A MICROBIOLOGICAL ASSAY TECHNIQUE FOR PANTO- THENIC ACID WITH THE USE OF PROTEUS MORGANII

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1 A MICROBIOLOGICAL ASSAY TECHNIQUE FOR PANTO- THENIC ACID WITH THE USE OF PROTEUS MORGANII BY MICHAEL J. PELCZAR, JR., AND J. R. PORTER (From the Department of Bacteriology, College of Medicine, State University of Iowa, Iowa City) (Received for publication, January 24, 1941) Several methods for the assay of pantothenic acid have been reported in the literature. They have been based on the growth response of yeast (l), bacteria (2, 3), and chicks (4, 5) to the vitamin. The relative merits of these methods have been discussed in an earlier paper (3). In a previous publication (6) it was shown that pantothenic acid is one of the growth factors required by Proteus morganii. Subsequent experiments in which synthetic crystalline calcium pantothenate (Merck) was employed in a chemically defined medium indicated that the organism responded to exceedingly low concentrations of this substance; raising the calcium pantothenate level within limits resulted in a corresponding increase in growth. On the basis of this reaction to pantothenate a technique has been developed for its assay. Since the method possesses certain desirable features, its application to routine assays of pantothenic acid in natural materials may be warranted. EXPERIMENTAL Test Organism-A strain of Proteus morganii designated as Strain 21 was used in this study. The stock culture was carried on nutrient agar slants and transferred at 2 week intervals. Preparation of Inoculum-A trace of growth from a 24 hour agar slant culture was taken up on the end of a wire transfer needle and * We wish to thank Merck and Company, Inc., for generously supplying the synthetic calcium pantothenate and the a-hydroxy+?&dimethyla-butyrolactonc employed in this study. 111

2 112 Assay of Pantothenic Acid inoculated into 10 ml. of the assay medium (described below) containing y per ml. of crystalline calcium pantothenate. After 24 hours incubation, 1 drop (approximately 0.05 ml.) of this first subculture, delivered from a 1.0 ml. pipette, was placed in 10 ml. of similar medium. Following a 24 hour incubation period the growth of this second subculture was comparable to the turbidity of a No. 1 McFarland nephelometer tube and it was used to inoculate the assay medium in test determinations. 1 drop of this culture, delivered from a 1.0 ml. pipette, was always used to inoculate tubes containing 10 ml. of media. Inoculation of the assay medium (lacking pantothenate) gave consistently negative results; growth could not be detected by any of the methods employed. Basal Medium-Previous investigations in this laboratory have shown that Proteus morganii can be cultivated in a chemically defined medium of the following composition: KHzPOd 4.5 gm., (NH&SO4 0.5 gm., (NH&l 0.5 gm. The reaction was adjusted to ph 7.4 and the mixture was tubed in 9.5 ml. quantities and autoclaved. Prior to inoculation the medium in each tube was supplemented with Fe(SO*) (NH&S04.6HzO ( M in 0.02 M HCl) 0.20 ml., MgS04.7HzO (0.4 per cent in HzO) 0.10 ml., glucose (50 per cent solution) 0.10 ml., nicotinic acid (low3 M in HzO) 0.10 ml., cystine (M/1500 in 0.1 M HCl) 0.10 ml.; calcium pantothenate (aqueous solution), 0.01 y, contained in 0.10 ml. Assay Medium-Although the organism grew well on serial subculture in the basal chemically defined medium described, it was apparent that the mass of growth, as well as the rate of growth, did not approach the level which was obtained with glucose-meat infusion broth, or other similar bacteriological media. Attempts were made to increase the growth of Proteus morganii by supplementing the basal medium with numerous substances known to serve as growth accessory or stimulating factors for bacteria. None of the media prepared, however, supported growth equal to glucose-meat infusion. Naturally, it is pertinent that a medium for the assay of a substance should be adequate in all respects except for the substance to be assayed. Since we were unable to produce such a substrate by supplementing a simple chemically defined medium with

