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Determination of vitamin A acid in human plasma after oral administration
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Determination Plasma
337-341
98,
of Vitamin A Acid in Human after Oral Administration’
LAWREPL’CE From
the Department
(1962)
of Medicine, Received
JURKOWITZ
The University March
of Chicago,
Chicogo,
Illinois
19, 1962
B quantitative method is described for detecting vitamin A acid in human plasma after oral administration. Levels of vitamin A acid ranging between 112 and 329 Fg./ 100 ml. were obtained, while vitamin A alcohol levels ranged between 33 and 63 pg./ 100 ml. Both antimony trichloride spectra and direct spect’ra obtained with this method are presented. Some of the difficulties in determining vitamin 9 acid quantitatively are discussed. INTRODUCTION
Interest in vitamin A acid has been stimulated recently wit’h the discovery by Dowling and Wald (1) that vitamin A acid mill serve the general tissue functions of vitamin A, but not its role in vision. Since the discovery of its biological activity (2)) investigators have attempted to detect vitamin A acid in tissues. Redfearn was able to find traces of what was probably the methyl ester in depot, fat after administration of t’he methyl ester of vitamin A acid (3). However, he and three others (1, 3-5) have failed to find vitamin A acid in rat tissues after the administration of very large doses of the free acid. This report will describe successful detection in human plasma, and outline some of the difficulties involved in the tissue det,ermination of vitamin -1 acid. EXPERIMENTAL ds described in detail below, all blood was collected in bottles containing SaF. It was found empirically that the presence of NaF in bloodcollect,ing bottles helped prevent appearance of compounds which interfere with the final spectrophotometric determination of vitamin A acid by the antimony trichloride renct,ion. The mechanism 1 This work was supported t)y :I research grant, 8-1528, from the National Institute of Arthritis and Metabolic Diseases of the National Inst,itutes of Health, U. S. Public Health Service. 337
by which NaF exerts this effect is unknown. The method involved preparation of an acetone extract of the plasma followed by saponification to form the potassium salt of vitamin A acid. Extraction with ethyl ether effected partial purification by removing vitamin A alcohol, carotenoids, and other lipids, while the vitamin A acid remained as the potassium salt in the lower layer. Following acidification, the vitamin A acid was extracted with ethyl ether. The ethyl ether was then evaporated and the extract dissolved in petroleum ether; it was next passed over a CaC03 column in order to partially adsorb compounds which interfere with calorimetric measurements. Of a variety of adsorbcnts tried, CaC03 was the best because it removed most of the interfering compounds without significantly affecting recoveries of vitamin A acid. Final determination of the vitamin A acid was conducted calorimetrically in chloroform by means of the antimony trichloride reaction. Direct spectra were obtained by further purification of the extract on acid-washed alumina. Vit,amin A acid was prepared for administration as follows. From 100 to 200 mg. of crystalline vitamin A acid (Distillation Products Industries), a powder was formed by dissolving the vitamin A acid in ethyl ether and evaporating the ether. The powder was then dissolved to the extent possible in a mixture of 15 ml. of Twcen 80 and 15 ml. of 0.1 211IGHPOI pH 9. More of the vitamin A acid was dissolved by adding four 15-ml. aliquots of buffer with stirring and n-arming to 37”C., the solution was well mised, and all was given. Four adult male subjects were used iu these esperiments. Each fasting ~ubjcct swallowed the mixture, and 3 hr.
