ANALYTICAL
BIOCHEMISTRY
Determination
77,
310-314
(1977)
of Linamarin
in Biological
Tissues
A new paper chromatography method for determination of intact cyanogenic glucoside linamarin is based on a reaction with p-anisaldehyde at 85°C; the reaction produces a pink color which is brightly fluorescent under long-wave uv light.
Linamarin, 2-hydroxyisobutyronitrile-p-D-glycoside, is a cyanogen widely occurring in many staple plant foods and feeds, such as cassava, flax, lima bean, peas, and white clover. It is often accompanied by its methyl derivative, lotaustralin, 2-hydroxy-2-methylbutyronitrile-P-Dglucoside ( 1). Extensive reviews have been written on the chemistry of linamarin (2), its biochemistry (3), analytical techniques (4), and toxicological significance (5); toxicological or nutritional observations clearly related to linamarin intake itself are meagre. Cyanide, released from linamarin through the hydrolytic action of linamarase is generally accepted as the toxic agent responsible for numerous cases of poisoning of humans as well as animals (6). It is also the analytical reactant for nearly all quantitative methods for linamarin developed to date (4), the most reliable of which employ either endogenous or exogenous linamarase for the liberation of cyanide. These methods and also that of Butler (1) based on measurement of the glucose component of linamarin are tedious, time consuming, and poorly suited for routine assays. This research note describes a simple quantitative procedure for the direct determination of the intact glycoside which was developed concurrently with studies on the fate of linamarin in feeding experiments with rats (7). The method obviates the need for acquiring a linamarase preparation and can be performed with the simplest laboratory utensils, provided that a few milligrams of pure linamarin are on hand for calibration. It is best suited for biological materials or in vitro systems known to contain linamarin as the principal cyanogen. Synthetic /3-linamarin is now available from Calbiochem (LaJolla, Calif.) and was used in evaluation of the new technique and for comparison with the natural p-linamarin from flax which was obtained pure and in mixture with lotaustralin through courtesy of G. W. Butler.’ The availability of synthetic p-linamarin made it possible to carry out extensive studies on extraction, isolation by ascending paper chromatography, and subsequent quantitation based on a rather specific reaction ’ Department of Scientific and Industrial Research, Palmerston North, New Zealand. 310 Copyright All rights
0 1977 by Academic of reproduction in any
Press, Inc. form reserved.
SHORT
COMMUNICATIONS
311
of p-linamarin with p-anisaldehyde. The reaction has a threshold of 0.3-0.5 pg of intact linamarin, and this corresponds to 0.03-0.05 pg of cyanide ion released by hydrolysis. Anisaldehyde is a common spottest reagent for simple carbohydrates and a host of other organic substances (8), however, linamarin was the only one which yielded both a pink to cherry red visible color coupled with a brilliant pink fluorescence viewed with a long-wave (A = 366 nm) uv light. Detection reagent. To 90 ml of ethanol are added, slowly in sequence, 2 ml of p-anisaldehyde, 6 ml of 85% o-phosphoric acid, and, dropwise, 3 ml of concentrated sulphuric acid. The clear reagent keeps well under refrigeration for at least 2 to 3 weeks, and even a 3-month-old reagent with faint yellow coloration was observed to produce a good reaction. Color reaction. Chromatography paper strips, developed as described below, are dried at 85°C for 20-25 min to remove traces of solvents and drawn through 5- 10 ml of reagent placed in a small porcelain dish. The strips are then lightly blotted and placed on a 20 x 40-cm glass plate in an 85°C oven. In 2 to 4 min, bright pink to reddish-purple spots due to linamarin appear on a grayish-pink background. Papers developed in an acetone or ethyl acetate solvent tend to produce a grayish-blue tone on heating. Overheating results in a grayish-blue background which partly masks the visible pink and obscures viewing in the uv light. Exposing the papers to water vapor, particularly if less than 2-3 pg of linamarin is present, will enhance the development of fluorescence and lighten the background. Steaming does weaken the visual pink and under certain conditions yields a light blue color with low concentrations of linamarin. Sample preparation and extraction. Plant or animal tissues are homogenized with the addition of aqueous ethanol or methanol to 80% volume concentration. Normally, 2- to 5-g samples are sufficient, depending on the expected linamarin concentration. In fresh plant tissues, endogenous linamarase must be inactivated by boiling or enzyme precipitation. This is unnecessary with animal tissues which are unlikely to contain the enzyme. Tests with whole blood, plasma, urine, and feces showed a minimum 90% recovery of added linamarin. These substrates did not inhibit hydrolysis of linamarin by added linamarase (5 mg in 3 ml of phosphate buffer, pH 5.5), in the presence of up to 1 ml of blood plasma, 3 ml of urine, or 650 mg of feces obtained from rats placed on a linamarin-containing diet. These tests have been made to ascertain the absence of linamarase inhibitors or, vice versa, the presence of linamarase-like activity in these tissues (7). In all recovery tests with animal tissues, the isolated linamarin spots were checked for cyanide evolution by spraying with linamarase (4 mg/ml) and reacting the freed cyanide with an adjacent picrate paper (37°C). Paper chromatography. Linamarin can be isolated on paper chromatograms by the use of several solvents reported earlier (2). Ascending
