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plication with an empirical factor. For antimony (both isotopes) this factor is 1.923, for gold it is 1.119. Values for 76As,'"Ag, "Mo(Tc), 72Ga,and 75Seare 1.652, 1.535, 1.802, 1.300, and 1.413 respectively. We carried out a flux determination run simultaneously utilizing Fe-Ru couples and cadmium measurements on gold. T h e resonance flux is definitely found lower from the gold data. Concentration calculations from the Fe-Ru flux data (and using empirical correction factors for isotopes with large R ratios) were better than on the basis of cadmium-ratio and -difference measurements on gold; however, our experience with cadmium measurements is far too limited for any sweeping statements. We do not claim that determination of the actual epithermal flux distribution via the high-lying resonances of the three ruthenium isotopes yields necessarily a more accurate value than can be obtained via the low-lying resonances of lS7Au. We have, however, developed an internally consistent approach which yields the correct concentration values for all elements. Parametric counting provides an easy way of determining molybdenum or certain rare earths in the presence of uranium, utilizing the known relative fission yields for the isotopes in question.
ACKNOWLEDGMENT We are indebted t o Norman Holden and others a t Brookhaven National Laboratory and to Ronald Fleming of the National Bureau of Standards for very helpful discussions.
Thanks are due to Richard Cashwell of the Nuclear Engineering Department a t the University of Wisconsin for performing the irradiations carried out there, and for permission to use his measuring equipment. Appreciation is expressed to L.D. Blitzer, B. E. Prescott, and D. L. Malm for quantitative analysis of the Al(Au) and the iron foil monitors, and to A. M. Mujsce for the thermogravimetric analysis of the gelatin samples.
LITERATURE CITED P. F. Schmidt and D. J. McMillan, Anal. Chem., 48, 1962 (1976). D. H. Anderson, J. J. Murphy, and W. W. White, Anal. Chem.. 40, 116 (1 976). D. H. Anderson, J. J. Murphy, and W. W . White, Eastman Org. Chem. Bull., 49, l ( 1 9 7 7 ) . P. F. Schmidt and J. E. Riley, Jr., "Determination of the Natural Abundance of Iron-58 by Neutron Activation Analysis", Anal. Chem.,see paper in the Aids for Analytical Chemists section. P. F. Zweifel, Nucleonics, 18, ( l l ) , 174 (1960). R. Kirkland, Georgia Institute of Technology Research Reactor, private communication. K . W. Geiger and L. van der Zwan, Mefrologia, 2, 1 (1966). T. B. Ryves and E. B. Paul, J . Nucl. Energy, 22, 759 (1968). T. 8. Ryves, Metrologia, 5 , 110 (1969). P. Schumann and 0. Albert, Kernenergie, 8, 88 (1965). J. W . Connolly, A. Rose, and T. Wall, AAECITM 191 (1963). T. Bereznai, D. Baizs, and G. Keomley. J . Radioanal. Chem., 36,509 (1977). T. Bereznai and T. D. MacMahon, University of London Reactor Centre, Research Report, August 1977.
RECEIVED for review May 17, 1978. Accepted November 3, 1978.
Comparison of the Determination of Cobalamins in Ocean Waters by Radioisotope Dilution and Bioassay Techniques G. M. Sharma," Henry R. DuBois, Albert T. Pastore, and Stephen F. Bruno New York Ocean Science Laboratory, Montauk, New York
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Application of two radioisotope dilution techniques to the direct determination of cobalamins in 1 mL of seawater is described. These techniques give results which are approximately 4-10 times higher than the results obtained by the standard microbiological assays. The disparity in results obtained by the biological and isotopic methods is explained by suggesting that the later technique measures both biologically active and inactive cobalamins indiscriminately. The combined sensitivity range of the two isotopic methods described in this paper is 0.5-400 pg B,JmL. The accuracy ( X , k)of individual values at the 1.0 pg/mL level was found to lie in the range of 0.1-0.2 P9.
