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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<rue 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.
EXI'EUlMENTAL l'HOCEDUHE
Interfering subAances are present in largest amounts in foods which have undergone either sume heat treatment or prolonged storage. Since canned foods are processed products likely to have uniform composition, especially If all cans are from the same lot, and are convenitmt for handling and storage, they were selected for expel'imentation. Preliminary chemical tests carried out on nine different fruits and vegetables canned in tin showed that . peaches and sauerkraut were too low in ascorbic acid content for convenient analysis by bioassay, pineapple in heavy sirup dark ened excessively with the phenylhydraZIne reagent, and the red pigment in raspberries made photocolorimetric readings difficult. Tomato juice (two brands), "dextrose-sweetened" on~~!!e juice, "sugar-sweetened" orange-grapefruit juice (two brands), grape fruit segments in heavy sirup, and spinach proved to be suitable for the study. Suflkient amounts of these canned foods for the entire investi gation were purchased at one time on the Washington, D. C. market. Four cans of each product wel'e opened upon arrival at the laboratory. Two of these were analyzed individually and two were combined and analyzed for ascorbic acid, dehydroascorbic acid, and reductones, and these results were averaged. Since chemical tests showed that reductones were entirely absent from the jukes and a small quantity only was present in spinach, as received from the market, 24 to 30 unopened cans of each product were stored at several elevated temperatures for different lengths of time in order to produce the interfering suhstances it was designed to study. Tomato juice was stored at room temperature (229 -35° C.) for 1,19, and 30 weeks; all the foods were stored in constant-temperature oven8 held at 45°,60°, or 73° C. for intervals varying from 3 days to 6 weeks, depending upon the particular experiment. To equalize the effect of temperature gradients within • the own, the positions of the cans were interchanged at weekly VITAMIN C-CUMI'AIUSON 0'" CHEMICAl. AN)) 1II0V)GICAL ASSAY 5
intervals during the long storage pel'iods and daily during the short periods. In order to hold the samples unchanged for bioassay or chemical analysis, an equal number of cans of each food was quiek-frozen as purchased and then held in a freezer at _400 C. until needed. The cans that had been stored at elevated temperatures were similarly frozen and held for assay after the required time in storage had elapsed. All five of the canned products were analyzed by the indophenol method of Bessey and King (1), including H~S reduction to measure both ascorbic acid and dehydroascorbic acid (15), and with the addition of the Mapson formaldehyde procedure (22). The Roe-Kuether phenylhydrazine method (dU), in which norit is an oxidizing and adsorbing agent, was applied in preliminary studiei;, but wai; replaced by the Roe-Oesterling (RO) (:18) proce dure, which s\lbstttutes bromine for 110rit and thm; eliminates incomplete elution of ascorbic acid from norit ai; a source of error. Parallel samples of stored and uni;tored orange-grapefruit juice, tomato juice, and spinach were a!'iHayed biologically for vitamin C value, for comparison with the chemical analyses. In addition, these three products were analyzed by the Roe-Mills-Oesterling Damron (37) modification (RMOD) of the phenylhydrazine method which differentiates between the ascorbic, dehydro ascorbic, and diketogulonic acids present in animal or plant tissues and which ,vas published while this study was in progress. • CIIEMICAL PIIOCEIIl!ItE EXTRACTION.-The contents of the cans of juices to be analyzed were thoroughly mixed in a beaker under nitrogen. A sample was removed and extracted under CO.,;' in a Waring blendor with 200 ml. of a mixture of 5 percent-HPO;: and 10 percent glacial acetic acid at pH 1.8. The quantity of juice taken was in the proportion of one part of :i uice to five or seven parts of acid, depending lIpon the ai-icorbic acid content of the food. A weighed portion of the slurry was filtered through glass wool into a volumetric flask, and diluted to volume with the acid mixture. The amounts of food and degree of dilution were calculated so that 1 ml. extract contained approximately 6 to 50 Itg. (micro grams) of ascorbic acid; the final ratio of acid to food varied from 7 to 40 parts to 1. Aliquots of this extract were used for analysis by both the indophenol and RO phenylh~rdrazine methods. For analysis by the RMOD method, extracts were prepared by thoroughly stirring' in a beaker ('qual pal·ts. by weight. of the juice or slurry and a lO-percent solution of Snel:! in 5 percent HPO;;, filtering the mixture through glass wool, and adding Rufficient
5 percent HPO:1 to give approximately a 0.5 percent concentration of SnCI:!, and diluting to a convenient volume for analysis.
'CO:! was preferred 1.0 nitro~en for its cooling effect, which helped offset hesting cllused by hlclldor. 6 TECHNICAL nULLETIN No. 1023, U. S. DEPT. OF AGRICUI.1'URE
It was necessary to sample the grapefruit sections and spinach .... in a different manner, The juice was first thoroughly drained ... from the solid and both l)ortions were weighed; samples of the solid and juice were ana YZt1d separately, Spinach was analyzed also by combining aliquots of the juice and leaves in a Waring blendor in a ratio corresponding to their original weights, Cen trifuging was substituted for filtering in separating the extract from the solid material. 2,(;-DICHLOIWPHENOLINDOPJl"~NOL METIIOD.-Ascorbic acid was measured by the method of Bessey and King (.l) and dehydro ascol'bic acid by reduction with H::S, as described by Harris and Ol1ivel' (.15). The Mapsoll procedure (:!2) as modified by Hewston and coworkers (17) was uHed to correct theHe values for the presence of reductolles. 'I'he chief modification consisted in the replacement of H:!SO'I with HPO;; for al'l'esting the formaldehyde reaction. Experiments of MapRon (22) had shown that iodine would oxidize cCl'tain intel'fel'ing substance:; that tended to increase the indophenol value and that were not amenable to the fOJ'maldehyde correction. J<'ollowing this suggestion, an additional procedure was introduced in these analyses by which bromine vapor, the oxidant employed by Roe and otherH, was substituted for iodine as an oxidant and the extract Willi then reduced with H.,S before titrating- with indophenol. This is, in effect, the principle-employed in the RMOD procedure, in which the final step, however, is color production with phenylhydrazine, • The dye solution waH freshly prepared euch week and stored in the dark at 30 C. Standardization of the dye with sodium thio SUlphate (4, i, ;!S) instead of ascorbic acid proved to be accurate and more economical of both time and matel"ial. 2.4-DI NITIWPH ENYLHYIJRAZI NE l\fETHODS.-The Roe~Oesterling method (8) waH employed without modification, . The Roe-MiIlR-Oestcrling-Damron method (7) wafol used for diffel'entiating between diketogulonic acid, dehydroascorbic acid, and ascorbic acid. These mlthors recommend a 6-hour incubation of the extracts with the phenylhydl'azine reagent. Either a shorter or longer period was more convenient in this laboratory, and pre liminary tests after 3 and 23 hoUl"s' incuiJation showed that the 23-hoUJ' pel'iod was satifolfactory, ThE.'refore, 23 hours were sub stitut£!d for the 6 hours of the originHI method. The Evelyn photoelectric colorimeter, with the 540 m", filter, was used for most of the spectJ'ophotometric readings: later, when a Beckman spectrophotometCl", Model DU, became available, it wus employed. Compal'ison of the two instruments, with identical samples, showed that they could be used interchangeably with no error.U
• Typical rl'sult!! with identkal sample!! read with both Evelyn colorimeter, and Reckrnan spectrophotollleter: OrangL'-grapefruit jUice as purchased: E reuding, :18.2 mg.; R rending, 37.7 mg. Stored orange-grapefruit jlJiee: • .E reuding, 10.0 mg.; n reading, 9.5 mg. Stored tomato juice: E l'eading, !1.4 mg.; B n:uding, ,10.2 mg. VITAMIN G- ('OMI'AIUSON OF ,'lIt:MICAI. ANI) 1lI01.0GlCAI. ASSAY 7
