
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650
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PLASMA RUBIDIUM
10 8
4 Z
3
6 4
80
1 00
120
140
~g/100
2
,IL
,I,
I
160
180
200
20
ml
Fig. 6 - - Similar to figure 4 e~ccept for Zn and Cu levels ire whole plasma samples from healthy adult modes. Detection
I
[
30
40
I
50
60
/zg/ 100 ml Fig. 7 - - Similar to figure 5 except for Br and Rb levels in blood plasma samples.
Limits
L o w e r l i m i t s of d e t e c t a b i l i t y w e r e e s t i m a t e d to ensure that meaningful trace element analyses were possible over a r e l e v a n t r a n g e of e l e m e n t a l c o n c e n t r a tions. T h e c a p a c i t y of E D X R F to m e a s u r e t r a c e quant i t i e s of e l e m e n t s is d e t e r m i n e d by t h e a r e a of t h e o b s e r v e d s p e c t r a l p e a k above t h e b r e m s s t r a h l u n g backg r o u n d . T h e m i n i m u m d e t e c t a b l e l i m i t h a s been def i n e d as t h e e l e m e n t a l c o n c e n t r a t i o n g e n e r a t i n g a peak a p p r o x i m a t e l y equal to t h r e e t i m e s t h e backg r o u n d v a r i a n c e c o n t a i n e d in t h e full w i d t h a t h a l f m a x i m u m of t h a t peakC'L T a b l e 3 p r e s e n t s d e t e c t i o n l i m i t s c a l c u l a t e d in t h i s m a n n e r f o r t h e p r e s e n t E D X R F s y s t e m . C h r o m i u m , an e s s e n t i a l e l e m e n t in i n t e r m e d i a t e c o n c e n t r a t i o n in b i o l o g i c a l m a t e r i a l , is included to show t h a t t h i s s y s t e m d e s c r i b e d h e r e i n is TABLE 2
not s u f f i c i e n t l y s e n s i t i v e to be used in t h e a n a l y s i s of e l e m e n t s at c o n c e n t r a t i o n s much lower t h a n t h e ones chosen. DISCUSSION T h e m e a n s of t h e o b s e r v e d e l e m e n t a l c o n c e n t r a t i o n s f o r h e a l t h y s u b j e c t s a r e in good a g r e e m e n t w i t h rep o r t e d values ' ' ' i ' . Small d e v i a t i o n s m a y be a t t r i b u t e d to e n v i r o n m e n t a l and g e o g r a p h i c f a c t o r s . A d d i t i o n a l discussion, h o w e v e r , is r e q u i r e d in t h e i n s t a n c e s of blood a n d p l a s m a Cu in f e m a l e s u b j e c t s a n d p l a s m a Rb results. T h e m e a n v a l u e s o b t a i n e d f o r blood a n d p l a s m a Cu levels in a d u l t f e m a l e s a r e c o n s i d e r e d s e p a r a t e l y f o r those on oral c o n t r a c e p t i v e t h e r a p y a s h i g h e r levels a r e expected ~'~. T h e n e a r l y b i m o d a l d i s t r i b u t i o n obTABLE 3
REGRESSION AND CORRELATION ANALYSIS OF BLOOD-PLASMACONCENTRATIONS FROM THE SAME SUBJECT
LIMITS OF DETECTION
Regression Equation: Y(plasma) = aX(blood) + b Element Variate
n
a
Copper Zinc Bromine Rubidium
69 69 68 70
0.56 -0.05 1.02 0.051
S2 0.0086 0.0026 0.0029 0.0004
r
P
0.59 -0.12 0.85 0.31
<0.01 >0.10 <<0.01 --~'0.01
Cr . . . . . . . . . . Cu . . . . . . . . . . Zn . . . . . . . . . . Br . . . . . . . . . . Rb . . . . . . . . . .
Blood Plasma (~g/100 ml) 25 17 16 8 (1.0 t~ Eq/litre) 15 (1.8 I~ Eq/litre)
Whole Blood (txg/100 ml) 34 26 22 10 (1.3 t~ Eq/litre) 26 (3.0 ~ Eq/litre)
132
STUMP, CARRUTHERS, D'AURIA, APPLEGARTH AND DAVIDSON CONCLUSION
--- 300 O0
000 /
E 280 Cb
o
r = 092
o~ 240 ::L. 220
u_
nx 200 >" m 180 rr 160 uJ
13_
n
. /
"
~"
.:;/+
140
0
o
/
y = 6.1 +0.93x
260
The potential value of the E D X R F technique described as a fast, efficient, relatively accurate multielemental analytic tool has been illustrated. While the present system was applied for measuring only four elements, in principle the elements P, C1, Ca, S. K and Fe could easily have been included. It is anticipated that with more sensitive E D X R F systems concentrations of such elements as Cr, V, Ge, Se and Cd could also be determined quantitatively.