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T h e concentration of cobalamins in ocean waters is usually measured by microbiological techniques ( I ) . Although these techniques are highly sensitive (range: 0.05-3 ng B,,/L) they are technically tedious and time consuming. Furthermore, the precision of the bioassays depends largely upon the presence or absence of inhibitors in the samples being analyzed. Because of these drawbacks of the biological methods, there is need for a technique for the determination of cobalamins in seawater which should not only be rapid and highly sensitive but must also be immune to the presence of 0003-2700/79/0351-0196$01 OO/O
inhibitors in the medium. Several radioisotope dilution techniques developed for the determination of vitamin B12 in plasma have been shown to be endowed with these characteristics (2, 3). Recently one of these techniques has been used for the determination of benzyl alcohol extractable cobalamins from ocean waters ( 4 ) . In this paper, we report our experience and results obtained in applying two radioisotope dilution techniques to the direct determination of cobalamins in a small volume (1.0 mL) of ocean waters. The two isotopic methods tested for the determination of cobalamins in ocean waters are called competitive binding and sequential saturation analysis ( 5 , 6). The B,,-specific binder used in the experiments was the porcine intrinsic factor (IF). Comparison of these IF-based radioassays with a standard biological assay ( I ) revealed that the former techniques give substantially higher values for the concentration of cobalamins in ocean waters. One explanation of unexpectedly higher values obtained by isotopic methods would be that these techniques measure the sum of the concentration of all cobalamin and cobalamin-like molecules. The cobalamin-like molecules may have been produced by environmental degradation of various naturally produced BI2 molecules.
EXPERIMENTAL Apparatus. Radioactivity was measured with a Beckman Gamma 8000 Counting System. This instrument has a factory c 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2 , FEBRUARY 1979
installed program to give % bound radioactivities directly. Reagents used in the experiments were pipetted with a Schwarz/?vlann biopipet. Polypropylene tubes (12 X 75 mm), logit-log graph papers, and a Cornwall 2 cm3 syringe for adding aqueous charcoal suspension were purchased from BectonDickinson Co., Mountain View Ave., Orangeburg, N.Y. 10962. Whatman GF/C filter pads and 0.45-pm membranes for filtering seawater were obtained from VWR Scientific Company and Amicon Company. Chemicals. Norite A (decolorizing charcoal) for stripping seawater and Human Serum Albumin (HSA) of cobalamins was purchased from Fisher Scientific Co. The unlabeled vitamin B12 was from Sigma Chemical Co. Lyophilized porcine intrinsic factor (IF),RIA grade charcoal, and aqueous solutions of j'Co-labeled Bl, containing 0.01'7~KCN were purchased from Becton and Dickinson Co. The concentration of KCN in labeled B,, solutions was considered to be sufficient to release cobalamins from endogenous protein binders when mixtures of 200 pL of labeled-B12 and 1.0-mLaliquots of the unknown samples were heated at 100 "C for 15 min. The 30% sterile HSA solution was purchased from Calbiochem, San Diego, Calif. 92112. Reagents. Vitamin-freeSeauater. One liter of seawater was obtained from the same area where samples to be analyzed were collected. This seawater was made vitamin-free by shaking with h'orite A as described in the literature ( 7 ) . Vitamin-free HSA. T o 3.0 mL of 30% HSA placed in a well cleaned plastic tube, 24 mL of st.erile distilled water and 3.0 mL of 0.48 N HCl were added. The resulting solution was heated at 100 "C for 15 min. The reaction mixture was brought to room temperature and 0.5 g of Norite A added to it. The suspension was vortexed and left at room temperature for 30 min. After centrifugation,the clear HSA solution was decanted and filtered through a 0.22 pm niillipore filter. The filtrate was stored aseptically a t 4 "C. Standard tests revealed that the 3 7 ~HSA solution thus prepared contains neither vitamin B12 nor B,, binding factors. Churcoal Susprn,>ion. The RIA grade charcoal, 1.25 g, was added to 100 mL of sterile distilled water. The suspension was stirred a t 4 "C for 30 min before and during use. The unused suspension was stored at 4 "C for later use. 0.01 N KCiV arid 1 h' HL'I. These were prepared from reagent grade KCN and concentrated hydrochloric acid using sterile distilled water. The solutions were stored in glass bottles at room temperature. C'nlubeled B,, Stundurd Solutiona. Serial dilutions of stock solution containing 100 mg B,,/L with sterile vitamin-free seawater gave working solutions containing 0.5-400 pg Bl,/ niL. The concentration of the stock solution was routinely checked by measuring extinctions at 278, 361, and 550 nm. Competitive Binding Experiments. ( A )Preparation uf t h e Standard Curce. For