The Crampton bioassay procedure (6) which relates odonto blast growth to vitamin C intake, was used. MANAGEMENT OF ANIMALS.-Weanling male guinea pigs weigh ing 200 to 250 gm. were purchased from one breeder as required. They were given a basal scorbutogenic diet supplemented with cabbage and kale until they reached 300-310 gm. in weight.T During this period, which varied in length from 1 to 3 weeks, de~ending upon how readily the animals accepted the new ration, dally weighings were made. When the required weight had been attained, the assay period was begun. At this time assay groups of six to eight animals were given a modified basal diet, with measured supplements of either the food under investigation or pure ascorbic acid instead of cabbage and kale. DIETS.-The basal ration used was a commercial pellet rat dietg found to be free of ascorbic acid by chemical tests, supplemented with 21 mg. alpha-tocopherol in cottonseed oil,1! and percomorph oil to supply 2,770 IV (International Units) vitamin A and 350 IV vitamin D per week. This basal diet, although not identical with the one used by Crampton, was very similar to it. Yellow corn meal, which was absent from the Crampton diet, was present in this one, and beet pulp and fish meal, present in his, were absent from this diet. Crampton used "dried, long-stored grass clippings" as roughage, which he regarded as essential. Filter paper was used as rough age (5) in these experiments; one 2- by 3-inch strip per day per • animal was eaten readily, and produced the desired effect upon fecal consistency. Guinea pigs fed this diet with a daily supple ment of either 100 gm. fresh cabbage and 50 gm. kale or 5 mg. ascorbic acid grew steadily for periods up to 6 weeks and on autopsy showed no signs of scurvy, such as joint hemorrhages, softness of bone, rough hair, or lack of muscle tone. Two conditions, which have been troublesome to other investi gators who have attempted to feed dry experimental diets to guinea pigs (.7, 5), were also encountered in the pre~~ent study: Refusal to eat, and dialThea of irregular occurrence, when dietary ascorbic acid was not present in optimum amounts. 'Therefore, it was found nece8Rary to furnish additional concentrates with the basal diet, in order to stimulate appetite, and control diarrhea in
T Crampton prefers to place guinea pigs on the ~corhutogenic diet at 28 ± :l days of age; however, sinc(' the animlll~ lIsed in this study were not raised in this Ilihorarory, ;iOO-:310 gm. weight appeared to be II more constant criterion for beginning the assay. • Percent composition of commercial rat diet: Whole ground yeIJow corn, 20; whole pulverized oats, 10.25; whole ground wheat, 20.5; linseed oil meal, 5; soybean melt!. 5: dehydrnh~d beef meal, 5; beef liver meal, 2; powdered American cheese, 4; skim milk powder, 7; dried cheese whe~', 3; alfalfa leaf meal (U. S. No. L sun-cured, carotene content not less than 60 pg. per gm.), 10; irl'ltdiated yeast. 0.25; iodized NaCI, 0.5; CaCO:1, 1; vacuum processed hone meal. 0.5; manganous sulphate, 36.8 gm. percent per ton of reed; and cold-pl'esBed wheat germ oil, 8 oz. per ton of .feed. • Alpha tocopherol acetate (Merck) in We!l!lon oil. 8 TECHNIC."I. IIULLE'!'IN No. IO~3, U. !:i. UEI''!', OF AGWGUlll'UH!o;
the assay animals, 'I'he following f;upplements were given b~' s~Tingc: (1) Approximah'ly 0.4 gm, brewer's yeast'" as an aqueous f;uf;pcmlion, daily; (2) 0.45 ml. liver extract" twice a week; (3) 100 mg. rutin'~ weekly tc animals under 400 gm, • in weight and twicc thifi amount to the heavit'r animals, The rutin appeared to be particularly effective in prcventing diarrhea, PREPARATION AND FEEDING 01-' ASSAY SUPPLEMtJNTS.-During the assay the vitamin C supplements wel'c fed to the animals thl'ice wcekly (.~5). As a standard of refercnce with each assay, ascorbic acid was fed at three levels-3.5, 7,0, and 14.0 mg, per week. It was freshly prepal'ed af; an aqueous solution beforc each feeding period; feeding was complett'(\ within 15 minutes of the time the solution W,:\·'! made, to avoid oxidative destruetion of the vitamin. The foods uncleI' aSf;ay were al';o fed at thrce levels, calculated to supply vitamin C valuc equivalent to thc three ascorbic acid le\"els. Six to C'ight guinE'a pigs for each af;say were fed the scorbutogenic diet only, as negative controls, The juices prf'senl:l.'d no unusual problems of sampling and feeding. t;sualiy one or two cans, depending upon the number of animal::-; to be fed, were opened under nit.. o~en every othe.. feeding day; once a wcek samples we ..e withd ..awn for chemical anal~'sis by both the indophenol and phenylhyd..azine methods, and the juicE' for the r1('xt feeding was st:QI'ed in hottles under nit..ogen at ~)' C. The amounts of juice to be fed were calculated from the total ascorbic add content of the food, as measured by the .indo phenol I·eactioll, co .... ected 1'0" reduct0lw::,;. Canned spinach presented certain problem::.; in the bi()a::.;sa~·. • It was planned originally to w('igh the ::.;pinach leaf directly into [('('ei CLIpS and allow the guinea pi~s to ('at it \'oluntaril~·. Howevel', they had no liking for spinach, and frequently did not eat the sUPj)lement for s(>\'eral hOllrs. Experiments showed that ascorbic aCl( was lost from the leaf rapidly under these conditions. There fore, feeding by syrin~e became a necessity. A ::-;Iurr,v consisting of a weighl'd amount of the leaf and a proportional amount: of drained liquid, which had been made 3 percent acid with I-IPO:;. was prepared in the Waring blendor, Sinec further dilution was necessary in order that the slurry would flo\\' freel,\' in the (e('ding syringe, a quantity of 5 per'cent mC'taphosphoric-l0 percent acetic- acid mixture equivalent in weight to the acid juice in the sluI'ry was added. An aliquot of this l:Hlspension was weighed, adjusted to pH4 with 2 N XaOH and diluted with 0.05 N pH4 citrate to a volume such that 25 ml. of the suspension was (·quivalent to .15 !,'1l1. of the diluted slurry. This pI'ocedureresulted in a supplement of relatively low vitamin C content, of which it was necessary to feed a large volume and
1. A suspension of 10 gm. brewer's type 200-B dry yeast, in 25 ml. dis tilled \\'ater. and. 1 ml. 95 per<;'t'nt ethyl alcohol, II Lederle's concentrated liver extrad (injectable), ;) U.S.P, units per ml. U Solution of 8 gm. rutin in 40 ml. propylene glycol, and 20 drops 50 peret:nt sucrose Bolution. • VITAMIN C-C.OMI'AJUSON .W CJU;MIGAL AN)) BIOLOGICAL ASSAY 9
which contained HPOa in appreciable amounts. In order to reduce the volume of a sJingle feeding, one-half of the ascorbic acid value required by the assay was given as pure ascorbic acid and the other half as spinach slurry. The supplements were fed five times a week instead of three, ns in the other assays. In order to validate this procedure, the ascorbic acid controls for the spinach assay nlso were fed five times weekly, and six guinea pigs w()re fed a quantity of the slurry sufficient to assure that they would get the equivalent of 1 mg. of ascorbic acid per day as their sole source of vitamin C. This was the largest amount of the acid slurry received by any of the animals on the spinach assay. The addition of a small amount of sucrose to the feeding solution increased the acceptability of spinach to the guinea pig. The same amount of sucl'ose was fed to both the ascorbic acid reference animals and to the negative controls. Also, eight; guinea pigs were given 7 mg. ascorbic acid per pig per week fol' (j weeks, with the addition of pure glucOl'eductone, equivalent to 7 mg. of ascorbic acid. 13 The glucoredllctone was prepared in the labol'atol~Y accOI'ding to the method of Euler and MartiuH (!J). HISTOLOG'/CAL PROCEDUKE.-At the end of 6 weeks, the experi mental klnimnls wel'e killed by chloroform, autopsied, examined for gross signs of scurvy, and the lower incisor teeth removed for histological examinatiOIl. The teeth were fixed, stained, and sec tioned as r1i!commended by Crampton (U). The final measurement of odontoblast length was facilitated by projecting 11 brilliant • image of the section, enlarged approximately 1,600 times, to a high-glaze! white paper. This was done by mounting a rig-ht-angle prism to the 10 X ocular of the compound microscope, using the 4-mm. objective. One of the newer powerful microscope spot lamps" was used directly beneath the condenser, The inner and outer ma'l"gins of the odontoblast layer in the critical region of mature odontoblasts (u) were tmced by hand on the paper; the height of this layer was then measured with a centimeter ruler to 0.05 mm. at five locations. The places selected for measurement were chosen at random, except that when the cells differed markedly in height, care was tak~~n to include both long and short cells in those measured. The ayerage odontoblast length of each section was finally converted to microns by means of a fador obtained by projecting a stage micrometer scale to the same magnification as the sections. It was possible to measure the actual height of the odontoblast layer to 1 micron. Thl,'! projection method proved supeJ"ior to the filar micrometer usually employed. and res lilted in a great saving of time and eyest;rain. with no sacrifice of accuracy.
II On thc bllsis of til(' indophenol tit.ni:ion value, 0.79 mg. giucul"cductOjlC ill equivalent t() 1 1lI~. aseorhk add. . . I' "FrCijl1cl hiJfh-inlcnsit}' Spot-lit,·, dl,;'gk 1110dt'l, 10 TECHNICAl. BULLETIN No. lO~:J. U. S. ))EI'T. (JF AGHICULTUHE
Four sections of each of the two lower incil;or teeth of each animal were examined and the results averaged, in evaluating • responses to the vitamin C supplements.
HESULTS AND DISCUSSION Data from the ehemical analyses of the foods before and after SUbjection to a variety of stomge conditions are summarized in tables 1 and 2 and compared with the biological evaluation,r. in table 3. CANNED 'FOODS, AS PUlWIIASED.-Both dehydroascorbic and ascol:bic acids were present in the canned foods amllyzed soon after plII'chafie, with the exception of spinach, which showed no evidence of dehyci"oaHcorbic acid. The dehyd"oasco"bic acid of canned fruit!;; and juices rCI)I'esclltcd a somewhat larger fraction of the total ascorbic acid than is usually reported for fresh foods UfO).
