A Modified Micro-Fermentation Method for the Estimation of Thiamine EDWARD S. JOSEPHSON AND ROBERT S. HARRIS, Massachusetts Institute of Technology, Cambridge, Mass.
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HE following improved procedure for t h e estimation of thiamine by t h e micro-fermentation method, using Rarburg-Summerson equipment (3) and special flasks (1) has proved to be highly accurate i n t h e measurement of thiamine in plant and animal tissues and fluids. M a n y steps
Preparation of Samples Clear liquid samples require no further preparation. Suspensions of solid samples may be made readily using a Waring Blendor. Preliminary tests must be made by diluting the sample with distilled water until a 1-ml. aliquot, made up to 100 ml., stimulates the production of carbon dioxide by yeast to an extent equivalent to 10 to 20 millimicrograms of crystalline thiamine (1 millimicrogram = 10-9 gram). To one of two aliquot samples in 100-ml. glass-stoppered flasks, add 5 ml. of 4 per cent sodium sulfite and 20 ml. distilled water. To the second aliquot sample add 25 ml. of distilled water. Adjust the pH of both solutions with 15 per cent sodium hydroxide or 8 per cent sulfuric acid to 5.2 using buffered methyl red as indicator. After placing cotton plugs in both flasks, heat in flowing steam a t 100" C. for 30 minutes, cool, and destroy excess sulfite with 3 per cent hydrogen peroxide, using acidified starch iodide solution ( 5 per cent potassium iodide, 1 per cent soluble starch, and 32 per cent sulfuric acid) as an outside indicator (spot test, b), and making certain that all sulfite is destroyed by adding a 2-drop excess of 3 per cent hydrogen peroxide. This is a critical step in the procedure, because the presence of traces of undestroyed sulfite inhibits carbop dioxide production, thus preventing the correction for nonthiamine carbon dioxide-stimulating substances. This procedure is best carried out by preparing in a spot plate a number of solutions containing 1 drop each of the acidified starch-iodide indicator. To test for sulfite destruction after the addition of 3 per cent hydrogen peroxide drop by drop, transfer a drop of the test solution to the spot plate by means of a stirring rod. Destruction of sulfite is indicated by the appearance of a purple color in the spot plate. Adjust both the treated and untreated test solutions to pH 6.2 with 15 per cent sodium hydroxide using neutral litmus as indicator and make up to 100-ml. volume. The concentration of carbon dioxide-stimulating substances in the untreated solution should be equivalent to 10 to 20 millimicrograms of thiamine per ml.,
i n this new procedure are identical with those in previous publications (1, 2) and are repeated here so t h a t t h e complete method may be available in one publication.
Reagents The following aqueous solutions are necessary for this analysis : SOLUTION A, 1 liter to contain 70 grams of c. P. citric acid (C~H807.Hz0)and 90.8 grams of c. P. dipotassium phosphate. SOLUTION B, 1 liter to contain 200 grams of c. P. anhydrous dextrose, 2.2 grams of c. P. monopotassium phosphate, 1.7 grams of c. P. calcium chloride (CaC12.2HzO), 10 grams of c. P. magnesium sulfate ( MgS04.7H20), 0.01 gram c. P. manganous sulfate (MnS04.4HzO), 0.01 gram of c. P. ferric chloride (FeCla.6H20, phosphorus-low), and 0.067 gram of crystalline nicotinic acid. AMMONIUMSULFATE SOLUTION, 15 PERCENT,prepared so that 1liter of final solution contains 150 grams of ammonium sulfate. GELATINSOLUTION,1 PER CEKT, prepared so that 1 liter contains 10 grams of elatin (Difco). THIAMINE STOCK~ O L U T I O S . Dissolve 50 mg. of thiamine hydrochloride (previously dried to a constant weight in a desiccator over phosphorus pentoxide) into a mixture of 26.4 ml. of undenatured 95 per cent ethyl alcohol, and 0.42 ml. of concentrated hydrochloric acid, and make up to 100 ml. with distilled water (4). This solution is stable for several months if kept a t 4' C. in a glass-stoppered flask. Immediately prior to each run, substock and standard thiamine solutions are prepared from the stock thiamine solution as follows: THIAMINE SUBSTOCK SOLUTION, 500 ml. to contain 5 ml. of 1 per cent gelatin and 1 ml. of thiamine stock solution. (This is best prepared by placing 1 per cent gelatin in a 500-ml. volumetric flask with 100 ml. of distilled water and shaking to wet the entire surface, then adding thiamine stock solution and distilled a ater up to volume.) Final concentration of thiamine is 1 microgram per ml. THIAMINE STANDARD SOLUTION A, 100 ml. to contain 1 ml. of 1 per cent gelatin and 1ml. of thiamine substock solution. (This solution is prepared by placing 1 per cent gelatin in a 100-ml. volumetric flask with 25 