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Separation of vitamin A from carotenoids in micro samples of serum
ANALYTICAL
BIOCHEMISTRY
(1965)
10
SHORT COMMUNICATIONS
Separation
of in Micro
Vitamin Samples
A
from of
Carotenoids Serum
Severe vitamin A deficiency (xerophthalmia) is an important cause of blindness in young children in many parts of the Near and Far East. Repeated determinations of serum vitamin A levels on these sick children necessitate micro samples. The method of Bessey et al. (l), in which the optical density of vitamin A is measured at 328 mp, is subject to many errors (2). A more recent method (3)) suitable for micro samples, depends on the blue color formation with trifluoroacetic acid resulting from both carotenoids and vitamin A, with a correction applied for the amount of carotenoids present. As this correction is derived from a pure /?-carotene standard, the accuracy of the value obtained for vitamin A depends on the carotenoids being p-carotene, since other carotenoids give varying intensities of color with the chromogen. It is known that blood carotenoids are a reflection of dietary carotenoids (4). Foodstuffs vary widely in their carotenoid composition. We have encountered cases showing only minimal clinical evidence of vitamin A deficiency with extremely low serum levels of the vitamin in the presence of high levels of carotenoids. With large quantities of material it is possible to separate vitamin A from the carotenoids by column chromatography, but-on a micro scale-separation by paper or thin-layer chromatography has resulted in low recoveries. A simple and rapid method of separation is described here and its usefulness illustrated in the study of human vitamin A deficiency being undertaken in this laboratory, the results of which will be presented elsewhere. Method. Capillary glass tubes, 100 x 2 mm, of the type used for melting point determinations, were partly closed at one end by a sintered glass plug formed by filling each capillary tube to a depth of 2 mm with glass powder and heating it in a cool flame for a few seconds. Excess glass powder was tapped out. About 100 such capillaries were tightly packed into a glass tube, one end of which was closed with a cork. The space above the capillaries was filled with the absorbent and the tube vibrated in a vertical position by holding it against a rapidly rotating drill. Complete filling with uniform packing was achieved in about 10 min. The 156
SHORT
157
COAIMVKICATIOS8
adsorbent found most useful for separating the individual carotenoids was alumina (200 mesh). Silicic acid (325 mesh) gave better separation of vitamin A from the carotenoids. Serum (0.1 ml) and 0.1 ml of 5% alcoholic potassium hydroxide were pipetted into a 50 X 5 mm tube, and thoroughly mixed. The tube was then placed in a water bath at 60°C for 20 min. After cooling, 0.15 ml of petroleum ether (boiling range 40-60°C) was added. The contents were thoroughly mixed by holding the tube against a rapidly rotating drill for 1 min, after which the tube was centrifuged; 0.1 ml of the petroleum layer was transferred to another tube and t,he volume reduced under a stream of nitrogen at room temperature to about 0.01 ml. When the sintered glass end of the capillary column was placed into the solution the sample was absorbed on the capillary column. The walls were washed with 0.01 ml of petroleum ether and the washing similarly absorbed into the column. The column was developed by standing it upright in a small tube containing petroleum ether. The solvent front reached the top of the adsorbent in about 10 min. When using silicic acid (325 mesh), vitamin A was concentrated in a narrow band near the bottom of the column (Rf 0.05) while @-carotene traveled to the top of the column (Rf 0.8). Other unidentified yellow pigments were frequently found between these two bands. The section of the capillary column containing the vitamin A was cut off and extracted with chloroform, and vitamin A determined by reading the intensity of the blue color produced with trifluoroacetic acid reagent (3). Recoveries from capillary columns of known quantities of vitamin A under such conditions in the presence of varying amount,s of p-carotene were better than 90% (see Table 1). This procedure was carried out on 12 samples of serum, 6 containing high levels of carotenoids and low levels of vitamin A, and the other 6 ‘L‘M3LE
RECOVERY
1
OF VITAMIN
A FROM CAPILLARY COLUMNS OF VARYING AMOUNTS OF ~-CAROTENE
Vitamin A content of sample, wz
~-Carotene content of sample, Irg
0.032 0.055
0 0
0.110
0
0.023 0.057 0.065 0.128
0.045 0.075 0.075 0.150
IN
Vitamin .4 recovered, Ia
0.030 0.054 0.111 0.023 C.056 0.063 0.124
THE PRESENCE
70 Recovery
94 98 101 100
98 97 97
158
SHORT
COMMUNICATIONS
containing low levels of both carotenoids and vitamin A as measured by the method of Neeld and Pearson (3). The values for vitamin A found by these two methods are compared in Table 2.
