Author Institution: Department of Chemistry, Ohio State Universit

Brode, Wallace R.

Magill, Mary A.

The Ohio Journal of Science

KnowledgeBank at OSU

A COMPARISON BETWEEN THE SPECTROPHOTO-METRIC AND BIOLOGICAL ASSAY FOR THEVITAMIN A CONTENT OP FISH-LIVER OILS.MARY A. MAGILL AND WALLACE R. BRODE,Department of Chemistry, Ohio State University.A standardized method of color analysis for Vitamin A (1)by the antimony trichloride reaction has been applied tocommercial samples of fish-liver oils. A comparison betweenthe spectrophotometric values and those obtained by biologicalassay would indicate that there is a considerable error in theantimony trichloride test.The fish-liver oils which were used in this series of experi-ments were furnished by the Parke Davis and Company,the Abbott Laboratories, the Eli Lilly Company, the E. R.Squibb and Sons, the Mead Johnson Company and the UpjohnLaboratories. Each of these companies gave feeding tests ontheir oils so that data could be obtained on the relative valuesand agreement between the chemical and biological assays.The Parke Davis and Company, through the courtesy of Dr.A. D. Emmett, furnished in addition, colormetric data ontheir oils as obtained by the Carr Price method, which weremade over a period of several years. The data furnished acheck on the relation between the colormetric and spectro-photometric methods, as well as the influence of time on theseoils. All of the samples used in the tests described in thispaper were kept in a refrigerator at 5° C.The oils have been divided into two sets, (a) those givingwith the antimony trichloride reagent an absorption bandat 608 mix (Table I) and (b) those giving an absorption bandat 622 ran (Table II). Ordinary "cod-liver" oil sold inpharmaceutical trade is made from the genus of fish knownas the gadus morrhua, and generally not exclusively from thecod fish. This group includes the cod, haddock, hake andpallock. All of the oils in Table I were designated as cod-liveroils. They included both domestic and foreign oils. Recordson some of the oils would indicate that they had been in storagefor some time, while others were quite fresh. There was noapparent relationship between the variations in the colortest—biological assay values and the age or source of the oil.127128 MARY A. MAGILL AND WALLACE R. BRODE Vol. X X X I I IIn certain cases where a marked difference was shown betweenthe values obtained from the two .methods of assay, bothtypes of assays were repeated and confirmed this difference.TABLE I.COMPARISON BETWEEN THE SPECTROPHOTOMETRIC AND BIOLOGICAL ASSAY DATA ONCOD-LIVER OILS (BASED ON THE ABSORPTION BAND AT 608 my).CompanyIIIIllIVVVISample No.123456789101112131415161718192021222324252627282930313233BiologicalAssay5735001000500625625-500400-500450-550600-666625+1000320080070060019008001350180019001000100010004000450013006507005006501200500850SpectrophotometricAssay (Referred toSample No. 11 as aStandard)590780640850800590690750770920100027908206706701060590173019301500119011708505300550013001220*152011001500260011001700Q/2 Spectrophoto-metric Values)610*7605507501300550850*NOTE.—In Groups V and VI it would seem necessary to multiply the spectro-photometric values by a factor of }/% to make the values obtained by this methodagree with those obtained by the biological assay.No. 2 VITAMIN A IN FISH-LIVER OILS 129In Table II are recorded the results obtained from halibut-liver oils. Here, again, there was found to be a wide variationbetween the two methods of analysis. These oils were forthe most part concentrates of the unsaponifiable fractionobtained from the crude oil. In certain cases (samples 34and 35) the concentrated oils were diluted with sesame oil.No explanation is offered for the necessity of this factor,of ]/2, as applied in groups V and VI in Table I, though aTABLE II.COMPARISON BETWEEN THE SPECTROPHOTOMETRIC AND BIOLOGICAL ASSAY DATA ONHALIBUT-LIVER OILS (BASED ON THE ABSORPTION BAND AT 622 m/t).CompanyVIIVIIIIXXSample No.343536373839404142434445Biological Assay50025003000037,500-50,00037,500-50,00025,000-37,5001300050003250050001750062500Spectrophotometric Assay(Referred to the Height ofthe Extinction Coefficientof No. 11, Table I, as aStandard)650231037800647001138007840029300170009480038007320084000justification