~ 112,8 mil 2,5 ~ iii 1112,S Iii ... IHIf_ Iii IIIIiiiiI :: Iii 1111/2.2 :: Iii IIm2.2 I.lol ~ UH~ I.lol ~ um_ III~u~.... "I"~ III~u~.... IIIII~ 1111,1.8 ""'~ 1111,1.25 1111,1.4 "'"~ II11,M ""'1.6 ""'1.6 MICROCOPY RESOLUTION TEST CHART MICROCOPY RESOLUTION TEST CHART NATIONAL BUREAU OF STANDARDS-1963-A NATIONAL BUREAU OF STANDARDS-1963-A' """,., ''''·'I'''~' .--. "- . Teclanicnl Bulletin No. 1023 • January 11TACKS Cpnlparison of the 2,6-Dichlorophenolindo­ phenol and 2,4-Dinitrophenylhydrazine Methods with the Cranlptoll Bioassay for Detenuining Vitanlin C Values in :Foods 12 By ELIZABETH M, HtW'STON, 1;"1.'/11;111., MUIIIIAY ~'ISIIER, biologist, a'tld ELSA ORENT-KEII.ES;' /Ullrilill'l/ I;/II'II,isl, 8/0'1'(/'/1. of Hilma'll. Nutrition and Home ECOIIO'II,.ics, AgricuUu'I"n[, Rt'HCIU'ch Adm'inist.Tution CONTENTS Jlagl: , Page Introduction ~""""""". 1 J Appendix A, summary of data ,Experimental dll'ocedure , , , , ' , 4 ' from the bioassay . " , ... , 22 Chemical pI1lScedure . """" 5 Appendix B. interference of glu- Biological Pi:ocedure ' , 7 ('oreductone in the RO and SUltS and dllkussion , , 10 1 RMOD 2,4-dinitrophenylhydra­ umm~ .. ,,;;!, ......... .. 18; zine methods , ... ,.,' 26 iteratoe citad "',',",. 19 I~ a:> ...- \. en ::I v C'l ~ ( 0::: ~ .INTRODUCTION t d'. .. ~ Th6l5.Rppli'tf\tion of chemical methods to the measurement of 8scor6Tc acicFin animal and vegetable tissues is often complicated by the presEfuce of interfering substances. The most troublesome of these are~he ascorbic-acid-Iike compounds which react in many respects Iik&ltrue m,corbic acid with the two most commonly used, reagents, 2,6-dichlorophenolindophenol and 2,4-dinitrophenylhy­ drazine. Since these substances are entirely inactive in the animal body as antisc(ll'butic agents, however, (8, H)" they give rise to estimates of vitamin C potency which are falsely high when values are calculated from chemical amllYHefl, The reductones (9, 10), which are derivatives of the carbohydrates and which may be present in plants and animals under natural conditions (29, 44), unquestionably reduce indophenol and may also react with phenyl­ 1 SUbmitted for pUblication May 31, 1950. , This research was donc as part of 1\ project Rupported by an allotment made by the Secretary of Agriculture from Special Research Funds (Bankhead-Jones Act of June 29, 1935), 'The authors are indehted to James F, Conch, Bureau of Agricultural and Industrial Chemist.ry, for his generosity in supplying the rutin used in this investigation, • Italic numbers in parentheses refer to Literature Cited, p, 19. 2 TECHNICAl. IlULLI<:'1'IN No. 102:1. lJ. 1:;. DEI'T. (W AGIUCUlJl'UHE hydrazine (:J.1). The othcr intcl"fcrence which ii' encountered most frequently is caused by 2,3-diketogulonic add, a biologically inac- • tive oxidation pr.oduct of ascorbic acid, This substance combines with phenylhydrazine to give the same characteriRtic red color by which ascorbic acid is measured in this reaction, but is entirely inactive with indophenol (31, :Ii). Both reductones and diketogulonic acid are generally thought to be absent from fresh fruits and vegetables; however, their presence has been demonstrated even in fresh products by several lIlvestigators (12, 44). Animal tissues-Hver, blood, and adrenals -have been shown to contain diketogulonic acid (92). It is also found in small quantities in normal urine, a.id may be present in conRiderable amounts in both blood and urine following injectionR of either dehydroascorbic acid or diketogulonic acid (.'12). Reduc­ tones have been found in the adrenalR (2.9). 1'he products which contain the largest amounts of interfering substances, howevet:, and in which the greateRt analytical prob­ lems are encountered, are fruitR and vegetable~~ that either have undergone some form of processing that employs heat, or have been stored for a considerable period of time. Reductones have been identified in many such foods (2.1,22, 2fi, 3/i, 4;1. 44). Diketo­ gulonic acid has recently hf'en identified in a number of both fresh and stored foods by Goldblith and Harris (.12), and discrepancies in aSRay results have been attributed, probably rightly, to diketo­ gulonic acid by other investigators (.1 fI, .'14) . Mapson (22) was able to correct successfully the ascorbic acid • values determined by the indophenol method for the presence of reductones by treating the food extracts with formaldehyde, which condenses with the ascorbic acid. In an attempt to eliminate the effect of interfering substances other thu'_ reductoneR (glutathione, cystein, cystine, etc.) from the ascorbic acid analyseR of urine, blood, and other animal tissues, and to diRpense with the URe of H:!S, the 2,4-dinitrophenylhy­ drazine method was introduced by Roe and his coworkers (20. 