540 CUTHBERTSON, PEGLER, QUADLING AND HERBERT [Vol. 76 The Assay of Vitamin Part v Some Substances that Interfere with the B12 Response of an Escherichia coli Mutant to Vitamin B12 BY W. F. J. CUTHBERTSON, H. F. PEGLER, C. QUADLING AND VALERIE HERBERT (Presented at the meeting of the Biological Methods Group on Tuesday, December 19th, 1950) The effects of various substances on the Escherichia coli plate assay of vitamin B,, have been investigated and permissible limits have been defined. For satisfactory assay C must be less than KN, where C is the maximum permissible concentration of the interfering substance expressed as a per- centage, K is a constant depending on the nature of the interfering substance and N is the concentration of vitamin B,, in pg per ml. Some reagents increase while others decrease the apparent vitamin-B,, response.IN a preliminary communication1 from these laboratories, it was shown that a mutant of Escherichia coli could be used as an assay organism for the measurement of vitamin B12. A more detailed description of the precise procedure recommended is in course of preparation ; meanwhile it may be of assistance to others working in this field if we give an account of some essential preliminary work, for without its completion a routine method could not have been established. This work was concerned with the effect of various reagents on the response of the mutant (which we call variant M 200) when used in the cup-plate assay for vitamin BI2. The reagents tested were chosen for a number of reasons; ( a ) they mightSept., 19511 THE ASSAY OF VITAMIS Bit.PART V 541 TABLE I EFFECT OF INTERFERING AGENTS ON RESPONSE TO VITAMIN B,, Solutions containing 0.2 pg of vitamin B,, per ml Response decreased Added Apparent ethylene glycol, vitamin B,, content, A I \ % v/v Pg Per 0.0 0.3 25 0.168 50 0.145 75 0.133 Response increased CuS0,.5H20, vitamin B,, content, I h 7 Added Apparent PQ Per ml 0-2 %&+ 0.01 0-226 0.05 0-236 1.0 0.258 be used as preservatives for vitamin B,, concentrates, (b) they might be used in the preparation of extracts, and (c) they might be expected to replace vitamin B,, in the nutrition of the test organism. Different concentrations of the reagents studied were placed on the assay plates in absence or presence of the vitamin. When the reagents were tested in its presence the concentration of the vitamin used was always kept at 0.2 pg per ml, Le., the same as that TABLE I1 Substance VALUES OF CONSTANT, Qualitative action A r In absence of In presence of vitamin B,, vitamin BIZ Response Alcohol slight reduces response reduced Acetone nil 9 9 Ethylene glycol nil 9 9 Propylene glycol nil 3, Ascorbic acid inhibits at 5% 9 , Formalin, 400,6 inhibits at 0.2y0.91 v/\- Values of K for \. 2 per cent. error error 5 per cent. 5 12.5 0.2 0-5 1 2.5 0.00," 0.0053 0.5 1.0 10 30 Response Butanol unaltered Toluene at concentra- Sodium formate tions tested MnS0,.4H20 FeS0,.7Hz0 Choline Betaine nil nil nil below 0.204 stimulates at nil up to O.Olyo stimulates at stimulates at 0.5 9.6 ; inhibib above 1% nil up to 20% 0.003 to o*lyo 0.05 yo nil - - nil - - nil below 0.2% > 1 > 2 nil up to 1.0% > 5 > 10 nil up to 0.01% > 0.05 > 0.1 nil up to 0.5% > 2-5 > 5 nil up to 0.5% > 2.5 > 5 nil up to 20% > 100 > 100 Response Methionine stimulates causes faint very very increased outer growth variable, variable,.zones - 0.007 - 0.015 Thioglycollic acid inhibits above increases response 0.5 1.7 0.5% Phenol inhibits above 5% 9 9 CuS04.5H,0 stimulates at Sodium chloride no effect below 3, o*oo5~0 10 to 20% 0.25 0-5 0.005 0.015 10 20 t o 40 Effect variable Homocystine stimulates at variable 0.2 0-5 0.2:; ; inhibits a t 1 t o 2% 0.2 to 0.5 0.2 to 0.5 Potassium inhibition above inhibition of cyanide 0.1% growth Cyanide in concentrations above 0.1 per cent. causes inhibition and prevents growth over the whole plate.542 NOAKES: THE DETERMINATION OF SELENIUM [Vol.76 used in the provisional assay technique. In all experiments the growth zones were compared qualitatively with those caused by the vitamin at concentrations of 0.02 and 0.2 pg per ml on the same plate. If the test mixtures produced growth qualitatively similar to that obtained with the vitamin alone then the diameters of the growth zones were measured and the apparent concentrations of vitamin B,, in the test mixtures were calculated from them. In this way the effect of interfering agents on the apparent activity could be determined. In some instances the interfering agent caused an apparent decrease in response, in others an increase. Typical examples of each kind of effect are shown in Table I.From these and similar results it is possible by graphical interpolation to determine the permissible concentration of the different substances allowable for a specified error. In the assay procedure used all solutions are diluted to approximately 0.2 pg per ml before assay ; clearly, the concentration of interfering agent producing a given error increases with the concentration of the original vitamin B,, solutions. Thus the allowable concentration of interfering substances may be expressed in the form C = KN per cent. where C is the permitted concentration in the test solution expressed as a percentage, N is the vitamin B,, concentration (in pg per ml) and K is a constant depending on the interfering substance. In Table I1 are given the values of K at levels leading to a 2 per cent. or a 5 per cent. error. This table shows whether the interfering agents cause an increase or a decrease in the apparent vitamin-B,, activity of the solution; the effects of the agents in the absence of the vitamin are also shown. A number of amino-acids have been tested by Bessell and Lees, to determine whether they interfere in this assay. Histidine, arginine and isoleucine tested at a level of 5 mg per ml were found to promote growth corresponding to 0.002, 0.001 and less than 0.005 pg of vitamin B,, per ml, respectively; none of these substances at this concentration interfered with the assay of solutions containing more than 0-2 pg of vitamin B,, per ml. REFERENCES 1. 2. GREENFORD, MIDDLESEX Bessel, C. J., Harrison, E., and Lees, K. A., Chem. and Ind., 1950, 561. Bessel, C. J., and Lees, K. A., private communication. GLAXO LABORATORIES LTD.