338
JURKOWITZ
later 50-60 ml. blood was drawn into a heparinized syringe. The blood was placed into bottles containing NaF (0.5 ml. of 2% NaF in water for each IO-12 ml. blood and dried in a desiccator). The blood was centrifuged, and the separated plasma was centrifuged a second time to remove remaining cells. Six milliliters of plasma was set aside for carotene and vitamin A alcohol determinations according to the method of Moore (6). The remaining 20-25 ml. plasma was cooled in the refrigerator, mixed with 3 vol. of cold acetone, and allowed to stand in the refrigerator for 1 hr. The resultant mixture was centrifuged, and the supernatant acetone-water extract was saved ; the sediment was re-extracted with 30 ml. acetone for 10 min. on a mechanical shaker to obtain any residual vitamin -4 acid, and centrifuged. The two acetone extracts were combined and evaporated under gentle suction until foaming occurred (60-70 ml.). Then 45 ml. of 2 AV KOH dissolved in 95% ethanol was added, and saponification was carried out at 35-40°C. for 30 min. under a nitrogen stream. The volume at this point was 60-65 ml. Forty milliliters of water was added, and the water-ethanol mixture (most acetone evaporated during saponification under a nitrogen stream) was extracted twice wit,h 75 ml. ethyl ether. The combined ether extracts were washed five times with 15 ml. water, and the washings were added to the water-ethanol mixture. The ether ext,ract after washing had a volumr of 145155 ml. and was discarded. The water-ethanol fraction was cooled, and 50 ml. of cold 2 S HCl was added. This mixture was then extracted twice with 100 ml. ethyl ether, and the combined ether extracts were washed three times with 20 ml. water. The combined ether extract, (185 ml.) was saved, while the washings were added to the water-ethanol-HCl mixture. The water-ethanol-HCl washings were extracted a third timrl with 100 ml. ethyl ether before being discardrd. The third ether extract was washed three t,imcs with 10 ml. water and saved (final volume 95 ml.). The combined ether extracts wrre cooled to -2O”C., and excess water was removed in a scparatory funnel. They were then evaporated to dryness, the final stages being conducted under nitrogen. The dried rstract was inzmedintcly dissol\-ed in 6 ml. pet,roleum ether (Raker’s b,p. 3060”) and poured onto a 1 cm. X 0.5 cm. CaCO, (Baker’s) column which had been previously heat,ed 4 hr. at 200°C. Three-milliliter aliquots of petroleum ether were used to extract the contents of the flask and were poured ont,o the column until 24 ml. in all were used. The petroleum ether eluate was evaporated with nitropcn, and the residue was imnzdin/el~/ dissol\-cd in 2-3 ml. CHCh
All spectrophotometric measurements were made in a room maintained at 23°C. Determination of the vitamin A acid was carried out in a Beckman recording spectrophotometer using l-ml. samples of the chloroform extract, 3 drops acetic anhydride, and 2.7 ml. of 25% (w/v) SbCl, (Baker’s Analyzed Reagent) in CHCL All measurements were made against a chloroform blank. For each unknown the time of maximum color intensity (ahout 80 sec. with freshly prepared SbCL) was first determined by following the change in optical density at 572 rnp with time. Then a complete absorption curve was rapidly recorded so that the 572-rnw peak was reached at, the time of maximum color intensity. Duplicate determinations agreed within 5% because the time of maximum color intensity was known for each sample and because the optical density is relatively constant for 10 sec. on either side of the maximum. Calculations were made from absorption at, 572 rnp using a st.andard curve calibrated with crystalline vitamin A acid. These curves are linear in the O-30 pg. range with 25% (w/v) &Cl, Usually about 14 hr. of continuous work elapsed from the drawing of the blood to the vitamin A acid determination wit)h the &Cl, reaction. It was thought that best results would he obtained if the experiment were carried through to completion in the shortest possible time. This concept was supported by some experiments in which the final tissue rxt,racts in chloroform were stored 48 hr. at -20°C. and lost 15% of the vitamin A acid (this loss occurs more slowly when the crystalline compound is dissolved in chloroform). -After passage through C!aCOR , the extracts contain compounds which make it impossible to identify the direct ultra\-iolrt’ spectrum of vitamin A acid. Much of this interfering material can be removed by further purification on acid-washed alumina. To obtain tlirc,rt spectra, nonquantitativo c~xperiments were donr using plasma from onr of the subjects. The prorrdurr was the same as that described ahore, but nftrr passage through the CnCO., column the pet.roleum ether was poured onto :an unheated alumina (Mrrck acid-washed) column. The ah~minn column was 3.5 X 0.5 cm., lm~parctl from a petroleum ether slurry, and deactivated with 2 drops water. After the 24 ml. prtroteum ether had run through, 10 ml. of 5% acetone in petroleum ether was run through, fotlowed by 15 ml. of 20% acetone in petroleum ether. The 20% aretonc fraction was saved, evaporated to dryness with nit,rogen, taken up i?nmct%iatell/ in petroleum ether. and the rhromatography repeated. The chromatography was done a third time before the extract in 20% acetone was t,ransfcrretl to CHCL , and the direct spectrum was measured in tlrcx Bcrkmnn DK-1 spect,rophotometer.