120
ACKNOWLEDGEMENTS
< o3
< _-I
100
,~,l".'i"
80
n
60
1 O0
' ,- , , , , , , 140
180
220
260
300
PLASMA COPPER BY A . A . ( p . g / 100 mls) Fig. 8 - - Displayed here are the observed conce~ztrations 'of Cu in the same set of blood plasma specimens as measured by x-ray fluorescence and by atomic absorption techniques. Also included are the observed experimental errors for each technique. A correlation factor (r) of 0.92 was calculated indicating good agreement between these sets of values. The regression equation for this set of data was found to be y = 6.1 + 0.93X (S z : 0.004). The line shown in this figure represents Y----X.
served for blood (Fig. 4) clearly reflects this expected increase. The means of the values obtained for females not using oral contraceptives is in good agreement with reported values"". The observed differences in copper concentrations could be used to assess the relative reactivity of female subjects to oral contraceptives ~,G~. Plasma and blood Rb values are difficult to measure by other analytical techniques, at least to this degree of sensitivity and convenience. Earlier studies ''~ do indicate slightly lower values for plasma Rb. The E D X R F technique is fairly sensitive to the element Rb and such data could ultimately be used for research and therapy in psychiatric disorders. This element has been found to exert positive effects in the treatment of manic-depressives ''~). Total bromine (as opposed to bromide ion) is easily determined by E D X R F and, although no clinical applications yet exist, application of E D X R F to analysis of this element may prove valuable in the future. The variation from a high detection limit for chromium to a low limit for bromine reflects the characteristic limiting feature of x-ray fluorescence analysis. Where a range of elements is to be analyzed while exciting the sample with essentially monochromatic radiation, the limit of detection invariably decreases with decreasing atomic number. The detection limit for chromium could be increased to about that of copper by utilizing a selenium secondary target. Even this, however, would not yield a limit of detection for which Cr could be determined in biological material using the present system.
The financial support for this interdisciplinary project from the Medical Research Council of Canada, the Vancouver Foundation, and the National Research Council of Canada is gratefully acknowledged. We are also greatly indebted to P. M. MacLeod, L. Wong, and A. Wu for clinical assistance without which the study could not have been completed. REFEREN C ~ S
1. Underwood, E. J., Trace Eleme)~ts in Human and Animal Nut'ritio~ (Academic Press, New York, N.Y., 1971). 2. Schroeder, H. A., and Nason, A. P., Clinical Chem., 17, 461 (1971). 3. McCall, J. T., Goldstein, N. P., & Smith, L. H., Fed. Proc. 30, lOll, 1971. 4. Agarwal, M., Bennett, R. B., Stump, I. G., and D'Auria, J. M., Anal. Cem., 47, 924 (1975). 5. Ong, P. S., Lund, P. K., Litton, C. E., and Mitchell, B. A., Adv. X - R a y Anal., ed. by L. S. Birks, 16, 124 (1972). 6. Bearse, R. C., Chase, D. A., Malanify, J. J., and Umbarger, C. J., Anal. Chem., 46, 499 (1974). 7. Goulding, F., and Jaklevic, J. M., Ann. Rev. Nuc. Sci., 23, 45 (1973). 8. Flint, R. W., Lawson, C. D., and Standel, S., J. Lab. Clin. Meth., 85, 155 (1975). 9. Routti, J. T., and Prussin, S. G., Nucl. Instr. Meth., 72, 125 (1969). 10. Sunderman, Jr., F. W., H u m a n Pathology, 4, 549 (1973). 11. Ziegler, C. A. ed., Application of Low Energy X a~d Gamma Rays, p. 317, Gordon and Breach (1971). 12. Fernandez, F. J., and Kahn, H. L., Clin. Chem. Newsletter, 3, 24 (1971). 13. Zuwaylif, F. H., General Applied Statistics, second ed., (Addison Wesley Publishing Co., Inc., Don Mills, Ontario, 1974). 14. Anspaugh, L. R., Robinson, W. L., et al, Compilations of published Information on Elemental Concentration in Human Organs in Both Normal and Diseased States., U.C.R:L. 51013 (microfiche) (1971). 15. Schenker, J. G., Jungries, E., Polishuk, W. Z., Int. J. Fertil., 17:28 (1972). 16. Horwitt, M. K., Harvey, C. C., Danum, C. H., Amer. J. of Clin. Nutr., 28; 403 (1975). 17. Wood, O. Lew, Bioehem. Med., 3, 458 (1970). 18. Fieve, R. R., Meltzer, H., et al. Amer. J. Psychiatry, 130(1): 55, (1973).
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