3 M. J. Pelczar, Jr., and J. R. Porter 113 numerous growth factor substances of known identity, it was necessary to resort to more complex materials. 10 per cent solutions of casein, yeast extract, liver, and proteosepeptone were prepared in N NaOH and autoclaved at 15 pounds pressure for 1 hour to effect complete inactivation of pantothenic acid. Each solution was then neutralized with glacial acetic acid, filtered to remove the precipitate, and finally autoclaved at 15 pounds pressure for 10 minutes. These served as stock solutions from which additions were made to the basal chemically defined medium. After several preliminary experiments in which the basal medium was supplemented with various quantities of the above solutions, it was found that if 0.1 ml. of the alkali-treated proteose-peptone solution was added per 10 ml. of basal media a substrate was obtained which supported growth equal to that obtainable in glucose-meat infusion broth. Not only was growth markedly increased by the addition of 0.1 per cent alkali-treated proteose-peptone to the medium, but the test organism could now initiate growth at a level of y of calcium pantothenate per ml. rather than at y, as was the case when the chemically defined basal medium was employed. Consequently, the medium selected for use in assaying pantothenic acid was the basal medium described above in which the calcium pantothenate was omitted, and to which the alkali-treated proteose-peptone was added to give a final concentration of 0.1 per cent (0.1 ml. of stock solution added to 10 ml. of media). This will be referred to as the assay medium. The assay medium was prepared in liter quantities and then dispensed in 10 ml. amounts into sterile cotton-stoppered test-tubes. Results Response of Proteus morganii to Added Pantothenate in an Otherwise Pantothenic Acid-Free Medium-Repeated experiments conclusively demonstrated that inoculation of the basal medium without pantothenate or the assay medium gave no detectable growth. However, the addition of calcium pantothenate in small amounts permitted moderate growth to take place in these media. The response of Proteus morganii to various concentration levels of calcium pantothenate was determined by three methods: (1) by measuring the increase in turbidity of the medium after a 24

4 114 Assay of Pantothenic Acid hour incubation period with the aid of a Klett-Summerson photometer; (2) by determining the amount of bacterial nitrogen in 50 ml. of media with a micro-kjeldahl technique; (3) by measuring the ph of the substrate after 24 hours incubation with a Leeds and Northrup universal potentiometer. The results obtained by these methods are presented in Table I, in which are also included corresponding determinations ob- TABLE I Response of Proteus morganii to Calcium Pantothenate in Pantothenic Acid-Free Media Amount of~d;~tothenate y per?nl. 0 (Uninoculated control) Response of test organism &s d&mined by Turbidity o- 0: o- o* 7.4 l I PH Bacterial N * Duplicate determinations. The turbidity was measured on the Klett-Summerson photometer. The values for bacterial N are in mg. per 50 ml. tained with the basal chemically defined medium. These data show that growth is absent in the pantothenate-free media. However, when as little as y of calcium pantothenate was added per ml. of the assay medium, a growth resulted which was demonstrable macroscopically and measurable by the turbidimetric method. Growth at this same level could not be detected by either ph or bacterial nitrogen determinations; consequently, turbidity readings were chosen for assay purposes in subsequent experiments. In the chemically defined basal medium the first

5 M. J. Pelcaar, Jr., and J. R. Porter 115 detectable growth occurred when y of pantothenate was added per ml. A standard curve for use in assaying materials was obtained by determining the relationship between growth and the concentra % c FIG. 1. Relationship between the turbidity and the concentration of calcium pantothenate in a chemically defined medium plus 0.1 per cent alkali-treated peptone. tion of calcium pantothenate ranging from to y per ml. of the assay medium. Values for this relationship are tabulated and plotted in Fig. 1. The curve lends itself very well for the evaluation of the pantothenic acid content of extracts from various materials. It is necessary, however, to establish such a curve each time a group of assays is made, since it has been ob-

6 116 Assay of Pantothenic Acid served that slight deviations in the curve result on different days. Application of Method for Assay of Extracts of Natural Materials -Aqueous extracts of natural materials were prepared as previously described (3, 7). The material was autoclaved for 30 minutes at 15 pounds pressure, after which any voluminous precipitate was removed by filtration through paper. The relatively clear filtrates were then autoclaved for 10 minutes at 15 pounds pressure and t,he sterile solutions served as the stock material for assay. A preliminary assay of each extract was first carried out by a titration technique to obtain the approximate pantothenate concentration in the specimen. This was done as follows: lo-fold dilutions were prepared from each extract and 0.1 ml. of each dilution was incorporated in 10 ml. of the assay medium; the tubes were inoculated and after 24 hours incubation were observed macroscopically for evidence of growth. In previous experiments with synthetic crystalline pantothenate, it was observed that y of calcium pantothenate per ml. of assay medium afforded growth of the test organism which was evident by macroscopic observation. Half of this amount, or y per ml., resulted in no growth detectable macroscopically. Consequently, in the preliminary assay of a substance if the tube containing 0.1 ml. of a 1: 100 dilution showed growth but 0.1 ml. of the 1: 1000 dilution was negative, it would indicate that 0.1 ml. of the lower dilution contained at least y, but less than 0.02 y, of pantothenate. Furthermore, it follows that the 0.1 ml. of the 1: 1000 dilution contained less than y of pantothenate. To obtain more exact information two or three amounts of either of these two dilutions were selected which by interpretation of the above results should give turbidity readings between 20 and 60 on the standard curve. These tubes were then inoculated, incubated for 24 hours, the turbidity read, and the pantothenate concentration calculated from the curve. In most instances no interference has been encountered by the opacity or color of the extracts, since the dilutions used have been sufficient to eliminate this error. However, when the amount of extract does impart either opacity or color to the assay medium, it is necessary to clarify the extract, previous to making an assay, by employing an adsorbent such as kieselguhr.

