ISSN:0003-2654    

DOI:10.1039/AN95176FX009

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176BX011

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176FP021

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176BP027

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

MARCH, 1951 Vol. 76, No. 900 THE ANALYST EDITORIAL A REFERENCE BOOK FOR ANALYSTS FOR many years a growing need has been felt by analysts engaged in industry and private practice for a reliable guide to the best standardised methods for the determination of specified constituents in a wide and ever-widening range of materials. To tell the searcher after information of this kind to go to a library and look it up is no solution to the problem; for the search usually creates the further difficulty of choosing from an unwieldy mass of references the method most likely to be best suited to the problem in hand; specialised knowledge combined with critical ability is necessary for a successful solution of the problem. To meet this need, the Analytical Methods Committee of the Society and a band of enthusiastic and devoted helpers have drawn up lists of tried and tested methods for the analysis and testing of some hundreds of commercial products, ores, metals and miscellaneous materials.These have now been collected and published in the form of a book, under the auspices of the Society, with the title “Bibliography of Standard, Tentative and Recommended or Recognised Methods of Analysis.” The book conforms in size and style of cover to the usual bound volumes of The Analyst, to the files of which it is expected to prove an indis- pensable addition. It is sold at a price that is causing considerable anxiety to our Honorary Treasurer-and one that bears no relationship to its real value. As knowledge accumulates and new and approved methods are devised for the deter- mination of this, that and the other constituent of a sample, further editions of this work will doubtless become necessary and desirable. In the hope that any future edition shall be of even more use to analysts than is the present volume, the compilers welcome suggestions for better methods than those now listed, or additions to the present list; these should be sent to the Honorary Secretary of the Analytical Methods Committee, at the Society’s office. The Analyst accords a hearty welcome to this new companion to its file of volumes in the full assurance that it will soon become an indispensable and a valued helper in furthering the improvement and perfection of analytical chemistry. 125

ISSN:0003-2654    

DOI:10.1039/AN9517600125

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

126 PROCEEDINGS [Vol. 76 PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS DEATHS regret to record the deaths of Kenneth Stuart McManus Frederick William Richardson Frank Robert Stephens William Heaton Thorns. SCOTTISH SECTION THE Sixteenth Annual General Meeting of the !Section was held in Glasgow on January 24th, 1951, and the following office bearers were‘ elected for the year:-Chairman-Mr. H. C. Moir. Vice-Chairman-Mr. R. S. Watson. Hon. Secretary and Treasurer-Mr. J. A. Eggleston, Boot’s Pure Drug Co., Ltd., Motherwell Street, Airdrie, Lanarkshire. Elected Committee Members-Messrs. A. R. Campbell, A. Dargie, H. Dryerre, J. M. Leitch, M. Pyke and J. Sword. Hon. Auditors-Messrs. J. Andrews and J. Gray. MICROCHEMISTRY GROUP THE Seventh Annual General Meeting of the Microchemistry Group was held at the Sir John Cass College, London, E.C.3, on Friday, January 26th, 1951.The following Officers and Committee Members‘ were elected for the ensuing year :-Chairman-Dr. Cecil L. Wilson. Vice-Chairman-Dr. A. M. Ward. Hon. Secrdary-Mr. Donald F. Phillips, 10, Richmond Road, Blackpool, N.S., Lancs. Hon. Treasurer-Mr. Gerald Ingram. Elected Committee Members-Dr. W. T. Chambers, Dr. J. G. A. Griffiths, Dr. G. F. Hodsman, Mr. R. F. Milton, Mr. G. H. Osborn and Dr. J. Sandilands. After an interval for tea a Symposium on “Radiochemical Techniques in Micro- chemistry” was presented by members of the staff of the Atomic Energy Research Establish- ment, Harwell, at which the following papers were read: “The Quartz Ultramicrobalance in Radiochemistry,” by Dr.J. K. Dawson ; “Micromanipulation of Radio-active Gases,” by Dr. W. J. Arrol; “The Determination of Trace Quantities of Elements by Radio-activation,” by Mr. A. A. Smales. An exhibition of microchemical apparatus, arranged by Messrs. R. F. Milton and D. W. Wilson, was held in the Microchemical laboratory; 14 members and firms contributed a very wide range of apparatus, consisting almost entirely of items not previously exhibited. PHYSICAL METHODS GROUP THE Sixth Annual General Meeting of the Group was held at 6 p.m. on Tuesday, November 28th, 1950, at the Iron and Steel Institute, 4, Grosvenor Gardens, London, S.W.l. Mr. B. S. Cooper, the Chairman of the Group, presided. The Group Officers and Elected Members of the Committee for the forthcoming year are lis follows:-Chairman-Mr.B. S. Cooper. Vice- Chairman-Dr. W. F. Elvidge. Hon. Secretary-Mr. R. A. C. Isbell, Hilger & Watts Ltd., Hilger Division, 98, St. Pancras Wav, Iandori, N.W.l. Membevs of Committee-Messrs. C. H. R. Gentry, J. Haslam, G. H. Osborn, J . E. Page, A. A. Smales and F. R. Williams. Hon. Auditors-Messrs. C. A. Bassett and TI C. Garratt (re-appointed). Votes of thanks were accorded to tiw two retiring members of the Committee, Messrs. L. A. Haddock and J. A. C. hlcClelland, for tlieir services during the past two years, and to the Hon. Auditors for their work during the past year. The Annual General Meeting was followed by the Twenty-eighth Ordinary Meeting of the Group, at which Dr. H. Irving delivered a lecture entitled “A Mid-Century Review of Physical Methods of Analysis”; this was followed by a discussion.March, 19511 ANALYTICAL METHODS COMMITTEE i87 BIOLOGICAL METHODS GROUP THE Sixth Annual General Meeting of the Group was held a t 6.15 p.m.on Tuesday, December 19th, 1950, in the rooms of the Chemical Society, Burlington House, Piccadilly, London, W.l. Mr. N. T. Gridgeman was in the Chair. The following were elected as Officers and Members of the Committee for the forth- coming year :-Chairman-Mr. N. T. Gridgeman. Vice-Chairman-Dr. H. 0. J. Collier. Hon. Secretary-Mr. S. A. Price, Walton Oaks Experimental Station, Vitamins Ltd., Dorking Road, Tadworth, Surrey. Elected Committee Members-Dr. W. F. J. Cuthbertson, Professor C. H. Gray, Dr. L. J. Harris, Dr. J. Raventos, Dr. G. F. Somers, Mr. G. A. Stewart and Ex-oficzo Members, the President, Honorary Treasurer and Honorary Secretary of the Society and the Editor of The Analyst. Hon. Auditors-Mr. D. M. Freeland and Dr. J. H. Hamence. The Annual General Meeting was followed by an Ordinary Meeting at which the following papers were presented and discussed: “Assessment of ACTH Activity,” by I. D. K. Halkerston and M. Reiss; “EugZena gradis as an Assay Organism for Vitamin BIT,” by G. E. Shaw; “The Effect of Interfering Agents on the Vitamin B,, Plate Assay (E. colz Mutant Method),” by W. F. J. Cuthbertson, Valerie Herbert, H. F. Pegler and C. Quadling.

