588 JONES: A PLATE ASSAY TECHNIQUE; FOR INOSITOL IN YEAST [Vol. 76 A Plate Assay Technique for Iaositol in Yeast BY A. JON.ES A Petri-plate technique is proposed for the assay of inositol in yeast by means of Saccharonzyces carlsbergeizsis; and, for one yeast, the results are compared with those of the h7eurosPora method. INOSITOL has been assayed in various products with the yeasts Saccharomyces cereviseae172 and Saccharomyces ca~lsbergensis~ and also with the inositol-free mutant 37401 of NeurosPora c~assa.~ Plate assays have been used previously for assaying several of the vitamins of the B complex5s6s7s8 and the possibility was therefore investigated of using a similar technique for inositol to that used for the assay of vitamin B6.9 In these laboratories the Neurospora assay for inositol was found to be unsatisfactory and a more reliable method, such as a plate assay with its simple apparatus, was desirable.EXTRACTION OF INOSITOL- Beadle4 and WoolleylJo used hydrolysis with either 3 per cent. sulphuric acid or 16 per cent. hydrochloric acid. The latter appeared to give a better extraction of inositol from dry brewer's yeast.4 Several methods of extraction were investigated in the plate assay, i.e., hydrolysis with N , 2 N , 3 N and 5 N hydrochloric acid and N , 3 N and 5 N sulphuric acid, and also with takadiastase plus papain, in which method 0.3 g of takadiastase and TABLE I METHODS OF EXTRACTION OF INOSITOL FROM YEAST Inositol content, mg/g A f \ Extraction method HCl N .. 2 N .. 3 N .. 5 N .. %SO, N .. 3 N .. 5 N .. Takadiastase + papain .... .. .. .. .. .. .. Yeast 129 r-A-, Mean 2.75, 2.75 2.75 2.94, 3.06, 2-76, 2.76, 2-96, 2.61, 2.50, 2-53, 2.72 2.48, 2.70, 2-66, 2.67, 2-70, 2.75 1.95, 2.40, 2.22 2.33, 2.19 2-36, 1-97, 2.06, 2.18, 2-16, 1.70, 1.95 1.78, 1-83, 1-70, 1.76 0.99, 1.08 1.04 Yeast 885 Yeast Y r+ Mean Mean 1.44 1 4 4 1.44 1.44 1.99, 2-04, 2.75, 3.33, 2-04, 1.83 1.98 3.08, 3-44, 3-37, 3.36, 3.27, 3.39, 3.14 2-79, 2.70, 3.00, 3.12, 3.19, 3.12, 3.23 0-98, 1.10 1.04 0.90 0.90 0.3 g of papain were added to 2.0 g of yeast in 15 ml of acetate buffer at pH 4.5 and incubated overnight at 37" C. When either hydrochloric or sulphuric acid was used, the yeast samples were hydrolysed with 10 times their weight of the appropriate acid. After hydrolysis by autoclaving at 15 lb pressure for 2 hours with sulphuric acid, or for 1 or 2 hours with hydro- chloric acid, the pH value of the sample was adjusted to 4.6 with sodium hydroxide.When hydrochloric acid was used, heating in an autoclave for 2 hours appeared to extract no more inositol than heating for 1 hour.OCt., 19511 JONES: A PLATE ASSAY TECHNIQUE FOR INOSITOL I N YEAST 589 The high concentrations of sodium chloride in the solutions after neutralisation of the yeast samples hydrolysed by 5 N hydrochloric acid did not appear to have any effect on the growth of S. carlsbergensis on the plates, but when 5 N sulphuric acid was used the sodium sulphate formed on neutralisation crystallised out readily and led to error in assessing the volume of the samples. No more inositol appeared to be released by 5 N than by 3 N hydrochloric acid, but the definition of the growth-zones after extraction with 5 N acid was generally better than with 3 N .Takadiastase plus papain appeared to release little inositol from yeast; this is in agreement with the findings of Beadle.4 Hydrolysis with hydrochloric acid gave higher results for yeast than did sulphuric acid. The results of these different methods of hydrolysis are shown in Table I. BASAL MEDIUM- The basal medium was essentially that of Atkin, Schultz, Williams and Frey,ll with the addition of 2.5 per cent. of agar. The higher concentration of agar was found necessary during the summer months, when growth zones tended to be less sharp with 2 per cent. of agar, as was shown with the basal mediumg used for vitamin B,.METHOD Distribute the medium (Table 11) in volumes of 20 ml in boiling-tubes and sterilise by steam for 20 minutes. TABLE I1 BASAL MEDIUM Glucose . . .. .. .. 50g Aneurine hydrochloride . . 250 pg Potassium dihydrogen phosphate 0.55 g Calcium pantothenate . . 1000 pg Citric acid .. .. .. 1.0 g Pyridoxine hydrochloride . . 1000 pg Casein hydrolysate . . .. 4.0 g Salt solution*. . .. .. 25ml Volume made up to . . . . 900ml pH value adjusted to . . .. 4-6 Agar .. .. .. . . 22.5 g .. .. 8 CCg Potassium citrate .. .. 