VITAMIK
A BCID
IN
HUMSN
339
PLASMA
RESULTS
The spectrum of the antimony trichloride reaction of crystalline vitamin A acid (Fig. 1) compared with that of tissue extracts (Fig. 2) indicates that with the described method substantial levels of vitamin A acid appear in human plasma after oral administration. Evidence that this represents vitamin A acid is provided by the character of the curve obtained. There are two main peaks, one at 572 mp which decreases in intensity with time, and another at 470 rnp which increases in intensity with time. Both the location of t’he maxima and the character of the decay curve are t,he same as those obtained with crystalline vitamin A acid. Neither the 572-rnp peak nor the 470-rnp peak appeared when Tween 80 buffer solution is administered without vitamin A acid. There are, however, differences between the antimony t,richloride spectra of crystalline vitamin A acid and those obtained with tissue extracts containing vitamin A acid. The crystalline compound shows a minimum at 495-500 mp and a lower base line below 525 mp. In addition, the crystalline compound has a low broad peak with a maximum at approximately 378-380 mp which decreases in intensity with time. This peak I I I I I I
1
I
I
I
I
I
350
400
450
500
550
600
WAVELENGTH
650
Imp)
FIG. 1. Absorption spectra of antimony trichloride reaction of crystalline vitamin B acid. Spectrum at maximum color intensity (62 sec.). -- - Spectrum when color intensity is decreasing (100 sec. after maximum). To 25.9 pg. of crpstalline vita,min A acid in 1 ml. chloroform were added 3 drops acetic anhydridc and 2.7 ml. of 25% (w/v) antimony trichloride.
I 350
I 400
I 450
I 500
I 550
1 600
t 650
WAVELENGTH
FIG. 2. Absorption
spectra of antimony trichloride reaction of vitamin A acid isolated from human plasma. ~ Spectrum at maximum color intensity (70 sec.). ---Spectrum when color intensity is decreasing (100 sec. after maximum). The spectra shown above were run on 1.0 ml. of a 2.9ml. chloroform extract, prepared as described in the text from 22 ml. plasma taken from an adult man 3 hr. after administration of 150 mg. vitamin A acid. The quantitp of vitamin A acid in this 1.0 ml. sample was calculated to be 21.1 fig.
is more prominent at earlier times than are shown here. These diffcrcncrs probably represent a failure to complet,ely remove unknown compounds which absorb at short’cr warelengt,hs or which react with ant’imony trichloride to give absorption at shorter wavelengths. The direct spect,rum (,Fig. 3) proTides further confirmation of the assumption that vitamin A acid is present in the extracts. A Iteak appears at 362 mp when vitamin A acid is administered which is not present when the Tween 80 buffer solut,ion is administered alone. In another experiment the peak was at 363 mp. The maximum of crystalline vit,amin A acid in chloroform i:: 366 inp, so there is a 3-4-mp difference bctwcen the extracts and the crystalline compound. This probably represents a failure to completely remove compounds which absorb at shorter warelengt.hs, as can be seen from the asymmetrical character of the direct spectrum of t’hc extracts. Table I provides a comparison of tlrc levels of vitamin A acid wit,h simultaneously determined vitamin ,4 alcohol levels. It can be seen that when vitamin A arid is
340
JCRKORITiS
FIG. 3. Direct absorption spectra of extracts of human plasma read in chloroform. -o-•-•-Spectrum after administration of vitamin A acid. -O-O-O-Spectrum after administration of Twecn 80 buffer solution alontl. -1 method for obtaining nonquantitative direct spcvtr’a. is described in the text. TABLE
I
VITAMIN A ACID .&ND VITAMIN A AI~~OHOI~ LEVEI.B Is
HUMAN
PLASMA TRATION
Dose of vitamin m.
100 150 150 150 200 200 * Corrected
A acid
3 HR. OF
AFTER
T’ITAMIN
Vitamin pg.‘flOO
ORAL A
Vitamin A acid _.___ ml. p1as712a ~y.:lov
112 171 200 278 247 329
ADMISIS-
ACID A alcohol” ml. plasma
03 45 43 50 33 35
for cnrotenoids.
given orally, the lcwls of vitamin A acid can far exceed those of vitamin A alcohol. The levels recorded probably represent only about 74% of the actual vitamin A acid present. This conclusion is reached because six experiments in which 100 pg. vitamin A acid in acetone were added to 25 ml. plasma gave recoveries of 69-78s with an average recov-cry of 74%.