TAI:IL"~ 1.·-E'Jj'cct of stO/'n{le at, severol fCmpCl'(lt'iwe.'l1lpon (L.';corln:c acid t' '\;;'8;;(;' Apj'H:ndix A for di:;tW;sion of dduils of bioussay and calculaliollil of • yit;alllill C pot.('l1cy. VITAMIN (",.. cnMI'MHf;ON OF ('In;~fI(·'''. ANIi mOJ.O(;JCAI. AilSA:\' 11 TABLE I.-Effect nf .qtomoc at .q(',l)(wal tnnpcmt1l.1'c.'J u.pon (t,scorbic acid va.lue.'1 (m,!/. lW1' .100 om.) u,lId 'i1It(l1'f(~1'ino substances in 5 canned products. (IS wW(I.';IIl1'ed b11 the 2,6-dichlorophmwlindo phenol and 2,~-di1l'it)'Ophenylh1ld1'Uzine methods-Continued Phenyl I ndUllhenol ' hydrnzine Produet and a10rnlte c:ondit ionR nt!K:ll'y-I{in~ Act~r IIr.. Hud - Ro..... Il,!forc Aftlll' II~!-; tr'(!nt- O~~fltl!rlinll' IbS II~:; mcnt Canned ornnge-grapefrult juil'e ("l!\\'I,·('t. ened with sugar") : Sample 1: At purchalle ;,12.0 3,1.8 36.3 3S.8 Stored 7 days at HOoO. 28.0 33.G 34.2 2S.8 Stored 10 day;; at, (iO°C. 24.:1 :n.:1 33.1 30.7 Stored 7 days at 7:.1°0. 8.9 8.1 10.2 (10.8) (18.3 ) Sampl;J'2: At purchase , ;19.5 42.8 33.G 37.3 Stored 7 days at (iOoO. 2G.4 aa.5 33.3 30.9 Stored 10 days at GOoO. 2:l.5 31.5 30.S 30.0 Oanned tomato juice: Sample 1: At pUJ'chase ,15.4 W.O 17,4 lS.0 Stored 4G days at room tel11pera turc (22°_35°0.) 1(i.O Stored 1:1:.\ days at J'()0111 tPI11Jlera ture (22°-:35·0.) Hi, 0 18.7 17.9 19.7 • Stored 210 day!> at l'OO))) t.CI11Jll'I·H' ture (22°_::5·0.) 18.5 15.6 17.0 (22.2) (22.0) Storcd 14 day~ at: 45"('. 11.1l 13.2 14.9 16.9 ( 1:1.8) (15.4 ) ( 18.4) Stol'ed 28 days lit 45"C. 14.1 1(i,8 Hi.5 13.2 Stm'cd 5 days lit 7:1·0. (i.0 13.0 11.4 7.2 (7.!i ) ( 14.9) (.14.6 ) Samplc 2; AtpUJ'chase .17.(i 20.0 22.0 20.0 Stored 5 days at 73°0. .13.1 11l.8 17.6 17.8 (lS.i!) Canned spinach (drained leaves): At purchase 17.7 15.1 J9.8 22.5 (I n.O) Storcd 21 days at 45°0. 14.7 18.a 18.2 19.0 (1M) Stored ,l2 days at .t1l"0. 20.5 20.(i 17.1 22.6 (::!-i.(l) (:2(;.9) ,Jtored 10 day!; at (iOoO. lli.2 18.4 22.9 18.4 (17.2) (20.3) (23.5) Stored 7 days at 73°0. 12.1l 10.5 (15.5) (22.6) (Hi.7) I All values determined by indophenol titration have hcen corrected for interfering substances by the l\fapson fOJ'mllldehyde condensation: when only 1. figure is given, the cOlTection is zero, indicating that no reductones were present; when a subtraction has been made for the presence of reductones, the uncorrected "apparent aSCOI ~ic acid" figure is in parentheses. ~ ~ TABLE 2.-Effect of tempc1'(lillre alld storage on total a.sco·rbic aci{l, ascQ/'bie acid, dehydroascorbic aeid and. ;J Q reductone content in 5 canned foods as memmred by the 2,6_(/-ichlorophenoii11dophenol-jonnaldehydc 'method :x; Z (8essey-King-Mupson procedure) (3 > Reductone interference t" Total ascorbic acid Distribution of total us(orLic add = , c: Tot.al a~corbicacid eo Product and I ~ All{larcnL I Aftc: Percent or storat:e oonditions Contlt!nt Chang-e Ascorbic acid DchydrO:lscorbic acid , correctIon apparent. Z l)f:'T 100 durinl: aRcorbic Z ~m. ~tonlg'p Content Cont.ent acid ? l"}r )00 I..,r 100 o ,;tn. I j..'III. ,..-.., -,,-- -._-- -~---- J/:;. Pen'CHt My./ j Pcrcolt Mg'; Percent Jig. JIg. P<:rccnt. != Grapefruit: 100 gill. : 100 gm. :r. Segments: e At purchase 27.S 22.6 I 81 5.2 19 27.8 27.8 0 to! o -3(; 82 :t2 18 23.5 17.9 24 Stored 10 days at 60 e. 17.9 14.7 I ~ Stored 5 days at 73°e. 5.5 -80 5.5 100 0 0 11.5 5.5 52 Drained liquid: ~- At purchase .. :. o I 15.6 13 C'l Stored 10 days at 60 e ..... 15.6 14.9 96 .7 4 l'7.9 ::e Stored 5 days at 73°e ..... 6.3 6.5 100 0 0 13.3 6.3 53 is Orange juice: c: Sample 1: :.; 36.3 36.3 0 c: At purchase ...... 36.3 28.2 78 8.1 22 I ::e Stored 21 days at 45"e ..... 30.0 -17 23.7 79 6.3 21 '27.2 23.7 13 l': Stored 5 days at 73°e ..... 15.4 -58 10.1 66 5.3 34 16.6 15.4 7 Sample 2: At purchase ...... 38.2 :14.9 91 3.3 9 I 38.2 38.2 0 Stored 3 days at 73°e 24.9 -35 18.1 73 6.8 27 31.5 24.9 21 ..... I • • • Orange-grapefruit juice: Sample 1: At purchase a4.8 az.o 92 2.8 8 34.8 34.8 o ~ 0 Stored 7 days at 60 C. aa.13 3 ~8.0 83 5.6 17 33.6 33.6 o 0 Stored 10 days at 60 C. :.11.3 ---10 24.3 78 7.0 22 31.3 31.3 o < 0 :; Stored 7 days at 73 C. 8.1 -~,77 8.9 100 o o '10.8 _ 8.9 18 > Sample 2: ll: .) 'f At purchase 42.8 39.5 fl2 ~.~ 8 42.8 ; o Z 0 42.8l Stored 7 days at 60 C. :33.5 -22 26.4 79 7.1 21 a3.5 o ~ c a3.5j·, Stored 10 days at 60 C. :n.5 - -26 23.5 75 8.0 25 :31.5 31.5 o ! Tomato juice: e- "" Sample 1: ll: ~ At purchase ..... 16.0 15A fli5 .6 4 16.0 16.0 o 0 Stored 14 days at 45 C. 13.2 18 11.5 87 1.7 13 15.4 13.2 14 ~ Stored 28 days at 45°C. 16.8 o 14.1 84 2.7 16 I 16.8 16.8 o ~ Stored 5 days at 73 0 C. 13.0 ·19 6.0 46 7.0 54 14.9 13.0 13 i Sample 2: :::. At purchase 20.0 17.6 88 2.4 12 20.0 20.0 o "'! Stored 5 days at 73 0 C. 15.8 21 15 13.4 85 !!.4 15 .18.5 15.8 ~ Spinach: ~ Drained leaves: ll: . At purchase ...... 15.1 17.7 19.0 15.1 20 > Stored 21 days at 45DC. 18.3 +21 14.7 80 3.6 23 '16.0 14.7 8 ~ 0 Stored 42 days at 45 C. 2D.6 -~36 20.5 100 .1 o 24.6 20.6 16 0 > Stored 10 days at 60 C. 18.4 -~22 16.2 88 2.2 12 20.3 18.4 I 9 z Stored 7 days at 73 0 C. 12.5 :-17 12.5 22.6 45 -' ::: t Reductones in this product were destroyed by II;!S treatment; this value represents reductones present before H;!S treatment. g g to '£> 1'1>> 0( ~ w ··7~ . ~ TABLE 3.-Comparisun of the 2,6-dichiol'uphellolin ----,----~---.-.. - ----~1 ~ Cramllton;; \\·f"'\,k BeHHey·Ki n~ ~ hioas,,~s D.~t"(,'I'if1tion(If sample f;r Aft"r Roe-Millo- ----.~---..... I'!1 ...·": - Br:,!-H::S Ro ,Ot>sterlinJ;-" hio:H;!-\ay _-_.- Befnt°t.' After" treatment j Oest.;>dm!-!"l Damron c H~ H~ . I ~ ------~------. --_.- -.._---- l'l Orange-grapefruit juice: I 31.7 30.8 I 30.5 30.0 33.1 :3z Sample 2: I (35.1) Held at -40· C. fo), I8!) days hefon' ;.;tart of ;\ 29.5 32.61 29.4 . 30.7 35.5 r assay. I (34.5) 36.0 31.6 • 30.4 28.0 (33.6) . (30.0) ~ 41~ c I 30.5 I Average :l3.1 30.4 32.2 1:13.0 (89-162 percent) ;r. I 30.3 '== (332) , After storage at -no C. for 5 days, held at 1 10.7 17.9 I 11.6 I 17.6 13.0 ~ I -; __40° C. 189 days before start of aSiiay. (27.5) j (24.4) (13.1)1' 3 6.6 11.5 I 10.7 i 17.4 11.8 c· (9.4) (21.7) (23.6) I (12.8) -.; :: 4 7.5 13.2 I 9.5 16.6 15.5 I :> (12.7) , (24.2) , (23.3) (16.3) ; Average 8.3 14.2 ; 10.6 ; 17.2 13.4 f !l.6 (75-136 percent) S 1 I (14.1)\ ~-=~ Tomato juice: 23.5 1 18.0 I 18.3 I 22.4 ~ Sample 2: i 0 (23.6) Held at -40 C. for 1 day before start of assay. 2 17.1 22.3 19.8 I 20.1 19.9 ! (21.1) j 4 17.1 19.4 15.6 21.8 15.0 I (20.3) I (15.9) . • • • • • ti 15.9 i 21.7 1!I.7 19.7 ' 15.9 ' ( 17.0) 8 15..l : 20.2 18.3 20.1 14.2 ; t (14.2 ) <: Average ----16.7 I 21.4 18.3 r---ZO-:8"I'-16.2 : 19.4 (75-180 percent) ~ ( 17.1) ! z After storage at 73" C. for 5 days, held at 1 ! I 10.7, [ 8.6/ _40" C. for 1 day before 5ta,·t of assay. (21.'i ) (20.0) f 2 I 9.0 1.4.8 14.0I 13.0 (14.0)'''.5, 8 I i!: !J..J ; 4 I 10.9 8.6 12.7 9.9 "CI I (14.2) (12.6) (10.5) > I i!! 9.-J 9.4 9.7 12.9 10.2 I y: 6 ! o (13.5) (13.9) (10.9) J z 8 I !L5 12.1 1:3.5 ]1.1 8.5 (]3.9) (9.4) c· ------j "! 9.2 ]:(8 10.51 U.(i (92 -170 pcn·t:nt) C A.verage ...... ,...... 13.2 L ,12.1 I ==toJ ---.---~--- (11.2) ::C='--- .. c--:-= :: Spinach: ~-.. 1 1 14.:l 14.6 13.9/- - 18.6 I 18.1 ;;- Leaves and liquid: (17.5) ; ( 18.8) (18.4) I ( 19.2) r- Held at 40" C. for 5\; days befOl'C start of assay. 5 1:3.7 16.2 17.2 r > Average 14.2 I --~I 14.6 ]8.6·~]8.1 122.1 (fl6-221 percent) ~ Aftel sturage at 45" C. for 42 days, held at ' J--t ]3.0 13.2' 12.4 19.