312
SHORT COMMUNICATIONS TABLE
1
COLORREACTIONSWITHP-ANISALDEHYDE
AT
85°C
Color reaction Compound (10 pg) KCN KSCN Ferricyanide Linamarin Amygdalin Mandelic acid Sinigrin Arbutin PhloridzinD Phloretin” Salicin Inositol Solanine Chlorogenic acid Glucose Fructose Galactose Sucrose Xylose
Visible NIL white dark blue pink pink pink pink purple orange blue-purple pink light purple gray-purple purple-pink red-purple deep purple red-purple deep purple light purple
uv Light
Intensity
-
-
white dark blue bright pink weak pink purple bright pink purple-blue purple purple reddish-purple blue purple deep purple -
intense intense very intense weak very weak strong intense very intense strong strong weak weak very strong very weak intense very weak strong very weak
-
a Very fast reaction, 5- 10 sec.
chromatography on 12 x 360-mm strips of Whatman No. 1 paper was found most expedient for routine assays. Paper strips are suspended in 50 x 400-mm test tubes with a paper clip on a wire hook inserted in bottom of a rubber stopper and developed after 1-hr equilibration with 8 ml of a 8: 1:2, 1-butanol:ethanol:water system, 5 hr, to a height of 15- 17 cm (R,:0.52-0.54 for linamarin, 0.58-0.61 for lotaustralin). A fast-moving alternate solvent is a 4:5: 1 system of ethyl acetate:acetone:water which rises 20 cm in 3 hr (R,:O.71-0.73 for linamarin, 0.82-0.84 for lotaustralin). The latter solvent is preferable for mixtures of linamarin and lotaustralin. Visual estimation. Linamarin concentration can be estimated visually by comparison of fluorescence in unknowns with a set of calibration standards. A set of standards (developed chromatogram spots cut out and fastened on a small cardboard or in a filing folder) will remain in usable condition for 4 or more weeks. Unknown solutions are brought down to threshold detection range of 0.5 to 10 pg in l/10, l/20, l/40 or other convenient dilutions (three to four dilutions are usually sufficient). Once a set value is established within the calibration range, the linamarin content of an extract can be easily calculated with a -+5% error; with experience, an even smaller error can be attained.
SHORT COMMUNICATIONS
313
For calibration, a stock solution containing 20 mg of p-linamarin (2 CLgIpl) in 10 ml 80% ethanol is used for a series of dilutions, 0.4, 0.8, 1.2, and 1.6 ml, made up to 2 ml volume in 15 x 4%mm vials; 5 ~1 of each of these dilutions is run through chromatographic development and detection treatment to provide 2- to IO-pg standards for comparison with the unknowns. A similar lower dilution series using 0.4 ml previously prepared standards diluted to 2.0 ml will give an additional range covering a 0.4- to 2.0-pg range. The visible pink color can be detected from 1 pg, whereas fluorescence at A = 366 nm lowers the detection threshold to 0.3 to 0.5 pg. The visible pink color fades in time, but the fluorescence of spots containing in excess of 5 pg of linamarin will persist for 4-6 months. Interferences. Some 25 substances, a few of which are listed in Table 1, were tested for reaction with p-anisaldehyde. Of the various carbohydrates, glycosides, and phenolics, only sinigrin showed a strong color reaction similar to that of linamarin. Amygdalin, another cyanogen, has shown also a similar reaction with about one-tenth of linamarin intensity. Most of the tested carbohydrates showed Rf values below 0.35 in the two employed solvents and therefore would not interfere with linamarin isolation. Pure methyl-linamarin (lotaustralin) was not available in sufficient quantity for the test, however, in a mixture with linamarin (G. W. Butler (Ref. l)), methyl-linamarin separated on chromatograms showed only a very weak reaction (approximately 5 pg in a sample), a quickly fading orange color without any fluorescence. Extracts from flax seedlings and from rat feces have shown a number of weak purple spots at R, values below 0.35, possibly sucrose at Rf .31, but none corresponding to glucose (Rf:O. 18) in 8: 1:2 1-butanol:ethanol:water solvent. The proposed method does not require elaborate sample purification, is suitable for rapid routine tests and is applicable to systems where any interfering substances or other cyanogens can be chromatographically eliminated. The procedure was successful in detecting and measuring intact linamarin in urine of rats dosed with the cyanogen (7). ACKNOWLEDGMENT The research was carried out with the aid of a grant from the International Research Centre, Ottawa, Canada.