COMPARISON
TABLE A ESTIMATIONS
OF VITAMIN
ON CAPILLARY Method SaiE?e
1 2 3 4 5 6 7 8 9 10 11 12
CS~O$ll&,
of Neeld
2 BEFORE COLUMNS
and Pearson
AND AFTER
SEPARATION
Method
described
Vitamin A. Pb %
Vitamin A, .w %
0 0 1 3 3 2 0 11 19 0 3 7
19 6 13 4 14 8 1 8 16 5 5 7
70 94 70 107 81 143 14 17 14 24 11 26
These results show that the carotenoids present in serum can cause falsely low values to be calculated for vitamin A. This effect is most apparent and the error is more serious when the vitamin A content is low. The method is also being applied to the separation of micro quantities of other lipids present in serum. ACKNOWLEDGMENT The financial support of U. S. Public Health Service Grant AM-05285 acknowledged. REFERENCES
is gratefully
1. BESSEY, 0. A., LOWRY, 0. H., BROCK, M. J., AND LOPEZ, J. A., J. Biol. Chem. 166, 177 (1946). 2. UTELY, M. H., BRODWSKY, E. R., AND PEARSON, W. N., J. Nutr. 66, 295 (1958). 3. NEELD, J. B., AND PEARSON, W. N., J. Nutr. 79,454 (1963). of the Carotenoids,” p. 229. 4. GOODWIN, T. W., “The Comparative Biochemistry Chapman and Hall, London, 1952. 2. L. AWDEH Nutrition Research Laboratory School of Medicine American University of Beirut Beirut, Lebanon Received June 1, 196.-f+
BIOCHEMISTRY
(1965)
10
SHORT COMMUNICATIONS
Separation
of in Micro
Vitamin Samples
A
from of
Carotenoids Serum
Severe vitamin A deficiency (xerophthalmia) is an important cause of blindness in young children in many parts of the Near and Far East. Repeated determinations of serum vitamin A levels on these sick children necessitate micro samples. The method of Bessey et al. (l), in which the optical density of vitamin A is measured at 328 mp, is subject to many errors (2). A more recent method (3)) suitable for micro samples, depends on the blue color formation with trifluoroacetic acid resulting from both carotenoids and vitamin A, with a correction applied for the amount of carotenoids present. As this correction is derived from a pure /?-carotene standard, the accuracy of the value obtained for vitamin A depends on the carotenoids being p-carotene, since other carotenoids give varying intensities of color with the chromogen. It is known that blood carotenoids are a reflection of dietary carotenoids (4). Foodstuffs vary widely in their carotenoid composition. We have encountered cases showing only minimal clinical evidence of vitamin A deficiency with extremely low serum levels of the vitamin in the presence of high levels of carotenoids. With large quantities of material it is possible to separate vitamin A from the carotenoids by column chromatography, but-on a micro scale-separation by paper or thin-layer chromatography has resulted in low recoveries. A simple and rapid method of separation is described here and its usefulness illustrated in the study of human vitamin A deficiency being undertaken in this laboratory, the results of which will be presented elsewhere. Method. Capillary glass tubes, 100 x 2 mm, of the type used for melting point determinations, were partly closed at one end by a sintered glass plug formed by filling each capillary tube to a depth of 2 mm with glass powder and heating it in a cool flame for a few seconds. Excess glass powder was tapped out. About 100 such capillaries were tightly packed into a glass tube, one end of which was closed with a cork. The space above the capillaries was filled with the absorbent and the tube vibrated in a vertical position by holding it against a rapidly rotating drill. Complete filling with uniform packing was achieved in about 10 min. The 156
SHORT
157
COAIMVKICATIOS8