{j 20.6 o 40' C. for 51l days before start of assay. (16.8) (17.9). (18.9) (22.7) 5 13.0 J 4.4 I 16.0 ~ ~ '-. Average t ~-I 13.4 I 13.1 1~6-I-Zo.6 IIG.l (82-187 PH~~nt)r > 1 All values determined by indophenol titration have been corrected for interfering substances by the Mapson formaldehyde con ~ densation: when one figure is given, this correction is zero, indicating that no reductones were present; when a subtraction has ~ been made for the presence of reductones, the uncorrected "apparent ascorbic acid" figure is in parentheses. . : The figures in parentheses represent ascorbic-dehydroascorbic acid before the correction for diketogulonic acid has been made. 'Figures in parentheses indicate exact fiducial limits for 95 percent of the cases...... 01 16 ,'ECHNICAL HUl.LETIN No, 1023, U, S, DEPT. 01-' AGIUCULTURE The juices and fruit gave no evidence of reductones by the Mapson formaldehyde correction. Spinach, on the contrary, con • tained small amounts of reductones following treatment with H!,!S. Smythe and King (Sf)), and Miller (2U) have shown that reducing substances may actually be formed in plant or aTlimal m~terial by reaction of H~S with many carbonyl compounds, including intermediates of carbohydrate metabolism, and a num ber of qui nones. Not all compounds of this nature are differen tiated from ascorbic acid by the Mapson procedure. Only traces of diketogulonic acid were indicated (table 3) in the freshly purchased canned foods that were analyzed by the RMOD method (27). The vitamin C value of the unstored juices measured by bioassay agreed well with the chemical measure ments, and the chemical methods agreed among themselves. It appears from these experiments that the original Bessf;y-King method, omitting even reduction with H~S, is the simplest and most practical chemical procedure to employ in routine analyses of foods, and at the same time accurately represents their vitamin C value, provided it is first ascertained that the proportion of dehydroascorbic acid is relatively small, and that no interfering reducing substances are present. The vitamin C value of the canned spinach, measured by bio assay, was higher than the figures obtained after taking into account the reductones and diketogulonic acid present. The reason for this is not clear, and none of the data explain this discrepancy. CANNED FOODS, STORED AT ELEVATED TEMPERATURES.-Changes • in both ascorbic acid and a:.;corhic acid-like substances occurred in these canned foods during storage at elevated temperature:.; for short periods of time. The higher the temperature, the more marked a.nd extensive were these alterations. These data are presented in table 1 and fmmmarizetl in table 2. Tomato juice was the most Htable of the products studied. Spinach was unique in that there appeared to be actual increases in the ascorbic acid during holding at a moderately high tempera ture (tables 1 and 2) ; it was only when held at 73° C. for 7 days that a loss of 17 percent occurred. Table 2 also demonstrates that falsely high ascorbic acid retentions would have been re ported for approximately half of the stored samples if no correc tion had been made forreductoneR, and emphasizes the necessity for measuring reductones in foods which have been subjected to heat or storag-e. The proportion of dehydroascorbic acid in the total ascorbic acid increased at elevated temperatures except in the grapefruit, and orange-grapefruit juice held at 73°. In these instances, dehydroascorbic acid was ausent, preHumably because destruction of ascorbic acid was proceeding so rapidly (table 2) that dehydro ascorbic acid was disappearing as soon as formed. Although the results of the several chemical methods agreed reasonably well with one another and with the bioassay when the unstored foods were analyzed, this uniformity did not occur • VITAMIN C-COMPARISON 0.' CHEMICAL AND DlOLOGJCAL ASSAY 17 in the stored foods, except tomato juice, in which agreement among all the methods was good throughout. ' The Roe-Oesterling procedure gave somewhat high values, espe cially for the grapefruit segments, orange-grapefruit juice, and spinach: in some instances the RO values agreed more clostlly with the uncorrected than with the formaldehyde-corrected vaiues. From this it is concluded that certain reductones for which the Mapson method makes correction are not completely removed by bromine oxidation. In other instances the RO value was lower than the corrected Bessey-King figure. The most obvious explana tion for these reactions is that under some circumstances, reduc ing substances are formed in foods for which correction is made more adequately by bromine oxidation than by formaidehyde condensation, However, the fact that "reductone:;" differ among themselves quantitatively in the intensity of color produced with the phenylhydrazine reagent, makes interpretation of these figures ditlicult. Penney and Zilva (31) have published results comparing the chromogenic activity of the compounds formed with phenylhydra zine by a number of such SUbstances, including glucoreductone, reductic acid, and ascorbic acid, Further, these investigators showed that there were also differences among these reducing substances in their reactivity toward indophenol. In the present study, comparison has been made between ascorbic acid and glucoreductone in their reactions with both phenylhydrazine and indophenol (see Appendix B) . In geneml, the results obtained using the Br2-H:?S treatment • agreed better with the indophenol than with the phenylhydrazine values; however, this was not invariably true. The vitamin C activity of the stored orange-grapefruit juice indicated by bio assay was lower than the ascorbic acid values obtained by any of the conventional chemical methods, but agreed well with those given by the Br~-H~S procedure, Stored tomato juice presented .fewer problems in chemical analysis than did orange~grapefruit JUice, possibly because of ibl lower sugar content. Likewise, the bioassay agreed well with all of the chemical methods. Spinach, when tested biologically, showed a potency higher than that given by the two indophenol methods, and lower than the two phenyl hydrazine procedures. The reductones present in the canned foods had no vitamin C activity. Bioailsay of pure glucoreductone served as further evi dence of this fact (table 4). This is in agreement with the obser vations made byE~ller and Klussmann (8) and Cruickshank as cited by Harris and Mapson (H) that "reductones" do not pre vent scurvy in guinea pigs. The problems presented by the vitamin C analysis of processed or stored vegetables and fruits are complex. Neither 2,6-dichloro phenolindophenol nor 2,4-dinitrophenylhydrazine is specific for ascorbic acid. 'rhere are many reducing compounds other than ascorbic acid which are formed in foods during preparation for • eating, and which read with the two substances more or less 18 TECHNICAL BULLETIN No. IO~3. U. S. DEPT. OF ,\GIUCULTURE readily, depending upon the conditions under which the analyses are performed. Some of these "reductones" are artifacts produced • by the reagents used in the analysis. There is no single chemical procedure which can be employed routinely for the ascorbic acid analysis of fruits and vegetables in which reducing substanees are present which behave like ascorbic acid chemically but not biologically. The bioassay, par ticularly the Crampton odontoblast-growth method, is specific for vitamin C activity and although less precise than chemical tests, remains the only sound method for the evaluation of vitamin C potency of such foods. SUMMARY 1'he mcmmrcment of biologically active ascorbic acid in foods by chemical means is often made di/licult by the presence of biologically inactive substances which react with 2,6-dichloro phenolindophenol and 2.4-t\initl'ophenylhydrazine, the two com pounds commonly ufo;ed in this meafo;urcment. 