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An improved colorimetric assay for polyols
ANALYTICAL
BIOCHEMISTRY
81,
An Improved
Calorimetric
SONG HAE Department
of Nutrition
18-20 (1977)
BOK’
and Food Cambridge,
Assay for Polyols
AND ARNOLD
L. DEMAIN
Science. Mussachusetts Massachusetts 02139
Institute
of Trchnologv,
Received November 8, 1976; accepted March 28, 1977 A new assay for polyols was developed that minimizes interference by sugars. It is based on oxidation of alditols to formaldehyde by acidic periodate, reduction of the excess periodate with L-rhamnose. and a short-time determination of the formaldehyde with the Nash reagent. When glycerol was added to various crude biological materials and was assayed, recovery was 90-106%.
The analysis of polyols in crude mixtures by periodate oxidation is difficult because sugars also are oxidized and contribute to the formation of the reaction product. formaldehyde. In 1966, Samuelson and Stromberg (1) described a method applicable to the analysis of alditols after the polyols had been separated from sugars by partition chromatography on ion-exchange resins. These investigators made an important contribution by discovering that acidic periodate (pH 1.0) reacted to a much greater extent with polyols than with aldoses. The unreacted periodate was reduced to iodate or iodide (depending on the pH) by addition of sodium arsenite. The formaldehyde was then determined calorimetrically with the Nash (2) reagent, which is a neutral solution of acetylacetone (pentane-2,4-dione) and ammonium salt. The Nash reagent reacts with formaldehyde to produce the yellow 35diacetyl-l:4-dehydrolutidine, which has an absorption maximum at 412 nm. In 1969, Vaskovsky and Isay (3) reported that it was much more convenient to use L-rhamnose than arsenite to remove excess periodate. We have combined the methods of Samuelson and Stromberg (1) and Vaskovsky and Isay (3) into a modified technique for polyol determination in the presence of sugars. MATERIALS
AND METHODS
Nush reagenr. Nash reagent (2) was freshly prepared by mixing 1.50 g of ammonium acetate, 2 ml of glacial acetic acid, and 2 ml of pentane2,4-dione and bringing the mixture to 1 liter with distilled water. Polyol assay. One-milliliter samples containing polyol were placed in 1 Present address: A. E. Staley Manufacturing
CopyrIght C lY77 by Academx Prr\s. Inc. All rtght? of reproductmn m any term rerrrvsd
Co., Decatur, Ill. 62.525.
ISSN noWXY7
POLYOL
ASSAY
CONCENTRATION
19
(@/ml)
FIG. 1. Absorbance curves for various polyols in the polyol assay. A Beckman DB spectrophotometer was used. Symbols: (0) glycerol: (ml erythritol: (0) arabinitol; (A) ribitol: (0) mannitol and glucitol.
test tubes (18 x 150 mm), and then 1 ml of 0.015 M sodium metaperiodate in 0.12 M HCI was added. After mixing and allowing to stand for 10 min at room temperature, 2 ml of a 0.1% L-rhamnose solution was added to each test tube to remove excess periodate ion. After further mixing, 4 ml of Nash reagent was added. Color was developed for 15 min in a water bath at 53°C. After cooling, the absorbance was measured at 412 nm with a spectrophotometer. Standard calibration curves for polyols were made in the range of O-25 pgiml. CvLtde biologicul materials. Bovine serum albumin (Sigma Chemical Co.) was used at 3.5 mgiml. Soil extract was prepared by autoclaving 1000 g of soil in 1 liter of distilled water for 30 min. After centrifugation, the supernatant fluid was filtered through a 1.2-pm Millipore filter and was adjusted to 1 liter. Malt extract, yeast extract, nutrient broth (all from Difco Laboratories). and urine were used at 350 kg/ml. For the recovery experiments, these materials, alone and mixed with 15 Fg of glycerol/ml, were assayed against a glycerol standard curve. RESULTS
Standard curves for various polyols are shown in Fig. 1. On a weight basis, glycerol produced the strongest color reaction, followed by erythritol, arabinitol, ribitol, and, finally, glucitol (sorbitol) and mannitol, which were equivalent.
