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ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
Competitive Intrinsic Factor Binding Assay Technique for Cobalamins in Natural Waters Robert A. Beck Department of Chemistry, Framingham State College, Framingham, Massachusetts 0 170 1
A competitive intrinsic factor binding (CIFB) method for the determination of 1O-'* g/mL concentration levels of benzyl alcohol extractable cobalamins in natural waters has been developed. Purified cobalamin extracts assayed by the CIFB protocol were 76.9 % of those concentrations determined for the same samples using high pressure liquid chromatographic analysis, and demonstrated a 10.98% relative standard deviation for a series of 32 CIFB assays. Application of the CIFB technique to natural waters indicated a sensitivity ranging from 9.98- 187.9 8 pg/mL.
T h e environmental occurrence of cobalamins in natural waters may be significantly implicated with the productivity of phytoplankton and dinoflagellates ( I , 2 ) . Traditional methods for the routine assessment of cobalamins in environmental systems have relied on bioassays ( I - T ) which are tedious and may be potentially inaccurate in the milieu of biochemically complex samples (8, 9). A more direct analysis of environmental cobalamins using high pressure liquid chromatography (HPLC) has been developed for the quantitation of 1.0-10.0 wg of benzyl alcohol extractable cobalamins (IO). Although the HPLC method has a n analytical capability for the assay of cobalamin-rich environmental samples, this method clearly lacks the required sensitivity for determining pg/mL concentrations which normally occur in natural waters. Because of the limitations of both bioassay methods and the HPLC technique, a more sensitive assay method for benzyl alcohol extractable cobalamins in natural waters has been developed which utilizes the basic principles involved in the measurement of an endogenous substrate by competitive protein binding. For this analytical method, the thermolabile glycoprotein, intrinsic factor (IF),serves as a nondiscriminative binding reagent for both radioactive 57Co-cyanocabalamin (vitamin BL2)and nonradioactive cyanocobalamin as well as its derivatives. Consequently, in the case of a fixed concentration of radioactive cyanocobalamin, an inverse relationship is observed between the amount of radioactivity bound to IF and increasing concentration levels of nonradioactive cyanocobalamins. This principle permits the determination of cyanocobalamin concentrations in samples ranging from 50-1600 pg/mL. Clinical applications of this competitive intrinsic factor binding (CIFB) technique have been widely reported ( I 1-13) but the reliability of the method for quantitating environmental cobalamins has not been described.
EXPERIMENTAL Reagents. All reagents for the CIFB assay of cobalamins including 57Co-cyanocobalamin, IF, and hemoglobin coated charcoal were obtained from RIA Products, Inc., Waltham, Mass. Standard concentration levels of cyanocobalamin used for HPLC quantitation of cobalamin extracts were prepared from cyanocobalamin obtained from Sigma Chemical Co., St. Louis, Mo. All other reagents, including benzyl alcohol, chloroform, sodium cyanide, sodium chloride, and sodium sulfate, were analytical reagent grade. 0003-2700/78/0350-0200$0 1.OO/O
Instrumentation. Radioassay of 5'Co-cyanocobalamin, bound to IF during the CIFB protocol, was determined by using a Beckman LS-1OOC Liquid Scintillation Counting System under conditions detailed previously by Gutcho et al. (14). Low potassium glass scintillation vials were used in conjunction with a cocktail of Ready-Solve GP obtained from Beckman Instruments, Irvine, Calif. Procedure. Eualuation of CIFB Assay Technique. Evaluation of the CIFB assay technique for quantitation of environmental cobalamins was based on the preparation of cobalamin-rich extracts derived from activated sewage sludge (10). The cobalamin extracts were purified by triplicate acetone precipitations ( 1 5 ) prior to quantitative HPLC analysis ( 1 0 ) . These extracts were subsequently diluted with 0.970 saline solution, through a multiplicity of serial dilution schemes, to a speculated concentration of less than 1600 pg/mL. The CIFB assay for cobalamins was performed according to the protocol outlined by RIA Products, Inc. (16),which is essentially the method reported by Lau et al. (11) and others (12, 17). All quantitative assays of cobalamin extracts were based on duplicate assays of eight replicative cyanocobalamin standards representing seven different