1'hese interfering reducing substances are most often encountered in veg'~tables or fruits which have been processed or stored and may give rise to crr'oncow;I~' high values when vitamin C is measured by chemical methodi'\. I!1 the cxperi!11cnt:-; rep?rted here. five canned pr?,ducts-g~ape frUIt segmenti; In heavy sIrup. "dextrose-sweetened orange JUIce, "sugar-sweetened" orange-grapefruit juice, tomato juice, and • spinach-wcI'e analyzed for a!;corbic acid, dehydroascorbic acid, and interl'cl'ing substances behaving like a:-;corbic acid by methods which employed both indophenol and phenylhydrazine. lnter ference was removed by either formaldehyde condensation, bro mine oxidation. 01: both. The canned foods were analyzed as received from the market, and after storage at room temperature and at elcvatC!d tempemturcs for relatively short periods of time. Parallel bioassays by the Crampton odontoblast-growth method were performed with three of the foods-canned orange-grape fruit juice, tomato juice, and spinach-before and after storage. The studies indicated that these canned foods, as received from the market, contained the largest proportion of their ascorbic acid value as ascol'bic acid, although more was present in the dehydro form than is usual in the fresh products. Interferences, rnmovable by either MapRon's formaldehyde condensation procedure or bromine oxidation, were absent. The results of all the chemical methods, as well as the bioassay, were in good agreement. Canned spinach Was exceptional in that it contained no dehydroa!'!corbic acid and gave evidence of redl/ctones by the formaldehyde reac tion. It is concluded that lise of the relatively simple Bessey-King indophenol method i!-! ju!-!tified as a routine procedure in the analysis of fruits and vegetables for biologically active ascorbic acid, if pr:eJiminary test:.; have first eliminated the presence of other reducing substances as a source of error. Even reduction with H ~S may he omitted as part of the routine. provided it is • first ascertained that the proportion of dehydroascorhic acid is negligible. Storage of the canned foods at elevated temperatures "cl'ulted in losses of al;corbic acid and inc..eases in the dehydroascorbic acid fraction, except when dest..uction of asco..bic acid was accel erated. Relatively la ..ge amounts of "reductones" were also pro duced. If no co ....ection had been made fo.. the biologically inactive "reductones," the vitamin C ..etentions would have appeared con siderably higher than those shown by bioassay. However, the Mapson formaldehyde condensation, 01' b..omine oxidation, or a combination of the two, g-ave chemical estimates of vitamin C value which agreed well with the bioassay in two of the foods that were analyzed by all of the procedu..es. Orange-grapefruit juice, which was relatively high in sug-a .. content, decreased more in vitamin C value and gave evidence of the production of a mOI'e complex a ..ray of inte..fering· reducing compounds than did tomato juice. Canned spinach behaved in an anomalous manner, the reaSons for which are not apparent. The bioassay value of the canned product, as pu..chased, was higher than even the unco....eded chemical measu..ements; sto..age at high tempe..ature decreased only slightly both ascorbic acid (chemical) and vitamin C value (biological). These stUdies indicate that when extraneous reducing sub stances are present, no single chemical pl'ocelllll'e as ~ret devised can be applied to all types of foods to obtain an acc:u ..atc measure of vitamin C potency, Neither the .2,(i-dichlorophenolindophenol nor the 2,4-dinit..ophenylhydrazine reagent is specific for aHcorbic: • acid, but each reacts with a vHI'iety of reducing compounds, under the conditions of the tCflb,. No sint\'le method or combination of methods used in these studies eliminated all intel'ference under all circumstances, Under some conditions, the oxidizing ~lI1d reducing reagents themselves reacted to form additional interfering compounds. A further complication is introduced in that the various reducing substances react quantitatively to different degrees both in the titration of indophenol and the production of color with phenyl hydrazine. Bioassay remains the only specific measure of vitamin C in the presence of interfering redlldn~ Huhstances, since no existing chemic:al procedure is entirely adequate JOI' this purpose. 1.ITER'\TLIRE CITED (1 ) BESSEY. O. A" and KING. C. G. 193:3.· TilE IH!':TIURUTION O~' VITAMIN C IN PLANT AN.D ANatAL TISSUES, ANI) ITS 11En~IIMINATION. Jour. BioI. Chem. 103: 687-698. (2) BLISS. C. I., and MAIIKS. H. P. 1939. Tilt: RIOJ.(I(;II~AI, ,\SS.H OF INSULIN. II. TilE ESTIMATION OF DRUG POTENCY FROM A I:RAIlEJl RESPONSE. Quart. Jour. Pharm. and Pharmacol. 12: 182-205, illus. (3) BOOTII, A. N .. ELVEIIJEM. C. A., lind HAIIT, E. B. 1949. TilE IMPORTANCE (W RIJLK IN TilE Nl!TIIITIf):>; (W THE GUI:>;EA PIG • • Jour. Nutr. 37: 21;:1-274. 20 'I'tx;tfNICA1. JlUJ,{.fo..'·IN No. 1023. U. S. VEI''I'. (II' MiIUCUL'I'Ullt: (-I) HUCK. R. E., and RITCIIIE, W. S. 1 !I:iS. A NEW METHOD f'OR Til.: $TANJlAIIJlI1.ATIO;'I: O~· Tilt: I.n: USEII FuR Til.: DETERMINATIIlN "" rF.VITAMIC ACIJl (VITAMI!\: c). (Ab stract) Indus. lind Eng-in. Chern., Analyt. I-~d. 10: 2fi. (5) COllEN, H., lind MENIIEL, L. B. • 1918. EXPERIMENTAL SCURVY (IF THE GUISEA PH: IS ItELATIOS TO THE DIET. Jour. BioI. Chern. 35: 425~453. (6) CRAMPTON, E. W. 1947. TilE IiR(lWTH (It. TilE ODONTOBLASTS OF Til.: INCISOIt ToOTH AS A CIUTERION tit· THE VITAMIN C I!\:TAKE Of' TilE IiUINEA PIG. Jour. Nutr. ;1:.1: 491.504, illus. (7) DICK, H. .\!I:.I1. Disscrtlltion (Pl'illlkJurt) quoted 1.9:1:1 in Handbuch del' Pflan zenlinalY!le. cd. G. Klein. Viennll. (8) EULER, H .., WiN. and KLUSSl\lANN, E. Ifl33. /'11 YSIOLOmSCIi t: VEItSU<;IIE \)Bf:1t VITAMIN f' (ASCOItHINS'AURE) UN!) RE!)UKTON (ENOL-TAItTI""N AUIEHYII). Hoppe-Seylers Ztschr..f. Physic.!. Ch(!rn. !.! 17 : Hi7-17(;. (9) --- and MARTIUS. C. \!I;!:'l. ·(JHf:lt. £1 N HOCIiItEIIUCIEItEN()ES Zl.ICI..:ItIl.:ItIVAT (II.:IIUKTON). Svcnsk. Kern. Tidskr. 45: 7:.1 74. (10) ---- and MAIITIUS, C. 19:13. UDEll REIIUKTON (EN(lI.-TAItTIlONALm:HYIJ) UNIJ ASCC/KHL"'SAURE. Justus Liebigs Ann. del' Chern. 505: ia-87. (1I) FEASTEIt, J. F .• TOMPKINS, M. D., and P.:ARO:, W.E. 1949. E.·.·ECT (It' STORAGE ON VITAMINS AND QUALITY IN CA:-'-NIW t'VOIIS. Food Res. 14: 25· :.19, iIIus. (12) GOLUBLlTIf, S. A .• and HARItIS, 'R. S. 1948. ESTIMATlllN OF ASCORBIC ACID IN FOOD PREPARATIONS. Analyt. Chern. 20: 649-651. ( 13) GUERRANT, N. B., VAVIGII. M. G., and DUTCllf:It, R. A. 1945. NUTRITIVE VALUE OF CANNEU H'(lJlS. INFLUENO: OF n:MPERA TUII.: AN!) Tun: Of' STOItAI1E ON VITAMIN CONTENTS. Indus. and Eng-in. Chern., Indus. I-:d. :37: 1240-124:1, illus. (14) HARRIS. L. J., und MAPSON, L. W. 1947. DETERMINATWN ot' ASCOIIIIIC ACIII IN PRESf:NCE OF INTERf'EIUNf: • SUBSTANCES BY TilE 'CONTINUOUS-.'I.(,W' METHOD. Brit. Jour. NutI'. 1: 7-.:18, illus. (15) --- and OLLIVER, M. .1942. VITAMIN METHODS. 3. THE RELIABILITY OF TilE METlW[) FOR ES TIMATING VITAMIN C BY TITRATION AGAINST 2 :6-OICHWRO PHENOLINDOPHENOL. 