MS Measurement of Stable Isotopes of Selenium - American

7 G C / M S Measurement of Stable Isotopes of Selenium

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For Use in Metabolic Tracer Studies CLAUDE VEILLON U.S. Department of Agriculture, Beltsville Human Nutrition Research Center, Building 307, Room 215, Beltsville, MD 20705 Numerous t r a c e elements a r e known t o be nutritionally e s s e n t i a l in man. I n order to assess t h e essentiality, dietary availability, and m e t a b o l i c f a t e o f t h e s e , means o f labeling f o r subsequent identification are needed. I n animal s t u d i e s , r a d i o i s o t o p e s a r e o f t e n used f o r this purpose, but their use in human s t u d i e s is g e n e r a l l y c o n t r a i n d i c a t e d due t o t h e radiation h a z a r d s . An a l t e r n a t e method is to use s t a b l e i s o t o p e s o f the elements, which overcomes this limitation. A method will be d e s c r i b e d f o r c o n v e n i e n t l y measuring the s t a b l e isotopes o f selenium, p e r m i t t i n g their use a s m e t a b o l i c tags in tracer s t u d i e s . Using one s t a b l e i s o t o p e as t h e tracer and another as internal standard, one can quantitatively identify in a sample t h e tracer, n a t u r a l (unenriched) selenium present w i t h it, and total s e l e n i u m . Some o f t h e k i n d s o f information o b t a i n a b l e from m e t a b o l i c tracer s t u d i e s will be discussed. Selenium has been recognized as an e s s e n t i a l t r a c e element in the d i e t s o f man and animals f o r many y e a r s (1). Another s t r o n g i n d i c a t i o n o f i t s e s s e n t i a l i t y is the f a c t t h a t it is an e s s e n t i a l component o f the enzyme g l u t a t h i o n e peroxidase (2). R e c e n t l y , s c i e n t i s t s from the P e o p l e s Republic o f China demonstrated t h a t Keshan d i s e a s e (a cardiomyopathy in c h i l d r e n ) was c o r r e l a t e d w i t h low d i e t a r y selenium i n t a k e s °uld l a r g e l y be prevented w i t h supplementation. S i m i l a r l y , poor selenium n u t r i t i o n in p a t i e n t s r e c e i v i n g total p a r e n t e r a l n u t r i t i o n has been l i n k e d to muscular discomfort (4) and cardiomyopathy (5). These s t u d i e s i n d i c a t e t h a t a much b e t t e r understanding o f the r o l e o f selenium in human n u t r i t i o n is needed. The types o f questions to be answered more fully are the b i o l o g i c a l a v a i l a b i l i t y o f selenium from foods, the f r a c t i o n o f d i e t a r y i n t a k e t h a t is absorbed, the metabolic fate o f absorbed selenium, b o d i l y 1

a

n

d

c

This chapter not subject to U.S. copyright. Published 1984, American Chemical Society

Turnlund and Johnson; Stable Isotopes in Nutrition ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