concentrations ranging from 0 to 1600 pg/mL. On the basis of these data, a standard regression line plot of logit B / B o values (ordinate) vs. the log of standard cyanocobalamin concentrations (abscissa) was determined. The logit B / B o values were calculated for standard and unknown cobalamin concentration levels using the standard radioassay formula of In {(B/Bo)/(l-B/B,)J,where B is the counts per minute (cpm) observed for ' Co-cyanocobalamin bound to IF during the radioassay and Bo represents the cpm for 57Co-cyanocobalamin bound at the "zero" cyanocobalamin concentration level. Assay of Total Cobalamins in Natural Waters. Vitamin BI2 related cobalamins in natural waters were prepared for CIFB assays by mixing a water sample with sodium nitrite and sodium cyanide in a ratio of 0.5:0.2 g/lOO mL and the pH was adjusted to 4.0 using concentrated hydrochloric acid. The sample was then boiled for 15 min under a well ventilated hood, sodium sulfate (20% w/v) was dissolved in the sample, and it was subsequently extracted three times with one-tenth volume of benzyl alcohol. The combined benzyl alcohol extracts were mixed with one-half volume of chloroform and the resulting solvent phase was extracted three times with one-tenth volume of water (IO). As previously reported, radioactive 5'Co-cyanocobalamin tracer studies have indicated cobalamin extraction efficiencies of 92.3 to 802% for natural water samples having respective volumes of 100 and 500 mL (10). Emulsions generated during the course of the previous extraction steps were easily eliminated by centrifugation for 10 min at 2500 X g. After concentrating cobalamins in a combined aqueous phase, this sample was further concentrated by lyophilization, only when necessary, to achieve a preferred analytical cobalamin concentration of 200-800 pg/mL. The salinity of the aqueous cobalamin extract was adjusted to 0.970 by adding sodium chloride and a 200-pL aliquot of this solution was assayed by the CIFB technique as previously described. Because of the analytical sensitivity of this procedure, all portions of the assay must be carried out in scrupulously clean, acid washed glassware.
RESULTS AND DISCUSSION Quadruplicate HPLC analyses of three independently prepared cobalamin extract preparations from activated C 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
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Table I. Quantitative Comparison of Cobalamin Extract Concentrations Based o n HPLC Analyses and CIFB Assays Competitive intrinsic factor binding assay data for samples Calculated concentrations Percent of for a series of cobalamin Cobalamin calculated concenNo. of Re1 std dev tration based on rich extracts based on concentration HPLC analysis (mean pg/mL i 2 s ) assays ( n ) (percent i 2 s ) HPLC analyses 549.1 436.6 314.0
415.69 336.67 244.41
i i i
24.85 21.66 18.76
8
16 8
8.63 t 4.23 13.25 I4.59 11.08 5 5.43
75.7 77.1 77.8
Table 11. Quantitative Comparison of Cobalamin Concentration Levels in Filtered and Unfiltered Natural Water Samples Based on CIFB Assays Filtered natural water samples’ Unfiltered natural water samples Mean cobalamin Mean cobalamin concentration No. of Re1 std dev, 7% concentration No. of Re1 std dev, % Sample origin (pg/mL i 2 s ) assays ( n ) (mean i 2 s)b (pg/mL F 2 s ) assays ( n ) (mean 1 2 s l b Universitv of Massachusetts Marine Field Station, Rockport, Mass. 37.91 i 1.84 7 6.54 i 3.42 19.40 i 2.21 7 15.39 i Boston Edison Nuclear Power Plant Breakwater, 8 15.02 i 7.36 9.98 i- 0.91 8 9.35 * Plvmouth. Mass. 30.55 i 3.18 Harior Breakwater Plymouth, Mass. 187.98 i 52.54 12 34.93 i 19.76 33.40 li 2.49 8 10.75 i Sudbury Dam, Fayville, 8 12.37 k 6.06 10.33 i 1.24 8 12.26 z Mass. (oligotrophic reservoir) 34.47 i 2.95 Hager Pond, Marlborough, Mass. (eutrophic pond) 61.26 5.05 8 11.90 i 5.83 18.81 i 0.80 8 4.35 t Treated sewage effluent, Easterlv U‘ater Pollution Controi Facility, Marlborough, Mass. 67.92 i 5.03 8 10.69 i 5.24 16.42 i 1.58 8 13.86 i a Samples were filtered through 0.22 Wm Millipore G.S. filters. Between assays reported for each set of samples. sewage sludge were determined to have concentrations of 10.98 h 0.24, 8.73 i 0.19, and 6.27 f 0.11 pg/lO pL. A series of variable dilution schemes for each extract, effecting a dilution of 1:20 000, resulted in respective anticipated cobalamin concentrations of 549.10, 436.60 and 314.00 pg/mL. The CIFB assays for these samples accordingly resulted in concentrations of 415.69 24.85, 336.67 21.66, and 244.41 f 18.76 pg/mL (Table I). These concentrations represented 76.9 % of the respective