Development
REFERENCES 1. Butler, G. W. (1%5)Phytochemistry 4, 127-131. 2. Seigler, D. S. (1975) Phytochemistry 14, 9-29. 3. Conn, E. E. (1973) Biochem. Sot. Symp. 38, 277-302. 4. Zitnak, A. (1973) in Chronic Cassava Toxicity (Nestel, B., and MacIntyre, R., eds.), pp. 89-%, Canadian International Development Research Centre, Ottawa, Canada. 5. Nestel, B., and MacIntyre, R., (eds.), (1973) Chronic Cassava Toxicity, Canadian International Development Research Centre, Ottawa, Canada.
314
SHORT COMMUNICATIONS
6. Montgomery, R. P. (1969) in Toxic Constituents of Plant Food Stuffs, (Liener, E. I., ed.), pp. 143-157, Academic Press, New York. 7. Barrett, M. D. P. (1976) Dietary Cyanide, Linamarin and Nutritional Deficiencies. Ph.D. thesis, University of Guelph, Canada. 8. Feigl, F. (1%6) Spot Tests in Organic Analysis, 7th ed., Elsevier, Amsterdam/New York.
A. ZITNAK~ D. C. HILL J. C. ALEXANDER~ Departments University Received
of Horticultural Science and Nutrition of Guelph, Canada May 24, 1976; accepted August 2, 1976
* Address reprint requests to A. Zitnak, Department of Horticultural of Guelph, Guelph, Canada, NlG 2Wl. 3 Department of Nutrition, University of Guelph.
Science, University
BIOCHEMISTRY
Determination
77,
310-314
(1977)
of Linamarin
in Biological
Tissues
A new paper chromatography method for determination of intact cyanogenic glucoside linamarin is based on a reaction with p-anisaldehyde at 85°C; the reaction produces a pink color which is brightly fluorescent under long-wave uv light.
Linamarin, 2-hydroxyisobutyronitrile-p-D-glycoside, is a cyanogen widely occurring in many staple plant foods and feeds, such as cassava, flax, lima bean, peas, and white clover. It is often accompanied by its methyl derivative, lotaustralin, 2-hydroxy-2-methylbutyronitrile-P-Dglucoside ( 1). Extensive reviews have been written on the chemistry of linamarin (2), its biochemistry (3), analytical techniques (4), and toxicological significance (5); toxicological or nutritional observations clearly related to linamarin intake itself are meagre. Cyanide, released from linamarin through the hydrolytic action of linamarase is generally accepted as the toxic agent responsible for numerous cases of poisoning of humans as well as animals (6). It is also the analytical reactant for nearly all quantitative methods for linamarin developed to date (4), the most reliable of which employ either endogenous or exogenous linamarase for the liberation of cyanide. These methods and also that of Butler (1) based on measurement of the glucose component of linamarin are tedious, time consuming, and poorly suited for routine assays. This research note describes a simple quantitative procedure for the direct determination of the intact glycoside which was developed concurrently with studies on the fate of linamarin in feeding experiments with rats (7). The method obviates the need for acquiring a linamarase preparation and can be performed with the simplest laboratory utensils, provided that a few milligrams of pure linamarin are on hand for calibration. It is best suited for biological materials or in vitro systems known to contain linamarin as the principal cyanogen. Synthetic /3-linamarin is now available from Calbiochem (LaJolla, Calif.) and was used in evaluation of the new technique and for comparison with the natural p-linamarin from flax which was obtained pure and in mixture with lotaustralin through courtesy of G. W. Butler.’ The availability of synthetic p-linamarin made it possible to carry out extensive studies on extraction, isolation by ascending paper chromatography, and subsequent quantitation based on a rather specific reaction ’ Department of Scientific and Industrial Research, Palmerston North, New Zealand. 310 Copyright All rights
0 1977 by Academic of reproduction in any
Press, Inc. form reserved.