ISSN:0003-2654    

DOI:10.1039/AN9517600126

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

March, 19511 ANALYTICAL METHODS COMMITTEE i87 Analytical Methods Committee REPORT PREPARED BY THE ANEURINE PANEL OF THE SUB-COMMITTEE ON VITAMIN ESTIMATIONS The Chemical Assay of Aneurine in Foodstuffs THE Analytical Methods Committee has received from its Sub-committee on Vitamin Estimations the following Report based on the work of its Aneurine Panel. The Report has been approved by the Analytical Methods Committee and its publication authorised by the Council. INTRODUCTION The following workers assisted in the preparation of this Report: E. R. Dawson (Chairman), F. Wokes (Secretary), D. C. M. Adamson, R. G. Booth, W. F. Elvidge, G. E. Foster, J. Greenbaum, R. A. C. Isbell, J. King, W. Martin, F. W. Norris, T. L. Parkinson, S. A. Price, H. N. Ridyard and S. Y . Thompson. Assistance in some of the investigations was also given by D.J. Finney, J. Houston, B. C. P. Jansen, G. F. Lothian, H. Monk, F. Clermont Scott and Miss S. Weiner. The Report is concerned with the chemical assay of aneurine in certain specified classes of foodstuffs, namely: cereals and cereal products; malt and malted products; yeast and yeast products; meat and meat extracts. This Report deals with the determination of aneurine by the thiochrome method. In this method the aneurine is oxidised by alkaline ferricyanide to thiochrome, which fluoresces in ultra-violet light. Under standard conditions the net fluorescence of the thiochrome is directly proportional to its concentration over a given working range. The method originated with Jansen,l and has undergone numerous modifications.Many of these are described in “Methods of Vitamin Assay,”2 which was found by the Panel to be specially helpful in devising the fluorimetric method described below. This method was based on much experimental work too lengthy to describe in detail. MAIN POINTS STUDIED Extraction-The Panel experimented with digestion with pepsin, papain, “Taka-diastase” and “Clarase,” combined with extraction with diluted hydrochloric acid, sulphuric acid or sodium acetate solution. Purification of the extract-Washing with isobutanol was found to be less effective than adsorption on base-exchange silicate for removal of interfering substances, as shown by the fluorescence of the blanks and the percentage recoveries of added aneurine. The recovery128 ANALYTICAL METHODS COMMITTEE : THE CHEMICAL v o l .76 could vary widely for a given sample according to the level of addition of ane~rine.~ This led the Panel to adopt a cautious attitude to the use of recovery methods to correct for the presence of interfering substances. Oxidation of aneurine to thiochrome-The use of a premixed alkaline ferricyanide solution gave more uniform results than separate additions of alkali and ferricyanide. Water- saturated isobutanol was found more satisfactory than dry isobutanol for extracting the thiochrome from the aqueous reaction mixture. A limit has been suggested for the amount of non-specific fluorescence in the isobutanol. Measurement of juorescence and calculation of results-Calculation of the net fluorescence is preferred to the use of a calibration curve.This calculation facilitated measurement of the fluorescence of the blanks, and was found to be effective with fluorimeters of the null-point type, at present mainly used in this country, as well as with fluorimeters of the direct-reading type. RESULTS OBTAINED BY THIS RECOMMENDED METHOD Table I summarises results obtained by the collaborating laboratories on samples of malt extract, wheat germ, wheat flour, yeast extract and dried yeast. As samples of malted barley and of malt and oil had previously been found to give satisfactory results with the assay methods applied to malt extract, they were not included-in the series. All the laboratories agreed in finding no significant amount of aneurine in the meat extract sample, which is therefore not included in Table I.Eiecause of the difficulties that the Panel had encountered when assaying yeast preparations, three samples of dried yeast differing widely in potency were examined. Table I1 contains results obtained for the samples by biological and microbiological methods. The results obtained by the proposed method showed a degree of agreement between different laboratories and with biological and inicrobiological results that seemed to justify the Panel recommending it as a method suitable for use by analysts to determine aneurine in the foodstuffs tested by the Panel. Further trials of the method are needed before it can be finally put forward as an official method of the Society. The Honorary Secretary of the Analytical Methods Committee (Dr.D. C. Garratt) will be pleased to receive comments and criticisms from any analyst using the method. The results in Tables I and I1 were obtained in the following laboratories: Boots Pure Drug Co., Nottingham; Cereals Research Station, St. Albans; Distillers Co., Ltd., Research Department, Epsom; the laboratories of D. W. Kent-Jones and A. J. Amos, London, W.5; Glaxo Laboratories, Greenford ; Government Laboratory, London, W.C. 1 ; Lyons Laboratories, London ; National Institute for Research in Dairying, Shinfield ; Nederlandsch Institut voor Volksvoeding, Amsterdam ; Ovaltine Research Laboratories, King's Langley ; Roche Products, Welwyn Garden City; Vitamins Ltd., Research Laboratories, London; Wellcome Chemical Works, Dartford. TABLE I COLLABORATIVE RESULTS BY THE RECOMMENDED METHOD AneuIine found, pg per g Malt Wheat flour Yeast f A \ Laboratory extract germ (85%) extract Sample 1 Sample 2 Sample 3 A 2.7 22 55 29 17 45 D 4.0 21 3.8 54 20 47 E 24 14 54 F 15 G 3.9 21 52 27 I 32 3.7 21 3-8 61 28 3.9 20 47 27 19 57 L M 3.5 23 3.8 39 N 3.6 37 47 Means 3-62 21.3 3.75 61.3 29.1 16.8 60 ~- A r -7 Wheat Dried yeast 16 k The data of Table I have been examined by Dr.E. C. Wood with a view to assessing the precision of the method. As the amount 'of aneurine in the various samples differed soMarch, 19511 ASSAY O F ANEURINE I N FOODSTUFFS 129 widely, it was first necessary to convert all the individual results into percentages of the mean result for each sample in turn. This has been done in Table IA, omitting references to particular laboratories.TABLE IA COLLABORATIVE RESULTS EXPRESSED AS PERCENTAGES OF THE CORRESPONDING MEAN RESULT Malt extract 74-7 110.6 107.9 102-3 107-9 90.8 Wheat germ 103.1 98-5 98.5 98-5 93.7 107.8 Wheat Yeast flour extract 101.3 107-1 101-3 105.2 101-3 101-3 96-0 118.8 91.6 76.0 7 Sample 1 99.5 82.4 92.6 109.8 96-1 92.6 127.0 Dried yeast A -l Sample 2 Sample 3 101.0 93.3 118.8 97.4 83-2 11 1.9 89.1 97.4 95.1 112.9 It is apparent that the agreement between laboratories is much better for some materials tested than for others. The coefficient of variation, which is the standard error expressed as a percentage of the mean, is in fact 4-9 per cent. for the two wheat products together, as compared with 13.5 per cent. for the four yeast products together.The appropriate test shows a high probability that this implies a real difference in the precision of the method when applied to these two classes of substance. The mean coefficient of variation for all the samples together is 11-9 per cent., though it is doubtful whether this pooling of all the results is justified. It is safer to say that when a sample of wheat flour or germ is being examined in different laboratories, 19 out of 20 of the results obtained should be found in the long run to lie within &lo per cent. of the mean for that particular sample; but if a yeast product is being examined the corresponding range will be &27 per cent. Agreement between replicate results in the same laboratory will of course be closer than this; the data available provide no evidence for assessing the “within laboratory” precision.TABLE I1 RESULTS BY BIOLOGICAL AND MICROBIOLOGICAL METHODS Aneurine found, pg per g Labora- tory Method used K Ratgrowth N Bradycardia P Ratgrowth F Yeast fermentation J Lactobacillus fermenturn Mean value of the colla- borative fluorimetric results (from Table I) r h -I Wheat Dried yeast A Malt Wheat flour Yeast f 7 extract germ (85%) extract Sample 1 Sample 2 Sample 3 20 3-9 21 3.3 45 27 38 17 64 4.1 23 68 3.62 21-3 3-75 51.3 29.1 16.8 50 RECOMMENDED METHOD APPARATUS- Base-exchange tubes-These are to be made of glass, the upper part being not less than 15 cm long and 0-8 to 1.0 cm in internal diameter and the lower part being narrow-bore tubing of suitable length. The lower end may be fitted with a tap or other method of con- trolling the rate of flow. A reservoir to contain at least 30 ml may be attached to the upper part.Oxidation vessels-These may be stoppered measuring cylinders, stoppered bottles, separating funnels or large boiling tubes of about 100ml capacity. Fluorescent grease must not be used to lubricate taps; glycerin or silicone may be used. Fluorimeter-This may be of the direct reading type, measuring in deflections, or the null- point type, measuring in densities.* The exciting radiation must be within the range 300 1 *Some null-point type instruments read also in “transmission.” Here transmission = density‘130 ANALYTICAL METHODS COMMITTEE : THE CHEMICAL vol. 76 to 400 mp; it is most conveniently obtained at suitable intensity by the use of a high-pressure mercury-vapour lamp, type MB, in conjunction with a primary filter.A secondary filter* transmitting mainly light between 400 and 450 mp is placed between the fluorescent solution and the photo-cell. REAGENTS- All chemicals should be of analytical reagenk quality or the purest otherwise obtainable. EthanoZ-Redistilled in all-glass apparatus. Sodium hydroxide, 15 per cent. solution-1)issolve 15g of sodium hydroxide in water and dilute to 100ml. Potassium ferricyanide, 1 per cent. solution-Dissolve 1 g of potassium ferricyanide in water and dilute to 100 ml. This reagent has been found to be stable for at least a week i f kept cool and in the dark, preferably in a brown bottle. Alkaline potassium ferricyanide solution-Dilute 3 ml of 1 per cent.potassium ferricyanide $0 100 ml with cool 15 per cent. sodium hydroxj.de solution. Hydrochloric acid , approximately 0.2 N soldution-Dilute 17 ml of concentrated hydro- chloric acid to 1 litre with water. Sodium acetate, 2.5 M solution-Dissolve 205 g of anhydrous sodium acetate (CH,COONa) or 340 g of CH3COONa.3H,0 in water and dilute to 1 litre. isoButyl alcohol (water-saturated)-Steam distil commercial isobutyl alcohol in an all-glass apparatus. The fluorescence of the distillate should be not more than that of a 1 in 100 .dilution in approximately 0.1 N sulphuric acid of the quinine standard (see p. 131), that is, of a diluted solution containing 0.01 pg of quinine sulphate per ml. Enzyme solution-Prepare a fresh solution daily from a suitable source of ph0sphatase.t Suspend, with thorough shaking, 6 g of the enzyme preparation in 2-5M sodium acetate solution and dilute to 100 ml with additional sodium acetate solution.Each batch of material used as a source of phosphatase should be tested for aneurine by this fluorimetric method and the necessary correction applied. Potassium chloride, 25 per cent. solution-Dissolve 250 g of potassium chloride in distilled water and dilute to 1 litre. The reagent is stable indefinitely. Acid potassizcm chloride solution-Dilute 0.5 ml of concentrated hydrochloric acid to 1 litre with 25 per cent. potassium chloride solution. The reagent is stable indefinitely. Base-exchange silicate-This consists of an. artificial zeolite in the form of a granular powder of 60 to 90 mesh size, tested for its suitability for adsorbing and eluting aneurine under the given conditions (at least 90 per cent."recovery" of aneurine should be obtained under the given conditions) .