adsorbent found most useful for separating the individual carotenoids was alumina (200 mesh). Silicic acid (325 mesh) gave better separation of vitamin A from the carotenoids. Serum (0.1 ml) and 0.1 ml of 5% alcoholic potassium hydroxide were pipetted into a 50 X 5 mm tube, and thoroughly mixed. The tube was then placed in a water bath at 60°C for 20 min. After cooling, 0.15 ml of petroleum ether (boiling range 40-60°C) was added. The contents were thoroughly mixed by holding the tube against a rapidly rotating drill for 1 min, after which the tube was centrifuged; 0.1 ml of the petroleum layer was transferred to another tube and t,he volume reduced under a stream of nitrogen at room temperature to about 0.01 ml. When the sintered glass end of the capillary column was placed into the solution the sample was absorbed on the capillary column. The walls were washed with 0.01 ml of petroleum ether and the washing similarly absorbed into the column. The column was developed by standing it upright in a small tube containing petroleum ether. The solvent front reached the top of the adsorbent in about 10 min. When using silicic acid (325 mesh), vitamin A was concentrated in a narrow band near the bottom of the column (Rf 0.05) while @-carotene traveled to the top of the column (Rf 0.8). Other unidentified yellow pigments were frequently found between these two bands. The section of the capillary column containing the vitamin A was cut off and extracted with chloroform, and vitamin A determined by reading the intensity of the blue color produced with trifluoroacetic acid reagent (3). Recoveries from capillary columns of known quantities of vitamin A under such conditions in the presence of varying amount,s of p-carotene were better than 90% (see Table 1). This procedure was carried out on 12 samples of serum, 6 containing high levels of carotenoids and low levels of vitamin A, and the other 6 ‘L‘M3LE
RECOVERY
1
OF VITAMIN
A FROM CAPILLARY COLUMNS OF VARYING AMOUNTS OF ~-CAROTENE
Vitamin A content of sample, wz
~-Carotene content of sample, Irg
0.032 0.055
0 0
0.110
0
0.023 0.057 0.065 0.128
0.045 0.075 0.075 0.150
IN
Vitamin .4 recovered, Ia
0.030 0.054 0.111 0.023 C.056 0.063 0.124
THE PRESENCE
70 Recovery
94 98 101 100
98 97 97
158
SHORT
COMMUNICATIONS
containing low levels of both carotenoids and vitamin A as measured by the method of Neeld and Pearson (3). The values for vitamin A found by these two methods are compared in Table 2.
COMPARISON
TABLE A ESTIMATIONS
OF VITAMIN
ON CAPILLARY Method SaiE?e
1 2 3 4 5 6 7 8 9 10 11 12
CS~O$ll&,
of Neeld
2 BEFORE COLUMNS
and Pearson
AND AFTER
SEPARATION
Method
described
Vitamin A. Pb %
Vitamin A, .w %
0 0 1 3 3 2 0 11 19 0 3 7
19 6 13 4 14 8 1 8 16 5 5 7
70 94 70 107 81 143 14 17 14 24 11 26
These results show that the carotenoids present in serum can cause falsely low values to be calculated for vitamin A. This effect is most apparent and the error is more serious when the vitamin A content is low. The method is also being applied to the separation of micro quantities of other lipids present in serum. ACKNOWLEDGMENT The financial support of U. S. Public Health Service Grant AM-05285 acknowledged. REFERENCES
is gratefully
1. BESSEY, 0. A., LOWRY, 0. H., BROCK, M. J., AND LOPEZ, J. A., J. Biol. Chem. 166, 177 (1946). 2. UTELY, M. H., BRODWSKY, E. R., AND PEARSON, W. N., J. Nutr. 66, 295 (1958). 3. NEELD, J. B., AND PEARSON, W. N., J. Nutr. 79,454 (1963). of the Carotenoids,” p. 229. 4. GOODWIN, T. W., “The Comparative Biochemistry Chapman and Hall, London, 1952. 2. L. AWDEH Nutrition Research Laboratory School of Medicine American University of Beirut Beirut, Lebanon Received June 1, 196.-f+