STABLE ISOTOPES IN NUTRITION


92

requirements, s t o r e s and n u t r i t i o n a l s t a t u s o f i n d i v i d u a l s , and factors affecting these. S t a b l e i s o t o p e s o f selenium (as w e l l as those o f o t h e r elements) can p r o v i d e a means o f addressing these questions by their employment in m e t a b o l i c tracer s t u d i e s . The same i n f o r mation can be obtained as when employing r a d i o t r a c e r s in animal s t u d i e s , but without the a s s o c i a t e d r a d i a t i o n hazards in human studies* F o r example, s t a b l e i s o t o p e s o f selenium can be b i o l o g i c a l l y i n c o r p o r a t e d i n t o t e s t foods and these used to monitor selenium b i o a v a i l a b i l i t y ( 6 , 7 ) . Described h e r e i n is a convenient, a c c u r a t e , s e n s i t i v e and r a p i d method f o r measuring s t a b l e i s o t o p e s o f selenium in b i o l o g i c a l m a t e r i a l s . A r a p i d sample p r e p a r a t i o n technique is used which does not r e q u i r e p e r c h l o r i c a c i d , w i t h i t s a s s o c i a t e d dangers. Based on i s o t o p e d i l u t i o n techniques and i s o t o p e r a t i o measurements, the method employs one enriched s t a b l e i s o t o p e as the i n t e r n a l standard and another as the metabolic t a g . T h i s permits the q u a n t i t a t i v e measurement o f the e n r i c h e d tracer, unenriched ( n a t u r a l ) selenium present w i t h it, and total selenium in the samples. Some examples o f the type o f i n f o r m a t i o n t h a t can be obtained w i t h these techniques will be d e s c r i b e d . Isotope D i l u t i o n The technique o f s t a b l e i s o t o p e d i l u t i o n permits one to determine v e r y a c c u r a t e l y the t r a c e element content o f a p a r t i c u l a r sample as w e l l as u s i n g s t a b l e i s o t o p e s as m e t a b o l i c t r a c e r s . The concept o f i s o t o p e d i l u t i o n f o r t r a c e element determinations is both simple and e l e g a n t . A known q u a n t i t y o f an enriched s t a b l e i s o t o p e is added to the sample t o be a n a l y z e d . By measuring the amount o f t h a t i s o t o p e (added) r e l a t i v e to another i s o t o p e (not added) o f the element, one can e a s i l y c a l c u l a t e the amount o f a n a l y t e present o r i g i n a l l y in the sample. Thus, the normal r e l a t i v e amounts o f the two i s o t o p e s ( n a t u r a l abundance) have been a l t e r e d , or " d i l u t e d " . Advantages. L e t us assume for the moment t h a t the requirements f o r most t r a c e element a n a l y s i s methods are a l s o met h e r e . These a r e : t h a t a known amount o f an enriched i s o t o p e o f the element in q u e s t i o n ( s p i k e ) has been added t o the sample; t h a t subsequent chemical p r o c e s s i n g renders the s p i k e and endogenous a n a l y t e in the same chemical form; and t h a t contamination is under c o n t r o l . The second assumption, e q u i l i b r a t i o n o f a n a l y t e and s p i k e , can be a c r i t i c a l one, in t h a t l a c k o f e q u i l i b r a t i o n c o u l d c o n t r i b u t e systematic e r r o r s . One major advantage o f s t a b l e i s o t o p e d i l u t i o n methodology is t h a t q u a n t i t a t i v e recovery o f the a n a l y t e is not r e q u i r e d . Both the a n a l y t e and the added s p i k e are i d e n t i c a l c h e m i c a l l y , so incomplete r e c o v e r i e s will a f f e c t both a n a l y t e and s p i k e in the same way. I n this regard the enriched s p i k e serves not o n l y as


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Stable Isotopes of Selenium

93


an internal standard for the determination, but also constitutes an ideal internal standard. Another major advantage of the method is that it has the potential of being an absolute method, that is, unlike most other methods, it is not necessary to calibrate instrument response against known standards. It is simply a ratio measurement, of two chemically identical species. Limitations. About 17 elements are mononuclidic, i.e., only one isotope of the element exists in nature, and several of these are of interest biologically, such as F, Na, ΑΙ, Ρ, Mn, Co, and As. In studies where radioisotopes can be used, using stable iso topes is usually less convenient and more time consuming. For trace element determinations, other methods like atomic absorption spectrometry are usually faster, more convenient and require simpler instrumenation. Other than for tracer studies in humans, stable isotope dilution methods perhaps serve best in establishing the accuracy of other methods. Basis of the Method In this method, samples are spiked with a known amount of an enriched isotope of selenium ( Se in this case) and wet digested to destroy the organic matter and render a l l of the selenium into the +4 oxidation state. Next, the selenium is reacted with 4-nitro-o-phenylenediamine (NPD) to form the nitropiazselenol (Se-NPD) which is extracted into chloroform. Aliquots of the extract are then introduced into the mass spectrometer (MS) via a gas chromatograph (GC) and individual ions measured to determine isotope ratios of the various selenium isotopes. From the observed isotope ratios and the known amount of 82s spike added, the amounts of enriched tracer (e.g., 7%e, 74se, etc.), unenriched selenium and total selenium in the samples can be calculated. Let us now look at each of these steps in detail. 2