$ Activate the base-exchange silicate as follovvs: Place a convenient quantity (100 to 500 g) of the base-exchange silicate in a suitable beaker, add sufficient hot 3 per cent. acetic acid solution to cover the material and maintain the temperature a t about 100" C for 10 to 15 minutes, stirring frequently. Allow the mixture to settle and decant the supernatant liquid. Repeat the washing three times with hot 25 per cent. potassium chloride solution, and finally wash with boiling water until the last washing gives no reaction for chloride. Dry the material at approximately 100" C and store in a well-closed container.Stock aneurine soZution, 100 pg per ml-Prepare this from the British Standard Prepara- tion, or a sub-standard of equal purity. Dissolve a weighed amount of aneurine hydrochloride equivalent to 50 mg of the International Standard in sufficient 0.2 N hydrochloric acid to make 500 ml. This solution is stable for several months if stored in a refrigerator (i.e., below Standard aneurine solution-Dilute 5 ml clf stock aneurine solution, warmed to room temperature, to 100ml with water. Transfer 10ml of this dilution to a flask containing 200 ml of approximately 0.1 N sulphuric acid and 126 ml of sodium acetate solution and dilute to 250 ml with distilled water. The final concentration of aneurine is 0.2 pg per ml. This solution is stable for at least a week if stored in the refrigerator.Prepare just before use. 15" C). * Chance 0x1, 1-5 to 2.0 mm thick, has been found suitable for the primary filter, and Chance OB2 t Taka-diastase (diluted with lactose) (Parke Davis & Co., London) and Clarase (Takamine Laboratories, (blue), 1-6 to 2.0 mm thick, for the secondary filter. Clifton, N. J., U.S.A.), have been found suitable. Decalso F, supplied by the Permutit Co., Ltd., London, has been found suitable.March, 19511 ASSAY OF ANEURINE IN FOODSTUFFS 131 Stock quinine sulphate solution (100 %per mZ)-Dissolve 0.025 g of quinine sulphate, B.P., in sufficient 0.1 N sulphuric acid to make 250 ml. This solution is stable for twelve months if stored in a dark brown bottle at a temperature below 5” C.Qzhine standard (1 pg per mZ)-Dilute 10 ml of stock quinine sulphate solution to 1 litre with 0.1 N sulphuric acid. This solution is stable for three months if stored in a brown bottle at a temperature below 5” C. Any solution that has been exposed to ultra-violet light in the fluorimeter should be discarded. Acetic acid, 3 per cent. solution-Dilute 30 ml of acetic acid to 1 litre with distilled water. Bromocresol green indicator-Triturate 100 mg of bromocresol green with 7.2 ml of 0-06 N sodium hydroxide and dilute with sufficient water, free from carbon dioxide, to make 200 ml. Nitrogen gas in cylinders-If desired, an air current can be used instead. PROCEDURE EXTRACTION- (a) Sampling-The material to be assayed, if solid, should pass a No. 30 B.S.sieve or a finer sieve, and should be well mixed just before withdrawal of the sample, to ensure homogeneity. If liquid, the material should be well stirred before sampling. (b) Accurately weigh or pipette into a large boiling tube a sample (not more than 6 g) estimated to contain not more than 50 pg of aneurine. Add 65 ml of approximately 0.1 N hydrochloric acid or sulphuric acid. Digest the sample for 30 minutes in a bath of boiling water, with frequent mixing. The liquid must remain at a pH below 4.5 during the digestion. If a t the end of the digestion it is not distinctly acid to the bromocresol green indicator, the extract should be discarded and a further quantity of the sample extracted with more concentrated acid. (c) Cool the extract to below 50” C and adjust the pH to between 4 and 4.5 by addition of 2-5 M sodium acetate solution, using bromocresol green as external indicator.Add 5 ml of freshly prepared enzyme suspension, mix, and incubate at 45” to 50” C for 3 hours, or a t 37” C overnight with addition of a drop of sulphur-free toluene. (d) Cool to room temperature, centrifuge the mixture until the supernatant liquid is clear and quantitatively transfer the supernatant liquid to a 100-ml volumetric flask. Wash the residue by centrifuging successively with 10 ml, 10 ml and 5 ml of 0.1 N hydrochloric acid or sulphuric acid, Add the washings to the supernatant liquid and dilute the whole to 100 ml with water. (a) Plug the bottom of an adsorption column with glass wool, which should be lightly packed, and fill the column with 6 g of activated base-exchange silicate suspended in water.Allow the water to drain almost entirely, but leave enough to cover the base-exchange silicate, and pour in 5 ml of 3 per cent. acetic acid. Allow to drain as before. (b) Transfer 25ml of the “original extract” to the column by means of a pipette. Discard the filtrate that has percolated through the column. Wash the column with three successive portions, about 10 ml each, of boiling water and discard the washings. (c) After washing the column, pour through 10 ml of almost boiling acid potassium chloride solution from a supply kept hot in boiling water. Collect the eluate in a stoppered 25-ml graduated cylinder. Add a second 10-ml portion when all of the first portion has entered the base-exchange silicate and collect the eluate in the same cylinder.When this second portion has drained through, cool the eluate to room temperature, dilute to 25 ml with acid potassium chloride solution and mix well. This is the “sample eluate.” This is the “original extract.” PURIFICATION- OXIDATION TO THIOCHROME- source of ultra-violet light must be avoided. In this and all subsequent stages undue exposure of the solutions to direct daylighi or other (a) Pipette 5 ml of sample eluate into each of two oxidation vessels. (b) Start a stream of nitrogen or air bubbling through the solution in vessel number 1, add 5 ml of alkaline potassium ferricyanide solution and then add 25 ml* of water- saturated isobutyl alcohol, the current of nitrogen or air still being continued. Shake (or continue vigorous bubbling) for 90 seconds.* See footnote on p. 132.ANALYTICAL METHODS COMMITTEE : THE CHEMICAL [vol. 76 Start a stream of nitrogen or air bubbling through the solution in vessel number 2, then add 5ml of 15 per cent. sodium hydroxide solution, followed by 25 ml* of water-saturated isobutyl alcohol, then continue as in step (b). This is the “unknown blank.” ( d ) Repeat steps ( a ) , (b) and (c) with 5 ml of standard aneurine solution in place of the sample eluate. CAUTION. To avoid changes in experimental conditions the oxidation of all solutions used Similar precautions must be 132 (c) The solution from step (c) is the “standard blank.” ht a given assay should be carried out in irnmedide succession. taken in the reading of their JEzcorescence.SEPARATION OF THIOCHROME SOLUTION AND MEASUREMENT OF ITS FLUORESCENCE- ( a ) After the solutions have stood for a few minutes to allow complete separation, add 1 ml of ethanol to the upper layer in each vessel and stir the upper layer gently until it is clear, taking care to avoid disturbing the aqueous layer. (b) Take off each upper layer into a cuvette and measure its fluorescence against that of the quinine standard, if a null-point fluorimeter is being used, or as direct deflec- tions if a deflection instrument is being used. The blank should exhibit only faint fluorescence. “RECOVERY” EXPERIMENT- Repeat the above procedure, including the steps of extraction, purification, conversion to thiochrome, separation of thiochrome solution and measurement of fluorescence, with a “recovery” experiment, made by adding, to another portion of the sample that is the same weight as that previously taken, a volume of the stock aneurine solution containing an amount of aneurine similar to the amount expected in that weight of sample.CALCULATION- convert all densities into antilogs and take reciprocals of these.? If the fluorescence has been measured on an, instrument provided with a density scale, Let U = reciprocal for unknown; U, = reciprocal for unknown blank; S = reciprocal for standard; S, = reciprocal for standard blank; V = volume of original solution put through base-exchange silicate. If the fluorescence intensities have been measured as deflections, these are used instead of the reciprocals of antilogs.Then the aneurine content of the sample in pg per g = U-Uu, 1 25 100 S - S, 5 V g of sample taken x - x - x - The factor 1/5 converts the reading to pg per ml instead of pg per 5-ml aliquot. Since the final volume of eluate is 25 ml, the factor 25/V corrects for volume changes during adsorption and elution. If the suggested 25 ml is adsorbed, this factor becomes unity. Note-This calculation assumes that the fluorescence of the thiochrome solution in the unknown is If it is not, the assay should be repeated using a smaller less than that of the quinine standard. amount of the material. USE OF RECOVERY EXPERIMENT DATA- Calculate the percentage recovery of the ad.ded aneurine from the following fomula- x 100 A, = aneurine content of sample in pg per g calculated as above; A, = aneurine content of sample with added aneurine calculated as above; A, = pg of aneurine added to each gram of sample. A, - A, A* where- * If 25 ml of water-saturated isobutyl alcohol doe:; not provide sufficient solution for conveniently filling the particular cuvettes in use, this volume can be increased to some precise higher figure, e.g., 30 ml, which must then be maintained throughout all experiments. t If the instrument reads “transmissions,” use those readings instead of reciprocals of antilog density; U, UB, S and SB are then the readings themselves. -March, 19511 ASSAY OF ANEURINE IN FOODSTUFFS 133 This percentage recovery provides an indication of the effect of disturbing factors, including the quenching effect of impurities, but should not be relied upon to make a satis- factory correction for interfering factors. In general, if the percentage recovery falls below 80, the result of the assay should be considered unsatisfactory. REFERENCES Jansen, B. C. P., Bec. Truv. Ckim. Puys-Bus, 1936, 55, 1046. “Methods of Vitamin Assay,” Association of Vitamin Chemists, Inc., New York, 1947. Ridyard, H. N., Analyst, 1950, 75, 634. 1. 2. 3.

