State of Vitamin A in Fish Liver Oils - Analytical Chemistry (ACS

Ed. , 1945, 17 (8), pp 499–503. DOI: 10.1021/i560144a012. Publication Date: August 1945. ACS Legacy Archive. Note: In lieu of an abstract, this is t...
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State of Vitamin

A

Oils

in Fish Liver

HENRY M. KASCHER AND JAMES G. BAXTER Research Laboratories, Distillation Products, Inc., Rochester,

A study has been made of the proportion of the vitamin A in a number of fish liver oils and distilled vitamin A ester concentrates which occurs in the ester and free alcohol form. The analytical methods used included distribution between petroleum ether and 83% aqueous ethanol b y the procedure of Embree and Kuhrt, and analytical molecular distillation. The assays indicated that 95% or more of the vitamin A was present as esters. Little or no free vitamin A alcohol was found. The fluorescence method for estimating vitamin A esters, proposed b y Sobotka, Kann, and Winternitz, has been examined. Their procedure indicated that large amounts of free vitamin A alcohol are present in fish liver oils and distilled concentrates. The authors' results indicate that this conclusion is incorrect because the fluorescence method described is unsuitable for this determination in fish liver oils and distilled concentrates. The possibility of developing an improved fluorescence procedure i s discussed.

A

LARGE number of molecular distillations done in this laboratory have indicated that the vitamin A in fish liver oils and in their distilled vitamin A ester concentrates is nearly all in combined form, being esterified with the higher saturated and unsaturated fatty acids. Less than 5% of the total has been found in the free or alcohol form. Recently, however, a method for determining vitamin A esters in fish liver oils by fluorescence measurements has been described (9),from which it appeared that large amounts of the alcohol occur in U.S.P. reference oil, halibut liver oil, oleum percomorphum, and distilled ester concentrates. This discrepancy has induced the authors to determine the percentage of the total vitamin A occurring as eaters in these and other fish liver oils using the older, well tested analytical methods and also the more recent fluorescence procedure. METHOD 1. DISTRIBUTION BETWEEN SOLVENTS

The first method studied was that of distribution between petroleum ether and 83% aqueous ethanol. By distribution of fish liver oils and their nonsaponifiable matter between immiscible solvents it was concluded ( 7 ) that the vitamin A in fish liver oils is substantially all eiterified. Quantitative procedures for determining the proportion of the vitamin A esterified in fish liver oils (6) and in blood ( 8 ) have since been described. The modified method used in this work was developed by N. D. Embree and N. H. Kuhrt of this laboratory, to whom the authors are indebted for permission to describe a hitherto unpublished procedure which is used routinely here. I t is based on the multiple extraction of a petroleum ether solution of the oil sample with 83% aqueous ethanol (by volume). After five extractions of crystalline vitamin A alcohol 97% was found in the combined ethanol extracts. Similar experiments on a distilled concentrate indicated that 3y0 of the vitamin A esters were extracted. Repetition on a refined cottonseed oil solution of crystalline vitamin A palmitate showed that 3% of the ester passed into the ethanol extract. The following analytical procedure was based on these observations.

N. Y.

The five ethanol extracts are combined and the volume is adjusted to 100 ml. A suitable aliquot (2 ml. if a high ester content is expected in the sample) is evaporated a t 40' in a current of nitro en in an Evelyn colorimeter tube and the residue is dissolvet in 1 ml. of chloroform for assay by the antimony trichloride reagent (3). The petroleum ether layer is adjusted to a volume of 100 ml. and a further dilution (usually 1 to 10) is made. One milliliter of this final dilution is evaporated under nitrogen in an Evelyn colorimeter tube. The residue in 1 ml. of chloroform is assayed by the blue color method. This method was standardized by a distilled concentrate whose biological potency (units per gram) had been calculated by multiplying the extinction coefficient ( E 328 mp) by the conversion factor of 2000. A calibration curve was constructed relating galvanometer readin s to units in the aliquot tested. Since 3% of the vitamin alcohol remains in the petroleum ether layer and 3y0of the esters are extracted by the 83% ethanol, the number of units of esters, E, and of alcohol, A , in the sample can be calculated by the formulas:

tFm.

