6 26
Stable Mg for Dietary Magnesium Availability Measurements RUTH SCHWARTZ Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853
Magnesium has only one short l i v e d radiotracer Mg (T 1/2=21.3 h ) . Thus stable Mg (11.01%) offers not only a radiation-free alternative to Mg, it can be used as a second, non-decaying tracer in single and dual tracer studies on magnesium metabolism. So f a r , only a fraction of the potential of Mg has been r e a l i z e d . Magnesium-26 can be analyzed by neutron activation analysis (NAA) as well as by various mass spectometric techniques. Mass spectrometers which are most accessible to investigators in the b i o l o g i c a l sciences utilize electron impact ionization (EIMS). This has been the preferred a n a l y t i c a l technique for Mg in n u t r i t i o n a l studies to date since it is more sensitive and precise than NAA. The usefulness of Mg is limited by its r e l a t i v e l y high natural abundance. Magnesium-26 doses needed for its accurate detection in blood or tissues are too large for injection into the c i r c u l a t i o n . For t h i s reason, research using Mg has been limited to studies of Mg absorption and b i o a v a i l a b i l i t y . Magnesium-26 has been intrinsically incorporated into leafy vegetables. These were subsequently labelled e x t r i n s i c a l l y with Mg and tested for isotopic exchangeability in rats and in human subjects. In addition, true Mg absorption was measured by simultaneous administration of Mg (orally) and Mg (intravenously). The r e l a t i v e merits and limitations of Mg and Mg are discussed with emphasis on t h e i r utilization in dual tracer protocols. 28
26
28
26
26
26
26
28
26
28
26
28
Magnesium is the fourth most abundant element in the body and, after potassium, the most abundant i n t r a c e l l u l a r cation. I t is a co-factor or participant in numerous b i o l o g i c a l and physiological processes, encompassing such diverse functions as hydrolysis and 0097-6156/ 84/0258-0077S06.00/0 © 1984 American Chemical Society
78
STABLE ISOTOPES IN NUTRITION
s y n t h e s i s of ATP and a l l enzymatic r e a c t i o n s t h a t depend on ATP, maintenance of membrane i n t e g r i t y , c o n t r o l of muscle tone and c o n t r a c t i o n , and the f u n c t i o n i n g of the nervous system ( 1 ) . De s p i t e the w e l l e s t a b l i s h e d importance of magnesium in l i v i n g systems, i n f o r m a t i o n is scant on i t s v a r i o u s r o l e s in the body and t h e i r r e l a t i o n s h i p s t o h e a l t h and d i s e a s e . The Recommended D i e t ary Allowances formulated by the N a t i o n a l Research C o u n c i l have i n c l u d e d recommendations f o r magnesium s i n c e 1968 C2)» but the l e v e l s recommended are s t i l l under debate ( 3 ) . There are few r e l i a b l e i n d i c a t o r s of m a r g i n a l magnesium s t a t u s , and l i t t l e is known w i t h c e r t a i n t y of the adequacy of magnesium in normal d i e t s . One of s e v e r a l f a c t o r s t h a t have impeded r e s e a r c h on magne sium metabolism is l a c k of a s a t i s f a c t o r y i s o t o p e . The s h o r t l i v e d r a d i o t r a c e r M g (T^ - 21.3h) (A, 5) has been used in i n t a c t animals and man, p r i m a r i l y , t o estimate r a p i d l y exchanging magne sium pools ( 6 - 1 1 ) . Such s t u d i e s , which seldom exceed 24-48 hours in d u r a t i o n , have y i e l d e d u s e f u l i n f o r m a t i o n in magnesium-depleted p a t i e n t s before and a f t e r magnesium therapy Ç1Q,11). However, even w i t h h i g h i n i t i a l doses of M g that a l l o w r a d i o a c t i v i t y measurements to be continued f o r up to s i x days, l e s s than 20% of body magnesium can be accounted f o r by i s