1. CONTROL TESTS WITH PLANT TISSUES. Biochem. Jour. as: 155-182, ilIus. ( 16) HARTZLER, E. 1948. FALSE HWU VALUES f'OR ASCORBIC Aem IN GUAVA JIJICE. A NOTE ON TilE USE OF THE COLOIUMETRIC METHOD WITH 2,4-OINITRO PHE:-'-YLlfynRA1.INE. Jour. NutI'. :15: 419--424. (17) HEWSTON, E. M., DAWSON, E. H., ALEXANDER, L. M., and ORENT-KEILES. E. 1948. VITAMIN AND MINERAL CONTENT OF CERTAIN FOODS AS AFFECTED BY 1I0ME PREPARATION. U. S. Dept. Agr. Misc. Pub. 628, 76 pp., ilIus. ( 18) IRWIN, J. O. 1943. ON THE CALCULATION OF THE ERROR OF BIOWGICAL ASSAYS. Jour. H3'1. [.London I 43: 121-128, illus. (19) 1944. A STATISTICAL EXAMINATION OF THE ACCURACY OF VITAMIN A ASSAYS. Jour. Hyg. I, LondonJ 4a: 291-314. (20) KUETHER. C. A.. TELFORD, I. R., and ROE. J. H. 1944. THE RELATION ()f'THE Bwon LEVEl, 0 .. ASCORBIC ACID TO THE TIS SUE CONCENTRATIONS OF THIS VITAMIN AND TO THE HIS TOWGY OF THE INCISOR TEETH IN THE GUINEA PIG. Jour. NutI'• 28: 347-368, iIIus. • VITAMIN (' ('OMI'AIU!;ON tit' tJllf:MICAI. ANI) 1II01",'GII:AI. .ARRAY 21 (21) LUGG, J, W. H, .1942, TilE USt: OF ~'nIlMAI.mmYllt; ANII 2.6-HICIiLOIlOl'IIF.NOLINDII Pllt:NOL I N 'I'll t: Ef.iTI M ATION (I~' ARCOIUtl('ACm ANIl 11.;11 YDRtI ASCfllUIJC "CII), Allstral. .JOIII·. I~xpt. BioI. and 1\f('d, Sci. 20: 27:1.285, illus. (22) MAPSON. L, W, l!)4;1.\'IT";\IIN ~1t:Tllons. \'1. Tilt: t;8'rIMATION (W .ASCflIUlIC ACllJ IN THE PRt:St:NCt: m' 1It:IIIJCToNt;S ANII AI,l.IED SURSTAN(,ES. Soc. Chem. Indus, Jour. Tl'llns. and COll1l11un. (;2: 22:'1~2:12. ilIus. (2:!) MEN,\Kt;R, .M. H., and GUEIlRANT, N. B. I!I:.I8. STANI!AlUlI1.ATION Of' 2,(;-111('11 1.0I101'lIt:NOLl !IO[lOPIU:Ntll.. AN 11'01 1'ltOYEil ;\I t:T 11 (II), Indlls. and Engin, Chcm., Anal~'t. Ed. 10: 25 2(;, illlls. . (24) l\fll.um, E. S. 19:1.. , AI'I'LICATION (I~' (IU"NTlT,\TI\'~J Sl't:CTII,\L ,\NAI,Y"t;S 1'0 RINARY l\IIXTUItES OF TIIt~ ('0l\1l\10N (' AIIOn:N{lIDS. 1'111111; Physiol. 9: 681-78". (25) W:.IS, 1'1I0TOELfXTIIIC SI'fX:TlWI'W.TOMETIIY AI'I'Llt:1) 1'(' TII~J (IUANTITA- 1'IV': AN,\I.YS':S (lP CMIOTENom AND ('IILOIIOI'IIn.t. 1'H:;\n:NTS 1:-': n:II:o.:'AIIY,\NIl QI1,\n:IINAIIY sysn:;\Is. C('J'(~al Ch(!II1. Iii: aJO·· aI0, HillS. (2(;) )IIL"':II, M. C, In·II, IIt:IIU("fONf: INn;IIFEllt;Nn: IN t;STIM,\TIOS OF \'11''\.\1/:0.:' ('.Food Res. 12: :1"~la5!), illus. (27) MILLS, M. 11., n,\;\IIWN, C. M., lind ROt" .1. Ii, 1!l4!l. Mi('OUIlIC M'II), nEIIY(lUOAH(,OIUlIt' .,\CW, ANti IllKt:TOGULONIC ACIII IN FUESII "",(l I'IWC~;S;;EIl "'(lr.n~;. Analyt. Chcm. 21: 707-709, Hlus, (28) n nd ){Ot:, ,J. H. .\947, A ('I(('I'I('AI, STUD" (W l'II\II'OSf,l) ~lOnn'I('ATJ(l:-';S oI" TilE ItOE ANI) KU~:TIlt:U~";l'IJ()1) FOil 1'lIf: m;n:U;\IlNATION (W,\SCOItBIC "cm, • WI'I'II FUUTIIEII COI'TIUIIUTIONS TO Tilt; (,IIt:MISTII'" OF' TillS l'IWn:II!.llIf:. ;I(lur. BioI. Chclll. 170: 15!1 )(;1, illus, (29) OI'I·t'NIIEL\It:U, G.. 8n:IIN, K, G., and ROMAN, W., <:Ollllb. I n:l!l. IIJ(lLl.I(ac,\I, ox illATION. ~{I i pp. The Hl\g'lI~·. (:10 ) OSt:u, B. L. I!150. I~ood. Anlliyt.. Chcll1: 22: 221 ~227. (31 ) Pt;NSt;Y, ;1. R., lind ZII,VA, S, S. Hi4;3, Tilt: 1.1.:,..;11;\11 NATION (It' 2 :;{-lllln:To-I-!;\JLONIC J\('IJ). Ri(lchem. •Iour. ;1/: ;.I!l~44, illlls. (:12) _... " - .. anei ZII,\'A, S. S. I f14:1. 'I'lIt: ('IH:~lIe AI, liEII " ywun Ot' nt:IIYHltO-I-ASC(lRBIC ACIO IN VITIlI) ASH II' \,IVO. Bi{,lchcm, .IOllr. :"17: "O~I "17, illus. (:1:1) - anei Z11,\',\ , S, S. 19·15. IN'I'EHFEItING SI1I1STA,,'('t:S IN Tilt; IWt: '\:0.:'1) KUt:TII.:1t METlH.[l t'.,1t TII~; Ot:n:ItMIN,\TlnS III' MWOItIlI(: ,\CIIl. Rif,('hclll. ;I(.ur. 39: an2 .~197. (:l,U PIJO,\N, ~I., and Gt;IIJO\'ICII, H••1. 19·1(i. Tilt; USt] or 2,4-IIlNITnol'lIt:Nn,IIY()IIA1.I="E Fnlt Tllf: nt;TEItMINA TIM; Of' Mi('(.ItIIIG ACIIl. Science (n.s.) ]03: 202-20:1. (15) POLl,AIIIl. A .• KIESEI(, M. ~-:., and STF:t:n:\IAN, .J. 19-14. Tilt: M;(,OIUlIC ,\CIll ('ONn:="T 0.' SOME FItUIT SYltllPS ANII OTIIEII l'II01Hl(:TS, SII<:. ChclIl. Indll;;, .Jollr. 'I'mn5. lind Crolllllllln. 6:{: 215218, illll:l, (:lIj) ROE, .J. H .. IlndKIJf:TIIEIt. C. A. 194:.1. TilE UEn:UI\II=".ATION ot' .ASCOIIBIC ACII) IN ,,"II0LE IILOOI) ANI) URINE TIlItOI]GII TIfE 2.4-J)l:o.:'ITIIOPIfENYLHYIlRAZINE IlEItIVATIVE OF DEIIYDIlOASCORBIC ACIil. Jou!:. Riol. Chem, 147: 399-407, • lHus, (:17) MII,I.!;. M. H., OESTEIII.IN(;, M. J., and DAMIl"N, C. M. J!HFI, ,.11.: ...:n:IlMINATIfIN (It' IIJKt."(I-I-(WL(lNIC Acm. DEHYDRf,-I M1CflIlRI(' A(cm, ANI) I-ASCORBIC Acm IN TilE SAME TISSUE EX TRACT BY TilE 2,4-IIINITROPHENYLIlYDRAZINE METHOD. Jour. BioI. Chem. 174: 201-208, illus. • C:18) --- and OESTEItLING. M. J. 1944. TilE UETERMINATION (It. UEIIYDROASCORBIC ACID AND ASCORBIC ACUI IN PLANT TISSUES BY TilE 2,4-nJNITROPIlENYLHYDRAZINE METW)J). Jour. BioI. Chem. 152: 511-517. (39) SMYTHt:, C. V., and KING .• C. G . .1942. A STUDY ot' ASCORBIC ACJJ) SYNTHESIS BY ANIMAL TISSUE IN VITIIO. Jour. BioI. Chem. 142: 529-541. (4(1) Sl':t:ot:COIt, G. W. 1!140. STATISTiCAL METIJODS AI'PLIED 1'1; EXPEfUMENTS IN AGRICULTUIlE ,\Nri BI(H.O(;\'. F.d. a, 422 pp., illus. Ames, Iowa. (41) SNOW, G. A., and ZILVA, S. S. H14a. A (:IIITICAL t:XAMINATION Ot' LUGG'S METIIOD .·OR THE DETERMI NATION OP I-ASCOIUIIC M:JI). Biochem. Jour. 37: (i30-li40. illu~. (42) --- lind ZIL\'A, S. S, IH44. A ClIITICAt, EXAMINATION (W I.UGI:'S IIU:TIIOI) .'Olt TilE UETERMINA THIN Ot· I-AS('OIlRIC Aem. 2. Biochem. Jour. 38: 458-467, ill us. (-Ia) WOKES, F .. OnGAN, J. G., DUNCAN, J., and JACODY. F. C. 19·':3, AI'I'AnF.NT VITAMIN C IN .'(IOI)S. Biochem. Jour. 37: 695-702, illu~. (-1-1) - OIlI:A:-I, .1. G.. and JACOBY, F. C. 1!).1:1. TilE .:STIMATJ(\N 01' Al'l'AnENT VITAMIN-C IN Foons. Soc. Chem. I nllus. Jour. Trans. and Commun, 62: 2a2-2a6, illus. (-15) ZU,\'A, S. S. IS·U. TlU: INf"I,UENCt: Of" INTEIIMITTENT CONSUMPTION OF VITAMIN C ON Tilt: IIEVELUI'MENT 0.' SCUIIVY. Biochem. Jour. 35: 1240 12-15, ill us. .APPENDIX A DATA FilUM TilE BIOASSAY • SI'~I!\IAIl\' OF The expel'imental data .from whkh the vitamin C hioa~say valu!'s were dedvc:d 111'(' SUIlIIlHII'ized in table 4. Analysis of variance (~O) demonstrated that in t.he individual assay groups, variation from linear regression of (,dontohlast len~th on log-dose of ascorbic acid was im;ignificant and that the F values for: variation (:aused by differences in dose were above the 1 percent levd of signitkance in all groups. When the data for all of the guinea pigs IIsed in the as! j. ..' (' • c ,,: 'rypknl 'wi(lnllO,(I(lJ1U' UPtlP:1I1HW(' -t,C d,lont .. bl;h ... frum ~HiI1N' pigs nn Ulfl lnwt'!'t IUld hight·... ' k\l'b of U't'lltim' lind nud ornnI!{'-l!rnpdntlJ jl1lt'l' !'-lIpph'HW.IHS~ Ilwl !loUt tl )Wg-:,lll\p·('lIlltro) pUU1l'tI II!g~ (;uuwn 1111-: lin: .1. {L!i lUg. ttM'PI'Iiic+ lIf'h!.. 1J.. '! 1I111g, ll~l'orht(' ntHI; ('~ 0'111lt!I'" J!ntJitllflllt JUIl'(' p'lllt\nh'ul to n!"1 JUl!, !h('ul'hl~l ~lt'J.[' J), tl"llIlJ!'··J!nlpi (run Jtth'!' N.ltll\tll('tll to • 211111!!' H"'-turlUt und. r-:,. nOH!'o['orhH Hli,I~t1Jtlllf,-nHUl (IH:gntl\l'('Wnruh, X 1·lu. • • • TABLE 4.-Statisti(·al compari.'5on oj (I'l.'('/"{l!/e lel/fJth of o(i(i)/toblasts oj g/liw;a piUl) jed /J levels 01 vitamin C as pure ascorbic acid, canned t(l11l(lto juice, orcmge-gnlpe!i'uit juice, and spinach ...... _...... --_._------ R'.'''llOtl~to food Rf'!"IKmlM! to r~ft!T~nc~fttundnrcl. wtcorLk ndlt ,..,-.,.-~--~--P-~~~h~~ - ~ A"("orbl(; , ___ .-.,;f_·_,'~:hlf!_ N"um~r f.~ .."alue ...;,unl~r a: acid j Num},.:l' "'---.- .--~ ---"'-"~'-' of Otlonio- VarIation of Odonto vari,.ttion r or Odonto l-=-T:puriRtionZ or from ~lIinp:tblaKt from I Jo:uint,.n blnHt. from ~lIiJl·):thla:!t equi "'n I~nt I}Qfl.c I)(.~~ Jin{lur l.iJ:li I,·n.,nh J~ linear Jc-l1brth lint}ur 1)1.;.8 Icni;1h f~d j Ili5.:M rl..·J;n!~~lon ? ...... ~,~_•...,.,'"'_ I reM'~8iOD ,,1, __ __ rt:a:ression ('"J _____ ._" c Mg. I Jf ie/'Oll:' Tomato juice a: "II perc/au As Jlurchased Stored 5 days at 73° C. > r;ificro713 1 I MicroJls I e ~ 5 :z 0.5 4 28 ± 6' 26 .;.. 5 I LLt26 ± 3 '} 3.06 2'.l. .L 6 \. 8.(j3 0.88 35 ± 4 • 14.4S" 1.0 6 28 .~a 1}·16.54•• 4.81 -1 5, c 2.0 6 40::: 1 4 41 :!: 7 f G ! 