2

E = 1 . 0 3 2 ~- 0.0322 A = 1.0322 - 0 . 0 3 2 ~ where z = total units in ethanol phase = total units in petroleum ether phase The percentage of the vitamin A esterified in the sample is calculated by the formula:

% vitamin A esters

Using this procedure 12 fish liver oils and 3 distilled concentrates of varying potencies were analyzed (Table I). In the oils and distilled concentrates examined, 95% or more of the vitamin was esterified. The accuracy of the method waa tested by adding the pure alcohol to halibut liver oil and to distilled concentrate. Assay of the mixtures (Table I) indicated that the method is accurate to within 5y0.

Table 1.

Potency Unita/g.

Halibut No. 2 Oleum percomorphum U.S.P. reference cod Ling cod Soupfin shark Barracuda shark Mexican shark Whale Hake Pollack Tuna Dogfish crystalline Halibut No. 2 vitamin A alcohol (52% of total vitamin A)

+

1

2 3 1

+ crystalline vitamin A alcohol (10% of total A) @

~~~

Vitamin A Ester Analyses b y Petroleum Ether-83W Ethyl Alcohol Distribution Liver Oil

499

lOOE A + E

The presence of esters of low molecular weight, such IW the acetate, would lead to error in using these formulas, since their distribution properties are different. Molecular distillation of many fish liver oils, however, has indicated that only the higher fatty acid esters of vitamin A are present.

~~

A weighed sample of fish liver oil or concentrate containing a total of about 20,000 units of vitamin A is dissolved in 20 ml. of petroleum ether (Skellysolve F) in an amber glass separatory funnel (125 ml.). This solution is extracted five times with 20-ml. portions of 83% ethLnol (83 ml. of absolute ethanol plus 17 ml. of water) which has been previously saturated with petroleum ether. The two phases are shaken together for a t least one minute in each extraction.

=

By definition.

Fish Liver Oils 62,800 68,400 1.700" 57,400 95,400 28,600 64,000 6,000 6.700 3,500 6,750 13,000 129,500

Distilled Concentrates 192,000 534,000 59.200 211,000

Eaters Found

% 9s 100 100 99 100 99 99 100 100 100 96 97 50 (48 cslcd.)

100 99 100 90 (90 calcd.)

INDUSTRIAL AND ENGINEERING CHEMISTRY

so0

15 I

I

Vol. 17, No. 8

absorption a t 328 mp in the diluent oils. The percentage of vitamin occurring as ester in the sample was calculated by the formula:

% vitamin A ester

=

recovered units of vitamin distilling a t 150' to 270' C. recovered units distilling a t 100' to 270' The results for the fish liver oils in question and for three distilled concentrates are given in Table 11. Again it was found that 95% or more of the vitamin A was esterified. The accuracy of the method was tested by recovery experiments with the crystalline alcohol added to halibut liver oil and distilled concentrate. It appeared (Table 11) that the procedure is accurate to within about 5%. 100

150

PO0

Temperature, Figure 1.

O

250

C.

Elimination Curves

---Diatilied concentrate PO0 000 units pw n a m Sam. piui r1tamin)A dcohol, 8.3% 01 total vlbain A Table 11.

Vitamin

A

Ester Assays by Elimination Curve Method

Liver Oil

Halibut No. 2 Oleum percomorphum U.S.P. reference cod Halibut No. 2 crystalline alcohol (48% of total A)

+

1

2 3

1

1

+(5%crystalline alcohol of total A) +(21% crystalline alcohol of total A)

C H R O M A T O G R A P H I C ADSORPTION

Assays by a third independent method have supported the conclusion that substantially all the vitamin A in fish liver oils is esterified. Reed and co-workers (6) recently found by a chromatographic adsorption method that 94% of the vitamin in a sample of halibut liver oil and 98% in a sample of distilled concentrate was present as ester. Similar results have been obtained in this laboratory using essentially the procedure described in the reference.

Ester Found

Recovery of Vitamin A in Distillation

A S S A Y BY FLUORESCENCE

%

%