for its use can be seen in certain groups of oils,where all the members had to be corrected by the same factor.Both the Spectrophotometric and biological assays were re-peated on certain of these oils, the results of which only con-firmed the previously obtained values. Undoubtedly thesevariations, since they are so consistent, are due to some causeother than errors of observation, and may be ascribed to someinhibitor, activator or to alteration in the structural natureof the chromophore.In Table II are presented data on the halibut-liver oils,which gave a band at 622 m/x, or some 14 m/x further towardsthe red as compared with the color obtained from the cod-liveroils with antimony trichloride. This shift has been reported130 MARY A. MAGILL AND WALLACE R. BRODE Vol. X X X I I Iby Morton (2). Heilbron, Gillman and Morton (3) suggestthat the substance giving the 572 mju (our 578 mjn) band is anester and the substance producing the 583 m/n band (whichappears at the same time as the 622 mix band) is an alcoholderived from the former substance by saponification. Noneof the oils in Table II gave the 608 m/i band as far as couldbe observed by inspection of the absorption bands, and noamount of dilution with CHC13 shifted the position of the622 mjot band. There was a weaker band at 705 m/x whichapparently accompanies the 622 mju band.The existence of vitamin-containing oils giving markedlydifferent absorption bands (608 m/x and 622 mtx) leads one topostulate the theory that Vitamin A is not a definite chemicalcompound, but that the physiological effect produced by theso-called "A" vitamin is due to any one of a series of closelyrelated compounds, which must have certain structures incommon. As already stated Morton suggests that this differ-ence may be as simple as that between an alcohol and anester. However, the fact that carotene fades in a mannersimilar to the fading of fish oils suggests a more complicatedmechanism.IFIGURE 1. Graphical representation of the changes in absorption bands on fading.1 to 1' representing the change from 622 m/x to 560 HIM; 2 to 2' from 608 to 532 m/r,3 to 3' from 590 to 495 m/i; and 4 to 4' from 578 to 472 m/u.. The abnormalfading represented below the wave length scale indicates an apparent changefrom the original chromogen to the 578 form and then fading of the 578 to thefinal 472 form. This abnormal fading is slight in the case of the 608 m/xchromogen but is the principal reaction in the case of the 622 m/* chromogen.Following the diagrammatic outline (Figure 1) for the band shifts:(a) It can be seen that the cod-liver oils producing a band at 608mfi fade to give a band at 532 m/i (2 to 2') and those giving a band at578 niju fade to 472 van (4 to 4'). In case a rather concentrated solu-tion was used so as to give only the 608 m̂u and no 578 m̂u band, fadedbands were observed at both 532 m/* and 472 m/u (2 to 4'). This wouldindicate that there is a shift from 608 to 578 together with the shiftNo. 2 VITAMIN A IN FISH-LIVER OILS 131from 608 to 532 mn, the latter being more rapid than the former. Ifthe amount of chromogen is unusually large, some 608 changes to578 which undergoes its normal fading reaction to 472 rmt. In thiscase just considered, no 578 m/x band appears because the changefrom 578 to 472 m/x is more rapid than that from 608 to 578 mix.(b) The above hypothesis is supported by the fact that if morechromogen is present, both 608 and 578 m/x bands are produced in theblue solution, and both 532 and 472 m/x bands appear in the fadedsolution. In this case, the 578 m/x band is entirely extinct before the608 mix band is extinct, indicating that the amount of chromogen hasbeen reduced to the amount present in case (a) above. These con-siderations also explain why solutions of oil dilute enough to produceno 578 m/x bands must be used to obtain spectrophotometric valuesof the 608 m/x bands which are directly proportional to the amountof oil used.(c) In the case of the halibut-liver oils, a band is produced at 622m/x, which corresponds to the 608 m/x band produced in ordinary oils.Since the predominate band in the faded solution is at 472 m/x, mostof the 622 chromogen must change to the 578 chromogen, which thenundergoes its normal fading reaction. Other bands appear in thefaded solutions of these oil concentrates at 545, 560, and 580 m/t, onlythe one at 472 being strong and well-defined.