e

Sample Digestion. Samples (1-5 g or mL) are weighed or pipetted into 100 mL micro Kjeldahl flasks containing 2 glass boiling beads. Depending on sample size, 3-5 mL of concentrated HNO3, 1 mL of H3PO4, and a known amount of enriched are added and samples allowed to stand for 1 hr at room temperature. Then samples are heated to boiling and additions of 30-50$ H2O2 begun slowly. This is continued until the volume is reduced to about 1 mL. Samples are then cooled briefly, 1 mL formic acid added, and reheated. This reduces any residual HNO3 to ΝΟ2· Next, 2 mL concentrated HC1 are added and the samples boiled gently for 10 min to convert any Se (VI) to Se (IV). Ten mL of water are added and the samples allowed to cool. They are then extracted with 3-5 mL of chloroform to remove any lipids present.


94



Chelation. The mtropiazselenol is formed by adding 0.5 mL of 1% NPD in water and the samples allowed to stand for 1 hr at room temperature with occasional shaking. The resulting Se-NPD is then extracted into 2 mL chloroform with mechanical shaking for 15 minutes. The chloroform layer is removed, placed in small glass test tubes and evaporated in a vacuum oven ( 100 Torr) at 50°. Then 100 uL of chloroform are added, the tubes closed with polyethylene plugs, and aliquots of this solution (1-10 uL) injected into the GC/MS for analysis. Total Selenium. As mentioned earlier, stable isotope dilution is a powerful tool in trace element analysis. Let us first look at how it can be used to determine the total selenium content of a sample. In the following section we will develop the method further for stable isotopes in metabolic tracer studies. As it occurs in nature, selenium consits of six stable isotopes with the relative abundances shown in Table I. Table I. Relative Abundance of Se Isotopes in Natural Se Isotope 74 76 77 78 80 82

Abundance, atom % 0.87 9.02 7.58 23.52 49.82 9Λ9

When Se-NPD is introduced into the mass spectrometer, the most intense group of ions is that of the parent ion, Se-NPD . This is illustrated in Figure 1 for natural (unenriched) Se-NPD. Six peaks are observed, corresponding to Se-NPD containing each of he six Se stable isotopes. By adding a known amount of enriched Se (spike) to the samples, and monitoring the observed isotope ratio of, say, WSefàSe, we can readily calculate the amount of natural selenium that had to be present in the sample. The enriched 82 spike which we use has relative abundances as shown in Table II. Table II. Relative Abundance of Se Isotopes in Enriched 8%5e +

+

2

Se

Isotope 74 76 77 78 80 82

Abundance « atom % 0.13 0.19 0.30 0.60 1.96 96.81


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95 8o

82

Thus, we can set up an expression for the Se/ Se ratio as follows: 80

82

Se/ Se = R Se (0.0919) + Se (0.968) n

g2

8

Here the numerator is the sum of a l l sources ofSe, i.e., 49.8$ in natural selenium (Se ) and 1.96$ in the Se (Se82> spike (Tables I and II). Similarly, the denominatorisa l l sources of Se; 9.19$ in Se and 96.8$ in Sees* We wish to express the amounts of Se and Se82 weights (e.g., ng). Yet the mass spectrometer measures the number of ions at a given mass. Since and differ in mass, a correction must be made to Equation 1. Looking at this another way, let us assume that we had added enough Se to a sample so that the Se and Se peaks in Figure 1 were the same size. This would correspond to the same number of Se and Se ions but different amounts (weights) of each isotope. So, Equation 1 has to be modified: 82


n

82

n

a s

n

82

8o

82

82

8

Se (0.498) + 0.976 Se (0.0196) flo

K

u

" Se (0.0919) + 0.976 Se (0.968) n

82

'