BACKQROUND. Sobotka, Kann, and Winternitz (9) on the basis of fluorescence measurements reached an entirely different conclusion-viz., that only about 60% of the vitamin A in U.S.P. reference oil, halibut liver oil, and oleum percomorphum is esterified. Only about So% of the vitamin A in distilled concentrate was apparently there as ester. The authors have described considerable experimental evidence which indicates that these conclusions are incorrect. The fluorescence procedure was founded on the observation that vitamin A esters fluoresce strongly in ethanol while the alcohol is only weakly fluorescent. Thus, by using the crystalline acetate and alcohol as standards it was possible to construct calibration curves from which' the apparent percentage of the vitamin esterified in fish liver oils could be calculated from the potency and the fluorescence intensity of the oil. It appeared to the present authors that a t least two conditions had to be satisfied to use this procedure successfully: (1) on an equivalent basis the vitamin A esters in fish liver oils had to have the same fluorescence intensity as the acetate; (2) any difference between the equivalent fluorescence intensity of the oil and of the acetate had to be due to the presence of the alcohol. Evidence presented by Sobotka and co-workers appeared to indicate that the first condition was satisfied. From measurements on vitamin A palmitate, myristate, and laurate the authors concluded that the equivalent fluorescence of saturated fatty acid esters of vitamin A was the same as that of the acetate. From measurements on a specimen of the linoleate it was concluded that the unsaturated esters have lower equivalent fluorescence intensities than the acetate, but that this can be compensated for by a correction factor of about S%. No evidence was provided to indicate that the second condition was satisfied, The authors have repeated many of these fluorescence measurements in this laboratory.

Fish Liver Oils 95 97 98 51 (48 calcd.)

92 99 90 109

Distilled Concentrates 97 98 97 94

M E T H O D II.

M E T H O D 111.

95 99

104

98

(E calcd.) (79 calcd.)

99

M O L E C U L A R DISTILLATION

This conclusion was confirmed by assaying the fish liver oils by a second, independent method, developed by Hickman ( 5 ) and Gray and Cawley (4). The basis of the method is illustrated in Figure 1, which gives the elimination\curves for a distilled vitamin A ester concentrate and for the concentrate containing sufficient added crystalline alcohol to constitute 8.3% of the total vitamin present. The abscissa gives the elimination or distillation temperature; the ordinate gives the percentage of the total vitamin 'distilling a t that temperature. The curve for the distilled concentrate shows the characteristic ester peak at about 210" with no indication of an alcohol peak a t 127" C. In the sample containing added alcohol the peak due to this substance is evident. The curves illustrate the ease with which vitamin A alcohol can be detected by molecular distillation and indicate that substantially none vias present in the distilled concentrate. The elimination curve for halibut liver oil (Figure 2) likewise indicated that only a small percentage of the vitamin was there as the alcohol. By aasuming that all the vitamin A distilling above 159' is in the ester form, which is a close approximation, it was possible to determine quantitatively the content in a series of fish liver oils. Constant yield and residue oil were used and the distillation was standardized with the pilot dye, Celanthrene Red. Assays were done by the antimony trichloride method because of extraneous 1

I N s m v x E N T . A Lumetron (Model 402-EF) fluorophotometer was used with a constant-voltage transformer in the instrument circuit. During operation ultraviolet light from a mercury vapor lamp (Nazda, 100-watt, S-4, cooled n-ith fan) is rendered parallel by an optical system and passed thpough a primary filter (Corning S o . 5840 or 584, 2.55-mm. thickness) having maximum transmission a t 360 mF. The fluorescence exciting beam is then split into two parts. One part enters the sample holder or cuvette (25-ml. capacity, 22 X 37 X 47 mm.). The fluorescent light

ANALYTICAL EDITION

August, 1945

130

180 Temperature,

Figure 2.

230

270

C.

Elimination Curve for Vitamin Halibut Liver Oil

A Esters in