ml. of distilled water and shaking to wet the entire surface, then adding thiamine substock solution and water up to volume.) Final concentration of thiamine is 10 millimicrograms per ml. (1 millimicrogram = 10-9 gram). THIAMINE STANDARD SOLUTION B, 100 ml. to contain 1 ml. of 1 per cent gelatin and 2 ml. of thiamine substock solution. (This solution is prepared in the same way as thiamine standard solution A.) Final concentration of thiamine is 20 millimicrograms per ml. YEASTSUSPESSION,2 PER CENT, prepared so that 100 ml. contain 2.0 grams of commercial baker's yeast. SODIUMSULFITESOLUTION, 4 PERCENT,prepared so that 100 ml. contain 4.0 grams of c. P. sodium sulfite (Nak30s.7HzO). POTASSIUM IODIDE SOLUTION, 5 PERCENT,prepared so that 100 ml. contain 5.0 grams of U. S. P. potassium iodide. SOLUBLESTARCHSOLUTION,1 PER CENT, prepared so that 100 ml. contain 1 gram of c. P. soluble starch. SULFURIC ACIDSOLUTION, 32 PERCENT,prepared by diluting 33 ml. of 96 per cent c. P. sulfuric acid to 100 ml. with distilled water. SULFURIC ACID SOLUTION, 8 PER CENT,prepared by diluting 25 ml. of 32 per cent sulfuric acid to 100 ml. with distilled water. HYDROGEN PEROXIDE, 3 PERCENT. SonIunr HYDROXIDE SOLUTIOS.15 PERCENT,prepared so that 1 liter contains 150 grams of c. P. sodium hydroxide. Solution A, solution B, the 15 per cent ammonium sulfate solution, and the 1 per cent gelatin solution are plugged with cotton, covered with foil, and sterilized in flowing steam a t 100" C. for 30 minutes on three successive days. Once opened, the solutions should be stored a t 4" C. The 2 per cent yeast suspension and the 4 per cent sodium sulfite solution are made up immediately before use.
TABLEI. COMPARISON OF ASSAY RESULTS FOR THIAMINE BY REVISEDMICRC-FERMENTATION A N D THIOCHROME METHODS Sample Urine A Urine B Urine C Urine D Urine E IJrine F Urine G Urine H
Urine I Urine J Food
A
Food B Food C Food D Food E Food F Food G Food H Food I Food J
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Micro Fermentation Thiochrome Microgram per gram or ml. 0.0663 0.0652 0.0674 0.0620 0.0582 0.0607 0.236 0.224 0.232 0,0383 0.0403 0.0395 0.0299 0.0321 0.0309 0.0590 0.0620
0.0610 0.0300 0.0300 0.0391 0.0385 0.103 0.105
0.0573 0,0584 0.399 0.407 0.486 0.477 0.469 0.479 0.524 0.512 0.803 0.808 0.519 0.507 0.906
0.913 0.744 0.769 0.566 0.579 0.510 0.500
0.0284 0.0370 0.100 0.0609 0.386 0.497 0.464 0.502 0,808 0,507 0,866 0.739 0.553 0.471
INDUSTRIAL AND ENGINEERING CHEMISTRY
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whereas the concentration of these substances in the solution treated with sulfite may now be below this range. If the treated solution is below an equivalent of 10 millimicrograms, it is necessary to add 10 millimicrograms of thiamine ( 1 ml. of thiamine standard solution A) before making up to volume. Experience has shown this to be necessary generally with food samples but not with urine samples.
Fermentation Procedure Prior to each run, grease all ground joints, open the capillary gas valves to the cups, and set the water bath to 30" C. I n the main chamber of each reaction flask place 1 ml. of distilled water. In the inset place 1 ml. of the test solution or suspension which contains carbon dioxide-stimulating substances equivalent to 10 to 20 millimicrograms of thiamine, or 1ml. of thiamine standard solution A or B. Then prepare the buffer-substrate-yeast suspension by mixing in a 100-ml. volumetric flask 15 ml. of solution B, 10 ml. of solution A, and 7 ml. of 15 per cent ammonium sulfate followed by 25 ml. of 2 per cent yeast suspension. Elapsed time is measured from the time the yeast suspension is added. Make the buffer-substrate-yeast suspension to volume and add 1 ml. to the inset of each flask. Fix the flasks to the manometers, place in the water bath, and flush with nitrogen through a manifold. Ten minutes have usually elapsed a t this point if 12 Summerson units (24 Warburg units) are employed. Shake the flasks a t 140 oscillations (70 round trips) per minute a t an amplitude of 3 cm. Stop the passage of nitrogen after 50 minutes have elapsed, and close the capillary gas valves to the cups. Release excess pressure in each unit 5 minutes later. Make a zero reading a t exactly 60 minutes, close the system, and make a final reading 60 minutes later. Calculations are based on the comparison of gas evolved in the test solution after correcting for nonthiamine stimulating substances with the gas evolution due to standard thiamine solutions A and B, assuming a linear relationship between thiamine concentration and carbon dioxide evolution over the 10 to 20 millimicrogram range. The gas evolution due to standard thiamine solutions A and B must be determined at each run.