ISSN:0003-2654    

DOI:10.1039/AN9517600127

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

March, 19511 ASSAY OF ANEURINE IN FOODSTUFFS 133 The Assay of Vitamin B12 Part HI* Microbiological Estimation with Lactobacillus Zactis Dorner by the Plate Method BY W. F. J. CUTHBERTSON, H. F. PEGLER AND JOAN T. LLOYD (Presented at the meeting of the Biological Methods Group on Tuesday, May 23rd) 1950) The cup-plate method adapted by Bacharach and Cuthbertson to the rapid microbiological assay of aneurine and riboflavine has been found applicable to the estimation of vitamin U,, with a suitable strain of Lacto- bacillus lactis Dorner. The procedure involves the use of a medium similar to those found necessary for other strains of lactobacillus. Ascorbic acid is needed in the assay medium but not in that for preparation of the inoculum. The technique is similar to that described previously for the method.Factors affecting the “zone” response of this organism to vitamin B,, have been investigated; they include the effects of vitamin B12C and the deoxy- ribosides, times of incubation and standing before incubation, range of concentrations of the vitamin and density of the inoculum. As a result a standardised (2 + 2) assay design has been devised; this permits rapid estimates of potency over a range sufficiently wide and with fiducial limits sufficiently narrow for routine purposes, without the use of an inconveniently large number of assay plates. A GROWTH factor for Lactobacillus Zactis Dorner ATCC 8000 has been shown by Shorbf. to be present in highly refined liver extracts in amounts proportional to the anti-pernicious- anaemia activity. This factor has been isolated by Rickes, Brink, Koniuszy, Wood and Folkersz and by E.L. Smith,3 and shown to be responsible for the clinical activity of liver extracts against pernicious anaemia; it is now known as vitamin BIZ. We were unable to develop a satisfactory assay procedure based on the data published by Shorb. In our experience the growth responses of this organism were highly irregular and not capable of giving reproducible results in normal microbiological assay procedures. Lacto bacillus fermenturn P 36, which in our hands gave very irregular results in standard techniques, could be used to assay aneurine with fair precision by the cup-plate method of Bacharach and Cuthbertson.* We therefore attempted to apply this technique to the estimation of vitamin B,% with L.lactis. METHOD In the cup-plate method a suitable agar medium, deficient in the factor under investiga- tion and inoculated with the test organism, is poured into petri dishes and “cups” are cut out of the agar and filled with appropriate dilutions of standard or test solutions. On * For particulars of earlier papers in this series (not in The Analyst), see reference list, p. 140.134 CUTHBERTSON, PEGLER AND LLOYD: ASSAY OF VITAMIN B12 [Vol. 76 incubation under suitable conditions, growth of the organism may occur in sharply defined circular "zones of exhibition" around the cups. The diameters of these zones have been found to be related to the concentration of the ,added growth factor and can be measured and used in the determination of the amount of growth factor present in the test solutions.The method developed in the estimation of vitamin B, is very similar to the technique employed in the estimation of aneurine by Bacharach and C~thbertson,~ but modifications have been introduced in the light of observations by Cuthbertson and Lloyd5 on the mode of growth of L. Zactis and its response to vitamin B,,. MAINTENANCE OF ORGANISM- The culture of L. Zactis is maintained by weekly transfer in soya bean medium (10 per cent. suspension of whole soya beans macerated in a Waring Blenclor and strained through coarse muslin). The inoculum is prepared from this by subculture in 10 ml of complete basal medium. This medium is the same as that described in Table I except for the omission of agar and ascorbic acid and the addition of 0*OOc5 to 0-02 pg of vitamin B,, (as liver extract) per ml.The inoculum is allowed to grow for 15 -to 16 hours and is then centrifuged, washed once in saline and diluted with saline to corresponld to turbidity 4 on the Burroughs Wellcome turbidity scale. Cultures more than +7 to 18 hours old are frequently unsatisfactory for assay purposes. PREPARATION OF THE MEDIUM- The medium is of the composition shown in Table I. The ingredients, except salts, are dissolved in about 800ml of water; after adjustment of the pH to 6.8, the phosphates TABLE I COMPOSITION OF AGAR MEDIA DEFICIENT I N VITAMIN B,, 1 mg Acid casein hydrolysate . . 5 g Nicotinic acid .. .. Glucose . . .. .. .. l o g Riboflavine .. .. .. 2oopg Sodium acetate, A.R... .. 6€! Adenine . . .. .. 10 mg .. . . . . 10 mg L-Cystine .. .. .. 200mg Guanine . . .. .. 10 mg DL-Tryptophan . . .. .. 100mg Uracil . . Tomato juice . . .. .. 60 ml .. I g Pg Tween 80t .. Aneurine hydrochloride Biotin . . .. Folic acid .. 2 P.tg .. I g p-Aminobenzoic acid . . .. 100 p g Ascorbic acid .. Pyridoxamine .. . . . . 400 p g KH2P04 . . . . . . 0.6 g . . . . . . . . Salt solution C; . . .. 5 ml * . 20; pg Calcium-D-pantothenate . . 200 p g K,HP04 .. .. . . 0.6 g Water to make . . . . . . 1000 ml pH adjusted to . . .. .. 6-8 Agar (N.2.): . . .. .. to 15g * Salts C: a solution of 10 g of MgSO4.7H2O, 0-5 g of sodium chloride, 0.5 g of FeS0,.7H,O, 2.0 gof t This amount of Tween 80 may be replaced by 0.39 g of Estax 36 or 29 (Watford Chemical Co.). 3 From Davis Gelatine Ltd., 29, Mitre Street, E.C.3.MnS0,.4H20, and water to 250 ml. and salt solution are added. The precipitate formed during this procedure is removed by filtration through washed paper pulp on a Buchner funnel. The medium is made up to volume and adjusted to pH 6-8. The agar is added to this solution and dissolved by steaming for 1 hour. The medium is now filtered, while still hot, through washed paper pulp, distributed in 130-ml aliquots and kept in 250-ml flasks. The flasks are plugged and autoclaved a t 10 lb steam pressure for 10 minutes. At this stage the medium may be kept for several weeks in a refrigerator. PREPARATION OF ASSAY PLATES- The flasks of medium are steamed for 35 minutes to melt the agar and are then cooled to 4 5 O to 50" C in a thermostatically controlled e:nclosure at 45" C.One millilitre of 0.13 per cent. ascorbic acid solution is now added. Each flask is inoculated with 1 ml of a freshly prepared suspension of L. Zactis adjusted to opacity 4 on the Burroughs Wellcome scale. The inoculum is well mixed in the medium, which is then distributed in the petri dishes at the rate of 1 2 6 d per plate. The plates are cooled in a refrigerator for half an hour to harden the agar; they may be stored in a cold room overnight or even for a week if not neededMarch, 19511 WITH Lactobacillus Zactis DORNER BY THE PLATE METHOD 136 at once. The plates are cut with a sharp 8-mm No. 10 cork-borer; the resulting discs of agar are removed with a needle leaving the cups into which standard and sample solutions may be distributed.TYPE OF PETRI DISHES REQUIRED- For this technique it is essential to use perfectly flat petri dishes so as to obtain a layer of agar of uniform thickness. The diameters of the zones of exhibition vary widely with differences in thickness of the agar. TIME OF INCUBATION- After the plates have been prepared they are incubated a t 37" C overnight. Further incubation does not alter the size of the zones of exhibition, but on certain subnormal media further incubation may lead to some improvement in the density of growth within the zones. CONCEIGTRATION OF VITAMIN B12- The diameter of the zones of growth is proportional to the logarithms of the amounts of vitamin B,, placed in the cups; this is shown in Fig. 1. This relationship holds true over a wide range of concentrations.The clarity with which the rings are defined decreases 10 0005 0-02 005 0-1 0;2 0 Response of L. Eactis Dorner ATCC 8000 to Each point corres- Vitamin 812, pg per ml vitamin B,, in the cup plate assay. ponds to the average reading obtained from six cups Fig. 1. slightly as the concentration of vitamin B,, decreases; under our conditions well defined rings are obtained with concentrations of vitamin B,, in the range 0.01 to 0.5 pg of vitamin B,, per ml. TIME OF STANDING BEFORE INCUBATION- As in the assays of penicillin and streptomycin, the time the plates are allowed to stand before incubation but after distribution of sample and standard solutions in the cups affects the size of the growth zones obtained. This effect is variable from occasion to occasion, but typical results are shown in Table 11.EFFECT OF INOCULUM DENSITY- The inoculum used affects both the size and the appearance of the zones of exhibition. In general, the heavier the inoculum the more sharply are the rings defined and the smaller are the zones. The effect of inoculum density is shown in Table 111.136 CUTHBERTSON, PEGLER AND LLO’YD: ASSAY OF VITAMIN B,, pol. 76 TABLE :[I RELATIONSHIP BETWEEN ZONE DIAMETER AND TIME INTERVAL BETWEEN Two experiments performed 0171 two different occasions FILLING PLATES AND PLACING I N INCUBATOR Concentration of vitamin B,, solution r 1 Time 0.05 pg per ml 0.2 pg per ml Time interval, Zone diameter, Zone diameter, interval, minutes mm mm hours 0 17.8 20.2 0 16 18.5 20.2 1.5 20 19.5 20.4 2-5 3.5 15-0 Concentration of vitamin B,, solution 0-05 pg per ml Zone diameter, Zone diameter, mm mm 16.12 19.0 16-5 19.12 17.62 21.0 17-75 20-9 26.0 30-0 A r \ 0.2 pg.per ml Each diameter is the average from three or four cups.TABLE ‘I11 EFFECT OF INOCU LUM DENSITY Volume of inoculum (turbidity 4) per plate (12.5 ml of agar medium), ml 0.0 1 0.02 0.06 0.1 0.2 Diameter o:E zone of exhibition for vitamin B, concentrations of- 7 A 3 0-05 pg per ml, 0-2 pg per ml, mm mm 21.2 24.4 20.8 23.8 19.9 23.5 19-6 23.6 17.9 20.4 Each diameter is the average from four cups. ASSAY DESIGN AND RESULTS For assay purposes, dilutions of standard vitamin B,, and test solutions are chosen to fall within the range 0.02 to 0.2 pg of vitamin B,,, per ml, over which the relationship between log dose and zone diameter has been found linear (Fig.1). Experiments have shown that the variance between zone diameters for the sa.me concentration of vitamin B, was much less when the cups used were on the same plate than when cups on different plates were compared; the “within plate” variance was about one-tenth of the “between plates” variance. Owing to the size of the petri dishes (9 cm dia.meter) and the size of the zones (13 to 25 mm) normally encountered, not more than 5 or 6 cups may conveniently be used on a plate. Consequently, for routine assays, two cups on each plate are reserved for standard solutions, the concentration of vitamin B,, employed being 0.02 and 0-2pg of vitamin B,, per ml. Two cups are normally used for the test solutions, which are diluted to fall within the range of 0.01 to 0-5 pg of vitamin B,, per ml.Two different concentrations of test solutions are used in the ratio of 1 to 10. The plates are fdled in the following order- (;) Lower test concentration. (ii) 0.02 pg of vitamin B12 per ml. (iii) 0-2 pg of vitamin B,, per ml. (iv) Higher test concentration. Three drops, 0-1 to 0.15m1, of solution, measured with a standard dropping pipette (see Bacharach and Cuthbertson*) are placed in each cup; the same dropping pipette is used for all dilutions of test and standard solutions. After the cups have been filled, the plates are left for a t least 10 minutes before being put in the incubator. This procedure has been adopted to minimise the effects of standing before incubation and of any differences between dropping pipettes.After incubation the zone diameters may be measured with a ruler (transparent celluloid rules are suitable) or callipers, or after projection on to a screen. The normal procedures (Irwine) applicable to the calculation of the results of assays involving a linear relationship between log-dose and response may be applied and fiducial limits may be determined.March, 19511 Plate 1 2 3 4 5 6 7 8 Average WITH LactobaciZZus lactis DORNER BY THE PLATE METHOD TABLE IV ASSAY OF VITAMIN B1, I N LIVER EXTRACT Variance analysis according to method of Irwin6 ANALYSIS OF VARIANCE- Standard vitamin B,, (3 drops per cup) 1 0.02 pg 0.2 I G 13-0 20.0 15.0 22.0 14.0 21.0 14.0 2 1.0 14-0 21.0 13.0 18.5 13.0 19.0 13.5 20.5 13.7 20-4 Sum of D.F.squares .. 1 374.1 Source of variation Common linear reaession .. Difference due to kbstances . . .. 1 9.6 Bias due to plates . . .. .. 7 16.6 Error . . .. .. .. .. 21 4.0 Total . . .. .. .. .. 31 405.1 Departure from parallel regression . . 