7 M. J. Pelczar, Jr., and J. R. Porter 117 TABLE Assay of Pantothenate from Extracts of Natural Substances II Klett- Summer- Pant0thenat.e Amount of extract incxxporated 80 Pantothenic concentration in 10 ml. assay media nephe- acid equivalent per lg. I,oeamd substance 7 7 Bacto-pep- 0.4 ml., 1 :loo dilution 20-20* tone 0.8 ( 1:lOO ( Bacto-liver :lOO Bacto-yeast 0.2 I 1:lOOO extract 0.3 Cc 1:lOOO Casein 0.1 1:lO I: * Duplicate determinations. TABLE Recovery of Added Calcium Pantothenate from Several Substrates - - Amount 3f pantohenate in hnount 01 I TOtal Specimen to which Ca panto- pecimem :rystalline I slculated Amount thenate was added as deterpanto- I wovered mined by ct&hhep,&antteo- thenate by ==Y added content Bacto-peptone Bacto-liver Bacto-yeast Casein Alkali-treated Bacto-peptone Bacto-liver Bacto-yeast Casein extract specimens extract previous assay Y III Y Y Y Per cent recovery

8 118 Assay of Pantothenic Acid Quantitative Recovery of Pantothenate from Various Substrates- Experiments were carried out patterned after those previously employed (3) for the quantitative recovery of pantothenic acid. The natural pantothenate content of Bacto-peptone, Bactoyeast extract, Bacto-liver, and casein was first determined. Aqueous extracts of each were prepared and the assay was conducted as described. Results of these assays, which are presented in Table II, show a close agreement between values obtained with duplicate samples, as well as with those obtained with different amounts of the same sample. Following this, a known amount of calcium pantothenate was added to a sample of each of the above extracts and the resulting mixtures were then assayed. Similar assays were made in which calcium pantothenate was added to alkali-treated samples of the above extracts. The results are presented in Table III, and the agreement obtained between the assay values and the actual pantothenate content in all instances provides evidence for the consistency in duplication of results and for the specificity of the test. Additional evidence for the specificity of this assay procedure is that it has been impossible to replace the pantothenic acid with any one or a combination of known bacterial growth factors. Furthermore, the intact pantothenic acid molecule is required, since in experiments in which the pantothenate molecule was replaced by its two and cu-hydroxy-@,/3- dimethyl-a-butyrolactone) growth failed to occur. DISCUSSION In this assay procedure a test organism is employed which is capable of responding to a lower dosage of pantothenate than the organisms used in other methods described in the literature. When as little as y of synthetic calcium pantothenate is added per ml. of the assay medium, visible growth occurs. This makes it possible to determine the pantothenate content of extremely small amounts of natural substances, a fact which may be significant, since other non-specific factors which might affect the test, such as inhibitory substances and interference by color or opacity, can be avoided by dilution. Preparation of inoculum is extremely simple and there is little

9 M. J. Pelczar, Jr., and J. R. Porter 119 chance for contamination to occur. The composition of the assay medium approaches chemical definition and consequently lends itself to accurate duplication. SUMMARY A microbiological technique, in which Proteus morganii is employed, has been described for assaying the pantothenic acid content of natural materials. Evidence has been presented which indicates that the response of the test organism to pantothenate is highly sensitive and specific. The organism requires only about y of calcium pantothenate per ml. of medium to initiate visible growth. Results obtained with this test indicate that recovery of pantothenate is practically quantitative. BIBLIOGRAPHY 1. Williams, R. J., and Saunders, D. H., Rio&em. J., 28, 1887 (1934). 2. Snell, E. E., Strong, F. M., and Peterson, W. H., Biochem. J., 31, 1789 (1937). 3. Pennington, D., Snell, E. E., and Williams, R. J., J. Biol. Chem., 136, 213 (1940). 4. Jukes, T. H., J. Biol. Chem., 129, 225 (1939). 5. Mickelsen, O., Waisman, H. A., and Elvehjem, C. A., J. Biol. Chem., 124, 313 (1938). 6. Pelczar, M. J., Jr., and Porter, J. R., Proc. Sot. Exp. Biol. and Med., 43, 151 (1940). 7. Potter, V. R., and Elvehjem, C. A., J. Biol. Chem., 114, 495 (1936).

10 A MICROBIOLOGICAL ASSAY TECHNIQUE FOR PANTOTHENIC WITH THE USE OF PROTEUS ACID MORGANII Michael J. Pelczar, Jr. and J. R. Porter J. Biol. Chem. 1941, 139: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1