one drop of test solution is added to a drop of the reagent on an object slide. After the preparation assumes a dark green color, it is examined under a magnification of 50 to 100 diameters. A solution containing the equivalent of 0.2 of 1% of osmic acid will yield characteristic crystals (Figure 1). At this concentration one drop contains approximately 7 5 y of the metal. The presence of copper or iridium does not seem to inhibit crystal formation. Should the sample be too dilute to yield a positive test within 5 to 10 minutes, or if interfering ions are present, one can take advantage of the highly volatile nature of the osmic acid. I n this case an object slide (more conveniently a “half-slide,” measuring 1.5 X 1 inch)
bearing a hanging drop of reagent solution is placed over a microcrucible or microbeaker containing one or two drops of test solution. TTTith a sample containing 20 y of osmium sufficient fumes reach the reagent to yield a positive test within 5 to 10 minutes a t room temperature. The reaction may be hastened by gently warming the crucible or beaker. If the element present is potassium osmate, for example, a positive test is obtained by dissolving the sample in normal hydrochloric acid, and then applying the reagent solution as described. The crystals exhibit a deep red-green color, and may be opaque unless they are small. The preparation is di-
chroic; if the crystals are sufficiently small (and thin), the colors are green and light tan. The composition of the compound has not yet been determined, but observation of slide preparations indicates that the dried crystals are stable under ordinary room conditions. LITERATURE CITED
(1) Chamot, E. M., RIason, C. W.,
“Handbook of Chemical Xcroscopy,” Vol. 11, 2nd ed., pp. 297-8, Wiley, New York, 1940. RECEIVEDfor review June 19, 1958. Accepted January 5 , 1959. Division of Chemistry, 19th Annual Meeting, Kansas Academy of Science, Ottawa, Kan., May 1958. Work made possible in part by a Cottrell grant from the Research Corp.
Ultramicromethod for Assay of Pteroylglutamic Acid (Folic Acid) Activity WILLIAM N. PEARSON, EILEEN R. BRODOVSKY, E. RAYMOND CARNES, and WILLIAM J. DARBY Division of Nutrition, Departmenfs o f Biochemistry and Medicine, Vanderbilf University School of Medicine, Nashville 5, Tenn.
b A modification of the usual microbiological assay for pteroylglutamic acid effects a 100-fold increase in sensitivity. It is possible to measure titrimetrically 0.01 to 0.10 m y of folic acid in a total volume of 0.20 ml., with S. fecalis as the test organism.
I
of studies of pteroylglutamic acid (folic acid, PGA) metabolism, the need arose for a method of assaying this vitamin in extremely small quantities of sample and in samples of extremely low potency. For example, the study of blood folic acid levels in the rat required a method utilizing only 5 to 10 pl. of serum, especially if serial determinations were to be made and the animal could not be sacrificed. Accordingly, the usual macromethod for the assay of folic acid, utilizing S. fecalis as the test organism, has been modified to permit a submicro level of analysis. The adaptation of the riboflavin assay (an L. casei assay) to a submicro level has been reported by Lowry and Bessey ( S ) , and the general method followed is similar to theirs. The procedure described below permits the quantitative assay of as little as 0.01 my of folic acid activity, l/loo of that which can be measured by the conventional macromethod. N THE COURSE
APPARATUS AND REAGENTS
Gilmont ultramicroburet, capacity 1.O ml. (Emil Greiner Co., S e w York). Test tube racks t o hold 6 X 50 mm. tubes (Sorthern Tool and Instrument Co., 50-23 192nd St., Flushing, S . Y.).