(d) If the solution of cod-liver oil is made up so that only the 578m/x (and no 608 m/x) band is produced, the only absorption band in thefaded solution is at 472 m/j (4 to 4').(e) Carotene gives a band with the SbCl3 reagent at 590 m/x, whichfades to give a band at 488 m/x. (Fig. 1, 3 to 3').The linear equation (y = mx + b) can be used to predict theposition of the absorption bands in the faded solutions of thesesubstances.Let y = band shift expressed in cm"1;x = original position of the band expressed in cm"1; andm and b are constants to be determined.Using the band shifts, 578 to 472 m/x and 608 to 532 m/x, the constantsm and b can be found, and their values are 1.797 and -27,215 respective-ly. These values can be substituted in the equation, and the valueof "x" determined for each substance, from which the position of thefaded band can be calulated:Faded BandOriginal band ("x") Value of "y" Calculated Observed(a) Cod-liver oil608 m/x 2,348 cm-1 532 mix 532 m/x578 mn 3,879 cm-1 472 m/x 472 m/x(b) Halibut-liver oil622 m/x 1,683 cm"1 563 m/x 560 m/x(c) Carotene590 mix 5,249 cm"1 495 m/x 488 m/xThe values obtained are all within the limits of experimental accuracy.132 MARY A. MAGILL AND WALLACE R. BRODE Vol. X X X I I IIt is to be noted that if the "chromogen" in the oil concentrates reactsin what we might term normal fading we would expect to obtain aband at about 560 m/x, where a weak band was observed.The mechanism of the fading of the 622 m/x band to aband at 560 mix appears to be perfectly normal and subjectto the same explanations that might be applied to the fadingof the other solutions. As indicated by the strength of the560 m/i band as compared with that of the 472 m/x band formedin the same solution only a small part of the chromogen under-goes normal fading, while the principal reaction is the con-version of the chromogen giving a band at 622 m/i into onewith a band at 580 m/z which in turn fades to the substancewith a band at 472 mju. Due to the similarity of the bandsthere is very little doubt but that the 580 m/x band, whichwas not very well defined, is the same as the 578 m/x band.If, as is apparently the case, the speed of the fading reactionof the chromogen with a band at 578 xn.fi to one with a bandat 472 is equal to, or greater than the speed of the reactionconverting the chromogen with a band at 622 m/x to one with aband at 578 mju, little, if any, indication of the presence ofthe 578 m/x band would be apparent in the fading solution.It follows then that the chromogen with a band at 622 m/xmust either be more stable than the chromogen with a bandat 608 m/i, with regard to what we have termed the normalfading (see Fig. 1) or less stable than the chromogen with the608 m/i band with regard to conversion to the form with aband at 578 m/x, since the principal reaction on the fading ofthe chromogen with a band at 622 m/i is the formation of asubstance with a band at 472 mju rather than at 560 mju.Observations on the rate of fading of the halibut-liver oil andthe cod-liver oil colors showed that the colors formed by theantimony trichloride reaction with the halibut-liver oils weremuch more permanent than those colors formed with the cod-liver oils.Table III is presented to show the agreement over a periodof years of the spectrophotometric and colorimetric values.There is very little aging effect over the period indicated.One of the cod-liver oils was found to be highly flavoredwith peppermint oil. The spectrophotometric readings on thissample were not proportional to the amount of oil used unlessthe oil was comparatively dilute, (about a 3% solution inCHC13). The values obtained by this procedure agreed withNo. 