cobalamin concentrations calculated for t h e same samples based on HPLC analyses and demonstrated a 10.98% mean relative standard deviation for 32 samples assayed. Moreover, studies of t h e same cobalamin extracts conducted by a reference laboratory (RIA Products, Inc., Waltham, Mass.) resulted in concentration levels 90.3 f 9.6% of t h e values reported in Table I. I n addition to the results outlined above, a high degree of comparability was noted for regression line plots of logit B / B o values (ordinate) vs. the log of concentrations (abscissa) for both cyanocobalamin standards (slope: -2.15; y-intercept: 6.13) and benzyl alcohol extractable Cobalamins (slope: -2.55; y-intercept: 7.17) over a concentration range of 50-1600 pg/mL. The correlation coefficient for both regression lines was 0.99. Although benzyl alcohol extractable cobalamins exhibited a statistical logit plot which was almost identical to that calculated for a series of standard cyanocobalamin concentrations, the fact remains that CIFB assays for the cobalamin extracts averaged 76.9% of those levels calculated for the same samples using HPLC analysis. A conclusive rationale for this disparity has not been formulated, but it has been widely recognized t h a t analogues of t h e basic cyanocobalamin structure are distributed widely in nature. Furthermore, many of these analogues are devoid of biological and clinical activities associated with cyanocobalamin. On this basis it is tempting to speculate that a portion of the purified cobalamin
*
*
8.06
4.58 5.27 6.01 2.13
6.79
extracts studied here may consist of a minor cobalamin fraction which exhibits less quantitative interaction with IF than other cobalamins more closely reminiscent of the cyanocobalamin structure. T h e total cobalamin concentrations determined for unfiltered freshwater and ocean water specimens varied from 30.55-187.98 pg/mL depending on the origin of t h e sample (Table 11). R a t e r samples found to have the highest concentrations were obtained from a eutrophic freshwater pond (Hager Pond, Marlborough, Mass.) and ocean water sampled 800 m from a treated sewage effluent discharge point (Plymouth Harbor Breakwater, Plymouth, Mass.). The mean relative standard deviation for cobalamin concentrations in all unfiltered water samples was 15.24%. The 34.93% relative standard deviation displayed by Plymouth Harbor Breakwater samples was attributable to high population numbers of zooplankton and phytoplankton suspended in the water column a t the time of sampling. As a result of cobalamins bound to microorganisms and other organic material, all water samples filtered through a 0.22-pm Millipore GS filter (Millipore Corp., Inc., Bedford, Mass.) consistently displayed smaller cobalamin concentration levels than unfiltered water samples (Table 11). Bioassays have previously suggested (18)that up to 25.0 pg of free cobalamins contained in a water sample may be bound to this type of mixed cellulose acetate and nitrate filter. However, radioactive 5’Co-cyanocobalamin tracer studies conducted in this investigation demonstrated a maximum binding of only 16.0 pg. Therefore, all cobalamin concentrations reported for filtered water samples in Table I1 have been tabulated in view of this consideration. I t should also be recognized that all concentrations detailed in Table I1 were based on the analysis of aqueous cobalamin extracts in the concentration range of 200-800 pg/mL. This analytical range was preferred since logit plot regression lines
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
may be statistically dubious at higher or lower concentrations. For those instances where natural water samples failed to provide an aqueous cobalamin extract with a minimum 200 pg/mL concentration, the extract was lyophilized to dryness and reconstituted to a smaller volume with 0.9% saline solution prior to CIFB analysis. Aside from the fact that the CIFB assay method is less time consuming and tedious than bioassays, it permits the analysis of cobalamins in a more representative size water sample and substantially eliminates interference of noncobalamin compounds in quantitative assays through implementation of selective benzyl alcohol extraction and concentration procedures. Moreover, the CIFB assay method described in this paper provides a means for evaluating cobalamin concentrations in natural waters on a routine basis using a sensitive analytical approach other than traditional bioassays.
ACKNOWLEDGMENT Thanks are expressed to Charles Zapsalis and Richard F. Milaszewski for helpful discussions.