SHORT
COMMUNICATIONS
311
of p-linamarin with p-anisaldehyde. The reaction has a threshold of 0.3-0.5 pg of intact linamarin, and this corresponds to 0.03-0.05 pg of cyanide ion released by hydrolysis. Anisaldehyde is a common spottest reagent for simple carbohydrates and a host of other organic substances (8), however, linamarin was the only one which yielded both a pink to cherry red visible color coupled with a brilliant pink fluorescence viewed with a long-wave (A = 366 nm) uv light. Detection reagent. To 90 ml of ethanol are added, slowly in sequence, 2 ml of p-anisaldehyde, 6 ml of 85% o-phosphoric acid, and, dropwise, 3 ml of concentrated sulphuric acid. The clear reagent keeps well under refrigeration for at least 2 to 3 weeks, and even a 3-month-old reagent with faint yellow coloration was observed to produce a good reaction. Color reaction. Chromatography paper strips, developed as described below, are dried at 85°C for 20-25 min to remove traces of solvents and drawn through 5- 10 ml of reagent placed in a small porcelain dish. The strips are then lightly blotted and placed on a 20 x 40-cm glass plate in an 85°C oven. In 2 to 4 min, bright pink to reddish-purple spots due to linamarin appear on a grayish-pink background. Papers developed in an acetone or ethyl acetate solvent tend to produce a grayish-blue tone on heating. Overheating results in a grayish-blue background which partly masks the visible pink and obscures viewing in the uv light. Exposing the papers to water vapor, particularly if less than 2-3 pg of linamarin is present, will enhance the development of fluorescence and lighten the background. Steaming does weaken the visual pink and under certain conditions yields a light blue color with low concentrations of linamarin. Sample preparation and extraction. Plant or animal tissues are homogenized with the addition of aqueous ethanol or methanol to 80% volume concentration. Normally, 2- to 5-g samples are sufficient, depending on the expected linamarin concentration. In fresh plant tissues, endogenous linamarase must be inactivated by boiling or enzyme precipitation. This is unnecessary with animal tissues which are unlikely to contain the enzyme. Tests with whole blood, plasma, urine, and feces showed a minimum 90% recovery of added linamarin. These substrates did not inhibit hydrolysis of linamarin by added linamarase (5 mg in 3 ml of phosphate buffer, pH 5.5), in the presence of up to 1 ml of blood plasma, 3 ml of urine, or 650 mg of feces obtained from rats placed on a linamarin-containing diet. These tests have been made to ascertain the absence of linamarase inhibitors or, vice versa, the presence of linamarase-like activity in these tissues (7). In all recovery tests with animal tissues, the isolated linamarin spots were checked for cyanide evolution by spraying with linamarase (4 mg/ml) and reacting the freed cyanide with an adjacent picrate paper (37°C). Paper chromatography. Linamarin can be isolated on paper chromatograms by the use of several solvents reported earlier (2). Ascending
312
SHORT COMMUNICATIONS TABLE
1
COLORREACTIONSWITHP-ANISALDEHYDE
AT
85°C
Color reaction Compound (10 pg) KCN KSCN Ferricyanide Linamarin Amygdalin Mandelic acid Sinigrin Arbutin PhloridzinD Phloretin” Salicin Inositol Solanine Chlorogenic acid Glucose Fructose Galactose Sucrose Xylose
Visible NIL white dark blue pink pink pink pink purple orange blue-purple pink light purple gray-purple purple-pink red-purple deep purple red-purple deep purple light purple
uv Light
Intensity
-
-
white dark blue bright pink weak pink purple bright pink purple-blue purple purple reddish-purple blue purple deep purple -
intense intense very intense weak very weak strong intense very intense strong strong weak weak very strong very weak intense very weak strong very weak
-
a Very fast reaction, 5- 10 sec.