generated is filtered throu l- two secondary f i l k n (Corning No. 3389 or 038, 1.43-mm. thicgees) and registered on twin barrierlayer photocells. The other part of the beam is passed through a reduction late (the authors used a No. 7) and deflected by a mirror to t i e balance photocell which can be turned through 90' and was maintained a t a 45" angle during the work. The balance hotocell and the two measuring photocells are connected in a gridge circuit with a slide-wire resistance and with a galvanometer (General Electric Co. multiple reflection, high sensitivity) as the zero indicator. The intensity of the fluorescence varies linearly with the slide-wire resistance which is calibrated. The sensitivity of the instrument with standard quinine sulfate (1.0 microgram per ml. Concentration) averaged 18 galvanometer divisions per slide-wire division. MATERIALS. Halibut liver oils 1 and 2 and oleum percomorphum were commercial samples obtained locally. The U.S.P. reference oil was a freshly opened sample. The synthetic vitamin A esters assayed were prepared by C. D. Robeson of this laboratory. Their propertieswill bedescribed elsewhere, The acetate, heptylate, myristate, palmitate, and succinate esters were purified by crystallization. The others were not crystallized but were purified by chromatography. Absolute ethanol (Industrial Chemicals, Inc.) was the solvent used in the work. Undistiued selected samples frequent1 had low fluorescence values (slide-wire readings = 2 or less7 and were used directly. Other samples, however, had a marked blue fluorescence. I n this case fractionation through a packed rolumn after refluxing over silver nitrate and potassium hydroxide reduced the readin to a suitably low value (1.0). Quinine sulfate ?concentration 1.0 microgram per ml. in 0.1 N sulfuric acid) was used as a secondary standard. PROCEDURE. The following sequence was employed in determining fluorescence. 1. The instrument was adjusted to give a slide-wire reading of 100 for standard quinine sulfate solution. A volume of 25 ml. was used in cell 1. 2. The instrument was set to give a slide-wire reading of zero for 0.1 iV sulfuric acid in cell 1. 3. The slide-wire reading for ethanol was determined in cell 2. This cell was chosen to have the same transmission and fluorescence properties as cell 1. Two cells were used to avoid decomposition of the vitamin by traces of acid. 4. The fluorescence of the sample in ethanol was determined in cell 2. Measurements were made over time intervals varying from about 6 minutes (vitamin A concentration calculated as alcohol, 0.25 microgram per ml.) to 25 minutes (concentration, 3.0 micrograms per ml.). All concentrations reported in the fluorescence section of this paper are calculated in terms of the alcohol. The fluorescence of the fatty acid esters rose to a maximum and then decreased while the fluorescence of the alcohol was less and declined steadily from its initial value (Figure 3). The authors' findings thus confirm those of Sabotka, Kann, and Loewenstein (8). Slide-wire readings were taken until the value at the maximum was determined. The difference between the

50 1

maximum slide-wire value for the sample and for the b!ank ethanol gave the corrected reading for the solution. 5. The slide-wire reading for the standard was redetermined and if within 2% of its original value the reading for the test sample was considered acceptable. Considerable difficulty wm experienced a t first in maintaining readings for the standard which were as steady as this. The authors have been informed that this difficulty is also experienced with other fluorophotometers. Substitution of the 5-4 tube for the Uviarc originally supplied with the instrument was helpful, Certain sites in the laboratory were preferable to others. The instrument varied in steadiness during the day and selection of the best time to make the measurements was frequently necessary. A grounded wire screen about the instrument did not seem to help. No single cause was found for the disturbances but attention to each of the factors described eventually reduced them to the oint where few readings needed to be discarded. 6. The fPuorescence of vitamin A acetate was determined at least once daily a t one or two concentrations .