The 0.976 factor now corrects this equation so that Se and Se82 in terms of weights of each (80/82 = 0.976). In other words, for an equal number of Se and Se ions there is a greater weight of Se present, so this term must be reduced by the 0.976 factor. There is one more factor to consider here. We are not actually measuring Se+ and S e , but Se-NPD and 82 e-NPD+. The NPD portion of the ion contains six carbon atoms, three nitrogen atoms and two oxygen atoms. Carbon is mostly C, but 1.1$ of carbon is c. Likewise, nitrogen is 0.37$ and oxygen is 0.20$ 0 . The occurrence of these in the ions will alter the Se-NPD/ Se-NPD ratio slightly from that of Se/ Se. The natural isotope ratio of Se/ Se is 5.42. Correct ing for the contributions of the ligand isotopes, the ratio should be 5.36. This ratio is actually observed, indicating no instrumental bias in the measurements. Frew et a l . (8) have discussed these corrections in detail. So, Equation 2 must be corrected for this small effect of the ratio differences of a factor of 1.01 (i.e., 5.42/5.36), namely: n

a r e

8o

82

82

8o

82

+

8o

+

S

12

l8

8o

8o

82

82

8o

82

Se (0.498) + 0.976 Se (0.0196) flo

"

0

η ·

0

+

) °-976 Se (0.968) 82


96


The correction for the ligand isotopes is small because of the 2 amu separation of the peaks. Adjacent pairs would have larger corrections. Solving Equation 3 for Se , we get: n

Se (0.0191 - 0.954R) 82

S e

n

=

0.0928R - 0.498

Here R is the observed Se-NPD/ Se-NPD ratio, SeQ is the known weight of the added Se spike, and Se is the weight of natural selenium in the original sample in the same units as Seg2* This relationship can also be used to establish the accuracy of the Se spike concentration, which is most conveniently added as a solution. Employing accurately prepared samples of natural selenium (Se known) spiked with known amounts of the Se solution, R is then measured and Equation 4 solved for Seg2* This is basically an inverse isotope dilution procedure. When performed on the same instrument to be used for subsequent measurements, it has the added advantage of cancelling out any mass discrimination by the instrument. 8o

82

2

82


n

82

n

82

82

Tracer Studies. In addition to using Se as an internal standard as described above, a second enriched isotope of selenium can be used as a metabolic tag. Let us take as an example the use of enriched 76

S e

as a tracer. We have used a batch of 76se for this purpose, with the relative abundances shown in Table III. Table III. Relative Abundances of Se isotopes in Enriched 75se Isotope

Abundance > atom %

74 76 77 78 80 82

0.14 96.88 0.85 0.99 0.95 0Λ8

In this case, we will need to measure 2 isotope ratios, namely oO /82se (spiking samples with Se, as before) and 76s /° Se (using 76s as the metabolic tag). Proceeding as before, we can set up expressions for these 2 ratios using the coefficients from Tables I-III: 82

2

Se

e

e

80 R

l

=

82

Se (0.498) + Seg (0.0196) + Se (0.0095)

Se

n

=

2

g

Se (0.0919) + Se (0.968) + Se (0.00l8) n

g2

76


( 5 )

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97


and:

76 Se

Se (0.0902) + Se (0.0019) + Se (0.969) η 82 J6

(6)


R = = 2 82 Se (0.0919) + Se (0.968) + Se (0.0018) Se η 82 76 As before, we have to incorporate corrections for the ligand isotopes and mass differences, but we now have 2 equations with two unknowns which can be solved simultaneously. The algebraic manipulations are quite messy and will not be presented here. Many of the terms in the solution have small insignificant coefficients and can be eliminated. The solution simplifies to: 0.009R - 0.0184)

3β (0.9

2

82

(7)

0.482 - 0.090R, and: R (0.085 Se + 0.897 Se ) - 0.084 Se - 0.0018 S e 2

Se

76

=

n

g2

R

82

( g )

0.0018R- 0.9688 2

From 2 ratio measurements and the known amount of spike added, one can quantitatively identify: a) the f Se tracer present in the sample, and b) the amount of natural selenium present with it. As a bonus, one also gets the total selenium content of the sample, which is simply the sum of these. Note that the equations derived here are valid only for the enriched materials described in Tables II and III. Different coefficients would have to be used for other enrichments and isotopes, but in principle any combination of isotopes could be used for internal standrad and metabolic tag. Applications Let us now consider some of the types of information one can obtain using stable isotopes in metabolic studies. In essence, one can obtain the same information as with radioisotopes, but with less convenient measurement. Stable isotopes also have the advantage of an "infinite half-life, permitting long term studies. For an element like selenium with six stable isotopes, more than one can be used in the same experiment at the same time. 11