duplicate runs, 18 sequentially numbered polypropylene tubes were arranged in pairs. One milliliter of sterile vitamin-free seawater was added to each tube of pairs 1 and 2 . Using standard solut ions. progressively increasing amounts ( 5 , 10, 20, 50, 100, 200, and 400 pg) of unlabeled B,, were added to the tubes of pairs 3--9. Then 200 pL (50pg) of labeled B,, and 100 p L of 1 N HCl were added t u all tubes. After mixing, the tubes were allowed to stand at room temperature for 15 min. Final reagents to be added were: 400 GL of HSA to all tubes and 200 p L of intrinsic factor tu pairs 2 9 only. After centrifugation, the supernatants which contained only cornplexed B,, were decanted and counted in a Beckinan Gamma 8000 counting system to give 7'~ bound radioactivities directly. The logit-log plot of % bound radioactivities vs. pg unlabeled B,,jmL was a straight line (slope: -2.30; j-intercept: 4 20). 'l'he correlatiuri coefficient fur the regression line was 0.99. In a separate experiment, the contents of the tubes after the addition of 1 N HCI and labeled B,, were heated at 100 "C for 15 min and then reacted Lvith the intrinsic factor. The standard curve constructed frvm the data of this experiment was identical with the one obtained in the first experiment. ( B )Determinution of C'obuictr~unsirz Seawater by C'oniprtitiue Binding 'Z'ec,hriiyue. 'I'ht-se deteriniiiations were carried out simultaneously with the preparation of the standard curve. 'The seawater samples were cullected from the Peconic Bay estuary, Long Island, N.1'. T h e sanlphg atations were located throughout
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the estuary ranging from the mouth of the Peconic River (samples 1 and 2, Table I) to Cedar Point, East Hampton (samples 3-10). The samples were first filtered through GF/C filter pads and then through 0.45-pm Aminco filter membranes. One-milliliter aliquots of the filterates, 100 yL 1 N HCl and 200 yL labeled BIZwere placed in two sets of paired polypropylene tubes. After mixing, one set of tubes was kept at room temperature and the other set was heated a t 100 "C for the same period of time. The set heated at 100 "C was cooled to room temperature in a running water bath. Same amounts of HSA and intrinsic factor as used in the preparation of the standard curve were added to the tubes of both sets. After an incubation period of 30 min, '70radioactivities bound to the intrinsic factor were determined and the concentrations of cobalamins were read from the standard curve. Columns 1 and 2 of Table I represent the results of duplicate analyses of 10 seawater samples carried out by incubating the samples with cyanide ions (present in labeled BIZ)and HC1 at room temperature and 100 "C, respectively. Sequential Saturation Analysis. ( A )Standard Curfie. To obtain data in duplicate, 16 sequentially numbered polypropylene tubes were arranged in sets of two. One milliliter of vitamin-free seawater was added to each tube of sets 1 and 2. Using standard solutions, progressively increasing amounts of unlabeled BI2(0.5, 1,2,4, 6, 8, 12, and 15 pg) were added to the tubes of the remaining sets. Then 50 yL of 0.01% KCN and 200 pL of HSA solutions were added to all tubes. After mixing, the tubes were allowed to stand at room temperature for 15 min. Intrinsic factor, 50 pL, was added to tubes of sets 2-8 only and, after mixing, the tubes were incubated at room temperature for 30 min. Finally, 200-pL aliquots of "Co-BI2 solution (50 pg, cpm = 6200) were added to all tubes and, after mixing, the contents of the tubes were incubated at room temperature for 45 min. The uncomplexed vitamin B,, was removed by adding 0.8 mL of charcoal suspension as described under competitive binding studies. After centrifugation, the supernatants which contained both labeled and unlabeled vitamin BIZbound to the intrinsic factor were decanted and the bound radioactivities measured. The bound counts in supernatants of tubes 1 and 2 were averaged and subtracted from the bound counts of supernatants of all other tubes. The plot of corrected bound counts (ordinate) vs. pg-unlabeled BI2/mL (abscissa) was a straight line (slope: -126. cpm/pg B,,; y-intercept: 3495 cpm). The absolute value of the slope of the regression line showed a high degree of agreement with the specific activity (124 cpm/pg) of labeled BIZused in the experiment. (B)Determination of Cobalamins in Seau'ater Samples by Sequential Saturation Analysis. Seawater samples were diluted 1:10 with vitamin-free seawater. Aliquots, 50 pL, of the intrinsic factor were first reacted with 1.0-mL aliquots of the diluted samples and then with 200 pL of the 57Co-B,,solution according to the procedures described under preparation of the standard curve. Radioactivities bound to the intrinsic factor were measured and the concentrations were determined by interpolation of the standard curve. The results were multiplied by 10 to express data as amounts per mL of the undiluted samples (See Table I, column 3).