1 0.2 Test preparation (3 drops per cup diluted as shown) - 1/750 1/75 14.0 21.0 15.0 22.0 15.0 22-0 15.0 23.0 15.0 22.0 14.0 20.0 14.0 21.0 15.0 22.0 14.6 21.6 Mean Variance square ratio Significance 374-7 19-72 significant 9.6 50.52 significant 0.2 1.05 not significant 2.37 12.47 significant 0.190 137 Activity found: 21.64 pg per ml. Fiducial range (P = 0-95) : 19.49 pg per ml t o 24.20 pg per ml; 90 per cent. t o 112 per cent. A typical assay result is shown in Table IV, together with the calculated fiducial limits.On average, fiducial limits of 415 per cent. (P = 0.05) may be expected in assays of this type employing six plates, although narrower limits can be obtained with a greater number of plates. Assays by the plate method and by a tube technique employing L . Zeichmannii 313 (Lees and Emery') have given comparable results (see Cuthbertson, Lloyd, Emery and Lees3). INTERFERENCE FROM DEOXYRIBOSIDES Under our conditions L. Zactis responds to the deoxyribosides as well as to vitamin B, (see Smith and Cuthbert~on~). The response to thymidine is shown in Table V. Zones TABLE V RESPONSE OF LactobaciZZus Zactis TO THYMIDINE (PLATE TEST) Diameter of zone of Thymidine concentration, exhibition P*.g Per ml 1 6 10 20 50 no zone 24 mm approx., indistinct zone 25.9 mm 28.1 mm 34 mm Each diameter is the average from four cups of exhibition of less than about 25 mm are not formed and only solutions containing 6 pg or more of deoxyriboside per ml give zones of exhibition. Up to 5 pg of deoxyribosides per ml cause only very slight interference in the plate test.Table VI shows the diameters of the zones of exhibition produced by mixtures of thymidine and vitamin B12. Deoxyriboside zones are readily distinguished from vitamin B,, zones, for the growth is much fainter and more diffuse. If vitamin B,, is present an inner zone of dense growth may be seen, but when the concentration of thymidine is high this zone is not well defined and cannot be measured accurately.138 CUTHBERTSON, PEGLER AND LLOYD : ASSAY OF VITAMlN B12 [vol. 76 Samples containing deoxyribosides are readily detected by the appearance of the growth zones and also by the slope of the log-dose - response curve, which is much greater for deoxy- ribosides than for vitamin B,, as should be clear from Table VI and Fig.2. TABLE VI RESPONSE OF L. Zactis TO MIXTURES OF THYMIDINE AND VITAMIN B,, (PLATE TEST) Composition of solution r \ Vitamin BIZ, Thymidine, 0.02 0 0-02 1 0.02 6 0.02 10 0.02 20 0.02 60 0.2 0 0.2 1 0.2 5 0-2 10 0.2 20 0.2 50 A Per PLg Per ml Diameters of zones of exhibition 7 A \ Inner zone due to Outer zone due to vitamin B12, thymidine, rnm mm 13.1 no zone 13.45 no zone 13.25 24.1 not measurable 26.6 99 28.0 99 30.1 17-05 no zone 17.1 no zone 17.2 23.2 not measurable 25.9 99 28.0 99 29.9 USE OF CHROMATOGRAPHY Ih’ THE PLATE ASSAY Interference due to the deoxyribosides may be eliminated by a technique previously indicated by Smith and Cuthberts~n.~ Micro-drops of about 1 to 4 4 , containing 0.004 to 0.04 pg of vitamin BI2, of test and standard solutions are placed about 1 inch from one edge of a square sheet of Whatman No.4 filter-paper and about 1 inch apart. Micro-drops of saturated riboflavine solution are placed just beneath but not touching the positions occupied 1 10 20 50 Thymidine,yg per ml 2o 5 Fig. 2. Response of L. Zactis Dorner ATCC 8000 to thymidine in the cup plate assay. Each point corresponds to the average reading obtained from four cups 4 Vitamin BI1. pg Fig. 3. Response of L. Zactis Dorner ATCC 8000 to vitamin B,, after separation of deoxyribosides by partition chromatography.Each point represents the average zone diameter obtained with four aliquots applied to the filter paper by the standard and test spots. The paper is dried in the incubator and then rolled into a cylinder and held in this way by metal paper-clips. The filter-paper cylinder is now placed in a beaker containing a layer of water-saturated n-butanol about 1 cm deep. Upward development is allowed to take place for about 1Q to 2 hours, i.e., until the riboflavine has passed through and about 0.5 cm beyond the sites at which the vitamin B, and test solutionsMarch, 1951 J 139 had been placed. The paper is then removed and dried in the incubator, and when dried it is cut along the front reached by the riboflavine. During this brief development the vitamin B,, does not move from its site of application, while the deoxyribosides (except cytosine deoxyriboside) travel with and beyond the riboflavin e.When a strip of paper to which the test and vitamin B,, solutions have been applied is placed on an inoculated agar test medium and incubated overnight, circular zones of exhibi- tion are produced by the vitamin B,,. The type of response obtained is shown in Fig. 3. With only four 2 - 4 drops of each standard and test solution, fiducial limits (P = 0-05) of 60 to 150 per cent. were obtained in the assay of samples containing 3 to 5 pg of vitamin B,, per ml. WITH Lactobacillus Zactis DORNER BY THE PLATE METHOD ASSAY OF VITAMIN B12 CONCENTRATES FROM FERMENTATION LIQUORS In the assay of extracts from streptomyces the results by the plate and tube methods (Lees and Emery’) often differed widely, the plate technique almost invariably giving higher results; in some instances they were three times those of the tube assay.It was noticed that the zones of exhibition obtained with these materials were not as sharp as those normally encountered and it was at first suspected that the interference was due to some substance other than vitamin B12; however, it was not possible to separate the vitamin B,, activity from the postulated interfering agent. It has recently been shown that another red cobalt-containing clinically active compound, in addition to the previously described members of the vitamin B,, group, is present in streptomyces fermentation products (Smithlo). This substance, designated BlZc in accordance with the suggestion of Buchanan, Johnson, Miles and Todcl,ll itself yields poorly defined zones of exhibition in the plate test under conditions used for vitamin B,, assay; weight for weight it appears to be 3 to 4 times as active as vitamin B,,, although in tube assays and clinically (Ungley, Mollin and Dacie12) its activity is approximately the same as that of vitamin B12.We have not been able to overcome the difficulty that thus appears to be due to vitamin 1312c. Until a suitable technique becomes available for doing this, results of plate assays on fermentation liquors may be very unreliable. FAILURE TO ASSAY SOLID MATERIALS CONTAINING ADSORBED VITAMIN B,, Vitamin B,, is firmly adsorbed by fuller’s earth. The assay of such adsorbates presents some difficulties.It was not found possible to elute completely all the vitamin B,, taken up by this material. When fuller’s earth adsorbate was placed on plates prepared for vitamin B,, assay, growth was noticed around the sites of application. Attempts were therefore made to develop an assay from this observation. Plates were prepared and cut in the normal way and aliquots of aqueous dispersions of the adsorbate were placed in the cups. After incubation the zones of growth around the cups containing the fuller’s earth were found to be closely comparable in qualitative appearance to the zones around the cups in which standard vitamin B,, had been placed; further, the assay data showed that the nature of the response curves obtained with the fuller’s earth adsorbate were not different from those obtained with aqueous solutions of vitamin B,, and that assays were statistically valid when tested by the usual mathematical analysis.The results were, however, much lower than those obtained by chemical assay (Fantes and Ireland13) or by elution techniques, e.g., one adsorbate gave 25, 48 and 50 pg per g respectively when tested by the plate assay, an elution technique and the chemical method. These observations are reported because with adsorbates the plate method gives results that are very consistent and appear to be valid when checked by methods in frequent use; for instance, another sample of adsorbate was tested on two separate occasions and gave results all within the range of 11 to 13 pg per g, although on other grounds it was believed to contain at least 30 pg of vitamin B,, per g.A fuller account of the assay of adsorbates is to be reported later. DISCUSSION OF THE METHOD The main advantages of the cup-plate technique are its speed and simplicity. Zones of exhibition are formed overnight, but they appear and may be measured after incubation for as little as 6 hours in favourable circumstances, whereas the tube methods require a t least overnight incubation for L. Zeichmanniil* and 4 days for EugZena gracilis.ls With the cup-plate method samples need not be sterilised; in the tube methods strict asepsis is140 CUTHBERTSON, PEGLER AND LLOYD: ASSAY OF VITAMIN I312 essential and all samples must be sterilised by filtration through collodion or sintered glass.Autoclaving or seitz filtration may lead to loss of vitamin B,,. Samples containing less than 50 rnpg per ml cannot be assayed satisfactorily, whereas the tubc methods employing L. leichmannii and Euglena graczlis may be used for concentrations as low as 2-0 mpg or 2 ppg of vitamin B,, per ml, respectively, in favourable circumstances. Thymidine and the other deoxyribosides may replace vitamin B,, in the nutrition of L. ZactisfG or L. Zeichmannii.17 In the tube techniques growth due to these substances is not readily differentiated from that due to vitamin BI2, but in the plate method the appearance of the growth zones due to the different types of substances is a t once discernible, because the deoxyribosides give wide diffuse zones of exhibition , while vitamin B,, under our conditions gives dense sharply defined zones of growth; hence confusion does not arise and interference is noticed a t once.Similarly, though antibiotics and toxic substances in general may interfere in any microbiological test, such interference is readily detected in the plate test by the appearance of zones of partial growth or rings of growth inhibition, Pol. 76 The cup-plate method is relatively insensit-be. . SUMMAKY Lactobacillus lactis Dorner ATCC 8000 requires vitamin B,, for its normal nutrition and has been foundsuitable for assay of the vitamin. by the cup-plate method. Vitamin B, and the deoxyribosides interfere with the procedure. The effect of the latter, but not of the former, can be eliminated by combining the method with payer chromato- graphy.The presence of deoxyribosides is generally apparent from the type of zone produced. The combined chromatographic and microbiological procedures make it possible, with a (2 + 2) assay design, dose ratios of 10 to 1 and incubation overnight, to attain a satis- factory degree of precision on a few microlitres of vitamin B,, solution. The method gives results in good accord with those given by the tube method based upon growth of Lactobacillus leichmannii 313. Our thanks are due to Dr. T. G. Brady, Trinity College, Dublin, for a very generous gift of thymidine and to Mr. B. Basil, Glaxo :Laboratories Ltd., Greenford, for statistical calculations. 1. 2. 3. 4. 6. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. REFERENCES Shorb, M. S., J . Biol.Chem.. 1947, 167, 455. Rickes, E. L., Brink, N. G., Koniuszy, F. R., Wood, T. R., and Folkers, K., Science, 1948, 107, Smith, E. L., Nature, 1948, 161, 638. Bacharach, A. L., and Cuthbertson, W. F. J., Analyst, 1948, 73, 334. Cuthbertson, W. F. J., and Lloyd, J. T., J . (Yen. Microbiol., 1951, 5 , 416. Irwin, J. O., J . Roy. Statist. SOC., 1937, Suppl. 4, 1. Lees, K. A., and Emery, W. B., Biochem. J., 1949, 45, ii. Cuthbertson, W. F. J., Lloyd, J. F., Emery, W. B., and Lees, K. A., J . Pharnt. Pharmacol., 1949, Smith, E. L., and Cuthbertson, W. F. J., Bicchem. J., 1949, 45, xii. Smith, E. L., PYOC. Roy. SOC. Med., 1950, 43, 535. Buchanan, J. G., Johnson, A. W., Miles, J. A,, and Todd, A. R., Chem. and Ind., 1950, 426. Ungley, C. C., Mollin, D. L., and Dacje, J. V., Lancet, 1950, 1, 353; Proc. Roy. SOC. Med., inthe Fantcs, K. I<., Ireland, D. M., and Green, N., Biochcnt. J . , 1950, 46, xxxiv. Hoffmann, C. E., Stokstad, E. L. R., Franklin, A. L., and Jukes, T. H., J . Biol. Chem., 1948, Hutner, S. H., Provasoli, L., Stokstad, E. L. R., Hoffmann, C. E., Belt, M., Franklin, A. L., and Kocher, V., and Schindler, 0.. Intern. Rev. Vzt. Res., 1949, 20, 441. Snell, E. E., Kitay, E., and McNutt, W. S., .J. Biol. Chem., 1948, 175, 473. 396. 1, 705. press. 176, 1465. Jukes, T. H., Proc. SOC. Ex@. Biol. Med., 1949, 70, 118. NOTE-Reference 5 is to Part I of this series. GLAXO LABORATORIES LTD. GREENFORD, MIDDLESEXMarch, 19511 WITH Lactobacihs lacfis DORNER BY THE PLATE METHOD DISCUSSION 141 MR. I<. A. LEES confirmed that when vitamin BIZ, was assayed by the L. lactis plate technique the values obtained were 3 to 4 times as high as those obtained by the tube methods or physical chemical procedures. In Mr. Lees’ laboratories the zones of growth with vitamin BIzc in plate tests employing L. leichmannii 413 were poorly defined. The type of growth obtained could, however, be readily differentiated from the responses given by thymidine, although the edges of the zones were not sharp, because the growth within the zones was as dense as that observed with vitamin BI2. To overcome these disadvantages a plate method employing an E. coli mutant1 has been developed and all pure factors so far tested give clearly defined zones. The E. coli technique has the further advantagc that a very simple medium is employed. REFERENCE TO DISCUSSION 1. Bessell, Christine J., Harrison, Eleanor, and Lees, I<. R., “Assay of Vitamin B, with a Mutant of Escherichia Cola,” Chem. and Ind., 1950, 561.