98


S e v e r a l groups have been a c t i v e in recent y e a r s in employing s t a b l e isotopes o f v a r i o u s elements in tracer s t u d i e s i n v o l v i n g t r a c e element metabolism. Janghorbani e t a l . (9-11) have r e c e n t l y reported on metabolic s t u d i e s w i t h Zn and Se s t a b l e i s o t o p e s . C a r n i e t a l . (12) i n v e s t i g a t e d i r o n u t i l i z a t i o n u s i n g 58p j an elegant study, Harvey i n v e s t i g a t e d Cu uptake in f i s h (13)· Copper is an example o f an element for which no s u i t a b l y l o n g l i v e d r a d i o i s o t o p e s e x i s t . Turnlund et a l . (14) measured i r o n and z i n c a b s o r p t i o n in e l d e r l y men u s i n g 7°Zn and 58p Yergey and co-workers (15) have s t u d i e d c a l c i u m metabolism w i t h s t a b l e isotopes o f t h a t element, w i t h r e l a t i v e l y inexpensive i n s t r u m e n t a t i o n . Schwartz and Giesecke (16) i n v e s t i g a t e d magnesium metabolism u s i n g both s t a b l e " M g and the s h o r t - l i v e d r a d i o i s o t o p e M g . A number o f elements o f n u t r i t i o n a l i n t e r e s t , i n c l u d i n g Mg, C a , C r , F e , N i , C u , Cd and Z n , were i n v e s t i g a t e d by Hachey and co-workers (17). Johnson (18) i n v e s t i g a t e d m i n e r a l metabolism in human s u b j e c t s employing 54 57pe, 67zn, 70 65c . The method d e s c r i b e d h e r e i n has been d e s c r i b e d in p a r t e a r l i e r (19). The methodology was subsequently employed in m o n i t o r i n g the i n t r i n s i c l a b e l i n g o f chicken products w i t h ' Se (7). These endogenously l a b e l e d products were subsequently used in a c o n t r o l l e d metabolic study to measure selenium uptake in pregnant women (20). n

e#


e#

2

2 8

Fe>

Zn a n d

u

P o o l S i z e s . M e t a b o l i c t r a c e r s can in p r i n c i p l e be used to estimate the s i z e o f v a r i o u s body pools f o r t r a c e elements. It is somewhat analogous to making s p e c i f i c a c t i v i t y measurements in r a d i o t r a c e r experiments. Let us take as an example t h e f o l l o w i n g . We have a c o n t a i n e r o f a f i x e d s i z e o r volume ( P ) . I n t o this c o n t a i n e r water is f l o w i n g a t a constant flow r a t e (U) and f l o w i n g out a t the same r a t e through a r a d i o a c t i v i t y d e t e c t o r . L e t us assume t h a t the contents o f the c o n t a i n e r are c o n s t a n t l y and r a p i d l y mixed. I f a t time t s 0 we i n t r o d u c e a known amount o f s o l u b l e r a d i o a c t i v e m a t e r i a l i n t o the i n f l o w i n g stream, the c o n c e n t r a t i o n o f this m a t e r i a l in the o u t f l o w i n g stream would be a t a maximum ( r a p i d mixing assumed). The c o n c e n t r a t i o n would then d e c l i n e as a d d i t i o n a l water flowed i n t o the c o n t a i n e r , but in a p r e d i c t a b l e manner. I t would d e c l i n e e x p o n e n t i a l l y and a s y m p t o t i c a l l y approach zero as more water continued to flow in. I f we were to p l o t counts-per-minute (CPM) in the outflow versus time we would see something l i k e t h a t shown in F i g u r e 2. T h i s process is known as e x p o n e n t i a l d i l u t i o n and our h y p o t h e t i c a l set-up would c o n s t i t u t e an e x p o n e n t i a l d i l u t i o n f l a s k . This is a g e n e r a l procedure and can be used to c a l i b r a t e d e t e c t o r response in both l i q u i d and gaseous systems. The equation d e s c r i b i n g this e x p o n e n t i a l d i l u t i o n in F i g u r e 2 is: Ut (CPM).r (CPM) e " Ρ t ο


(9)

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99


80


Λ

CO

c φ

82

c ο

mass, amu Figure 1. Mass spectrum of Natural (unenriched) Se-NPD. (Repro duced with permission from Ref. 19. Copyright 1983, American Institute of Nutrition.)