20
BOK
Interference
AND
DEMAIN
by Sugars
A number of sugars were examined for color formation in the polyol assay. On a weight basis, aldohexoses gave only 1.5% the absorbance produced by glycerol. whereas aldopentoses yielded 3% interference. An aldotetrose such as erythrose interfered by 12%, whereas trioses produced 29% as much color as did glycerol. Ketohexoses interfered by 15%. Recovew Experimerlts There was excellent recovery of glycerol when it was added to various crude biological materials (90% for nutrient broth and urine; 100% for albumin, malt extract, and yeast extract; 106% for soil extract). DISCUSSION
The main difficulty in the assay of polyols has been interference by sugars. In our modified method, the influence of aldohexoses and aldopentoses is minimal. Only in the cases of ketohexoses, tetroses, and trioses is there significant interference. Despite this potential interference problem, good recoveries (90-106%) of glycerol were obtained when it was added to bovine serum albumin, soil extract, malt extract, yeast extract, nutrient broth, and urine. Thus, it is clear that our modified polyol assay method can be used for the analysis of glycerol or other polyols in the presence of complex organic materials. Protein seems not to interfere in the assay. However, if the complex sample contains high concentrations of trioses, tetroses, or ketoses as compared with glycerol, the interference could be considerable. When assaying the polyol content of a crude mixture, it is important to choose the predominant polyol to construct the standard curve. This can be done by paper chromatography of the mixture as we have previously described (4). Compared with mannitol, the mobility of other polyols was 1.65 for D-arabinitol, 1.69 for ribitol, 2.07 for mesa-erythritol, and 2.67 for glycerol. ACKNOWLEDGMENT The authors gratefully acknowledge Memorial Fund for making this work
the financial possible.
support
of the Lewis
REFERENCES 1. 2. 3. 4.
Samuelson. 0.. and Striimberg, H. (1966)Cavboh~I. Res. 3,89-96. Nash, T. (1953) Biochrm. J. 55, 416-421. Vaskovsky, V. E., and Isay, S. V. (1969)Ad. Biochenz. 30,25-31. Bok, S. H., andDemain. A. L. (1974)Biot~cllnolBio~n~. 16,209-130.
and Rosa Strauss
BIOCHEMISTRY
81,
An Improved
Calorimetric
SONG HAE Department
of Nutrition
18-20 (1977)
BOK’
and Food Cambridge,
Assay for Polyols
AND ARNOLD
L. DEMAIN
Science. Mussachusetts Massachusetts 02139
Institute
of Trchnologv,
Received November 8, 1976; accepted March 28, 1977 A new assay for polyols was developed that minimizes interference by sugars. It is based on oxidation of alditols to formaldehyde by acidic periodate, reduction of the excess periodate with L-rhamnose. and a short-time determination of the formaldehyde with the Nash reagent. When glycerol was added to various crude biological materials and was assayed, recovery was 90-106%.
The analysis of polyols in crude mixtures by periodate oxidation is difficult because sugars also are oxidized and contribute to the formation of the reaction product. formaldehyde. In 1966, Samuelson and Stromberg (1) described a method applicable to the analysis of alditols after the polyols had been separated from sugars by partition chromatography on ion-exchange resins. These investigators made an important contribution by discovering that acidic periodate (pH 1.0) reacted to a much greater extent with polyols than with aldoses. The unreacted periodate was reduced to iodate or iodide (depending on the pH) by addition of sodium arsenite. The formaldehyde was then determined calorimetrically with the Nash (2) reagent, which is a neutral solution of acetylacetone (pentane-2,4-dione) and ammonium salt. The Nash reagent reacts with formaldehyde to produce the yellow 35diacetyl-l:4-dehydrolutidine, which has an absorption maximum at 412 nm. In 1969, Vaskovsky and Isay (3) reported that it was much more convenient to use L-rhamnose than arsenite to remove excess periodate. We have combined the methods of Samuelson and Stromberg (1) and Vaskovsky and Isay (3) into a modified technique for polyol determination in the presence of sugars. MATERIALS
AND METHODS
Nush reagenr. Nash reagent (2) was freshly prepared by mixing 1.50 g of ammonium acetate, 2 ml of glacial acetic acid, and 2 ml of pentane2,4-dione and bringing the mixture to 1 liter with distilled water. Polyol assay. One-milliliter samples containing polyol were placed in 1 Present address: A. E. Staley Manufacturing
CopyrIght C lY77 by Academx Prr\s. Inc. All rtght? of reproductmn m any term rerrrvsd
Co., Decatur, Ill. 62.525.