(always a t 0.5 microgram per ml., frequently a t 3.0 micrograms per ml.) and used as a primary standard during the work. 7. The concentration of vitamin in the test solution was determined spectrophotometrically usin the Beckman instrument. This method was employed rather t f a n the antimony trichloride y d u r e , also used routinely in the laboratory, since experience as indicated that the s ctrophotometer gives more reliable assays for fish liver oils a n 8 i s t i l l e d concentrates having potencies r a t e r than 50,000 units per gram. Sobotka and co-workers, owever, used the blue color method exclusively. To determine the effect of this in calculating fluorescence intensities, the authors assayed vitamin A acetate, palmitate, halibut liver oils 1 and 2, oleum percomorphum, and distilled concentrate by both procedures. The deviation of the antimony trichloride from the spectrophotometric assays was, respectively, 0, $3, -3, -7, -3, and 0%. This agreement is sufficiently good so that the conclusions drawn in the paper should hold for assays done by either procedure. To obtain close agreement it may be necessary to check the blue color calibration curve repeatedly during the work. In the spectrographic assays, fish liver oils were dissolved in ethanol to give concentrations suitable for the spectrophotometer (a'pproximately 3.0 micrograms per ml.). An aliquot of each solution was immediately assayed. The stock solutions were then diluted to give the concentrations desired for the fluorophotometric measurements.

5 Figure 3.

10

Time, Minuter Variation of Fluorescence Intensity

15

(FJ with

Time

Conccnbation, approxinatrly 0.5 miaomam per ml. 1. Crystalline vitamin A mcatmta 9. Vitamin A alcohol

CALCULATIONS. No system hae yet been generally adopted for the quantitative expression of fluorophotometric data. Since some way of comparing the fluorescence of preparations containin different esters was needed, the following system was devised. slide-wire readings made with the Lumetron, as previously stated, vary linearly with the amount of fluorescent light falling on the photocells. Furthermore, within limits, the intensity of fluorescence of vitamin A is proportional to the concentration. Thus, within these limits, if the instrument is standardizede.g., by quinine sulfate a t a concentration of 1.0 microgram per m1.-the quotient of the slide-wire reading at maximum fluorescence and the vitamin A concentration (calculated as alcohol) should be ronstant. The quotients for two preparations are then

INDUSTRIAL AND ENGINEERING CHEMISTRY

502

was assayed rather than by using an aver me value of F, for the acetate. The avera e of the indicated n u a e r of determinations made was then calc3ated.

proportional to their fluorescence intensities. The authors have used in the text the term “equivalent fluorescence”, F., to designate this quotient. Therefore: F, =

0.1755 EP

FLUORESCENCE OF H A L I B U T LIVER O I L DISTILLATES

This fluorophotometric procedure was used to determine how well the two conditions considered necessary for the successful assay of vitamin A esters by the method of Sobotka et al. (9) were satisfied.

where8 = maximum slide-wire reading for the preparation in ethanol 328 mp) of the preparaE = extinction coefficient tion. The coefficient for vitamin A alcohol a t 328 mp is taken as 1750 P = % by weight of the preparation in ethanol

The preparations used consisted of halibut liver oil and fractions obtained by molecular distillation, taking cuts a t 20” intervals from 180’ to 240’. In Table 111, Column 4, is iven the equivalent fluorescence, F,, for the preparations. 8olumn 5 ves the quotient X 100 of the F , value for the fraction and for $e acetate. Column 5 thus gives the approximate apparent percentage of the vitamin present in esterified form, by fluorescence, using the acetate as a standard. The error introduced by neglecting the fluorescence of the alcohol is small.

CONCENTRATION RANGE FOR LINEAR RESPOME. The authors’ work indicates that the fluorescence intensity of vitamin A fatty acid esters in ethanol varies linearly with the concentration within the approximate ran e 0.025 to 0.55 microgram per ml. (calculated as alcohol). T%e data of Sobotka, Kann, and hewenstein for the acetate support this conclusion (8) and suggest that the operating characteristics of their *instrument (made by Pfaltz and Bauer, Inc.) are similar to the authors’. 100 1

I

J/

0.5 Figure 4.

1.0

,

1.5

2.0

2.5