The dosage of vitamin A acid given in tlwsc experiments represented about 1.5-3.0 mg./kg. of body weight. Since Sharman (5) gave 10 mg. to a single rat, and Redfearn (3) gave 1 mg. daily for as long as 24 days
to single rats, it seems unlikely that the failure of other investigators to find vitamin A acid in tissues relates entirely t,o the dosage used. Other possible explanations include species difference, the methods of administration of the vitamin A acid, and the amounts of tissue used for analysis. However, it seems likely that the success of the experiments pres,ented here relates at least in part to the method of preparation of tissue extracts. Rcdfearn (3), for example, extracted tissues with ether as one would do for vitamin A alcohol. When the standard ethanol-petroleum ether prow dure (6) for vitamin A alcohol is applied to plasma known to contain large amounts of vitamin A arid, the 572-mp peak cannot be detected with anv certainty, although there i:: a suggestion df the 470-mp peak in the decay curves. To obtain curves similar to thaw in Fig. 2, it, was necessary to scparatc vitamin A acid from compounds which interfcred in the antimony trichloride rcaction. For good quantitative recoveries, in addition to t#he lwocedure described and the usual precautions in handling vitamin A alcohol, several other factors should be mcntioncd. The antimony trichloridc reaction of vitamin ,1 arid behaves differently from that of vitamin A alcohol in an important respect. Whereas one can store 25% (w,!vj antimony trirhloridc in chloroform indefinitely and obtain the same absorption for a giwn amount of vitamin A alcohol, storage of the antimony trichloride for 2 weeks increased the amount of color produced with vitamin A acid by 10-15s without changing the wavclcngth of the maximum extinction. In addition, aging of the antimony trichloride for 2 weeks causes the peak of the color reaction with vitamin A acid to appear at 60 sec. instead of 80 sec. Longer periods of aging of the antimony trichloride can product greatw effects. These effects wrc looked for, but not found with vitamin A alcohol. Sor could they be explained by the presence or absence of acetic anhydride in the reaction with 25:L (w/v) antimony trichloridc in chloroform. In quantitative experiments on vitamin A acid, errors which could be introduced 1)~ these clffects arc avoided by performing cali-
VITBhiIPi
9 ACID
bration curves at the time of an experiment and const,antly rechecking t,hem. Another characteristic of vitamin h acid n-hich makes its detection more difficult t,han that of vit,amin -4 alcohol is the lower intensity of the color produced by t,he reaction wit,h 25% (w/v) antimony trichloride. One needs approximately five times as much vitamin A acid to obtain the same color intensity at 572 rnp that one needs for a given amount of vitamin L4 alcohol at 618 mp using freshly prepared 257; iw-/v) antimony trichloride. From considerations based on t,he intensity of the color reaction Cth vitamin A acid, t~lic shape of the ant,imany t,richloride spectrum wit’11 tissue extracts at low levels of vitamin A acid, and t,he percentage recoveries, it, is possible to make a rough t>st.imatc that, the plasma levels of vitamin h acid below 50 pg./100 ml. plasma would not be dctcctablc by this method. When one considers that about, 85% of normal humans have vitamin A alcohol levels between 24 and 84 /Lg./‘100 1111. (80280 I.U./lOO ml.) according to Moore (71, it is readily apparent that large and phrsiologically important amounts of vi&m& A acid could be easily overlooked even in a relatively simple t’issw like plasma. This has implications with respect to current concepts of the role of vitamin A acid. Other workers (1) have emphasized that. the failure to find Ctamin A acid may be a consequence of rapid conversion to an active met,abolite or a consequence of low tieSW levels. The technical considerations elaborated abow make it, clear that the presence of even relatively large quantit,ies
IS
HUMAN
PLASMA
3-I 1
of vitamin h acid may not have been tlrtccted. It is therefore apparent that more detailed invest,igation of a variety of tissues from many species with more sensitive methods will be necessary before it can be concluded that vitamin A acid is not, present in physiologically significant amount,s. It, is also clear that vit,amin A acid is not SOrapidly metabolized in man, because appreciahle amounts are found in human plasma 3 hr. after administration of a large dose.
The, author
wishes to thank
Dr. R. 1,. J,nntl:tu
ior llip advice and c~ncouragcmcnf,tlwing the course of this n-o&, ant1 for his counsel in the n-riting of this manuscript. Tllc? author is indebted to Dr. George W&l for st,imulating an early interest in vitamin 11, and sul~secprntly in vitamin A acid. He would also like to thank lhr Organic Rescarvh Laboratory of Distillation Products Industries, Rochester, X. Y.. for generous gifts of vitamin A acid.
J. E., AXD A$-ALD,G., I-'mc. Sat/. Aced. Sci. L-.,%46,587 (1960). .iRESS, J. G., AKD \-as Dow, D. A,, .v&wt’ 157, 190 ( 1946). REDFIMRS. E. K., A~lr. Bioclwln. Bio/,A!/s. 91, 226 (1960). HARRIS, I’. I,., in “Syml~osium on Nutrition” (R.. RI. Herriott, ed.), p. 71. John Hopkins Prc,.q Baltimorc~, 1953. ~H.4RMAN, I. bl., Bait. J. hTllfrifioil 3, viii (1949). &~OORF 3, T. * “T’itamin I4 >” 1). 586. Elscvier Pnhl. Co., Amsterdam, 1957. &lOORE, T., “Vitamin Ai’ p. 361. Elscvicr Pul,l. Co., Amslrrdam, 1957.
1. I~WLIX,
2. 3. 4.