o t o p e d i l u t i o n Ç9) · The short h a l f l i f e of M g f u r t h e r l i m i t s e s t i m a t i o n s of magnesium a b s o r p t i o n which may r e q u i r e f e c a l c o l l e c t i o n s f o r longer p e r i o d s than those o p t i m a l f o r M g d e t e c t i o n (9,12,13) by methods other than whole body counting (13). C l e a r l y , a second magnesium i s o tope, not l i m i t e d by r a p i d decay, would be u s e f u l in r e s e a r c h on magnesium metabolism. The f o l l o w i n g is an examination of the p o t e n t i a l of the s t a b l e i s o t o p e M g (11·01%) as an a l t e r n a t i v e and complement to M g . While the second minor i s o t o p e of magnesium, M g 0-0.00%), has a lower n a t u r a l abundance, M g has the advantage of being de~ t e c t a b l e by a n a l y t i c a l techniques that are c u r r e n t l y a c c e s s i b l e to i n v e s t i g a t o r s in b i o m e d i c a l f i e l d s (.14-16). These techniques have been p u b l i s h e d elsewhere (14,15) and w i l l not be d e s c r i b e d in d e t a i l here except where such d e t a i l s serve to u n d e r l i n e or i l l u s t r a t e the c e n t r a l theme of t h i s r e v i e w : to compare and c o n t r a s t t r a c e r c a p a b i l i t i e s of M g and M g . 2 8
2 8
2 8
2 8
2 6
2 8
2 5
2 6
2 6
Methods f o r the D e t e c t i o n of
2 8
2 6
Mg
The most accurate and p r e c i s e method f o r i s o t o p e r a t i o measure ments of metals a v a i l a b l e at present is thermal i o n i z a t i o n mass spectrometry (TIMS). The technique has been used s u c c e s s f u l l y f o r isotope d i l u t i o n measurements of magnesium Ç17), but no t r a c e r s t u d i e s w i t h magnesium isotopes u s i n g TIMS have been p u b l i s h e d to date. Thermal i o n i z a t i o n mass spectrometry is f a i r l y time c o n suming, even w i t h r e c e n t l y developed automated instruments (18). T h i s f a c t o r must be weighed a g a i n s t the s u p e r i o r p r e c i s i o n of TIMS, however, which g r e a t l y reduces the need f o r m u l t i p l e r e p l i cates. By f a r the g r e a t e s t d é t e r r a n t to the use of TIMS in in
6.
SCHWARTZ
79
Dietary Mg Availability Measurements
v i v o t r a c e r s t u d i e s w i t h s t a b l e i s o t o p e s t s the cost of the in struments which are seldom a c c e s s i b l e t o i n v e s t i g a t o r s in b i o m é d i c a l research u n i t s . Two methods t h a t have been used in t r a c e r s t u d i e s w i t h % g are neutron a c t i v a t i o n a n a l y s i s (NAA) and EIMS, mass spectrometry of a v o l a t i l e c h e l a t e w i t h e l e c t r o n impact i o n i z a t i o n 015,19,20). N e i t h e r technique can match TIMS in accuracy or p r e c i s i o n , but both may o f f e r g r e a t e r sample throughput. U n t i l more p r e c i s e techniques become more g e n e r a l l y a v a i l a b l e , r e s e a r c h u t i l i z i n g s t a b l e metal i s o t o p e s w i l l remain l i m i t e d to the c o n d i t i o n s i m posed by the a n a l y t i c a l f a c i l i t i e s a t hand. As w i l l be shown below, research w i t h s t a b l e magnesium i s o t o p e s to d a t e , has had to be t a i l o r e d to the l i m i t a t i o n s of a v a i l a b l e a n a l y t i c a l techniques. 2
Neutron A c t i v a t i o n A n a l y s i s . Magnesium-26 has a s m a l l c r o s s s e c t i o n of 0.03 b . The product of i r r a d i a t i o n w i t h thermal neutrons is M g (T^ * 9.5m). As shown in Table 1, s e v e r a l elements com monly present in b i o l o g i c a l m a t e r i a l s g i v e r i s e to r a d i o a c t i v e n u c l i d e s w i t h r a d i a t i o n s at energy l e v e l s c l o s e to those c h a r a c t e r i s t i c of M g . Neutron a c t i v a t i o n was used in the f i r s t t r i a l s of M g as an in v i v o t r a c e r when measurements were made w i t h a w e l l - t y p e N a l - T l c r y s t a l d e t e c t o r ( 1 4 , 2 1 ) . Under these c o n d i t i o n s the presence of sodium, aluminum and manganese in the samples in t e r f e r e d in the accurate d e t e c t i o n of M g , but c o u l d be reduced or e l i m i n a t e d by sample p u r i f i c a t i o n . 2 7