2 VITAMIN A IN FISH-LIVER OILS 133the animal assay. It was subsequently found that pepper-mint oil in the presence of certain reagents such as sulfuricacid, acetic anhydride, etc., and to a lesser extent with antimonytrichloride, gives an absorption band similar to that producedby the fish-liver oils. Perhaps of even more significance isthe fact that in the standard color reaction for peppermintoil (5) a blue color is produced with absorption bands at 622,580, 505 and a weak band at 705 m/x. It was the presence ofTABLE III.COMPARISON BETWEEN THE COLORIMETRIC AND SPECTROPHOTOMETRICASSAY OF COD-LIVER OILS.SAMPLE N O .111213141516171819202127282930D A T E OF OBSERVATIONColorimetric19281019105.96.5105.91510192892294.76.595.9151513127.591219311227107.57.5103.91616131210131013Spectrophoto-metric19311027.98.26.76.710.65.917.319.315.011.912.215.211.015.0this latter weak band in the more stable and concentratedcolor solutions of the peppermint oils that prompted a re-examination of the blue color produced with the halibut-liveroils, with the discovery of a similar band at 705 mp. (4) In somepeppermint oil solutions, particularly those to which mineralacids were added, a band was produced at 605 m î instead ofat 622 niju. The conditions under which either of these twobands (605 m/x or 622 m/x) might be produced were notdetermined.The nature of this blue material in the peppermint oiltest is a matter of uncertainty. It has been reported to beazulene, which in turn is said to be produced by the oxidationof cadinene (a member of the sesquiterpene family). Aninvestigation of the green color produced by cadinene, obtained134 MARY A. MAGILL AND WALLACE R. BRODE Vol. X X X I I Iby distillation of cade oil, showed bands at 605 and 670while a sample of German Chamomile oil in chloroform (ob-tained from Fritschie Bros, who obtained it from Schimmeland Co.) (the source of the material used by Ruczika in hisstudy of azulene (6) ) which was bright bule in color gavesharp bands at 608 and 660 m/i. Careful distillation of oilof peppermint produced a fraction, B. P. 60-65° (2-3 mm.)in which practically all of the color forming material is to befound. The blue solutions give a most brilliant red fluorescence.SUMMARY.A method for the testing of oils for vitamin A by the useof the SbCl3 reagent has been applied to a number of commercialsamples with some agreement between the colorimetric andbiological assay values within certain groups of samples.Differences which did occur were consistent in the samplesfrom any one laboratory or for a a given set of samples.The nature of the absorption bands produced by SbCl3on cod-liver oils, halibut-liver oils, and carotene and the result-ing faded bands would indicate that several closely alliedcompounds may be responsible for both the color reactionsand the vitamin properties.Oil of peppermint contains a color-forming compound whichhas to a rather marked degree the same type of chromogenas is to be found in certain oils known to contain vitamin A.REFERENCES.1. Brode, W. R,, and Magill, M. A. J. Bio. Chem., 92, 87 (1931).2. Gillam, A. E., and Morton, R. A. Biochem. J., 25, 1346 (1931).3. Heilbron, I. M., Gillam, A. E., and Morton, R. A. Biochem. J., 25, 1352 (1931).4. Morton, R. A., Heilbron, I. M., and Thompson, A. Biochem. J., 25, 20 (1931).5. Allen's Commercial Organic Analysis. P. Blakiston's Son and Co., 1925,Vol. IV, 106.6. Ruczika, L. Helv. Chim. Acta., 9, 118 (1926).Geology.This book, gotten out in the usual McGraw-Hill style, may well fill a placein the text-book field that has been neglected, i. e., "one-semester beginningcourse for college students," as the authors say in the preface. The generalarrangement and order of presentation would seem to be that which would do verywell in such a short course. The text is not over-burdened with needless detail,although some of the words are the less common ones of geologic literature. Theillustrations are from many sources; numerous of them are new to textbooks.While I cannot recommend it for longer courses, it should find use in those placeswhere Geology only receives one semester or one quarter of the time of astudent—WILLARD BERRY.Geology, by W. H. Emmons, G. A. Thiel, C. R. Staufer, and I. S. Allison,xii + 514 pp. New York, The McGraw-Hill Book Co., 1932.

A Comparison Between the Spectrophotometric and Biological Assay for the Vitamin A Content of Fish-Liver Oils

https://kb.osu.edu/bitstream/1811/2621/1/V33N02_127.pdf

A Comparison Between the Spectrophotometric and Biological Assay for the Vitamin A Content of Fish-Liver Oils

Abstract

Similar works

Full text

Available Versions

KnowledgeBank at OSU