Kith this modified procedure repeated estimations of the thiamine content of various plant and animal tissues and fluids have shown a n agreement within 2 per cent (Table I).
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Furthermore, these results agree within 5 per cent with those obtained b y fluorometric assays of the same samples (Table I). This fermentation procedure is preferred to fluorometric methods in t h e analysis of samples containing very small quantities of thiamine or when information is desired on t h e fate of thiamine during the handling and preparation of foods and in tissue metabolism. Over 1000 successful thiamine determinations have been performed by the authors using the method described.
Summary A revised procedure for the microestimation of thiamine in tissues and tissue fluids using the Warburg technique is presented. Interference b y nonthiamine substances is corrected by sulfite treatment. Residual sulfite is eliminated b y a peroxide treatment. This procedure is especially applicable to the assay of tissues and fluids low in thiamine (10-8 gram of thiamine per ml.) and in the measurement of thiamine degradation products in tissue metabolism and in the treatment of foods.
Acknowledgment Technical assistance from Elizabeth Weeks and Helen C. Scrufutis is acknowledged.
Literature Cited (1) Atkin, L., Schultz, A. S., and Frey, C. N., J. Bid. Cheni., 129, 471 (1939). (2) Schulta, A. S.,Atkin, L., and Frey, C. N., IND. ENG.CHEM., ANAL.ED., 14, 35 (1942). (3) Summerson, W. H., J. Bid. Chern., 131, 579 (1939). (4) U. S. Pharmacopoeia X I , 2nd Supplement, p. 131, 1939. FROMt h e Department of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Mass. T h i s research has been supported hy a grant from the Williams-Waterman Fund of Research Corporation.
Colorimetric Microdetermination of Iron C. P. SIDERIS, Pineapple Research Institute of Hawaii, Honolulu, T. H.
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HE observation of van Klooster (1) in 1921 t h a t nitroso R salt forms a green color with ferrous iron was later con-
firmed by the writer (2). Further studies on the reaction between ferrous iron and nitroso R salt, started in connection with various potassium analyses where iron occurred as a contaminant, suggested the possibility of using this reagent for the determination of ferrous iron, T h e method, as described below, has been found satisfactory for determining iron in plant tissues.
Reagents STAXDARD IROXSOLUTIOK.Dissolve over a hot late 1 gram of metallic iron C'rust-free" wire) in a 1000-ml. flasi containin 10 ml. of concentrated sulfuric acid and 100 ml. of water, coo$ and make to volume with water. Standards containing 0.2 t o 20.0 micrograms of iron per ml. are prepared from this solution. XITROSO R SALT, Place 0.5 gram of nitroso R salt in a 100-ml. volumetric flask containing 70 ml. of water, dissolve by gentle agitation, and then complete the volume with redistilled (ironfree) acetone. The reagent keeps well for many months. SODIUM ACETATE. Place 544.3 grams of sodium acetate (ironfree) in a 1000-ml. volumetric flask, then pour in 300 ml. of water, and shake gently a t intervals while heating on a hot plate until complete solution, Make to volume with water and filter.
HYDROXYLAMINE SULFATE.Place 10 grams of hydroxylamine sulfate in a 100-ml. volumetric flask containing 80 ml. of water, shake until complete solution, and complete the volume with water.
Procedure Ash about 5 grams of dry plant tissue in a platinum crucible. Dissolve the ash with 10 ml. of 5 N hydrochloric acid by heating slowly over a hot plate. Add to the platinum crucible approximately 20 ml. of water. Heat the mixture to about 90" C., transfer to a 100-ml. flask, cool nearly to room temperature, dilute to the mark, and filter. Place a 10-ml. aliquot of the solution in a test tube and neutralize with 5 N sodium hydroxide in the presence of a very small piece of litmus paper. Remove the latter with a glass rod and add 1 ml. of hydroxylamine sulfate, 1 ml. of nitroso R salt, and 2 ml. of sodium acetate. (Either 2 ml. of sodium acetate or 1 ml. of 1.5 N ammonium hydroxide can be used for adjusting the solution to pH 8 t o 10.) Make to 15 ml. in a graduated test tube and allow to stand for 20 mlnutes or longer. Transfer to a cell, preferably 2.5 mm. in thickness, and determine the color intensity with a photoelectric colorimeter, using a No. 47 light filter with transmission limits 445 to 505 millimicrons. A 2.5-mm. cell can be obtained by using a plunger 7.5 mm. thick in a 10-mm. cell as supplied with the Summerson-Klett photoelectric colorimeters. In case of formation of a precipitate by calcium with sulfate or