LITERATURE CITED (1) L. Provasoli, "Algal Nutrition and EvbophicakJn" in "Eutrophication: Causes, Consequences, Correctives", Proceedings of a Symposium, National
Academy Science, Washington, D.C., 1969, pp 574-593. J. Cairns, G. R. Lanza, and B. C. Parker, "Pollution Related Structural and Functional Changes in Aquatic Communities with Emphasis on Freshwater Algae and Protozoa", Proc. Acad. Nat. Sci. Philadelphia, 124 (5),79 (1972). A. F. Carlucci and S.B. Silbernagel, Limnol. Oceanogr., 11, 642 (1966). S. R. Hoover, L. Jasewicz, J. B. Pepinsky, and N. Porges, Sewage Ind. Wastes, 24, 38 (1952). H. Y. Neujahr, Acta, Chem. Scand., 9, 622 (1955). R. Strohecker and H. M. Henning, "Vitamin Assay-Tested Methods", Chemical Rubber Co. Press, Cleveland, Ohio, 1965. W. H. Sebreli and R. S.Harris, "The Vitamins", Vol. 11, Academic Press, New York, N.Y., 1968, pp 120-258. E. L. Smith, "Vitamin B,,", John Wiley and Sons, New York, N.Y., 1960. W. Shive, J. M. Ravel, and W. M. Harding, J. Bbl. Chem., 176, 991 (1948). R. A . Beck and J. J. Brink. Environ. Sci. Techno/.. 10. 173 (1976). K-S. Lau, C. Gottlieb, L. R. Wasserman, and V. Herbert, Blood, 26, 202 (1965). L. Wide and A. Killander, Scand. J . Ciin. Lab. Invest., 27, 151 (1971). C. Rosenbium, Talanta, 11, 255 (1964). S. Gutcho, J. Johnson, and H. McCarter, Clin. Chem. ( Winston-Salem, N.C.),19, 998 (1973). H. H. Fricke, U.S. Patent 2,582,589 (1952). RIA Products, Inc., "In Vitro Quantitative Measurement of Serum Vitamin B,, by Radioassay", Waitham, Mass., 1977. Y . K. Liu and L. W. Sullivan, Blood, 39, 426 (1972). 6.C. Parker, J . Phycoi., 5 , 124 (1969).
RECEIVED for review September 14,1977. Accepted November 21, 1977.
Determination of Chlorine Dioxide in Sewage Effluents J. Ross Knechtel" Wastewater Technology Centre, Burlington, Ontario L4R 4A6
Edward G. Janzen and Edward R. Davis University of Guelph, Guelph, Ontario N 1G 2 W 1
A spectrophotometric method is developed for the determination of chlorine dioxide in sewage treatment plant effluents. The decrease in absorbance at 550 nm of acid chrome violet K (ACVK) enables the direct spectrophotometric determination of C102 in sewage effluent samples. Centrifugation is employed to remove suspended solids. I n a NH4CI-NH3 buffer of pH 8.1 to 8.4, no Interference from active chlorine, hypochlorites, chlorltes, chloramines, and nitrites was observed. The results obtained using the ACVK technique were verified against electron spin resonance spectrometry.
T h e subject of this paper is the use and measurement of chlorine dioxide in sewage treatment plant effluents. There were three good reasons for the consideration of chlorine dioxide as an alternative to chlorine ( I ) . In the first place, chlorine dioxide is a much stronger oxidizing agent than chlorine. Because of this, a smaller dosage of chlorine dioxide than chlorine should be possible for disinfection purposes. Because a smaller dosage is possible, a lower residual could result. The second reason for the choice of chlorine dioxide over chlorine was that chlorine dioxide does not react with ammonia to form chloramines as does chlorine ( 2 ) . The reaction of chlorine with ammonia does two things. It reduces the oxidizing power of the oxidant and produces a toxic by-product. The third reason for the choice of chlorine dioxide was that it oxidizes the phenolic ring in phenols ( 3 ) ,whereas, 0003-2700/78/0350-0202$01 .OO/O
chlorine forms phenolic chlorides when it combines with phenols. This could produce taste and odor problems in potable water supplies. Several published methods (4-9) exist for C102 determination in water but not for sewage effluents. These techniques are not entirely specific for C102 because the measurement includes other chlorine species ( 7 ) . Modifications in some of these procedures are suggested to mask or remove these effects. In the effluent disinfection project itself, the chlorine dioxide residues were measured amperometrically (9, IO) using phenylarsineoxide (PAO) as titrant. The chlorine dioxide values reported were not verified by a referee method. Because of the uncertainties of this technique and the results, work was undertaken to examine an alternative method. The method selected (8)t o measure chlorine dioxide depends on the selective decolorization of a dye (acid chrome violet K). The relationships between the level of chlorine dioxide initially present and the amount of decolorizing which occurs obeys Beer's law up to 200 wg C102. The maximum absorbance of the complex formed occurs a t 550 nm. A study was undertaken to investigate the feasibility of using the acid chrome violet K technique for the measurement of C102 in sewage effluent samples. The results obtained on sewage effluent samples using this technique were verified using an electron spin resonance method. The chlorine dioxide molecule is the only stable chlorine species which contains an unpaired electron and which is capable of existing in
e 1978 American Chemical Society