3.0

Concentration, microgram per ml. Variation of Fluorescence Intensity with Vitamin Concentration 1. 2. 3.

Vol. 17, No. 8

A

Vitamin A acetatr Vitamin A palmitate Mixed vitamin A ertrrr of halibut liver oll

A plot illustrating thib linear relation for the acetate, palmitate, and vitamin A esters in halibut liver oil is given in Figure 4. In this range F , is independent of the concentration. A t higher concentrations F. decreased so that a t a concentration of 3.0 micrograms per ml. the value for the acetate was only 65% of the value in the l i n e s range. The authors believe that a comparison of the fluorescence of vitamin A preparations can best be made in the linear range and the conclusions drawn in this paper are principally based on such measurements. It is shown below that anhydro vitamin A may interfere with fluorescence measurements made a t higher concentrations. ERRORS.Accurate determinations of the equivalent fluorescence of vitamin A preparations are difficult to obtain at present. .In assay of a fish liver oil is likely, from the authors’ experience, t o be in error by 5% or more, depending on the potency. This includes the error of physical measurement and that due to s urious absorption at 328 mp in the sample. It seems probable tEat the error in a single fluorophotometric measurement is as large. In addition, errors may occur in preparing the extremely dilute solutions used. I t is thus not surprising that the fluorescence of even highly purified preparations varies apparently from day to day. For example, twenty-two independent determinations on vitamin A acetate during a 2-month period gave F , values varying from 43.9 to 56.9. The average value was 51.7 d t h a standard deviation of ~ 3 . 4 . The errors appeared to be iystematic to a lar e extent. Thus the daily ratio of the F. value for the acetate a n i palmitate over a period of time varied much less than the individual values for each ester. To minimize these difficultly avoidable errors the averages of as many independent determinations as possible have been reported (Tables I11 and IV). Account was taken of the systematic nature of the errors by calculating each ratio in the tables from the F , value for the acetate obtained on the day the other preparation

The undistilled halibut oil appeared to contain only 63% of the vitamin A as ester by this method, which agrees closely with the value of about 60% reported by Sobotka et al. This value is of course entirely out of line with assays made by the three independent methods described, but for the moment we shall assume that it is correct. If halibut liver oil does contain 40% of the vitamin A as alcohol the first distillate fraction should ‘assay, by fluorescence, nearly 100% alcohol since this substance distills a t low temperatures. Actually the first fraction appeared to contain only slightly less esterified vitamin (60%) than the original oil. Furthermore, subsequent distillate fractions which would be stripped free of the alcohol never assayed 100% ester. It therefore appears that the low equivalent fluorescence exhibited by vitamin A in halibut liver oil is not due to the presence of ,the alcohol. The similar Fa values for other fish liver oils (Table IV, A) suggests that this conclusion also holds for them. The reason why halibut liver oil has a low Fa value was next investigated. I t was found that one important reason for this is that vitamin A esters like or similar to those occurring in halibut liver oil have intrinsically lower equivalent fluorescence intensities than the acetate. The F1 value for the palmitate (Table 111) was only 79% of that of the acetate. The value for an ester synthesized from the crystalline alcohol and the mixed fatty acids from saponified halibut oil (iodine value = 143) and for an ester prepared from the same acids after catalytic hydrogenation with platinum oxide (iodine value = 6.3) was only 87% of that of the acetate. Thus, neither of the two basic conditions the authors consider necessary to make successful the fluorescence assay method of reference (9) appeared to be satisfied. The vitamin A esters in the fish liver oils investigated did not have the same equivalent fluorescence as the acetate. The examination