Bioassays of Seawater Samples. The 10 seawater samples analyzed by isotopic methods were also bioassayed using clones 3H and 13-1 of the diatom Thalassiosira pseudorianu. The procedure used was essentially the same as described by Carlucci ( 1 ) . Glassware used for bioassays was cleaned in 3 N HC1, rinsed with distilled water, baked in a hot air oven at 250 "C for 4 h, and autoclaved for 20 min a t 121 "C. Vitamin free seawater was prepared according to method described by Strickland and Parsons (7). Samples for B,, (and B12analogue) assay were dispensed in 6- or 10-mL aliquots into 50-mL microfernbach flasks (Bellco Inc.) with stainless steel caps. Each sample was diluted with 10 mL of vitamin free seawater (27-29%0) enriched with nutrients as described by Carlucci ( I ) . External standards (calibration curve) for the bioassay contained 0, 0.1,02, 0.4, 0.8, 1.0, 2.0, and 3.0 ng L of vitamin BIZ. Vitamin BIZwas added to duplicate sets of each sample, resulting in an addition of 1 ng L-'. These flasks served as internal standards to determine possible inhibition of the seawater to the assay organism. Flasks with samples, external standards, and internal standards were inoculated with =IO4 cells mL ' of B,? depleted T. pseudonana cells.
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The bioassay series was incubated in a temperature controlled room at 20 i 1 "C below a bank of fluorescent lamps emitting 6000 lux. The assay was terminated after 5 days and vitamin B12 (and B12analogue) concentrations were calculated on the basis of final cell density. Cell counts were made using a hemacytometer. The results of the analysis are collated in columns 4 and 5 of Table I.
RESULTS AND DISCUSSION T h e determination of vitamin B12by radioisotope dilution techniques has invariably been carried out in 0.9% NaCl solution (8). T h e salinity of seawater, on the other hand, lies in the range 2.8-3.570. Major contribution to this salinity comes from chloride, sulfate, magnesium, potassium, calcium, and sodium ions. T o evaluate the effect of the complex salt matrix of seawater on the precision and accuracy of isotopic methods, standard solutions of B12 prepared in charcoaltreated seawater were analyzed by competitive binding procedures (4). When experimentally determined 70-IF-bound radioactivities were plotted vs. pg BlZ/mL on a logit-log graph paper, a linear standard curve of slope -2.30 was obtained. The close correspondence of the slope of the experimental line to the theoretical value of -2.303 suggested (9) that the inorganic constituents of seawater do not interfere with the determination of vitamin B12by the isotopic method. At an average standard deviation of 1070,the detection limits of the competitive binding procedure were found to lie in the range 10-400 pg B12/mL. The relative standard deviation at 5 pg/mL was -30%. T h e competitive binding experiments designed to detect vitamin Blz at levels below 5 pg/mL were found to yield erratic results. T o develop a procedure capable of measuring the concentrations of B12at levels below 10 pg/mL, standard solutions containing 0.1-50 pg Blz/mL were analyzed by the techniques of sequential saturation analysis (6). After several trials, the procedure described in the Experimental section was found to give IF-bound radioactivities which were inversely but linearly related to the concentration of B12in the range 0.5-15 pg/mL. T h e regression line of IF-bound radioactivities, (M, vs. the concentration of Blz, (X), was almost identical with the theoretical curve given by the equation y = Bo - s X . In this equation, Bo represents cpm bound to the intrinsic factor when the concentration of the unlabeled Blz was zero and the slope, s, is the specific activity of labeled B12 used in the experiment. The correlation coefficient for the regression line was 0.99. By combining the results of the competitive binding and sequential saturation analysis, it may be concluded that seawater samples containing >0.5 pg cobalamins/mL can be directly analyzed for the concentration of this micronutrient by isotopic methods. In Table I, the results of the determination of cobalamins in 10 seawater samples by competitive binding, sequential saturation, and bioassay procedures are compared. Columns 1 and 2 of the table represent the data of the competitive binding experiments carried out by incubating the samples with KCN (present in the j7Co-labeled B12solution) at room temperature and at 100 "C, respectively. T h e samples were heated with KCN a t 100 "C to free cobalamins from endogenous protein binders. For sequential saturation analysis, the samples were diluted 1:lO with charcoal-treated seawater. The results were multiplied by 10 to express data in amounts per mL of the undiluted samples (column 3). T h e clone 3H of the bioassay organism Thalassiosira p s e u d o n a n a measures the sum of the concentrations of 5,6-dimethylbenzimidazole cobalamin (vitamin B12), desdimethylbenzimidazolecobalamin, 5-hydroxybenzimidazole cobalamin, and 5-methylbenzimidazole cobalamin. Under the assay conditions, the clone 13-1 of T . p s e u d o n a n a measures the sum of 100% concentration of cobalamins listed above, 50% concentration of Factor B (BIZwithout the nucleotide),
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Table I. Comparison of the Results of Cobalamin Determinations of Seawater Samples by Isotopic and Bioassay Techniquesa isotopic methods bioassay ______ T competitive binding sequenT. pseudotial satu- pseudo- nana, sample room temp. 100 "C ration kana, 3H 13-1 no. 6.4 11.2 1 41.5 56.0 39.5 4.8 10.4 52.0 38.0 46.0 53.0 2 13.8