5.0 g Biotin . . .. Nicotinic acid . . .. .. 1000 pg * The salt solution contains 1.7 g of KC1; 0.5 g of CaC4.6KO ; 0-5 g of MgS0,.7H20; 0.01 g of FeC1,.6H20 and 0.01 g of MnS0,.4H20 per 100 ml. PREPARATION OF THE INOCULUM- Maintain S.carlsbergensis by fortnightly sub-cultures on 4 per cent. malt agar, at a pH value of 5 to 5-6, in 1-ounce screw-capped bottles, with incubation for 18 to 24 hours at 28" to 30°C. Wash off the growth of yeast from each bottle with 10 ml of sterile saline, centrifuge, wash once with saline and resuspend the yeast growth in sufficient saline solution to make the opacity of the suspension correspond to tube No. 9 or 10 of the Wellcome standard opacity tubes. PREPARATION OF THE PETRI PLATES- Take five tubes for the standard and five for each test and hold at a temperature of 48" to 50" C. Add 1.0 ml of inoculum suspension to each tube, mix thoroughly before pouring and allow the plates to cool on a flat surface. Cut four holes in each plate by means of a 10 mm cork-borer and remove the agar discs.Just before use, heat the tubes in a bath of boiling water. PREPARATION OF THE YEAST SAMPLES- To 2.0 g of yeast add 20 ml of 5 N hydrochloric acid and sterilise at 15 lb pressure in the autoclave for 1 hour. Cool and adjust the pH value to 4.6 by the careful addition of approximately 50 per cent. sodium hydroxide solution. Adjust the volume to give an estimated concentration of approximately 400pg of inositol per ml, filter and dilute the filtrate 1 to 2, 1 to 4 and 1 to 8.590 JONES: A PLATE ASSAY TECHNIQUE FOR INOSITOL 1s YEAST [Vol. 76 Prepare standard plates to cover the rapge 50, 100, 200 and 400 pg of inositol per ml. Place 0-1 ml of each dilution of the standard and of the test solutions in the appropriate holes of the Petri plates, and incubate a t 28" to 30" C overnight. Yeast 12.5 25 50 100 mg per mi Standard 50 100 200 4 0 0 ~ per mi Fig. 1.Dose Dose - response curves for standard and for yeast Measure the diameters of the growth-zones in millimetres. For the standards, plot the mean diameters against the concentrations in pg per ml. For the samples, plot the mean diameters against the concentrations in mg per ml. As in the vitamin B, assay,n the effect of doubling the concentration of the substance being estimated is to increase the zone diameters by 2.5 mm. TABLE 11" ZONE DIAMETER READINGS FOR STANDARD INOSITOL AND A YEAST SAMPLE Inositol standard Concentration, pg/ml r 50 mm 25.0 25.5 25.5 25-0 25-5 100 mm 27.5 28.0 28-0 28.0 28.0 200 mm 30.0 30-5 30.5 30.5 30.5 3 400 mm 32.5 33.0 33-0 33.0 33.0 25.3 27.9 30.4 32.9 Yeast sample Concentration, mg/ml 12.5 mm 23.0 22-5 23.0 22-5 23.0 25 mm 25.5 25.0 25.5 25.0 25.5 50 mm 28.5 27.5 28.0 27.5 28.0 100 mm 31.0 30.0 30.5 30.0 30.5 22.8 25.3 27.9 30-4Oct., 19511 JONES: A PLATE ASSAY TECHKIQUE FOR INCSITOL IN YEAST RESULTS 591 Table I11 and Fig.1, in which the zone diameters are given to the nearest 0.5 mm, show typical results for a standard and for a yeast sample a t various dose levels, From these can be seen the spread of the readings for any given dose throughout the five plates, as also the linearity of the dose response to the standard and to the yeast sample. In a recovery experiment a yeast sample solution was taken, containing 200 ,ug of inositol per ml (mean value of four levels of assay), and to 10 ml of this solution were added (a) 10 ml of a solution containing 200 pg of inositol per ml and (6) 10 ml of a solution containing 400 pug of inositol per ml.They gave mean assay values of (a) 200 pg of inositol per ml and (b) 303 pg of inositol per ml. These mixtures were assayed at four levels. COMPARISON WITH THE Neuros$ora ASSAY- the same hydrolysed samples being used in both assays. of N . crassa was found to be essential for a reasonably satisfactory assay. in two separate assays are shown in Table IV. For yeast 129 a comparison was made between the plate and Neurospora methods, A heavy inoculum of the spores The values obtained TABLE IV A COMPARISON OF INOSITOL ASSAY VALUES BY S carlsbergensis AND Newospora Yeast 129 S. carlsbergensis Autoclaved 15 lb pressure for Xeuvospova assay plate assay 1 hour with 5 N HC1 Inositol, Inositol, mg/g mg/g First assay 1 .. .. .. 3.21 2-04 2 . . .. .. 3.33 3-06 Second assay 1 . . .. .. 3.83 2 .. .. .. 3-92 3 .. .. .. 3-89 4 .. .. .. 3.79 5 .. a . .. 