2 7
2 6
2 6
Table I . Metal 26
NAA C h a r a c t e r i s t i c s of Natural Abundance %
0.038
A1 Na
100 100
0.235 0.400
*°Ar Mn
99.6 100
0.610 13.3
2 7 2 3
5 5
1
M g and Common Contaminants
Cross S e c t i o n (b)
11.01
M g
2 6
Product Nuclide 27
Kajor Energies CMéV)
1
M g
9.5m
A1 *Na
2.2m 15h
^Ar Mn
1.8h 2.6h
2 8 2l
5 6
0,84 C70), 1.013 1.78 (100) 1.37 (100), 2.75 1.29 (100) 0.847 ( 9 9 ) , 1.81
I r r a d i a t i o n w i t h thermal neutrons
Samples were p u r i f i e d before a c t i v a t i o n u s i n g the s o l v e n t e x t r a c t i o n procedure of Hahn et a l . (22) w i t h t h e n o y l t r i f l u o r o a c e t o n e as the l i g a n d . An a c t i v a t i o n r o u t i n e was developed to a v o i d subse quent contamination w i t h aluminum, to a l l o w the excape of ^ A r , to monitor f l u x v a r i a t i o n s , and to f a c i l i t a t e spectrum s t r i p p i n g of r e s i d u e s of manganese, aluminum, and sodium (15). The advent of h i g h e f f i c i e n c y germanium d e t e c t o r s has s i g n i f i c a n t l y reduced the
80
STABLE ISOTOPES IN NUTRITION 5 6
i n f l u e n c e of contaminants other than M n , which has a f a r longer h a l f l i f e than M g . C o r r e c t i o n s f o r M n can be made by repeated counting a f t e r M g has decayed to non-detectable l e v e l s . Neutron a c t i v a t i o n a n a l y s i s has low s e n s i t i v i t y f o r the de t e c t i o n of M g . The best r e s u l t s were o b t a i n e d , in our hands, by i r r a d i a t i n g samples c o n t a i n i n g 200-300 yg n a t u r a l magnesium (2035 yg M g ) f o r one minute at a neutron f l u x of 1 0 c m " s e c followed by d e t e c t i o n w i t h a HP-Ge d e t e c t o r . Since it is u s u a l l y necessary t o run a c t i v a t i o n s in t r i p l i c a t e and to c a r r y out a d d i t i o n a l analyses f o r t o t a l magnesium by an independent method, such as atomic a b s o r p t i o n , NAA is not s u i t a b l e f o r specimens c o n t a i n i n g magnesium at low c o n c e n t r a t i o n s . I t was found s u i t a b l e f o r the analyses of f e c a l samples, m a r g i n a l f o r u r i n e and u n s u i t a b l e f o r plasma ( 1 5 , 2 1 ) . 2 7
5 6
2 7
2 6
26
1 2
2
- 1
Mass Spectrometry w i t h e l e c t r o n impact i o n i z a t i o n (EIMS). Mass spectrometers w i t h e l e c t r o n impact i o n i z a t i o n are w i d e l y used in b i o m e d i c a l r e s e a r c h , p r i m a r i l y f o r i d e n t i f i c a t i o n and q u a n t i t a t i o n of o r g a n i c molecules. A d a p t a t i o n of EIMS to i s o t o p e r a t i o mea surements of m i n e r a l s has, t h e r e f o r e , c o n s i d e r a b l e appeal f o r in v e s t i g a t o r s i n t e r e s t e d in u s i n g s t a b l e m i n e r a l i s o t o p e s as b i o l o gical tracers. Since EIMS is s u i t a b l e o n l y f o r r e l a t i v e l y v o l a t i l e mate r i a l s , most metals must be converted t o v o l a t i l e c h e l a t e s before they can be analysed by t h i s technique. The magnesium c h e l a t e found most amenable to EIMS is the d i k e t o n a t e M g ( 2 , 2 , 6 , 6 ' - t e t r a m e t h y l - 3 , 5 - h e p t a n e d i o n e ) (Mg(THD) ) (19). The c h e l a t e can be formed in aqueous s o l u t i o n s at pH >9 in the presence of excess THD (16). E x t r