of halibut liver oil distillates indicated that their lower fluorescence wm not due to the presence of the alcohol. It therefore appears that the method in its described form is unsuitable for determining the percentage of esterified vitamin A in fish liver oils and distilled concentrates. The authors believe that a satisfactory fluorescence method might be developed by using a differentstandard than the acetate, especially if it were used on high potency liver oils. The accuracy and simplicity of the solvent distribution methods now available, however, make it doubtful whether such a procedure would find more than limited application. FLUORESCENCE OF PURIFIED V I T A M I N A ESTERS

Such data aa the authors have indicate that the molecular weight and structure of the fatty acids with which vitamin A is esterified play an important role in determining the intensity of fluorescence. This is indicated in Table IV, B, where the ratios

August,

ANALYTICAL EDITION

1945

of the F. values for a series of purified vitamin A esters to that of the acetate are given. I n the linear concentration range there is a progressive decrease for this ratio in the series of straight-chain fatty acid esters including the acetate, propionate, butyrate, caproate, and palmitate. Comparison of the values for the butyrate and isobutyrate and caproate and isovalerate esters suggests that s i d e chain methyl groups may depress the fluorescence, but the evidence is not conclusive. Comparison of the equivalent fluorescence of esters prepared from the crystalline alcohol and the mixed fatty acids of halibut liver oil, before and after hydrogenation (Table 111), indicated, contrary to the findings of Sobotka et al., that unsaturation in the fatty acids with which the vitamin is esterified does not necessarily decrease the fluorescence. The equivalent fluorescence ratios in Table IV were determined both in the linear concentration range and in more concentrated solutions. The latter values were determined because much of the work of Sobotka and co-workers was reported as being done in the higher concentration range. I n this range, as previously mentioned, the F , values for the esters were lower than in the linear concentration range. The decrease in Fa value with increasing molecular weight wm less uniform than at lower concentrations. Nevertheless, the ratio of the F. values of the palmitate and acetate a t the higher concentrations w a substantially the same as the value obtained at the lower concentration. This appeared to be true also for the vitamin A esters in fish liver oils (Table IV, A). Therefore, the discrepancy between the authors’

Fluorescence of Halibut Liver Oil and Distillates in Ethanol (Vitamin A Concentration 0.4 to 0.5 y per ml. Standard, quinine sulfate,

Table 111.

1.00 Y per ml.) Diatillation Apparent Temgerature. (328mr) Approximate C. (Beckman) Fa % Esters 43.6 31.9 63 (3)”

‘ik.

Sample Ralibut liver oil No. 1 Distillate fraction 1 2 4 6 Vitamin A palmitate Ester of vitamin A and mixed halibut oil acids Eater of vitamin A and mixed, hydroeenated halibut oil mid8

..

180 200 220 240

30.3 32.3 33.7 34.3 43.3

60 (3)

..

209.0 255.0 132.5 21.4 907

..

809

41.5

87 (3)

..

865

42.5

87 (3)

% {ii

67 (2) 79 (5)

IV.

Fluorescence of Fish Liver Oils and Purified Vitamin Esters in Linear and Nonlinear Range Linear Rangea

Sample Distilled concentrate Oleum percomorphum Halibut liver oil No. 2 U.S.P. reference cod liver oil Vitamin A Acetate Propionate Butyrate Isobutyrate Isovalerate Caproate Heptylate LMyristate Palnutate Oleate a

b

(328mr) (Beckman) A. 95.4

Apparent % vitamin A esters

Fish Liver Oils 69(1) 35.7

24.4

72(1)

18.0

55(1)

60(1)

31.4

33.6

63(2)

19.2

61 (2)

30.1

58(1)

20.3

62(1)

1470 1420 1406 1329 1319 1290 1230 956 907 886

0.4 to 0.5 Y per ml. 2.9 to 3.5 Y per ml.

Nonlinear Rangeb

30.7

B.

A

Fa

Fa

Apparent % vitamin A esters

34.2 0.85

findings and those of Sobotka et al. cannot be attributed to differences in the concentration of vitamin A used. O T H E R POSSIBLE CAUSES F O R LOW FLUORESCENCE O F FISH LIVER OILS