4-01 2- 70 2-75 2.70 2.66 2.67 The agreement shown by the first assay is good, but in the second, the Neurospora method shows considerably higher values. These may be accounted for by the fact that the growth responses to the standard inositol in the Neurospora assay were almost identical in both assays, but the growth responses to the test samples were very much greater in the TABLE V RESULTS OBTAINED IN A (2 + 2) ASSAY OF INOSITOL I N YEAST Zone diameters in millimeters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Totals Means Standards lob pg/ml 31.5 29.5 32-0 31.5 31.5 31.0 31.0 31-5 31.0 31-5 31.5 31-0 31.0 31.0 31.0 31.0 ..498.5 1 400 pg/ml 36-5 35.0 37.0 36.5 36.5 36.0 36.5 36-5 36-0 36.5 36.5 36.0 36.0 36.0 36.0 36.0 579.5 7 1 to 60 30.0 29.0 30.0 29.0 29.0 29.0 30.0 29.5 29.0 29.0 29.0 28.5 28.5 29.0 29- 0 29.0 466.5 31.156 36.219 29,156 .. Yeast -7 A- 1 to 15 35.0 34.0 35.0 34.0 34.0 34.0 35.0 34.5 34.5 34.0 34.0 33.5 33.5 34.0 34.0 34.0 547.0 34.187592 [Vol. 76 second. Other Neurospora assays on yeast 129 failed through lack of growth in two-thirds of the flasks. Table V shows the results obtained in a (2 + 2) assay using 16 plates, with a dose-ratio for standard and yeast Y of 4 to 1. When these results were submitted to variance analysis, no significant difference was found between the slopes for standard and test sample; this confirmed the validity of the assay technique.The estimated potency from the 64 readings was 3.29 mg of inositol per g with limits of 3.05 to 3.54 mg per g at a probability level of 5 per cent. From the 20 readings of the first 5 plates, which showed the greatest variation in response, the estimated potency was 3-60 mg per g with limits of 3.12 to 4-20 mg per g (Table VI). JONES: A PLATE ASSAY TECHNIQUE FOR INOSITOL I N YEAST TABLE V I VARIANCE ANALYSIS OF (2 + 2) ASSAY AS IN TABLE v Treatment Degrees of freedom Variance Difference between samples . . 1 Linear regression .. .. 1 Unparallelism , . .. 4 . 1 Error . . .. .. .. 60 65-00 407.59 0.0039 0.27 Difference between samples . .1 17.112 127-51 { Error . . . . .. .. 16 0.28 Linear regression . . .. .. 1 5 Plates Unparallelism . . .. .. 1 0.0125 The average value, 3.14 mg per g, for yeast Y (Table I) obtained from a total of 76 plates, assayed on different days, is in good agreement with the value of 3.29 mg per g from the 64 plates in the (2 + 2) assay. SENSITIVITY OF METHOD- By this method amounts of inositol of 20pg per ml can be detected, but for greater precision the extracts should contain at least 60 pg per ml, which is equivalent to 0.9 to 1-0 mg of inositol per g of original sample. The sensiti.vity and range of the assay method are, possibly, not as great as could be desired, but, against that, it is rapid and simple. The Neurospora method has the advantage of a lower assay level, but the disadvantage of a long incubation period and irregularity in the mould growth in the liquid medium.This irregularity appears to be an inherent defect in all Neurospora assays, and it has been all too frequently encountered in these laboratories with the N . crassa mutants in the assays of inositol, choline and biotin, and with N . sitophila in the vitamin B, assay. The plate assay has also been used to determine inositol in molasses (2.80 mg per g). Thanks are due to the Directors of Beecham Research Laboratories, Limited, for permission to publish,^ to Dr. S. Morris for his interest and encouragement in this work, and to Mr. V. Thorne for assistance with the statistical analysis. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. REFERENCES Woolley, D. W., J . Biol. Chem., 1941, 140, 453. Williams, R. J., Stout, A. K., Mitchell, H. K., and McMahan, J. R., University of Texas Some, S., and Sobotka, H., Arch. Biochem., 1947, 14, 93. Beadle, G. W., J . Biol. Chem., 1944, 156, 683. Bacharach, A. L., Nature, 1947, 160, 640. Jones, A., and Morris, S., Analyst, 1949, 74, 333. Bacharach, A. L., and Cuthbertson, W. F. J., Ibid., 1948, 73, 334. Genghof, D. S., Partridge, C. W. H., and Carpenter, F. H., Arch. Biochern., 1948, 17, 413. Jones, A., and Morris, S., Analyst, 1950, 75, 613. Woolley, D. W., J . Biol. Cheun., 1943, 147, 481. Atkin, L., Schultz, A. S., Williams, W. L., and Frey, C. N., Ind. Eng. Chern., Anal. Ed., 1943, Publication No. 4137, 1941, p. 27. 15, 141. BEECHAM RESEARCH LABORATORIES LIMITED BROCKHAM PARK, BETCHWORTH, SURREY February, 195 1