Figure 2 . Illustration of exponential dilution from an exponen t i a l dilution flask.



100

where (CPM)t is the counts per minute a t time t ; (CPM) is the counts per minute a t time 0; U is the f l o w r a t e o f water i n t o the c o n t a i n e r ; and Ρ is the s i z e o f t h e c o n t a i n e r . We can extend this concept t o tracer s t u d i e s in humans as w e l l . L e t us take as an example t h e metabolic study o f Swanson et a l . ( 2 0 ) . I n this study, 3 groups o f women (non-pregnant, early-pregnant and l a t e - p r e g n a n t ) were placed on a c o n t r o l l e d d i e t c o n t a i n i n g 150 ug S e / d a y . On day 8, they a l s o ingested 150 ug S e , but 110 ug o f it was n a t u r a l (unenriched) selenium and 40 ug o f it was enriched 76$ i n c o r p o r a t e d i n t o egg products (7). So we now have the s i t u a t i o n i l l u s t r a t e d in F i g u r e 3 · Here we have a s i t u a t i o n analogous to our e x p o n e n t i a l d i l u t i o n f l a s k . The equation d e s c r i b i n g this s i t u a t i o n would be: 0


β

Ut (Se).= (Se) e " Ρ t ο

(10)

where: ( S e ) t is the ug o f 76$ a t time t ; ( S e ) is the ug o f 7%e a t time 0; U is the ug Se f l o w i n g in ( e . g . , 150 ug/day); and Ρ would be the " p o o l s i z e ( i n u g ) . I n this s i t u a t i o n , the most convenient time u n i t would be days. I f we look a t , s a y , u r i n a r y e x c r e t i o n (24-hour c o l l e c t i o n s ) u s i n g days as our time u n i t , then (Se) would be t h e ug o f 7bse in each 24-hour u r i n e , ( S e ) would be t h e ug o f Se for the day t h a t the f Se was i n g e s t e d ; U would be 150 ug Se/day; and Ρ would be their " p o o l s i z e " in u g . P l o t t i n g 24-hour u r i n a r y 76s e x c r e t i o n f o r these s u b j e c t s versus days y i e l d s curves very s i m i l a r t o F i g u r e 2 . However, there is a more i n f o r m a t i v e way to look a t these d a t a . I f we take the n a t u r a l l o g a r i t h m o f both s i d e s o f Equation 10 we g e t : β

0

w

Q

e

ln(Se) = l n ( S e ) t

o

-

(11)

Thus, a p l o t o f l n ( S e ) ( i . e . , I n o f t h e d a i l y T Se u r i n a r y e x c r e t i o n ) versus t ( i . e . , days) should y i e l d a s t r a i g h t l i n e w i t h a slope o f - U / P . S i n c e U is known in this c a s e , Ρ can be calculated. This is i l l u s t r a t e d f o r the non-pregnant s u b j e c t s in this example in F i g u r e 4 . The s l o p e f o r the f i r s t 3 days corresponds to a " p o o l s i z e " o f 230 ug S e . An i n t e r e s t i n g c o i n c i d e n c e is t h a t the plasma selenium c o n c e n t r a t i o n s and the estimated plasma volumes (very s i m i l a r in these s u b j e c t s ) correspond t o about 230 ug o f c i r c u l a t i n g plasma s e l e n i u m . T h i s may, o f course, be c o i n c i d e n c e , but s i n c e plasma and u r i n e can exchange t h i n g s in the kidneys one might s p e c u l a t e t h a t there is perhaps more to it than c o i n c i d e n c e . The l e s s - n e g a t i v e slope seen f o r days 11 and 12 i n d i c a t e s a l a r g e r a d d i t i o n a l p o o l w i t h p o s s i b l y a slower turnover r a t e than t h a t o f days 8-10.