a c t i o n i n t o e t h y l ether provided a simple method f o r s e p a r a t i n g the c h e l a t e from excess THD which otherwise i n t e r f e r e s in the MS a n a l y s i s of Mg(THD) . The ether l a y e r was t r a n s f e r r e d to a 10 ml g l a s s tube and allowed to stand at room temperature. As the ether evaporated, c r y s t a l s of Mg(THD) were deposited on the middle and upper i n s i d e surfaces of the tube, l e a v i n g a THD l a y e r in the w e l l . The l a t t e r was a s p i r a t e d and washed out w i t h 2 s u c c e s s i v e 200-300 y l a l i q u o t s of methanol. The c r y s t a l s were r e d i s s o l v e d in methanol at a c o n c e n t r a t i o n of 1-4 mg m l " Mg(THD) . Recovery of Mg as the c h e l a t e is about 25-45%. Reproducible mass s p e c t r a could be obtained by i n t r o d u c i n g 2-4 yg of the c h e l a t e by d i r e c t probe i n t o a F i n n i g a n 3300 quadrupole mass spectrometer u s i n g a h e a t i n g r a t e of 200° per hour (16). Under these c o n d i t i o n s , the most abundant i o n peaks seen were those of MgTHD w i t h peaks at m/z 207, 208, 209 corresponding to M g , M g , and M g r e s p e c t i v e l y . Carbon-13 c o n t r i b u t i o n s from *MgTHD r a i s e the r e l a t i v e i n t e n s i t y at m/z 208 to about t w i c e the l e v e l t h a t can be accounted f o r by the n a t u r a l abundance of M g , w h i l e the peak at m/z 209 is increased by l e s s than 8% by l i g a n d c o n t r i b u t i o n s of C and 0 . The r e l a t i v e i n t e n s i t i e s de termined in standards of known i s o t o p e c o n c e n t r a t i o n s d e v i a t e d somewhat from t h e o r e t i c a l v a l u e s and v a r i e d from day to day. Conf
2
2
2
2
1
2
+
2if
2 5
2 6
2
2 5
1 3
1 8
6.
SCHWARTZ
Dietary Mg Availability Measurements
81
sequently, a l l i s o t o p e r a t i o measurements in unknown m i x t u r e s were based on standard determinations i n c l u d e d in each set of a n a l y s e s .
Table I I .
Comparison of NAA and EIMS Analyses f o r
yg Mg per r e p l i c a t e D e t e c t i o n l i m i t , % excess of n . a . % p r e c i s i o n at d e t e c t i o n l i m i t Maximum p r e c i s i o n , (% S . D . ) 1
1
Z b
Mg.
NAA
EIMS
100 15-20 26 3-4
0.1-0.2 5 18 0»5
I n excess of n a t u r a l abundance
Table I I summarizes the d i f f e r e n c e s in EIMS and NAA f o r the d e t e c t i o n of M g in b i o l o g i c a l m a t e r i a l s . Sample p r o c e s s i n g f o r EIMS a n a l y s i s is s i m p l e r and more r a p i d ' t h a n t h a t in p r e p a r a t i o n f o r NAA. The a b s o l u t e s e n s i t i v i t y is increased by about two orders of magnitude, and p r e c i s i o n by a f a c t o r of 2 - 3 . Consequently, lower l e v e l s of M g excess can be detected w i t h confidence ( 1 6 ) . T h i s comparison suggests t h a t EIMS can be a p p l i e d to M g measurements in speciments l i k e l y to be low in t o t a l magnesium and M g excess, e . g . u r i n e and plasma. U n f o r t u n a t e l y , these e x p e c t a t i o n s were not fully realized. The comparisons summarized in Table I I do not take i n t o account the i n f l u e n c e of i n s t r u m e n t a l memory, a f a c t o r t h a t was observed d u r i n g e a r l i e r t r i a l s (15,16) but became i n c r e a s i n g l y troublesome as the volume of analyses i n c r e a s e d . The e f f e c t of memory can be minimized by grouping sample s e t s w i t h i n incremental ranges of 10-20% in excess of n a t u r a l abundance, and by adapting the mass spectrometer w i t h standards enriched w i t h M g to the l e v e l s a n t i c i p a t e d in a given s e r i e s of a n a l y s e s . Four to s i x r e p l i c a t e s were analysed r o u t i n e l y , but the number