ISSN:0003-2654    

DOI:10.1039/AN9517600133

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

March, 19511 WITH Lactobacillzds Zactis DORNER BY THE PLATE METHOD 141 The Assay of Vitamin B12 Part IV* The Microbiological Estimation with Lactobacillus Zeichmannii 313 by the Turbidimetric Procedure BY W. B. EMERY, K. A. LEES AND J. P. R. TOOTILL (Presented at the meeting of the Biological Methods Gvoup on Tuesday, M a y 23rd, 1950) Details are presented of a microbiological tube assay for vitamin B,, with Lactobacillus Zeichmannii 313 as test organism. The procedure employs materials that are readily available in this country. Statistical analyses of a (3 + 3) assay and a standard response curve are given and show that the method is sufficiently sensitive and accurate for routine use. ‘Two papers1 92 from these laboratories describe work on factors affecting the microbiological techniques used for assaying vitamin B12; the cup-plate method is described in detail else- where in this issue.3 In this paper we give the corresponding details for ,the so-called “tube assay’’ with a turbidimetric procedure for measuring the growth of the micro-organism.The conditions used are based on the work described in one of the papers cited above., As was also found by Cuthbertson, Pegler and Lloyd,3 it has been our experience that Lactobacillzcs Zactis Dornel-2 9 5 gives a very irregular growth response to graded doses of vitamin B12. We therefore carried out further work on the use of LactobncilZzcs k i c h m a m i i 313 in the vitamin B,, assay, this organism having been recommended by Skeggs, Huff, Wright and Bosshardt6; other workers have already reported on its use.’ As already briefly reported,* our assay method depends on the inability of L.leichmannii 313 to synthesise vitamin B,, under defined conditions, so that there is a direct relationship between the growth of this organism and the coiicentration of vitamin B,, in the test medium over a certain range. Attempts to devise an assay suitable for routine use, employing the medium of Snell, Icitay and M ~ N u t t , ~ were not successful, the chief difficulty being the production of a dense precipitate during the autoclaving of the medium. This medium was modified by the omission of enzymatic casein digest and reduction of the level of salts B. To prevent precipitation in the tube and consequent high blanks, the medium was preheated in bulk and filtered hot.Hoffman et aZ.7 reported that thioglycollic acid increased the growth response of L. leiclzmannii to vitamin B12. This was thought to be due to protection of the vitamin * For particulars of earlier papers in this series (txro not in The Analyst), see reference list, pp. 145-146.142 EMERY, LEES AND TOOTHILL: ASSAY OF VITAMIN B12 WITH [Vol. 76 during the autoclave cycle. Our own work on the effect of reducing agents and oxygenation,2 as well as the recent work of Koditschek, Hendlin and Woodrufflo on the nutrition of L. Lactis Dorner, indicates that vitamin B1, functions as an anti-oxidant for the test organism, and consequently there is a direct relationship between the E, of the medium and the vitamin B,, requirement. We have been able to show the mathematical relationship between the response of L.Zeichmannii and the depth of medium in the assay tubes.2 TUBE ASSAY TECHNIQUE PREPARATION OF MEDIUM- Solutions A and B are prepared according to the details in Table I and mixed as required, because it has been found that the complete medium does not keep well. TABLE I ASSAY MEDIUM Solution A (double strength)- Sodium acetate, A.R. .. .. . . . . .. Sodium citrate, A.R. .. . . .. .. .. .. .. .. ::it' ;}Snell and Wright" I ' - . . . .. Acid hvdrolvsate of casein (based on total solids) . . Tween-80 (purified for T.B.'test) Sodium oleate . . .. .. .. Glucose . . .. .. * . .. Glass-distilled water to . . .. . . OY Crill 10 (Croda Works, Goole) L-Cystine .. .. .. .. pH adjusted to . . .. .. . * Solution B (forty times single strength)- Adenine, guanine and uracil (of each) DL-Tryptophan .. .. .. .. p-Aminobenzoic acid . . . . .. Asparagine . . .. .. .. Calcium-D-pantothenate . . . . Aneurine hydrochloride . . .. Pyridoxal . . . . .. .. Pyridoxamine . . . . .. .. Nicotinic acid . . .. .. .. Riboflavine . . .. . . .. Biotin . . .. . . .. .. Folic acid .. .. . . . . Glass-distilled water to . . .. .. .. .. .. .. .. .. . . .. . . .. .. . . .. .. .. . . . . .. .. .. .. .. .. .. .. .. .. * . . . . . .. .. .. .. .. . . .. .. .. .. . . .. .. . . .. .. .. .. .. * . .. ,. * . .. .. .. .. .. * . .. .. .. .. .. The pH of solution A is adjusted to 6.8. The solution is then heated to boiling and I t is stored in a Solution B is stored in a refrigerator a t pH 3.0 to 4.0 and may be used for a period of filtered with the aid of kieselguhr and its pH is then re-adjusted to 6.8.refrigerator and may be used over a period of 5 to 6 days. 10 to 14 days. PREPARATION OF ACID HYDROLYSATE OF CASEIN- Five hundred grams of "vitamin-free" casein (Glaxo Laboratories Ltd.) are heated under reffux with 2-5 litres of 6 N hydrochloric acid for 9 hours. The hydrolysate is con- centrated to a syrup in vucuo and diluted to 4.5 litres with glass-distilled water; after the pH has been adjusted to 3.0 with solid sodium hydroxide, the mixture is filtered and treated twice with 50 g of Sutcliffe Speakman No. 5 charcoal, each absorption involving stirring for 30 minutes. The final solution is diluted to 51 litres. This procedure yields a hydrolysate containing 1.0 to 1-25 per cent.w/v of total nitrogen. We normally preserve the hydrolysate by freeze-drying to a dry powder; alternatively it may be stored in a refrigerator under toluene. Each batch of acid-hydrolysed casein is tested over a suitable range of concentration. We have recently found casein hydrolysate supplied by Allen and Hanbury Ltd. to be satisfactory .March, 19511 Lactobacillus Zeichmannii 313 BY TURBIDIMETRIC PROCEDURE 143 CLARIFIED TOMATO JUICE- Commercial canned tomato juice is filtered, with the aid of kieselguhr, through a Whatman No. 1 filter-paper supported on a No. 54 paper. The filtrate is a clear pale straw colour. The filtered tomato juice is stored in the refrigerator and is used over a period of 3 days only. Some brands of commercial tomato juice do not contain the factor required by L.Zeich- mannii. A large quantity of a tested brand is therefore bought and stored in the cold room: we have found “Crest” brand tomato juice to be satisfactory. MAINTENANCE OF TEST ORGANISM AND PREPARATION OF INOCULUM- I.. Zeichmannii is maintained by daily transfer in liquid medium (yeast extract, 1 per cent.; tomato juice, 5 per cent.; separated milk to 100 per cent.). More than three hundred transfers have not resulted in any detectable alteration in the sensitivity of the organism. TABLE I1 ANALYSIS OF DAILY RESPONSE CURVE Dose, mpgpertube 0.23 0.35 0.53 0.79 1.19 1-78 2-67 4.00 Totals Responses Rack I . . Sub Totals Responses Rack I1 .. Sub Totals Responses Rack 111.. Sub Totals Totals . . Orthogonal Coefficients Means ... . 0.15 . . 0.14 . . 0.29 . . 0.19 . . ’ 0.19 . . 0.38 . . 0.17 . . 0.16 . . 0.33 . . 1-00 { z . . 0.167 0.28 0.20 0.48 0.23 0.25 0.48 0.23 0.23 0.46 1-42 - 5 +l 0.237 0.36 0.36 0.72 0.34 0.37 0.71 0.33 0.38 0-7 1 2.14 -3 -3 0.357 0.51 0.68 0.53 0.63 1.04 1.31 0.54 0.64 0.45 0.61 0.99 1.25 0.57 0.65 0.49 0.68 1.06 1.33 3.09 3.89 -1 $1 - 5 -5 0.515 0.648 0.85 0.80 1-65 0.7 1 0.85 1.56 0-94 0.83 1.77 4.98 +3 -3 0.830 1.06 0.9 1 1.97 0.09 0.93 2.02 1.09 1.12 2.21 6.20 +5 +1 1.033 1.21 1.22 2.43 9.89 1-29 1.24 2.53 9.92 1.18 1.24 2-42 10.29 Regression coefficients 7-38 30.10 +0.627,083 + 7 + 77.88 +O-077,262 + 7 + 10.02 + 0*009,94O 1.230 Regression values 0-156 0.251 0.365 0.500 0.655 0.829 1.023 1.237 ANALYSIS OF VARIANCE FOR HOMOGENEITY- Sum of Mean Variance Source D.F.squares square ratio Significance Doses . . . . .. 