5. 6.
7.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Determination Plasma
337-341
98,
of Vitamin A Acid in Human after Oral Administration’
LAWREPL’CE From
the Department
(1962)
of Medicine, Received
JURKOWITZ
The University March
of Chicago,
Chicogo,
Illinois
19, 1962
B quantitative method is described for detecting vitamin A acid in human plasma after oral administration. Levels of vitamin A acid ranging between 112 and 329 Fg./ 100 ml. were obtained, while vitamin A alcohol levels ranged between 33 and 63 pg./ 100 ml. Both antimony trichloride spectra and direct spect’ra obtained with this method are presented. Some of the difficulties in determining vitamin 9 acid quantitatively are discussed. INTRODUCTION
Interest in vitamin A acid has been stimulated recently wit’h the discovery by Dowling and Wald (1) that vitamin A acid mill serve the general tissue functions of vitamin A, but not its role in vision. Since the discovery of its biological activity (2)) investigators have attempted to detect vitamin A acid in tissues. Redfearn was able to find traces of what was probably the methyl ester in depot, fat after administration of t’he methyl ester of vitamin A acid (3). However, he and three others (1, 3-5) have failed to find vitamin A acid in rat tissues after the administration of very large doses of the free acid. This report will describe successful detection in human plasma, and outline some of the difficulties involved in the tissue det,ermination of vitamin -1 acid. EXPERIMENTAL ds described in detail below, all blood was collected in bottles containing SaF. It was found empirically that the presence of NaF in bloodcollect,ing bottles helped prevent appearance of compounds which interfere with the final spectrophotometric determination of vitamin A acid by the antimony trichloride renct,ion. The mechanism 1 This work was supported t)y :I research grant, 8-1528, from the National Institute of Arthritis and Metabolic Diseases of the National Inst,itutes of Health, U. S. Public Health Service. 337
by which NaF exerts this effect is unknown. The method involved preparation of an acetone extract of the plasma followed by saponification to form the potassium salt of vitamin A acid. Extraction with ethyl ether effected partial purification by removing vitamin A alcohol, carotenoids, and other lipids, while the vitamin A acid remained as the potassium salt in the lower layer. Following acidification, the vitamin A acid was extracted with ethyl ether. The ethyl ether was then evaporated and the extract dissolved in petroleum ether; it was next passed over a CaC03 column in order to partially adsorb compounds which interfere with calorimetric measurements. Of a variety of adsorbcnts tried, CaC03 was the best because it removed most of the interfering compounds without significantly affecting recoveries of vitamin A acid. Final determination of the vitamin A acid was conducted calorimetrically in chloroform by means of the antimony trichloride reaction. Direct spectra were obtained by further purification of the extract on acid-washed alumina. Vit,amin A acid was prepared for administration as follows. From 100 to 200 mg. of crystalline vitamin A acid (Distillation Products Industries), a powder was formed by dissolving the vitamin A acid in ethyl ether and evaporating the ether. The powder was then dissolved to the extent possible in a mixture of 15 ml. of Twcen 80 and 15 ml. of 0.1 211IGHPOI pH 9. More of the vitamin A acid was dissolved by adding four 15-ml. aliquots of buffer with stirring and n-arming to 37”C., the solution was well mised, and all was given. Four adult male subjects were used iu these esperiments. Each fasting ~ubjcct swallowed the mixture, and 3 hr.