of r e p l i c a t e s needed was greater w i t h samples h i g h l y enriched w i t h M g . Theo r e t i c a l l y , the p r e c i s i o n w i t h which i s o t o p e excess is measured in creases w i t h i n c r e a s i n g l e v e l s of enrichment. Because of the memory problem, p r e c i s i o n expressed as % standard d e v i a t i o n was best at enrichment l e v e l s of 20-35% in excess of n a t u r a l abun dance. At these l e v e l s the r e l a t i v e standard d e v i a t i o n f o r r e covery of added M g was 3-5% of the mean. At higher enrichment l e v e l s NAA may provide b e t t e r p r e c i s i o n than EIMS. B u t . as w i l l be shown below, most enrichment l e v e l s encountered in °Mg t r a c e r s t u d i e s are l e s s than 40% in excess of n a t u r a l abundance. I t must be s t r e s s e d that both methods have d e f i c i e n c i e s . U n t i l b e t t e r techniques become a v a i l a b l e the method chosen by i n v e s t i g a t o r s w i l l continue to depend on the f a c i l i t i e s a v a i l a b l e to them. 2 6
2 6
2 6
2 6
2 6
2 6
2 6
2
82
STABLE ISOTOPES IN NUTRITION 26
P o t e n t i a l Uses o f M g as an a l t e r n a t i v e o r Complementary Tracer to 28Mg The obvious advantages of a s t a b l e t r a c e r over a s h o r t - l i v e d r a d i o a c t i v e i s o t o p e are l a c k of r a d i a t i o n hazards, f l e x i b i l i t y in t i m i n g of experiments independantly o f i s o t o p e production scher* d u l e s , and the p o s s i b i l i t y of s t o r i n g f o r i n d e f i n i t e p e r i o d s m a t e r i a l s enriched w i t h the t r a c e r . These f e a t u r e s add p o s s i b i l i t i e s t o r e s e a r c h t o magnesium metabolism that could not be c o n s i ^ dered when M g was the only a v a i l a b l e t r a c e r i s o t o p e , i f M g can, in f a c t , r e p l a c e o r complement M g as a t r a c e r . The extent to which t h i s is p o s s i b l e w i l l be examined by c o n s i d e r i n g the major uses of m i n e r a l t r a c e r s in metabolic research f o r : k i n e t i c s t u d i e s , determination of a b s o r p t i o n p a t t e r n s , measurements of t r u e a b s o r p t i o n , endogenous f e c a l e x c r e t i o n , and b i o a v a i l a b i l i t y from d i e t a r y sources. 28
26
28
K i n e t i c S t u d i e s . Magnesium-26 has a r e l a t i v e l y h i g h n a t u r a l abundance and thus does not s a t i s f y one o f the major requirements f o r a t r u e t r a c e r : t h a t it be d e t e c t a b l e in systems t o which it has been added in amounts that do not measurably a l t e r the steady s t a t e . Table I I I shows t h a t M g and M g can, t h e o r e t i c a l l y , be detected w i t h f a i r l y comparable p r e c i s i o n in the plasma o f a man f o r 12-24 hours a f t e r an i n j e c t i o n of 20 y C i M g o r 20 mg M g . However, w h i l e 20 y C i o f v i r t u a l l y c a r r i e r - f r e e M g have no d i s c e r n i b l e e f f e c t of plasma magnesium p o o l s , 20 mg M g are l i k e l y to i n c r e a s e t o t a l plasma magnesium by 25-35% and unbound magnesium by about 80%. K i n e t i c a n a l y s i s based on the r e s u l t i n g plasma i s o t o p e curve would l e a d t o erroneously h i g h estimates of ex changeable magnesium pools. C l e a r l y , M g is not s u i t a b l e f o r k i n e t i c s t u d i e s o r any other purpose such as e s t i m a t i o n of endo genous f e c a l magnesium f o r which it must be administered by i n j e c tion. 28
26
28
26
28
26
28
Table I I I .
Plasma Isotope Concentration a f t e r an I.V. of 20 y C i M g or 20 mg M g 28
28
Time (h) % Dose L "
1
cpm
1
26
Mg
4.6 1.7 1.1 0.9 0.7 0.5
4020 1300 850 500 300 90
Mg
% S.D. % e
0.5 4 8 12 24 48
Injection
Zb