7 6.12 1,62 Racks . . . . . . 2 0.006,20 0*003,100 1.251 none Interaction . . . . 14 0*029,87 0*002,134 0.861 none Error . , . . . . 24 0*059,50 0.002,479 Total . . . . . . 47 6*217,19 .2NALYSIS OF VARIANCE FOR REGRESSION- Sum of Mean Variance D.F. squares square ratio Significance Quadratic . . . . 1 0*099,60 0*099,60 40.178 very Linear . . .. . . 1 6*017,16 6.017,16 2427.656) high Deviation .. . . 5 @*004,86 0*000,97 0.391 none Doses . . . . . . 7 6*121,62 Standard error = 0.0498 The inoculum is prepared by two daily transfers in basal assay medium enriched with purified liver extract to give a final vitamin B,, concentration of 0.015 pg-per ml. Volumes of 0.5 ml are subcultured into 10-ml volumes of enriched basal medium in 6 inch x 2 inch tubes and the cultures are incubated at 37” C for 18 to 20 hours.The cells from the second144 [Vol. 76 transfer in inoculum medium are centrifuged, washed once in sterile physiological saline and re-suspended in sterile saline to Burroughs ’Wellcome opacity 4. This suspension is diluted 1 to 8 in a mixture of equal volumes of sterile autoclaved physiological saline and clarified tomato juice, the latter having been previously sterilised by filtration through a sintered-glass filter; 0-5 ml of this inoculum is used for each assay tube. EMERY, LEES AND TOOTHILL: ASS.4Y OF VITAMIN B12 WITH PREPARATION AND DISTRIBUTION OF TUBE MEDIUM- Solutions A and B are mixed in the following proportions and 84ml are introduced For 500 assay tubes the following are mixe(1- into each of several 6 inch x 2 inch standard tubes by means of an Ayling filler.Solution A . . .. .. .. .. . . . . 2500ml Solution B . . .. .. .. . . .. .. 125ml Glass-distilled water to a . .. .. . . . . 4250ml By the additions of 1 ml of suitably diluted test sample and 0.5 ml of inoculum, the volume is increased to 10 ml and the medium reduced to “single strength.” The pH is re-adjusted to 6.8 if necessary. 0~0002 u 00 0 2 04 0.6 0-8 1.0 1.2 1.4 Response Fig. 1. A typical I-esponse curve The assay tubes are covered by aluminium caps and autoclaved a t 151b pressure for 10 minutes, the pressure in the autoclave being released quickly and reduced to atmospheric pressure in 2 minutes. We have found that slight differences in the autoclave cycle pro- foundly influence the slope of the growth resportse curve.Increasing the autoclave cycle to 30 minutes decreased the accuracy of the test by reducing the range of growth available for the daily response curve. We have not fount1 that increased autoclaving will produce a medium in which L. Zeichmannii no longer requires vitamin B,,, as reported by Shawl2 for L. Zactis Dorner. The daily reference curve is obtained by six-tube replicates, each of eight doses of pure crystalline vitamin B12, logarithmically spaced in the range 0.00016 and 0.0027 pg per tube. A few blank tubes are set up as controls each day to indicate what growth, if any, occurs in the absence of vitamin BI2. The analysis of a daily response curve is given in Table 11; it shows that the standard deviation of a reading is approximately 0.05. Test samples are diluted according to the estimated potency and sterilised by filtration through sintered-glass filters.One millilitre of each dilution is added to the assay tubes and followed by 06ml of inoculum. The tubes are shaken to ensure thorough mixing of the inoculum and incubated in an incubator room at 37” C for 17 hours. Small laboratory type incubators have not been found suitable. After incubation, the growth is stopped by the addition of 2 drops of 40 per cent. formaldehyde solution to each tube or by steaming; the turbidities are read on a Spekker photo-electric absorptiometer in the normal way. Dr. W. F. J. Cuthbertson (personal communication) has now found it possible to replace the Tween 80 by Estax 36 (Watford Chemical Company) and the tomato juice filtrate by a A typical response curve is shown in Fig.1.March, 19511 LactobaciZZus Zeichmannai 313 BY TURBIDIMETRIC PROCEDURE 145 mixture of 250 pg of fumaric acid, 250 pg of sodium ethyloxalacetate* and 100 pg of DL-alanine per millilitre of single strength medium. These constituents may be incorporated in the “forty times strength” vitamin solution. Dr. Cuthbertson has also reported that certain batches of inoculum medium, i.e., basal medium enriched with vitamin BIZ, that failed to produce satisfactory growth after storage for a few days were greatly improved by the addition ANALYSIS OF (3 + 3) ASSAY Responses of standard (-.-h-.--..-- -7 0~0008 0.0012 0.0018 0.93 1.12 1.38 0.83 1.15 1.29 0.54 0.98 1-30 0.56 0.74 1.28 1 -06 1.12 1.28 0.86 1-05 1.32 (pg per tube) I Responses of unknown A > 4/9u 2/3u U 0.83 1-15 1.33 0.87 1-08 1.33 0-96 0.95 1.31 0.92 1.13 1.34 0.80 1.09 1.20 1.03 1.05 1.32 ‘Totals .. . . . . . . 5-10 6.16 7-85 5.41 6-49 7-83 38.84 Sample difference . . . . -1 -1 -1 +1 -!-1 i- 1 + 0.62 Linear regression . . . . --1 0 +1 -1 0 -t 1 +5*17 Departure from parallelism . . + 1 0 -1 -1 0 $1 - 0.33 Combined curvature . . . . $1 -2 +1 f l -2 i- 1 + 0.89 Opposed curvature . . . . -1 4- 2 -1 +1 - 2 i-1 -0.37 AXALYSIS OF VARIANCE- Source D.F. Sample difference . . . . 1 Linear regression . . . . 1 Departure from parallelism . . 1 Opposed curvature . . .. 1 Combined curvature . . . . 1 Error . . .. . . .. 30 Sum of squares 0.01068 1.11370 0.00454 0~01100 0~00190 0.33 173 Mean Variance square ratio Siplicance 0-01068 0.97 not Significant 1.11370 100.70 P < 0.001 0.00464 0.41 not significant 0.01 100 0.99 not significant 0.00 190 0.17 not signscant 0.01 106 Total .. . . .. . . 35 1.43755 Potency ratio: 1.07 Fiducial limits: (P = 0.95) 0.93 to 1.22 (93 to 122 per cent.) of small amounts of thioglycollic acid or ascorbic acid ; this suggested that an accumulation of peroxides had occurred. Excessive amounts of these reducing agents, however, led to the production of unsatisfactory inocula and consequently boiling and cooling of the inoculum tubes immediately before inoculation is recommended. We wish to acknowledge valuable technical assistance by Miss F. M. Ord and Mr.D. G. Goodinson. REFERENCES 1. 2. 3. 4. 5. 6. 7. Cuthbertson, W. F. J., and Lloyd, J. T., J. gew. Microbid., 1951, 5, 416. Lees, K. A., and Tootill, J. P. R., in pr~pnration. Cuthbertson, W. F. J., Pegler, H. F,, and Lloyd, J. T., Anrtlyst, 1951, 76, 133. Shorb, M. S., J. Biol. Chem., 1947, 169, 455. - , Science, 1948, 107, 396. Skeggs, H. R., Huff, J. W., Wright, 1,. D., and Bosshardt, D. K., J. Biol. Chem., 1948, 176, 1469. Hoffmann, C. E., Stokstad, E. L. R., Hutchings, H. L., Dornbush, A. C., and Jukes, T. H., Ibid., 1949, 181, 635. * Commercial brands have not been found suitable and laboratory preparation according to Wislicenus, -4 nnalen, 1888, 246, 31 5, is recommended.146 COATES, HARRISON AND KON: THE CHICK ASSAY OF 8. 9. 10. 11. 12. Lees, K. A., and Emery, W. B., Biochem. J., Pvoc. Biochem. Soc., 1949, 45, ii. Snell, E. E., Kitay, E., and McNutt, W. S., J . Bid. Chem., 1948, 175, 473. Koditschek, L. K., Hendlin, D., and Woodruff, H. B., Ibid., 1949, 179, 1093. Snell, E. E., and Wright, L. D., Ibid., 1941, 139, 675. Shaw, G., Nature, 1949, 164, 186. [Vol. 76 NOTES-References 1, 2 and 3 are to Parts I, I1 and 111 of this series. Since this paper was written the following two papers on the microbiological assay of vitamin B,, Thompson, H. T., Dietrich, L. S., and Elvehjem, C. A., J . Bid. Chem., 1950, 184, 175. Skeggs, H. R., Hepple, H. M., Valentik, I<. A., Huff, J. W., and Wright, L. D., Ibid., 1950, 184, have appeared- 211. GLAXO LABORATORIES LTD. SEFTON PARK STOKE POGES, BUCKS.