36). By this met.hod, all of the ascOi bie acid is oxidized to dehydroascorbic acid which is then treat.ed with the phenylhy­ drazine reagent.; the reRtllting precipitate is dissolved with 85 percent H:!SO.I and the color measured photocolorimetrically. Norit wa!'; the first oxidizing agent used b~' Roe and 0thers; later bromine water and finally bromine vapor (.17) were substituted for norit. By employing a modified pt'ocedure for extraction of the t.issue, Roe and Oesterling (i/8) adapted the method to the measurement of the vitamin C content of foods. These investi­ gators decided that it was unlikely that sufficient reductones occurred in processed foods to have an effect of any appreciable magnitude upon the ascorbic acid values obtained by their method, Snow and Zilva (41,42), however, had demonstrnted the pres­ ence of reductones in considerable amounts in processed foods. Furthermore, Penney and Zilva (33) prepared in the laboratory pure glucoreductone and mixtures of reductones, which reacted with phenylhydrazine under the conditions employed in the Roe­ VITAMIN C-COMPARISON 0.' CHEMICAL AND BIOLOGICAL ASSA1: 3 Kuether (86) method. Penney and Zilva had found also that diketogulonic acid interfered even more markedly than reductones in the reactions. III fact, they had published a method (~rl) for the quantitative measurement of 2,3-diketogulonic acid whi'ch used dinitrophenylhydrazine as the reagent and had employed it in a study of the chemical beh£vior of dehydroascorbic acid in vivo and in vitro (82). In the light of these experiments, Roe, Mills, CeRterling, and Damron (37) further modified the Roe-Oesterling procedure so as to differentiate between ascorbk, dehydroascorbic, and diketo­ gulonic acid8 present in the same tissue extract. No cognizance waR taken of the possible presence of l'eductones, whieh could interfere with the measurement of any or all of these llscorbic­ acid-like substances. During a study of the effect of home cooking procedures on the vitamin and mineral content of a series of foods (17), retentionR of 102-113 percent ascorbic acid were obtained in french-fried and country-fried potatoes, when the indophenol method of Bessey and King (1) was employed. Ascorbic acid retentions were re­ duced to 57 and 67 percent, respectively, when two of the same lots of potatoes were analyzed, using the Mapson formaldehyde modification (22) to correct for reductoneR. This indicated the presence of interfering substances, probably biologically inactive reductones, equal to about 45 percent of the apparent ascorbic acid content. Other investigators have reported anomalous asc;orbic acid values for canned fruit" and vegetables that had been kept at elevated temperaturel'l 1.)1' varying periods of tim€l. In 1945, Guerrant, Vavich, and Dutcher (13) observed losses of aRcorbic acid in samples of fruit juices held at 85° F. for 270 days, but increases in canned yellow corn and lima beans that had been expmled to 11 or' for the same length of time. The authors nttributed these increases "to the formation of nonascorbic acid compounds which reacted with the dye during the course of the assay." They were unsuccessful in their attempt to discover either the nature of the interfering substances or their probable concen­ tration by means of additional analyses by the method of Roe and Oestl1rlin~. Feaster, Tompkins, and Pearce (11) summariz(!d the available data on retention of ascorbic acid in canned fruits, vegetables, and :iuices; storage at 21 0 C. resulted in "small sacrifk~es of ascorbic acid" and even higher temperatures for a longer time, 27° for ] year, resulted in los!;es of only 5 to 15 percent. From their own experiments they reported 90-percent or betteI' retentions of ascorbic acid in canned tomato juice stored at temperatures of 4°, 10°, 16°, and 21°. It seemed logical to que~tion whether or not all figures for ascorbic acid retentions of food~ after prolonged storage might not. in reality. be partially attributable to the inadvertent inclusion of "interfering substance!';'" ir. the ascorbic acid measurements, although it was only when the interfering substances were present in amounts sufficient to give apparent 4 TECHNICAL BULLETIN No. 1028, U. S. DEPT. 0.' AGRICULTURE J' retentions of 100 percent or more that their presence became • evident. Only a bioassay of vitamin C value would prove whether or not the conclusions drawn from chemical analyses were true. A comparison of the indophenol tib'ation with the bioassay as measurements of vitamin C value of a number of fruits and vege­ tables was made by Harris and Olliver (15). They found good agreement between the results of the two methods and concluded that interfering substances were not found in any fresh fruits and vegetables, or various processed materials. No attempt was made by these investigators to show the presence of reductones 01' other interfering substances by chemical procedures. No data have been published making comparisons among the vitamin C values obtained by the indophenol method, the phenyl­ hydrazine method, and the bioassay when substances that inter­ fere with both chemieal methods are known to be present.