338
JURKOWITZ
later 50-60 ml. blood was drawn into a heparinized syringe. The blood was placed into bottles containing NaF (0.5 ml. of 2% NaF in water for each IO-12 ml. blood and dried in a desiccator). The blood was centrifuged, and the separated plasma was centrifuged a second time to remove remaining cells. Six milliliters of plasma was set aside for carotene and vitamin A alcohol determinations according to the method of Moore (6). The remaining 20-25 ml. plasma was cooled in the refrigerator, mixed with 3 vol. of cold acetone, and allowed to stand in the refrigerator for 1 hr. The resultant mixture was centrifuged, and the supernatant acetone-water extract was saved ; the sediment was re-extracted with 30 ml. acetone for 10 min. on a mechanical shaker to obtain any residual vitamin -4 acid, and centrifuged. The two acetone extracts were combined and evaporated under gentle suction until foaming occurred (60-70 ml.). Then 45 ml. of 2 AV KOH dissolved in 95% ethanol was added, and saponification was carried out at 35-40°C. for 30 min. under a nitrogen stream. The volume at this point was 60-65 ml. Forty milliliters of water was added, and the water-ethanol mixture (most acetone evaporated during saponification under a nitrogen stream) was extracted twice wit,h 75 ml. ethyl ether. The combined ether extracts were washed five times with 15 ml. water, and the washings were added to the water-ethanol mixture. The ether ext,ract after washing had a volumr of 145155 ml. and was discarded. The water-ethanol fraction was cooled, and 50 ml. of cold 2 S HCl was added. This mixture was then extracted twice with 100 ml. ethyl ether, and the combined ether extracts were washed three times with 20 ml. water. The combined ether extract, (185 ml.) was saved, while the washings were added to the water-ethanol-HCl mixture. The water-ethanol-HCl washings were extracted a third timrl with 100 ml. ethyl ether before being discardrd. The third ether extract was washed three t,imcs with 10 ml. water and saved (final volume 95 ml.). The combined ether extracts wrre cooled to -2O”C., and excess water was removed in a scparatory funnel. They were then evaporated to dryness, the final stages being conducted under nitrogen. The dried rstract was inzmedintcly dissol\-ed in 6 ml. pet,roleum ether (Raker’s b,p. 3060”) and poured onto a 1 cm. X 0.5 cm. CaCO, (Baker’s) column which had been previously heat,ed 4 hr. at 200°C. Three-milliliter aliquots of petroleum ether were used to extract the contents of the flask and were poured ont,o the column until 24 ml. in all were used. The petroleum ether eluate was evaporated with nitropcn, and the residue was imnzdin/el~/ dissol\-cd in 2-3 ml. CHCh
All spectrophotometric measurements were made in a room maintained at 23°C. Determination of the vitamin A acid was carried out in a Beckman recording spectrophotometer using l-ml. samples of the chloroform extract, 3 drops acetic anhydride, and 2.7 ml. of 25% (w/v) SbCl, (Baker’s Analyzed Reagent) in CHCL All measurements were made against a chloroform blank. For each unknown the time of maximum color intensity (ahout 80 sec. with freshly prepared SbCL) was first determined by following the change in optical density at 572 rnp with time. Then a complete absorption curve was rapidly recorded so that the 572-rnw peak was reached at, the time of maximum color intensity. Duplicate determinations agreed within 5% because the time of maximum color intensity was known for each sample and because the optical density is relatively constant for 10 sec. on either side of the maximum. Calculations were made from absorption at, 572 rnp using a st.andard curve calibrated with crystalline vitamin A acid. These curves are linear in the O-30 pg. range with 25% (w/v) &Cl, Usually about 14 hr. of continuous work elapsed from the drawing of the blood to the vitamin A acid determination wit)h the &Cl, reaction. It was thought that best results would he obtained if the experiment were carried through to completion in the shortest possible time. This concept was supported by some experiments in which the final tissue rxt,racts in chloroform were stored 48 hr. at -20°C. and lost 15% of the vitamin A acid (this loss occurs more slowly when the crystalline compound is dissolved in chloroform). -After passage through C!aCOR , the extracts contain compounds which make it impossible to identify the direct ultra\-iolrt’ spectrum of vitamin A acid. Much of this interfering material can be removed by further purification on acid-washed alumina. To obtain tlirc,rt spectra, nonquantitativo c~xperiments were donr using plasma from onr of the subjects. The prorrdurr was the same as that described ahore, but nftrr passage through the CnCO., column the pet.roleum ether was poured onto :an unheated alumina (Mrrck acid-washed) column. The ah~minn column was 3.5 X 0.5 cm., lm~parctl from a petroleum ether slurry, and deactivated with 2 drops water. After the 24 ml. prtroteum ether had run through, 10 ml. of 5% acetone in petroleum ether was run through, fotlowed by 15 ml. of 20% acetone in petroleum ether. The 20% aretonc fraction was saved, evaporated to dryness with nit,rogen, taken up i?nmct%iatell/ in petroleum ether. and the rhromatography repeated. The chromatography was done a third time before the extract in 20% acetone was t,ransfcrretl to CHCL , and the direct spectrum was measured in tlrcx Bcrkmnn DK-1 spect,rophotometer.