ISSN:0003-2654    

DOI:10.1039/AN9517600141

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

146 COATES, HARRISON AND KON: THE CHICK ASSAY OF [Vol. 76 The Chick Assay of Vitamin B12 and the Animal Protein Factor BY M. E. COATES, G. F. HARRISON AND S. K. KON (Presented at the meeting of the Biological Methods Group on Tuesday, May 23rd, 1950) A method of assay with chicks for vitamin B,, and the animal protein factor is described and its accuracy discussed. The method has been applied to several natural materials, and a comparison with microbiological results has been made. Evidence is presented for the existence of other factors besides vitamin B,, in the animal protein factor. Difficulty in obtaining satisfactory responses to crystalline vitamin R,, is reported. ALTHOUGH the method now described was originally designed to measure vitamin B,,, it has become obvious irom our results to date that the test is not specific for vitamin B,, alone and that other as yet unidentified vitamins produce a similar growth response in chicks.We prefer therefore to describe the test as an assay of the animal protein factor (APF), although the standard used so far has been a preparation of vitamin B,, (Examen, Glaxo) presumed to be free from other factors, and results have been calculated in terms of vitamin B12. Preliminary accounts of our method of assay have already been presented.lP2 The present paper describes the application of the method to the assay of a number of natural materials ; wherever possible, results have been compared with those obtained microbiologically and it is evident that the two methods are often not in close agreement. EXPERIMENTAL PRODUCTION OF THE CHICKS- As previously reported,l normal stock chicks proved unsuitable for this assay, as their reserves of animal protein factor a t hatching were high.Chicks were therefore hatched from hens given an all-vegetable diet.2 I t was of major importance in all this work that both hens and chicks should be kept on wire screens to prevent access to their droppings, which have been shown by Rubin, Bird and Rotlhchild3 to contain components of the animal protein factor, probably as a result of microbial synthesis after voiding. A deficiency of animal protein factor in hens results in lowered hatchability of their eggs,2s4 so that to obtain adequate numbers of chicks we found it necessary to include a small but sufficient quantity of vitamin B,, in the hen diet to maintain hatchability at a reasonable level without excessive storage in the chicks.About 3 pg of vitamin 13,, per 100 g of diet proved much too high for this purpose,, but 0.3 pg per 100 g was satisfactory. In 9 months, with the addition of this amount of vitamin B,,, hatchability of the fertile eggs from 30 hens never fell below 50 per cent., yet the chicks showed marked signs of deficiency of vitamin B,, a t an early age.March, 19511 VITAMIN BIZ AND THE ANIMAL PROTEIN- FACTOR 147 DESIGN OF THE ASSAY- The assay was designed on similar lines to those already in use here6 for other members of the vitamin B complex, except that chicks of both sexes were used. The basal diet, with a high content of vegetable protein, had the following percentage constitution.ground maize, 38.6 ; ground barley, 20 ; defatted soya grits, 35 ; dried grass, 3 ; bone meal, 1.5 ; limestone flour, 1 ; sodium chloride, 0.672; MnS0,.4H20, 0.028; arachis oil, 0.2. To each 100 g of diet were added 400i.u. of vitamin A, 50 B.S.I. units of vitamin D,, 0-15pg of ribofiavine and 1 pg of nicotinic acid; the doses of standard and test preparations were incorporated in the diet. With chicks produced as described above, a linear relationship was established between their body weights at 4 weeks of age and the logarithm of the dose of vitamin B12. This relationship held over a range of 0.5 to 3.0 pg of vitamin B,, per 100 g of diet. As far as possible assays were done with at least three dose levels of both standard and test material, with 5 to 10 chicks on each dose.Calculations were made as recommended in British Standards Specification 911 : 1940,6 and in most assays the graphs of response to standard and test preparation proved straight and parallel to each other. ERROR OF THE ASSAY- The error of estimation unfortunately proved to be high, owing chiefly to the great variation in response among the chicks at each dose level. As the number of. chicks available TABLE I REPRODUCIBILITY OF RESULTS FOR THE CHICK ASSAY OF THE ANIMAL PROTEIN FACTOR Pooled T.F.L. at P Sample Result P = 0.95 Xa Result Error b g of vitamin B,, per g pg. of vitamin BI2 per g Fish solubles.. .. . . (i) 1.27 0.36 to 2-28 (28 to 180%) (ii) 1.23 0.68 to 1-90 (55 to 155%) (iii) 1.01 0.42 to 1-80 (42 to 176%) Fuller’s earth adsorbates of ( i ) 134.5 68 t o 267 (50 to 198%) (61 to 144%) 1.17 0.99 to 1.37 (85 to 118%) 120.5 102 to 142 i 0*36 (72 to 118%) } 0.23 Streptomyces griseus liquor (ii) 113.9 70 to 164 is limited by the special conditions needed to produce them, we find it is impracticable to attempt to reduce the variance either by increasing the size of the test groups or by severely culling the chicks before the beginning of the test.It is possible that the accuracy could be improved if pedigree records of individual birds were kept ; the birds could then be distributed over the experimental treatments according to parentage, as is usual with the “litter mate” system adopted in rat assays. In spite of the error being so high, results were fairly reproducible, as can be seen in Table I.The results for three successive assays of fish solubles and for two of an adsorbate from Streptomyces griseus liquor agreed closely among themselves, although the error of each individual test was high. The value of x2 for each series of assays was very low indeed, and the pooled results had limits of error well within those usually accepted in a biological assay. COMPARISON WITH MICROBIOLOGICAL RESULTS Whenever possible microbiological assays were made of the samples of material tested with chicks. The results are shown in Table IT. We are very much indebted to our colleagues F. W. Wilby and J. E. Ford and to Dr. W. F. J. Cuthbertson, of Glaxo Laboratories Ltd., for these microbiological tests. It will be seen from the table that results by the two methods rarely agreed and that the chick assay gave on the whole considerably higher values.These discrepancies could be the effect of (a) destruction or incomplete extraction of vitamin B,, during preparation of the sample for microbiological assay, (b) the existence of vitamin B,, in a bound form available to chicks but not to micro-organisms or (e) the presence in the148 COATES, HARRISON AND KON: ‘THE CHICK ASSAY OF [Vol. 76 samples of other growth factors essential to chicks but not to EugZena gracilis or LactobaciZlus leichmannii. Two assays of fish solubles and two of whale solubles support the last as the most probable explanation for the following two reasons. (i) Samples 1 and 2 of the fish solubles were prepared from the same starting material, but of the two, sample 2 had been I20 TABLE I1 , 8 8’ -- - -----(y’ - COMPARISON OF THE CHICK ASSAY OF THE ANIMAL PROTEIN FACTOR WITH MICROBIOLOGICAL RESULTS Chick assay Microbiological assay r A - I A \ Sample Result T.F.L.at P = 0.95 Result, Organism pg of vitamin B,, per g pg of vitamin BIZ Per g 0.5 Euglena: gvacibis Fish solubles . . .. 1 1-17 0-99 to 1.37 2 0.30 0.16 to 0.46 0.4 97 3 1.63 1-10 to 2.60 0-7 17 Whale solubles . . . . 1 (a) 0-14 0.03 to 0.27 0.04 0 (b) 0.71 0.22 to 2.11 2 0.43 0-03 to 0.96 0.08 Lactobacillus leachmannii Whalemeat extract . . 0-38 0.110 to 0.61 0.1 Euglena gracilis Fuller’s earth adsorbates 1 120.5 102 to 142 about 60 Lactobacillus.. leichmannti 65 20 to 134 about 37 17 0 I Log Dose Interval Fig. 1. Assay of whale solubles.Doses of standard: 0.5, 1.0 and 2.0 pg per 100 g of diet. Response to standard - Response to whale solubles - - - - which agrees fairly well with the microbiological finding, was obtained with chicks from freshly-depleted hens ; the second, much higher, result was obtained in a simultaneous assay with chicks from hens that had received the all-vegetable diet for at least 12 months. The dose-response curves for these two assays are shown in Fig. 1. We suggest that the freshly- depleted hens, and consequently their chicks, were deficient only in vitamin B,,, but that the hens that had been longer on the diet had become d.epleted of other components of the animal protein factor as well, and that their chicks responded also to these other growth factors in the fish solubles.EXPERIENCE WITH CRYSTALLINE VITAMIN B,, Owing to the shortage of crystalline vitamin B,, it was impossible to use it regularly as a standard in chick assays and a microbiologically-standardised preparation of ExamenMarch, 19511 VITAMIN B12 AND THE ANIMAL PROTEIN FACTOR 149 was used instead. When, by courtesy of Glaxo Laboratories Ltd., a small quantity of the crystalline vitamin became available to us, we made several attempts to compare it with the potency of our standard preparation of Examen by chick assay. These attempts in- variably failed, as the response of the birds to graded doses of the crystalline material, and occasionally of the Examen, did not pass the test for linearity. The responses were haphazard and showed no particular trend, so it was unlikely that the non-linearity was due to instability of the vitamin in the diets.This lack of linearity has not been encountered with crude materials, although it sometimes happened with Examen. Similar difficulties were experienced with assays of crystalline vitamin B12c, also obtained through the courtesy of Glaxo Laboratories Ltd. I t may be that other factors of the animal protein factor complex, present in fish solubles and possibly to a slight extent in Examen, are necessary for the proper utilisation of vitamin B12. I t is also possible that vitamin B!, alone, without the rest of the animal protein factor complex, upsets the balance of the intestinal micro-organisms and interferes with their contribution to the nutrition of the host. DISCUSSION OF RESULTS This comparison of values found by chick and microbiological assay on crude materials containing vitamin B,, shows very clearly that the two techniques are not measuring the same thing.Although it is probable (but by no means certain) that the microbiological methods measure only vitamin BI2, it is evident that values found by the chick tests as used by us represent other growth factors as well. This marked difference in results is particularly interesting with assays using Euglena gracilis, which is believed to be more specific than Lactobacillus Zeichmannii in its response to vitamin BI2. Considerable evidence for the complex nature of the animal protein factor has already been given by Stokstad, Jukes, Pierce, Page and FranklinJ7 by Carlson, Miller, Peeler, Norris and Hewer8 and by Menge, Combs and S h ~ r b , ~ who demonstrated the necessity for other members of the animal protein factor complex in animal nutrition, but as yet little is known of the importance, if any, of these factors in human pernicious anaemia.In work concerning vitamin B,, and its allied vitamins, therefore, it will only be discovered by careful comparison with clinical results whether the microbiological test or the chick test gives the truer assessment of anti-pernicious- anaemia activity, although at present the chick assay is apparently the only means of measuring the whole animal protein factor complex. We are very much indebted to Glaxo Laboratories Ltd. for gifts of crystalline vitamin B,, and standardised Examen, to Dr.J. A. Lovern of the Torry Research Institute, Aberdeen, for the samples of fish and whale products, and to Messrs. D. J. G. Black and J. Getty of the Poultry Department of Reading University for the supply of specially bred chicks for this work. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. Coates, M. E., Harrison, G. F., and Kon, S. K., Biochem. J., 1950, 46, vii. Black, D. J. G., Getty, J., Coates, M. E., Harrison, G. F., and Kon, S. K., Ibid., 1950, 46, viii. Rubin, M., Bird, H. R., and Rothchild, I., Poult. Sci., 1946, 25, 526. Bird, H. R., Rubin, M., Whitson, D., and Haynes, S. K., Ibid., 1946, 25, 285. Coates, M. E., Kon, S. K., and Shepheard, E. E., Brit. J . Nutvit., 1950, 4, 203. British Standards Institution, 1940, Specijication No. 91 1. Stokstad, E.L. R., Jukes, T., Pierce, J., Page, A. C., and Franklin, A. L., J . Bzol. Chem., 1949, Carlson, C. W., Miller, R. F., Peeler, H. T., Norris, L. C.. and Heuser, G. F., Poult. Sci., 1949, Menge, H., Combs, G. F., and Shorb, M. S., Ibid., 1949, 28, 776. 180, 647. 28, 750. THE NATIONAL INSTITUTE FOR RESEARCH IN DAIRYING UNIVERSITY OF READING DISCUSSION DR. T. BARTON-MANN enquired whether Miss Coates had had any trouble, when depleting parent Did Miss Coates collect eggs for incubation over a period from these depleted His own experience showed that depletion of parents led to egg-eating and that collecting stock, with egg-eating. parents? eggs over a period resulted in very great unevenness in growth of chicks.150 LARKIN AND STUCKEY: SOME OBSERVATIONS ON THE p o l . 76 He also referred to the work of Miller and Groschke,’ who had abandoned the depletion of parents After incubation of the eggs the chicks were fed a depleted diet for 14 days MISS COATES replied that she had not herself encountered egg-eating b u t had occasionally had trouble In her laboratory eggs were not individually marked, so that Parent birds appeared to be thoroughly She agreed that culling the chicks after a preliminary depletion period might and fed them a normal ration. and then only those chicks with weights ranging from 75 t o 90 g were selected for assay purposes. with cannibalism in some groups of hens. growth of the chicks could not be related to the age of the egg. depleted after 6 t o 8 weeks. be advactageous, but had not had enough birds available to do so. REFERENCE TO DISCUSSION 1, Miller, D. C., and Groschke. A. C., Quad. BidI. Illichigan State Colf., 1950, 32, 279.

ISSN:0003-2654    

DOI:10.1039/AN9517600146

出版商:RSC    

年代:1951

数据来源: RSC