Some hypotheses to explain the low equivalent fluorescence of vitamin A in fish liver oils wqre investigated and found to be untenable. The hy othesis that the linear fluorescence response for the esters in &h liver oils might occur at lower concentrations than for the acetate was examined. Equivalent fluorescence values obtained a t a concentration of 0.025 to 0.55 microgram per ml. would thus be low and an erroneous value for the ester content of the fish liver oil would result. The data in Figure 4 indicate, however, that this hypothesis is incorrect. The range within which fluorescence intensity varied linearly with concentration was the same for the acetate, palmitate, and the halibut liver oil vitamin A esters. The ossibility that substances-.g., of phenolic nature (I)may infibit or quench the vitamin fluorescence waa studied. To test this, vitamin A acetate was added to halibut liver oil No. 1 to form a mixture having (328mp) = 84.5. At a concentration of 0.477 microgram per ml. the Fa value for the mixture waa 99% of that calculated from the F. values for the components. This is evidence that the low fluorescence of the halibut oil examined is not due to inhibiting substances. It provides no assurance, however, that inhibitors are absent from other fish liver oils. This is an additional source of uncertainty in attempting t o assay vitamin A esters by fluorescence. The activity of anhydro vitamin A as an inhibitor of fish liver oil fluorescence W M of interest, since this substance has an absorp tion band a t 365 mp which is almost exactly the wave length of the fluorescence exciting radiation. Furthermore, i t occurs in fish liver oils in amounts up to 2% of the vitamin A. The inhibiting action of crystalline anhydro vitamin A was studied by preparing mixtures of it and vitamin A acetate in ethanol to give solutions in which the percentage by weight of the anhydro compound to acetate was 1,5, and 10%: The equivalent fluorescence of these mixtures was determined in the linear (0.4 to 0.5 microgram per ml.) and nonlinear (2.9 to 3.2 micrograms per ml.) concentration ranges. In the linear range the F, values found for the mixtures were, respectively, 97, 94 (11, and 98% of the value for the pure acetate. I n the nonlinear range the values were, respectively, 97, 91, and 887 of the value for the pure acetate. It thus appears that anhycfro vitamin A interferes with the fluorescence of the vitamin esters only a t relatively high concentrations. This is an additional reason for making measurements in the linear concentration range. Anhydro vitamin A interferes, however, only when present in concentrations greater than are normally found in fish liver oils. ACKNOWLEDGMENTS

* Average of indicated number of determinations. Table

503

Purified Vitamin A Esters

51.7 48.0

46.7 37.2 42.5 42.6 43.6 41.8 43.3 44.6

33.9 29.1 29.3 25.2 27.9 28.6 29.5 26.9 25.6 28.4

The authors wish to thank C. D. Robeson for providing the purified vitamin A ester samples and coordinating much of the work; R. Fleisher and R. W. Lehman for performing analytical distillations; and S. Licata and L. Weisler for assays by the distribution method. R. Brauer, formerly of this company, rendered valuable assistance at the beginning of the fluorophote metric work. The authors are particularly indebted to S. E. Krewer, Photovolt Corp., New York City, for advice on fluorophotometric measurements and for materials needed to expedite the work. LIlERATURE CITED

(1) Bowen, E. J., Trans.Faraday Soc., 35, 19 (1939). (2) Clausen, S. W . , et al., Abstracts, 99th Meeting, AMERICAN CHEMICAL SOCIETY, p. B-9, 1940. (3) Dann, W.J., and Evelyn, K . A., Biochem. J., 32, 1008 (1938). (4) Gray, E.L e B . , and Cawley, J. D., J . L h Z . Chem., 134,397 (1940). (5) Hickman, K . C . D., IND. ENQ. CHEM.,29, 968, 1107 (1937). (6) Reed, G . , Wise, E . C., and Frundt, R. J. L., IND. ENG.CHEM.. ANAL.E D . , 16, 509 (1944). (7) Reti, L.,Compt. rend. sac. bid., 120, 577 (1935). (8) Sobotka, H . , Kann, S., and Loewenstein, E . , J . A m . Chen. Sac., 65, 1959 (1943). (9) Sobotka, H..Kann, S., and Winternitz, W., J . B i d . Chen., 152, 635 (1944). PRESENTED in part before the Division of Biological Chemistry at the 108th Meeting of the AMERICAN CHEMICAL SOCIETY, New York, N. Y. Communication No. 66, Distillation Products, Inc., 755 Ridge Road W a t , Rochester 13, N. Y.