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urine feces hair etc. 7e

Se spike Se in : 1SO fig/day 7 e

Figure 3 . al. (20).

S * spilt* : 40 fig (plu* 110 μ g S*)

Diagram of the metabolic tracer study of Swanson et

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t(day) Figure k. Plot of ln(.Se) vs. t for the non-pregnant subjects of Svanson et a l . (20). Similar plots for their early-pregnant and late-pregnant subjects gave increasing values of P.


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A similar plot for the late-pregnant group of subjects gave a larger Ρ value. However, the difference can not be attributed solely to the fetus, since a l l three groups of subjects were in slightly positive selenium balance.


Conclusions The method described herein provides one with an excellent means of determining the selenium content of biological samples and of measuring enriched stable isotopes of selenium in metabolic tracer studies. The sample preparation method is rapid and avoids the problems associated with HCIO4. The chelation with NPD combined with the separation capabilities of gas chromatography and the mass discrimination of the mass spectrometer result in an extremely specific method with virtually no chance of interfer ence by other elements in the sample. The method has proven valuable in endogenous food labeling and human metabolic studies with enriched stable isotopes of selenium. Its potential in assessing various selenium body pools is currently being further explored. Literature Cited 1. Subcommittee on Selenium, National Research Council, 1983, "Selenium in Nutrition, revised edition", National Academy of Sciences, Washington, D.C. 2. Rotruck, J.T.; Pope, A.L.; Ganther, H.E.; Swanson, A.B.; Hafeman, D.G.; Hoekstra, W.G. Science 1973, 179, 588. 3. Keshan Disease Research Group of the Chinese Academy of Medical Sciences. Chin. Med. J. 1979, 92, 471-482. 4. van Rij, A.M.; Thomson, C.D.; McKenzie, J.M.; Robinson, M.F. Am. J. Clin. Nutr. 1979, 32, 2076. 5. Johnson, R.A.; Baker, S.S.; Fallon, J.T.; Maynard, E.P.; Ruskin, J.N.; Wen, Z.; Ge, K.; Cohen, H.J. New England J. Med. 1981, 304, 1210. 6. Janghorbani, M.; Christensen, M.J.K.; Steinke, F.H.; Young, V.R. J. Nutr. 1981, 111, 817. 7. Swanson, C.A.; Reamer, D.C.; Veillon, C.; Levander, O.A. J. Nutr. 1983, 113, 793. 8. Frew, N.M.; Leary, J.J.; Isenhour, T.L. Anal. Chem. 1972, 44, 665; 9. Janghorbani, M.; Young, V.R. Am. J. Clin. Nutr. 1980, 33, 2021. 10. Janghorbani, Μ.; Young, V.R.; Gramlich, J.W.; Machlan, L.A. Clin. Chim. Acta. 1981, 114, 163. 11. Janghorbani, M.; Christensen, M.J.; Nahapetian, Α.; Young, V.R. Am. J. Clin. Nutr. 1982, 35, 647. 12. Carni, J.J.; James, W.D., Koirtyohann, S.R.; and Morris, E.R.; Anal. Chem. 1980, 52, 216.



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13. Harvey, B.R. Anal. Chem. 1978, 50, 1866. 14. Turnlund, J.R.; Michel, M.C.; Keyes, W.R.; King, J.C.; Margen, S. Am. J. Clin. Nutr. 1982, 35, 1033. 15. Yergey, A.L.; Vieira, N.E.; Hansen, J.W. Anal. Chem. 1980, 52, 1811. 16. Schwartz, R.; Giesecke, C.C. Clin. Chim. Acta, 1979, 97, 1. 17. Hachey, D.L.; Blais, J.C.; Klein, P.D. Anal. Chem. 1980, 52 1131. 18. Johnson, P.E. J. Nutr. 1982, 112, 1414. 19. Reamer, D.C.; Veillon, C. J. Nutr. 1983, 113, 786. 20. Swanson, C.A.; King, J.C.; Levander, O.A.; Reamer, D.C.; Veillon, C. Am. J. Clin, Nutr. 1983, 38, 169. RECEIVED January 31, 1984


MS Measurement of Stable Isotopes of Selenium - American

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