The nutrient broth used to culture the organism consisted of 2.0y0 yeast extract, 0.5% proteose peptone, 1.0% dextrose, and 0.2% potassium dihydrogen phosphate. The nutrient agar on which the culture was maintained with monthly transfer contained all the ingredients listed for the nutrient broth and 1.0% agar in addition. Both nutrient broth and agar mere sterilized by autoclaving a t 121” C. for 15 minutes. Charcoal-treated, vitamin-free casein hydrolyzate was prepared by adjusting the pH of 100 ml. of acid-hydrolyzed casein (loyocasein) (Nutritional Biochemicals Corp.) to pH 3.5 Rith sodium hydroxide. To this was added 2.0 grams of activated charcoal. The solution was shaken on a reciprocating shaker for 1 hour, filtered, and made to a volume of 200 ml. Solutions of purines, amino acids, vitamins, and salts were made as for ordinary microbiological assays. The basal medium used is given in Table I. It is similar to that used in the folic acid macromethod described by the Association of Official A.gricultura1 Chemists (1) except that charcoaltreated casein hydrolyzate was used, methionine was added ( d ) ,and potassium salts were substituted for the sodium salts employed in the macroassay. Folic acid standard solution was prepared by adding 50 ml. of 0.01N sodium hydroxide in 20% ethyl alcohol to 20.0 mg. of pteroylglutamic acid. As soon as the pteroylglutamic acid was dissolved, the pH was readjusted to 7 to 8 with hydrochloric acid, and the solution was made t o a final volume of 1000 ml. n-ith distilled water. This solution was then diluted to give working standard solutions containing 0.01, 0.02, 0.04, 0.06, and 0.10 my of pteroylglutamic acid per 100 pl, The solutions were
stored by freezing them in small portions, so that fresh aliquots could be thawed each time an assay was set up and the excess discarded. Desiccated chicken pancreas (Difco Laboratories Inc., Detroit 1, Mich.) was used as a source of folic acid conjugase. PROCEDURE
The assay was carried out in 6 X 50 nim. borosilicate glass test tubes in a total volume of 0.20 ml. The standard curve was set up by measuring in triplicate 100-PI. amounts of each of the five standards plus a distilled water blank with a Levy-Lang constriction pipet. The same pipet m-as used for all additions, but was thoroughly rinsed before a new standard was added. Each group of three tubes was covered with an aluminum cap and the rack of tubes was autoclaved for 10 minutes a t 121” C. along with a test tube containing 10 ml. of the double-strength medium. Addition of the basal medium and inoculation of the tubes were accomplished in a single step. The tube of sterile, doublestrength medium was inoculated with a single drop of a barely turbid, well washed suspension of a 24-hour culture of S.fecalis 29-21. (This wild strain gives excellent growth and has been used for routine macroassays in this laboratory for more than 4 years, but there is no reason to suspect that the usual organism specified for this assay, S. fecalis ATCC 8043, could not be used.) One-tenth milliliter of this inoculated medium was pipetted into each tube with a sterile, 1-ml. graduated pipet, the tip of which was bent and slightly constricted to permit better control of its contents. Usually one pipet was used for 18 to 20 tubes. Samples t o be assayed were prepared as recommended VOL. 31, NO. 6, JUNE 1959
1 1 13
bate at 37” C. for 18 hours, steamed for 10 minutes to inactivate the enzymes, filtered, and made to a known volume. Appropriate enzyme blanks were also run, but the folic acid activity of the pancreas preparation was found to be negligible. The values obtained by the two methods are shown in Table 11.
Table I. Basal Medium for Ultramicroassay of Pteroylglutamic Acid with S. fecalis
Amount for 100 M1. of DoubleStrength Medium 1000 mfi.
Component Casein (as charcoal-treated hydrolyzate) DLTryptophan 40 mg. DbMethionine 5 mg. LCysteine . HCl 50 mg. GAsparagine . Ht0 60 mg. Adenine sulfate 1 mg. Guanine . HCl 1 mg. Uracil 1 mg. Xanthine 2 mg. Thiamine . C1 . HCl 40 Riboflavin 100 Nicotinic acid 80 Y Pyridoxine . HCl 400 y p-Aminobenzoic acid 100 y Calcium pantothenate 80 Y Biotin 21. Magnesium sulfate . 7H20 40.0 mg. Potassium chloride 2 . 6 mg. 2 . 0 mg. Ferrous sulfate . 7H20 Manganese sulfate . HZO 21.5 mg. Tween 80 10 0 mz. Glutathione 0 . 5 mi. Potassium citrate H20 5 . 7 gram Dextrose 4 . 0 gram Potassium phosphate, 0 . 6 gram dibasic A11 reagents except manganese sulfate were mixed together and the pH was adjusted to 6.8 with NaOH. The manganese sulfate wm then added and the volume adjusted to 100 ml. with distilled water.