chromatography on 12 x 360-mm strips of Whatman No. 1 paper was found most expedient for routine assays. Paper strips are suspended in 50 x 400-mm test tubes with a paper clip on a wire hook inserted in bottom of a rubber stopper and developed after 1-hr equilibration with 8 ml of a 8: 1:2, 1-butanol:ethanol:water system, 5 hr, to a height of 15- 17 cm (R,:0.52-0.54 for linamarin, 0.58-0.61 for lotaustralin). A fast-moving alternate solvent is a 4:5: 1 system of ethyl acetate:acetone:water which rises 20 cm in 3 hr (R,:O.71-0.73 for linamarin, 0.82-0.84 for lotaustralin). The latter solvent is preferable for mixtures of linamarin and lotaustralin. Visual estimation. Linamarin concentration can be estimated visually by comparison of fluorescence in unknowns with a set of calibration standards. A set of standards (developed chromatogram spots cut out and fastened on a small cardboard or in a filing folder) will remain in usable condition for 4 or more weeks. Unknown solutions are brought down to threshold detection range of 0.5 to 10 pg in l/10, l/20, l/40 or other convenient dilutions (three to four dilutions are usually sufficient). Once a set value is established within the calibration range, the linamarin content of an extract can be easily calculated with a -+5% error; with experience, an even smaller error can be attained.
SHORT COMMUNICATIONS
313
For calibration, a stock solution containing 20 mg of p-linamarin (2 CLgIpl) in 10 ml 80% ethanol is used for a series of dilutions, 0.4, 0.8, 1.2, and 1.6 ml, made up to 2 ml volume in 15 x 4%mm vials; 5 ~1 of each of these dilutions is run through chromatographic development and detection treatment to provide 2- to IO-pg standards for comparison with the unknowns. A similar lower dilution series using 0.4 ml previously prepared standards diluted to 2.0 ml will give an additional range covering a 0.4- to 2.0-pg range. The visible pink color can be detected from 1 pg, whereas fluorescence at A = 366 nm lowers the detection threshold to 0.3 to 0.5 pg. The visible pink color fades in time, but the fluorescence of spots containing in excess of 5 pg of linamarin will persist for 4-6 months. Interferences. Some 25 substances, a few of which are listed in Table 1, were tested for reaction with p-anisaldehyde. Of the various carbohydrates, glycosides, and phenolics, only sinigrin showed a strong color reaction similar to that of linamarin. Amygdalin, another cyanogen, has shown also a similar reaction with about one-tenth of linamarin intensity. Most of the tested carbohydrates showed Rf values below 0.35 in the two employed solvents and therefore would not interfere with linamarin isolation. Pure methyl-linamarin (lotaustralin) was not available in sufficient quantity for the test, however, in a mixture with linamarin (G. W. Butler (Ref. l)), methyl-linamarin separated on chromatograms showed only a very weak reaction (approximately 5 pg in a sample), a quickly fading orange color without any fluorescence. Extracts from flax seedlings and from rat feces have shown a number of weak purple spots at R, values below 0.35, possibly sucrose at Rf .31, but none corresponding to glucose (Rf:O. 18) in 8: 1:2 1-butanol:ethanol:water solvent. The proposed method does not require elaborate sample purification, is suitable for rapid routine tests and is applicable to systems where any interfering substances or other cyanogens can be chromatographically eliminated. The procedure was successful in detecting and measuring intact linamarin in urine of rats dosed with the cyanogen (7). ACKNOWLEDGMENT The research was carried out with the aid of a grant from the International Research Centre, Ottawa, Canada.
Development
REFERENCES 1. Butler, G. W. (1%5)Phytochemistry 4, 127-131. 2. Seigler, D. S. (1975) Phytochemistry 14, 9-29. 3. Conn, E. E. (1973) Biochem. Sot. Symp. 38, 277-302. 4. Zitnak, A. (1973) in Chronic Cassava Toxicity (Nestel, B., and MacIntyre, R., eds.), pp. 89-%, Canadian International Development Research Centre, Ottawa, Canada. 5. Nestel, B., and MacIntyre, R., (eds.), (1973) Chronic Cassava Toxicity, Canadian International Development Research Centre, Ottawa, Canada.
314
SHORT COMMUNICATIONS
6. Montgomery, R. P. (1969) in Toxic Constituents of Plant Food Stuffs, (Liener, E. I., ed.), pp. 143-157, Academic Press, New York. 7. Barrett, M. D. P. (1976) Dietary Cyanide, Linamarin and Nutritional Deficiencies. Ph.D. thesis, University of Guelph, Canada. 8. Feigl, F. (1%6) Spot Tests in Organic Analysis, 7th ed., Elsevier, Amsterdam/New York.
A. ZITNAK~ D. C. HILL J. C. ALEXANDER~ Departments University Received
of Horticultural Science and Nutrition of Guelph, Canada May 24, 1976; accepted August 2, 1976
* Address reprint requests to A. Zitnak, Department of Horticultural of Guelph, Guelph, Canada, NlG 2Wl. 3 Department of Nutrition, University of Guelph.
Science, University