VITAMIK
A BCID
IN
HUMSN
339
PLASMA
RESULTS
The spectrum of the antimony trichloride reaction of crystalline vitamin A acid (Fig. 1) compared with that of tissue extracts (Fig. 2) indicates that with the described method substantial levels of vitamin A acid appear in human plasma after oral administration. Evidence that this represents vitamin A acid is provided by the character of the curve obtained. There are two main peaks, one at 572 mp which decreases in intensity with time, and another at 470 rnp which increases in intensity with time. Both the location of t’he maxima and the character of the decay curve are t,he same as those obtained with crystalline vitamin A acid. Neither the 572-rnp peak nor the 470-rnp peak appeared when Tween 80 buffer solution is administered without vitamin A acid. There are, however, differences between the antimony t,richloride spectra of crystalline vitamin A acid and those obtained with tissue extracts containing vitamin A acid. The crystalline compound shows a minimum at 495-500 mp and a lower base line below 525 mp. In addition, the crystalline compound has a low broad peak with a maximum at approximately 378-380 mp which decreases in intensity with time. This peak I I I I I I
1
I
I
I
I
I
350
400
450
500
550
600
WAVELENGTH
650
Imp)
FIG. 1. Absorption spectra of antimony trichloride reaction of crystalline vitamin B acid. Spectrum at maximum color intensity (62 sec.). -- - Spectrum when color intensity is decreasing (100 sec. after maximum). To 25.9 pg. of crpstalline vita,min A acid in 1 ml. chloroform were added 3 drops acetic anhydridc and 2.7 ml. of 25% (w/v) antimony trichloride.
I 350
I 400
I 450
I 500
I 550
1 600
t 650
WAVELENGTH
FIG. 2. Absorption
spectra of antimony trichloride reaction of vitamin A acid isolated from human plasma. ~ Spectrum at maximum color intensity (70 sec.). ---Spectrum when color intensity is decreasing (100 sec. after maximum). The spectra shown above were run on 1.0 ml. of a 2.9ml. chloroform extract, prepared as described in the text from 22 ml. plasma taken from an adult man 3 hr. after administration of 150 mg. vitamin A acid. The quantitp of vitamin A acid in this 1.0 ml. sample was calculated to be 21.1 fig.
is more prominent at earlier times than are shown here. These diffcrcncrs probably represent a failure to complet,ely remove unknown compounds which absorb at short’cr warelengt,hs or which react with ant’imony trichloride to give absorption at shorter wavelengths. The direct spect,rum (,Fig. 3) proTides further confirmation of the assumption that vitamin A acid is present in the extracts. A Iteak appears at 362 mp when vitamin A acid is administered which is not present when the Tween 80 buffer solut,ion is administered alone. In another experiment the peak was at 363 mp. The maximum of crystalline vit,amin A acid in chloroform i:: 366 inp, so there is a 3-4-mp difference bctwcen the extracts and the crystalline compound. This probably represents a failure to completely remove compounds which absorb at shorter warelengt.hs, as can be seen from the asymmetrical character of the direct spectrum of t’hc extracts. Table I provides a comparison of tlrc levels of vitamin A acid wit,h simultaneously determined vitamin ,4 alcohol levels. It can be seen that when vitamin A arid is
340
JCRKORITiS
FIG. 3. Direct absorption spectra of extracts of human plasma read in chloroform. -o-•-•-Spectrum after administration of vitamin A acid. -O-O-O-Spectrum after administration of Twecn 80 buffer solution alontl. -1 method for obtaining nonquantitative direct spcvtr’a. is described in the text. TABLE
I
VITAMIN A ACID .&ND VITAMIN A AI~~OHOI~ LEVEI.B Is
HUMAN
PLASMA TRATION
Dose of vitamin m.
100 150 150 150 200 200 * Corrected
A acid
3 HR. OF
AFTER
T’ITAMIN
Vitamin pg.‘flOO
ORAL A
Vitamin A acid _.___ ml. p1as712a ~y.:lov
112 171 200 278 247 329
ADMISIS-
ACID A alcohol” ml. plasma
03 45 43 50 33 35
for cnrotenoids.
given orally, the lcwls of vitamin A acid can far exceed those of vitamin A alcohol. The levels recorded probably represent only about 74% of the actual vitamin A acid present. This conclusion is reached because six experiments in which 100 pg. vitamin A acid in acetone were added to 25 ml. plasma gave recoveries of 69-78s with an average recov-cry of 74%.