ISSN noWXY7
POLYOL
ASSAY
CONCENTRATION
19
(@/ml)
FIG. 1. Absorbance curves for various polyols in the polyol assay. A Beckman DB spectrophotometer was used. Symbols: (0) glycerol: (ml erythritol: (0) arabinitol; (A) ribitol: (0) mannitol and glucitol.
test tubes (18 x 150 mm), and then 1 ml of 0.015 M sodium metaperiodate in 0.12 M HCI was added. After mixing and allowing to stand for 10 min at room temperature, 2 ml of a 0.1% L-rhamnose solution was added to each test tube to remove excess periodate ion. After further mixing, 4 ml of Nash reagent was added. Color was developed for 15 min in a water bath at 53°C. After cooling, the absorbance was measured at 412 nm with a spectrophotometer. Standard calibration curves for polyols were made in the range of O-25 pgiml. CvLtde biologicul materials. Bovine serum albumin (Sigma Chemical Co.) was used at 3.5 mgiml. Soil extract was prepared by autoclaving 1000 g of soil in 1 liter of distilled water for 30 min. After centrifugation, the supernatant fluid was filtered through a 1.2-pm Millipore filter and was adjusted to 1 liter. Malt extract, yeast extract, nutrient broth (all from Difco Laboratories). and urine were used at 350 kg/ml. For the recovery experiments, these materials, alone and mixed with 15 Fg of glycerol/ml, were assayed against a glycerol standard curve. RESULTS
Standard curves for various polyols are shown in Fig. 1. On a weight basis, glycerol produced the strongest color reaction, followed by erythritol, arabinitol, ribitol, and, finally, glucitol (sorbitol) and mannitol, which were equivalent.
20
BOK
Interference
AND
DEMAIN
by Sugars
A number of sugars were examined for color formation in the polyol assay. On a weight basis, aldohexoses gave only 1.5% the absorbance produced by glycerol. whereas aldopentoses yielded 3% interference. An aldotetrose such as erythrose interfered by 12%, whereas trioses produced 29% as much color as did glycerol. Ketohexoses interfered by 15%. Recovew Experimerlts There was excellent recovery of glycerol when it was added to various crude biological materials (90% for nutrient broth and urine; 100% for albumin, malt extract, and yeast extract; 106% for soil extract). DISCUSSION
The main difficulty in the assay of polyols has been interference by sugars. In our modified method, the influence of aldohexoses and aldopentoses is minimal. Only in the cases of ketohexoses, tetroses, and trioses is there significant interference. Despite this potential interference problem, good recoveries (90-106%) of glycerol were obtained when it was added to bovine serum albumin, soil extract, malt extract, yeast extract, nutrient broth, and urine. Thus, it is clear that our modified polyol assay method can be used for the analysis of glycerol or other polyols in the presence of complex organic materials. Protein seems not to interfere in the assay. However, if the complex sample contains high concentrations of trioses, tetroses, or ketoses as compared with glycerol, the interference could be considerable. When assaying the polyol content of a crude mixture, it is important to choose the predominant polyol to construct the standard curve. This can be done by paper chromatography of the mixture as we have previously described (4). Compared with mannitol, the mobility of other polyols was 1.65 for D-arabinitol, 1.69 for ribitol, 2.07 for mesa-erythritol, and 2.67 for glycerol. ACKNOWLEDGMENT The authors gratefully acknowledge Memorial Fund for making this work
the financial possible.
support
of the Lewis
REFERENCES 1. 2. 3. 4.
Samuelson. 0.. and Striimberg, H. (1966)Cavboh~I. Res. 3,89-96. Nash, T. (1953) Biochrm. J. 55, 416-421. Vaskovsky, V. E., and Isay, S. V. (1969)Ad. Biochenz. 30,25-31. Bok, S. H., andDemain. A. L. (1974)Biot~cllnolBio~n~. 16,209-130.
and Rosa Strauss