44 ± 25 I OJ: ('"J ______~______O_r_a_n~ge-gr~_ef_r_u_it~J~·u~i-ce--~~------~~_ j =tIJ ,___ As purchased Stored 5 days at 73° C. a: I 2.2 .;.. 2 <- t------, 6 I 25 ± 4 !} i5 .5 3 24 -:1.~;3 4 > a5 ~ 4 0.80 i 6 :35"~ 9.08" 0.31 r" 1.0 5 35 ::: 3 } 36.93" 6.89 G (55.0 7: 5 44 :" __ ..~,._. 1 (j . 38 ± 4 > 2.0 8 38 ± 4 3' __ i :z Spinach' t:! I --S-t-or-e-d--G-\-~'e-e-k-s-a-t-45-a-C-.--- As purchased == ~------.~ o ., 25 .;.. 4 · 3 '>4 + 1 } - s:: 2·1 ~'::J .5 6 I H ~ .09 (j ;n ::': (j 11.83 1.62 6 35 ~;:5 , 11.59 1.73 1.0 6 :~1± 6 }14.91*'" I I ! i5 (j 'I~~~~-!-' 3 > (j 41::': 6 I 6 40 ± 5 :-- 2.0 G ~li~ 4 I > The average length of odontoblasts of 8 guinea pigs fed 1.0 mg. of ascorbic acid plus 0.79 mg. glucoreductone was 34 ± 2' microns. rr. .. ~ _._--__------_. --: 1 Standnrd de\'intion. 2 Two a&teriHk!i indicate sh:nlficancf! at the I lh.'f'Ct"'nl h.·\"d (1.· nlJO,'e. 'Vitamin C fed. hulf U!4 Ill.rt! :l::;~'~~ruicacid und half as "pjnhl'h. • Vitnmin C f~ u!t Rilinltch 01-1)'. t'5 24 n:QIINICAL BULLETIN No, 1023. U, S, n~:p'I', OF AGrUCUI.TURE Bliss and Marks (t!), alld applied by Crampton (6) in his studies, because the guinea pigs in the vllrious gl'Oups to be compared completed lhe 6-week assay period in unequal nurnber[i, This formula relates logarithms of the mean doses of the standard and t.he unknown to theil' mean odontoblast responses lind to the average slope of thcrc3(>onse curve, The following • ('(Iuation is used: X .1. 11., 'y. 1\1 .;;; •• --b-, 1\1 is the logarithm of pcd:cncy of the food; X. and X" the mean log dosl's for the stlllldard and unknown; Jf. lind iT., the mean responses for standal'd and unknown; I" the IIvcragc slope of the CUl'Ve of rcsponsc, The eXlict fiducilll limits of each asslIY were also caltulatcd by the method of Irwin (/8). Only one of the neg-alivc COli trois survivt)d the (i-week assay period. and all showed gross si,,6ls of scurvy Oil autop.,y. There were many fewcr hemorrhag-es lind no ('xh ihition of lhe classic "faee-lItht)" position after rutin and yeaRl' WI'I'C int.rodutNI into the dict. A few animals thllt were l'I!cciving ascorbic add, t'specially lit tht· 0.5 mg. per day lcvel. di('d during- the assay period; a small propol'tion of lhese showed sign:; of scurvy. The!'t) we!'e /I few dcat.hs alllOIl~ lhe :iupplcnlC'lIt()d animals which wel'e attl'ibulable to respiratory infection, or refuSli1 to cat, rather lhan scurvy. COIllJ\lIrison of the weight gains of the alii mal:; at. different levels of ascorbic acid intake l'cv('aled 110 significant. difren'nees. Some of the animals that r('c:eh'ed 0.5 IIlg, (Jf aSCOl'bie acid ~~I'ew a~ well as those 011 the highcr levels. TI](I 11(ll1SPl'C'itkil~r of growth a~ II Illea"lll'e of vitamin C deficiency WIIS in mal'ked cont.rllst. to lll\' hig-h Spt·citic.:iLy of the odontoblast re:;;ponse, Figures I, ~, allli :l show g-llillea pigs that l'cc'dved the assay diet alone, the aSSily diet with cabbug-c and kale supplements, and the assay diet supple Il1cnt('d with a low level of ascorbic ucid, respectively. • F1GU"tJ J ,-ExpcrinWlIlal guinea pig al end of a,;say period: Neglltivc con- • t.l'ol, rcteivcd assay diet only. VI'I'AMIN I: t'tlMI'AWSON Of.' CIU~MH.!AI. AND 1II1)I.OGlC:\I. ASSAY 25 8 FIGUUE 2,-J~:xpel'illlcntal 1{1Iinl':1 pig' at cnd of as;:;ay pcriod: Received assay diet with f:uhhul{c unci knit' slIpprcmcnts, • FIGURE 3,-J APPENOlX .B I~TERFER.:NCE I,.. GI,UemU:ulJCTONE IN nn: RO AND RI\IOD 2,4.J)I~ITROPln:NYI.I'YORAZI~.: METIIODS • !n order to secure more complete information abOUL the RO reaction when applied to a product in which reductones were known to be present, complete absorption curves for this reaction with orange.grapefruit juice were measured between wave lengths ,!50 and 550 mil. Comparison of the curve for the juice with that of a solution of pure ascorbic acid which had also been subjected to bromine oxidation is shown in figure 4, The two differl·d con· siderably; the c;U1've for omnge.gl'apefruit juice absorbed markedly in the region of 450 to ,170 mil, which, according to data published by Mills and Roe (:!8) chal'llctt'rizes the phenylhydrazine del'ivutivc of "reductone." Simillir complete absorption curves wcre made for the diketol~ulonic, dehydroascorbic, ascorbic acid (DKA.DHA.AsA) aliquot of unstored orange.grapefruit juice alid the diketogulonic acid (DKA) aliquot of the stored product in the RMOD I'eaction (fig, 4, B), These curves demonstrate that reductones ruther than diketogulonic acid were prohably responsihle for much of the color produced in the diketogulonic acid (DKA) aliquot of the stored juice, Penlley and Zilva (:I.V) showed that not only dehydroascorbic acid, but also reductic acid, reductone, glucoreductone, and diketogulonic acid react with phenylhydrazllle reagent. as ulled in the Roe·Kuether method (36) for measur· ing a~corbic acid. Reductone dcveloped 50 percent, and reductic acid 92 pel'· cent of its final color after only 15 minuu.s at 25" C, For this reasor it was considc.red desh'ahle to detel'mine whether or not "reductones" were also likely to interfere under the conditions of the Roe.Mills·Oesterling·Damron modification (.17), whieh allows corn'ction to be made for diketoguloni(' acid, hut not for the other interfering substances mentioned, The action of H~S and Br~1 I'eagen!.); used in the RMOD method, on gluco reduci.one had not been thoroughly investigated, although Mapson U~) interpreted the results of his experiment.s with "reductones," produced by the action of alkali on either glucose 01' <:ane sugar, as indicating the pres('nce of two types of rcducton{', hoth of which were oxidized by iodine. Suhsequent • reduction with H~S completely regenerated one of the compounds in 75 min· utes, the seeond only after G hours exposure, Snow and Zilva (.U) reported that the Pl'opOl'tion of each form of reductone in solutions prepared from glucose and alkali, varied with the concentration of alkali and the heating ttme ('mployed, :\tATEIII,\LS, METllflOS, PRflCEOlJRI-:S Crystalline j!lucol'eductnne was prepared by the method of Euler and Martius (f), /0), omitting final recrystallization from ethyl acetate which gave the purest compound, but from which yields Were too small to be practicable for pl'cparing the IImounts of material necessary for u bioassay. The crudel' fl'llction, recrystallized from petroleum ether" and acetone, was used instead. The IInalytical techni'lues (If Roe, Mills, Oesterling, and Damron (37) for measuring ascorbic, dehrdroascnrhic, and diketogulonic acids in plant and animul tissues were applied to solutions of known amounts of the pure j!lucol'eductone, and 1I!;(:orbic acid; a and 2:1 hours of coupling were sub· stituted Jor the (i hours employed hy Roc and associates in order to observe both early and late changes taking place in t.he reaction mixtures; other minor modifrcation!; were introduced only to serve the purpo!;;es of the experinumts. Glucoreductone (solution I) and ascorbic acid (solution II) were dissolved in 5 percent HPO:I containing 0.5 percent SnCl~ in concentrations ranging from 5 to 200 Itg. pCI' ml. Three aliquots were taken from each solution and subjected to t.he analytical treatment employed in the RMOD method to measure t.he three ascorbic acid fractions, i.e, (A) diketogulonic and dehydl'D ,., .Puriticd Skcll~'solve, fraction F., h,p, 30°_60° C. • VITAMIN C--COMPARISON OJ<' CHEMICAL AND BIOLOGICAL ASSAY 27 ascorbic acid (DKA-DHA): (B) diketogulonic acid (DKA): (0) diketo gulonic, dehydroascorbic, and ascorbic acids (DKA-DHA-AsA). From these values the "true" ascorbic acid and dehydroascorbic. acid, as well as diketo culonic acid present in the original tissue or food extract are calculated. 