20-
1ReprerenIs Zpoinlr of
ldentlcol
volue
-)
Table II. Comparison of “Total” Pteroylglutamic Acid Values“ Obtained by Macro- and Ultramicroassay
UltraMacro- micromethod, method, Sample ?/Gram ?/Gram 21.5 22 8 Yeast extract 5 3 Mustard greens, dried 6 6 3.6 3 0 Turnip greens, dried 2 0 2.3 Soybean flour Samples had been stored for a relatively long time prior to assay; for thiE reason, these values are not considered comparable to “normal” values found in the literature. Table ill. “Free” Folic Acid Values of Human Serum Obtained by Macro- and Ultramicroassay
Donor
Macromethod, Mr/Ml. Serum
Ultramicromethod, hIylM1. Serum
A
0.2
0.0
E a 0.4 G 0.4 0.5 D 0.4 1.0 1.2 C 0.5 1.5 B 0.2 1.9 F 1.9 4.4 H 4.4 a PGA concentration too low to be measurable by macromethod. 11 14
ANALYTICAL CHEMISTRY
for the macroprocedure, with slight modifications only if very small amounts of material were available. One hundred-microliter aliquots were set up in triplicate and treated in the same way as the standard. The rack of inoculated tubes was placed in a desiccator containing some water-saturated cotton batting to provide a humid atmosphere to prevent evaporation of liquid from the tubes. The “desiccator” was closed and placed in a conventional incubator a t 37” C., and the tubes Kere allowed to incubate for 72 hours. At the end of this time, the tubes were removed from the desiccator and steamed for 10 minutes to stop growth, because the titration of a large assay takes more than several hours, during which time growth of the nontitrated tubes might otherwise continue. For titration, the contents of each tube were transferred with a fine-tipped pipet to a small beaker of about 2-ml. capacity, 0.02 ml. of 0.04% bromothymol blue indicator was added, and the sample was titrated with 0 . 2 3 sodium hydroxide added from an ultramicroburet. Stirring m-as accomplished n-ith a jet of carbon dioxide-free air. Foaming was minimal, and it was usually not necessary to add caprpl alcohol to control it. Results were calculated from the standard curve as in the macroassap. A usual curve is given in Figure 1. To check the reliability of this ultramicromethod, several natural materials were assayed for “total” folic acid by this procedure and by the usual macromethod. Each sample was suspended in 0.05M potassium dihydrogen phosphate buffer, p H 7.0 (200 ml. per gram of sample), and autoclaved a t 121” C. for 15 minutes. After cooling, desiccated chicken pancreas was added to each sample as a source of folic acid conjugase; 30 mg. of chicken pancreas per gram of sample was used, except for the high-potency sample, yeast extract, for which 100 mg. per gram was used. The samples were layered with toluene, allowed to incu-
Determinations were made by both methods of the “free” folic acid levels in the sera of several laboratory personnel. Five milliliters of serum was diluted with 20 ml. of water, the p H was adjusted to 6.8 with hydrochloric acid, and the samples were autoclaved at 121” C. for 7 minutes. The samples were filtered, the precipitate was discarded, and the filtrate was frozen for storage. Upon thawing, a flocculent precipitate appeared, so the samples were homogenized in a glass homogenizer, and centrifuged at 2000 r.p.m. for several hours. The samples were then filtered, the p H was adjusted to 6.8, and the solution was made up to a known volume. The results of macro- and microanalysis of serum are presented in Table 111. Only when the serum levels are relatively high is there reasonable agreement between the two methods. Because, in most cases, the folic acid content of serum was very low and the limits of sensitivity of the macromethod were exceeded, the folic acid activity of the serum could not be very precisely estimated by this method. By the ultramicromethod the folic acid content of low-potency material may be more accurately estimated. The performance of this assay has been generally good, although initially, occasional unexplainable erratic points on the standard curve were noted. Considerable effort was expended to eliminate erratic behavior, and this has been greatly reduced. As the folic acid macroassay does not usually have the consistency and precision of other microbial assays-e.g., niacin or riboflavin-this ultramicromethod can be applied with as much confidence as the macroprocedure. ACKNOWLEDGMENT
The strain of organism (8. fecal& 29-21) used in this laboratory for the assay of folic acid was kindly furnished by A. P. Harrison, Jr., Department of Biology, Vanderbilt University. LITERATURE CITED
(1) Assoc. Offic. Agr. Chemists, Washington, “Methods of Analysis,” 7th ed., p. 784,1950. (2) Brodovsky, E. R., Utley, hl. H., Pearson, W.N., Science 128, 307-8 (1958). (3) Lowry, 0. H., Bessey, 0. A., J . Bid. Chem. 155, 71-7 (1944). RECEIVEDfor review September 23, 1958. Accepted January 12, 1959. Investigation supported in part by FSA PHS Grant No. A-147 and by AEC Contract Eo. AT(40-1)-1033.