The dosage of vitamin A acid given in tlwsc experiments represented about 1.5-3.0 mg./kg. of body weight. Since Sharman (5) gave 10 mg. to a single rat, and Redfearn (3) gave 1 mg. daily for as long as 24 days
to single rats, it seems unlikely that the failure of other investigators to find vitamin A acid in tissues relates entirely t,o the dosage used. Other possible explanations include species difference, the methods of administration of the vitamin A acid, and the amounts of tissue used for analysis. However, it seems likely that the success of the experiments pres,ented here relates at least in part to the method of preparation of tissue extracts. Rcdfearn (3), for example, extracted tissues with ether as one would do for vitamin A alcohol. When the standard ethanol-petroleum ether prow dure (6) for vitamin A alcohol is applied to plasma known to contain large amounts of vitamin A arid, the 572-mp peak cannot be detected with anv certainty, although there i:: a suggestion df the 470-mp peak in the decay curves. To obtain curves similar to thaw in Fig. 2, it, was necessary to scparatc vitamin A acid from compounds which interfcred in the antimony trichloride rcaction. For good quantitative recoveries, in addition to t#he lwocedure described and the usual precautions in handling vitamin A alcohol, several other factors should be mcntioncd. The antimony trichloridc reaction of vitamin ,1 arid behaves differently from that of vitamin A alcohol in an important respect. Whereas one can store 25% (w,!vj antimony trirhloridc in chloroform indefinitely and obtain the same absorption for a giwn amount of vitamin A alcohol, storage of the antimony trichloride for 2 weeks increased the amount of color produced with vitamin A acid by 10-15s without changing the wavclcngth of the maximum extinction. In addition, aging of the antimony trichloride for 2 weeks causes the peak of the color reaction with vitamin A acid to appear at 60 sec. instead of 80 sec. Longer periods of aging of the antimony trichloride can product greatw effects. These effects wrc looked for, but not found with vitamin A alcohol. Sor could they be explained by the presence or absence of acetic anhydride in the reaction with 25:L (w/v) antimony trichloridc in chloroform. In quantitative experiments on vitamin A acid, errors which could be introduced 1)~ these clffects arc avoided by performing cali-
VITBhiIPi
9 ACID
bration curves at the time of an experiment and const,antly rechecking t,hem. Another characteristic of vitamin h acid n-hich makes its detection more difficult t,han that of vit,amin -4 alcohol is the lower intensity of the color produced by t,he reaction wit,h 25% (w/v) antimony trichloride. One needs approximately five times as much vitamin A acid to obtain the same color intensity at 572 rnp that one needs for a given amount of vitamin L4 alcohol at 618 mp using freshly prepared 257; iw-/v) antimony trichloride. From considerations based on t,he intensity of the color reaction Cth vitamin A acid, t~lic shape of the ant,imany t,richloride spectrum wit’11 tissue extracts at low levels of vitamin A acid, and t,he percentage recoveries, it, is possible to make a rough t>st.imatc that, the plasma levels of vitamin h acid below 50 pg./100 ml. plasma would not be dctcctablc by this method. When one considers that about, 85% of normal humans have vitamin A alcohol levels between 24 and 84 /Lg./‘100 1111. (80280 I.U./lOO ml.) according to Moore (71, it is readily apparent that large and phrsiologically important amounts of vi&m& A acid could be easily overlooked even in a relatively simple t’issw like plasma. This has implications with respect to current concepts of the role of vitamin A acid. Other workers (1) have emphasized that. the failure to find Ctamin A acid may be a consequence of rapid conversion to an active met,abolite or a consequence of low tieSW levels. The technical considerations elaborated abow make it, clear that the presence of even relatively large quantit,ies
IS
HUMAN
PLASMA
3-I 1
of vitamin h acid may not have been tlrtccted. It is therefore apparent that more detailed invest,igation of a variety of tissues from many species with more sensitive methods will be necessary before it can be concluded that vitamin A acid is not, present in physiologically significant amount,s. It, is also clear that vit,amin A acid is not SOrapidly metabolized in man, because appreciahle amounts are found in human plasma 3 hr. after administration of a large dose.
The, author
wishes to thank
Dr. R. 1,. J,nntl:tu
ior llip advice and c~ncouragcmcnf,tlwing the course of this n-o&, ant1 for his counsel in the n-riting of this manuscript. Tllc? author is indebted to Dr. George W&l for st,imulating an early interest in vitamin 11, and sul~secprntly in vitamin A acid. He would also like to thank lhr Organic Rescarvh Laboratory of Distillation Products Industries, Rochester, X. Y.. for generous gifts of vitamin A acid.
J. E., AXD A$-ALD,G., I-'mc. Sat/. Aced. Sci. L-.,%46,587 (1960). .iRESS, J. G., AKD \-as Dow, D. A,, .v&wt’ 157, 190 ( 1946). REDFIMRS. E. K., A~lr. Bioclwln. Bio/,A!/s. 91, 226 (1960). HARRIS, I’. I,., in “Syml~osium on Nutrition” (R.. RI. Herriott, ed.), p. 71. John Hopkins Prc,.q Baltimorc~, 1953. ~H.4RMAN, I. bl., Bait. J. hTllfrifioil 3, viii (1949). &~OORF 3, T. * “T’itamin I4 >” 1). 586. Elscvier Pnhl. Co., Amsterdam, 1957. &lOORE, T., “Vitamin Ai’ p. 361. Elscvicr Pul,l. Co., Amslrrdam, 1957.
1. I~WLIX,
2. 3. 4.
5. 6.
7.