100r---~~______~ 90 \ A \ 80 \ 70 '\-60,..9. ascorbic acid 60 x I I "x I , / x ',,-- / -A...... x--x/ '-Orange-grapefruit juice, stored 73·C.,5 days z Q en • !!! 90 B en~ Orange-vrapefruit juice, z80 as purchased (OKA + ct a:: OHA+AsA aliquot). ~ 70 ~ ~,/ / 60 '®, /ci 50 00.....00 " 40 -oo-oo-® 30 • Orange-grapefruit JUice, 20 ,-stored at 73°C.,5days (OKA aliquot>. 0 450 470 490 510 530 550 570 WAVE LENGTH -(mil) FIGURE 4.-Ahsorption curves: A. for Roe-Oesterling reaction with stored orange-grapefruit juice and with a solution of pure ascorbic acid; B. fer Roe-Mills-Oesterling-DamrG!l reaction with aliquots of unstored and stored • orange-grapefruit juice. 28 'I'E(JHNICAI. IIULLN'I'IN Nu. Itl~;j. U. S. IJEP'I'. OF' AGRIOULTURE AFTER 3HRS. COUPLING A 0.7 • BC 0.6 BAIBtl ·0 0.5 BS 0.4 0.3 0.2 0.1 0.0 ::L E 0 v AFTER 23 HRS. COUPLING B 10 O.B BC ~ 0.7 taU > JIa-o en~ z 0.6 UJ 0 0.5 • 0.4 0.3 0.2 0.1 ®--®Ascorbic acid,JIA @}---@Glucoreductone. (DKA aliquot) IA (DKA aliquot) II---K Glucoreductone, ....--.Ascorbic acid, I C (DKA +DHA+AsA JIC (OKA +OHA + aliquot) As A aliquot) FlGUlUiJ 5.-Roe·Mills·Uestcl'ling-Da/lll'Un reaction: Calibration curves for aliquots of glucol'cductone solution Bnd ascorbic acid solution. A. after 3 hours coupling with 2,4·dinitrophenylhydrazine; B, after 23 hours coupling with 2,4.dinitl:Ophenylhydrazinc. I Color produced varied unpredicb\bly from time to time. • VITAIIUN ~COMPAlUSON OF CHEMICAL AND BIOLOGICAL ASSAY 29 The percent transmission of the final solution contair.ing the phenylhy drazine chromogen was mea!:.ured with the Beckman spectrophotometer, model DU, at wave length 540 mIl. This is the wave length usually employed in ascorbic acid calibration curves. In addition, complete curves between wave lengths 450 and 580 mIl were read for at least one concentration of each aliquot of solutions I ancl II and their respective blanks. The same substance is responsible for the color in all of the ascorbic acid aliquots; it is apparently I:n osazone-type compound formed by the coupling of diketogulonic acid wit.\ 2,4-dinitrophenylhydrazine in the presence of H:!SO~. The preliminary trehtment to which each of the aliquots is subjected in the RMOD method determines which of the reducing compounds originally present will take pal·t in the final coupling reaction. A bioassay of the pure solutions was made also, in which eight guinea pigll were fed the ascorbic-acid-fl'ee diet supplemented with 1 mg. ascorbic acid and 0.79 mg. glucoreductone (equivalent to 1 mg. ascurbic acid, by the indophenol method) pel' day. In figure 5, A and H, are plotted the absorptions at 540 mIl for aliquots A, B, and C of solutions I and U in the concentl'ation range of 20 to 80 "g. per 4 m!. of reaction mixture, after coupling with phenylhydrazine for a and 23 hours, respectively. These are similar to the calibration I!UrVes usually prepared with a pUl'e ascorbic acid standard when measuring ascorbic acid by either the RO or RMOD reaction. The curves in figure 5 indicatc that glucol'eductone, if present in the reaction mixtul'c of aliquots A 01' C, would interfere appreciably with a reading made at 540 mIl after either a or 23 houl's' coupling; if present in aliquot B, glucoreductone would give no interference at 3 hours, and only u small amount, which would val'y unpredictably from time to time in these experiments, after 23 hours. It is noteworthy that glucoreductone subjected to the treatment given aliquot C intel·fered considerahly more than the same amount when present in aliquut A. Apparently dehydl'oreductones, analogous to dehydroallcorbic acid, which arc formed by the oxidation of reductones according to Snow and Zilva (.41), do not I'eact with phenylhydrazine pre cisely like glucoreductune. This circumstance further complicates calculation • of the various IIsCUI'bic acid del'ivatives in the pl'esenCIl of glucoreductone, and gives rise to uncel'tllinty abuut thc accul'llcy of the final values obtained. The ascorbic acid present in eithel: aliquot A or B interfel'es but little, even after 2a hours' coupling; this justifics one of thc fundamental assump tions of thc RMOD mcthod, i.e., that ascorbic acid participates in the I'eactioll with phcnylhydrazine only in aliquot C. Ahsol'ption curves betwccn wave iengths 450 and 570 m.... for the three aliquots of the RMOD reactioll as aPJllied to the purc glucol'eductone solution and aliquots A and C of the aSl!ol'bic acid solution after coupling for a and 2:1 hours are shown in figure (j. In aliquot A, only slight intcl'icl'cncc frulI! ascol'hic acid is indicatcd cven aftcr 23 puurs, with as much as 240 ....g. ascorbic acid per 4 m!. reaction mix tUI'c. Glucoredut;tonc, whcn Hli:dyzcd as the sanlc ailljuot, gavc cUl've;.; after both a and 2:{ hours whi!'h suggcst slight intcrfercncc of questiunable quanti tative significance at 540 Ill". Both solutions I and II in aliquot B reactcd slightly with phenylhydrazine after 2:.: hours; however, absorption at 540 mIl was affected but little. It may be significant that SnCb is the stabilizer employed in aliquot B, and thiourea in aliquots A and C.Figure 6, A, includes an absorption curve for glueo reductone in aliquot B, with thioul'ca substituted fOI' SnCI~. Under these conditions, which arc identical with those of the RO reaction, glucoreductone gives a characteristic absorption curve in the 45~70 mIl region of the spectrum, and absorption at 540 m .... is not negligible. This observation suggests that SnCI~ Illlppresses interference from glucoreductone and that the possible substitution of SnC!:! fOI' thiourea in aliquots A and C especially when glucOl'eductone is 11I'cllent, merits investigation. The absorption curves for solutions I and II following treatment as for aliquot C (DKA-DHA-AsA) are the most significant. After 3 hours' coupling, ascorbic acid shows the characteristic diketogulonic acid derivative curve; • glucoredllctone also gives a characteristic curve whose maxima and minima ao TECHNICAL BULLETIN No. 1023, U. S. nUT. 010' AGRICULTURE differ from those of ascorbic acid, although both derivatives absorb strongly in the same region of the spectrum. These observations suggest the possibility • of correcting quantitatively for substances like reductones whose phenyl hydrazine del'ivativc,; giv(' chul'acteristic reproducible absorption curves. This could he done hy lIIeasuring the specific absorptions of reductic acid. and reductone:; derived frolll other tYlles of lIuglirs. liS well liS tho!!e from glucose, and upplying the IlldhodfrelJuently used til calculate individual components of a mixture of carotenoid pigments (!!.4. !!:». A 100 AFTER 3HRS. COUPLING 90 80 I 70 I I i= 60 v I ~ .....'. x I ~ 50 W .....L&.- I I ~40 J. X Z Q 30 '. DC--' CJ) CJ) ~ ~ 20 UB·O CJ) lIA} ·0 ~ 10 Ie IBJI a: • .... 0 I.....I--'-.....~-"--I.-l--L...... '"'--I.....I~ 450 4)0 530 570 450 490 WAVE LENGTH (m~) l{--K Dehydroascorbic acid 00---<> Glucoreductone - I Bt , IC,40 ~g./4ml. ( I" thiourea) ,80 ala./4ml. ___ Glucoreductone-IC, 60 ~g. 14ml. FIGUUF. (,i.-Roe-Mills-Ot.·Slcrling-lJalllroll rcm:tioll: Abso/'pt'nn ("urVl'" be t.wecn \\'aVl' lengths 450 and 570 ml' for aliquots of glucorl'd"ctone solution and ascorhiC' acid solution. A, afte/' ;-l hours coupling with 2,4-dinitro phenylhydrazine; n, after 2il huurs coupling with 2.4-dinitrophenyl hydmzine. 1 Color III'odliecd vllried unprcdictahly .frull1 tilllC to time. {cu. S. \..QVE:HNM€NT '''HINTING OFFICE: U~!jo-tt92943 Fur !1nh i hy Ulll HlI'WThltf~nd(·nt or JhH'ultltlnt!5. U .....;. (iC.l\-·('!'nrn,}rlt; Prifltin~ fHlicf!. • \~lllshiligtHn ~». n. C. !i. • • . , I