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Interference of polyols with the Lowry-Lopez phosphorus assay: Reaction of polyols with Mo7O246−
ANALYTICAL
BIOCHEMISTRY
Interference
101,
148-153 (1980)
of Polyols with the Lowry-Lopez Phosphorus Reaction of Polyols with Mo,O& KAUKO
Assay:
K. MAKINEN
Department of Biochemistry, Institute of Dentistry, University of Turku, SF-20520 Turku 52, Finland Received May 3, 1979 Certain open-chain polyols were shown to interfere with the determination of phosphorus of the Lowry-Lopez method by forming a complex with Mo,O$;. The ability to interfere with the assay increased with increasing chain length of the polyols: Ethylene glycol and glycerol did not react at all; i-erythritol reacted to a small extent, but hexitols and perseitol formed stronger complexes. Depending on the polyol, interference occurred even at 0.2 mM (hexitols) or 2 mM (xylitol) concentrations. At these concentrations the polyols interfered only to a small extent with the phosphorus assays based on the use of Triton X-100 and molybdate. The comolex formation was exploited in the development of a calorimetric p0ly0l assay. -
It is a well-known fact that alditols form certain types of complexes with various inorganic polybasic acids or their salts and anhydrides in aqueous solutions (1). Consequently, complexes with boric, molybdic, tungstic, and other acids, as well as the oxides of antimony and arsenic, have been described. These complexes are believed to be true esters with one or more alditol molecules. A chelate type of structure may be involved in these complexes (1). As these complexes are often weak and form ideally under nonphysiological conditions involving either high salt concentrations or extreme pH values, they may have greater biological significance only in restricted cases. However, this reaction can be of significance in various chemical assays based on the formation of complexes between the above-mentioned inorganic acids, salts, or anhydrides, and the compounds to be analyzed if suitable alditols are simultaneously present. This communication describes a case in which polyols interfere with the determination of phosphorus with the Lowry-Lopez method which is based on 0003-2697/80/010148-06$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
148
the formation of a blue molybdenum-containing inorganic dye. MATERIALS
AND METHODS
Reagents. Phenazine methosulfate, nitro BT tetrazolium salt, and the polyols were obtained from Sigma Chemical Company (St. Louis, MO.),” except for xylitol which was a product of Xyrofin Ltd. (Baar, Switzerland). Hypoxanthine was obtained from E. Merck AG (Darmstadt, West Germany) and xanthine from Fluka AG (Buchs, Switzerland). Malachite green oxalate (C.I. 42000) and other reagents were from J. T. Baker Chemical Company (Phillipsburg, N. J.). Chemical methods. The effect of poly01s on the phosphorus assay was studied at 22°C in the original reaction mixture described by Lowry and Lopez (2), with small modifications. Consequently, the following reaction mixture (7.0 ml) was used: (a) 1.0 ml of a solution containing 1% L-ascorbic acid and 0.735 mM KH*PO,, (b) 4.0 ml of an aqueous polyol solution,
EFFECT
OF POLYOLS
ON PHOSPHORUS
1 =lO 2 = 20
3=30 4=40 5=60
10
1
MIN If
,, I' ,I
6 =120,
0
180,
240
20 [XYLITOL]
149
ASSAY
and
450MIN
30
40
mM
FIG. 1. Time dependence of the breakdown of molybdate-xylitol complexes in reaction mixtures of the Lowry-Lopez phosphorus assay method, plotted as the increase of E,,, versus xylitol concentration.
and (c) 2.0 ml of a solution containing 0.028 M acetate buffer, pH 4.0, 2.7 mM ammonium molybdate (VI) tetrahydrate 0.000167 N fW~,hMo,%-4&Ol, and
H,SO,. The reactions were started by adding (c) to a freshly made mixture of (a) and (b). The blue color which developed at different rates, depending on conditions,
0.1 a09 0.08
0.07 006 0.05
0.03 002 0.04
0
5
10
15 [XYLlTOq
20
25
30
35
mM
FIG. 2. Time dependence of the breakdown of molybdate-xylitol complexes. The data, obtained from Fig. I, were plotted on semilogarithmic paper. The tested reaction times fell into two categories which are represented by the two straight lines.
150
KAUKO
K. MAKINEN
TABLE
1
RELATIVE ABILITY OF CERTAIN POLYOLSTO FORM COMPLEXES WITH Mo,O$;” Time after mixing (min) Polyol
10
20
30
40
60
140
Perseitol Galactitol D-Mannitol D-Sorbitol L-ArabitoP Ribitol Xylitol i-Erythritol Glycerol Ethylene glycol Water
0.013 0.014 0.021 0.038 0.039 0.144 0.170 0.345 0.165 0.191 0.168
0.015 0.016 0.023 0.059 0.062 0.263 0.291 0.418 0.294 0.334 0.296
0.018 0.017 0.025 0.081 0.087 0.382 0.383 0.422 0.419 0.449 0.425
0.020 0.018 0.028 0.105 0.114 0.445 0.427 0.425 0.458 0.479 0.472
0.021 0.020 0.032 0.136 0.155 0.462 0.441 0.424 0.480 0.497 0.500
0.024 0.023 0.043 0.183 0.222 0.468 0.443 0.426 0.541 0.558 0.568
o The experiments were carried out as described in the Materials and Methods section. The concentration of each polyol was 4 mg in the 7.0-ml reaction mixture which in principle corresponded to the original method of Lowry and Lopez (2). The results are given as extinctions (700 nm) of the reaction mixtures as a function of time following mixing of the reagents. The values given for water show the development of the blue color in a normal mixture in which the reaction between phosphate and Mo,O$; proceeded freely. The lower values shown for the polyols indicate competition between phosphate and polyols for Mo,O%. * D-Arabitol yielded essentially similar results.
was determined at 700 nm with a Beckman Model 25 spectrophotometer equipped with a sipper system and a Beckman Model 3 115 printer. Other phosphorus assays were carried out according to Kallner (3) (malachite green method), Eibl and Lands (4) (Triton method), and See and Fitt (5) (calorimetric method). RESULTS
Preliminary studies showed that xylitol, at concentrations above approximately 2 mM, interfered with the Lowry-Lopez phosphorus assay. The interference appeared as a delay in the formation of the characteristic blue color. At very high xylitol concentrations the final color practically did not develop at all during the working day. More detailed experiments showed that the time dependence of the development of absorption at 700 nm followed the pattern shown in Fig. 1. The reaction was considered to have resulted from the formation of a complex between the Mo,O& aggregate and xylitol. The complex formed was not,
however, very stable under the conditions described (in 0.1 M acetate buffer, pH 4.0). The time dependence of its breakdown was linear only during a short period after mixing of the reagents, and particularly at low xylitol concentrations. During prolonged standing (longer than 20 min), the breakdown curve assumed a sigmoidal shape and thus the longer reaction times indicated in Fig. 1 resulted in virtually similar curves. Consequently, at a xylitol concentration of 25 mM, for example, the phosphorus assay according to the standard method was impossible. The standard method presupposes the measurement of the extinction 5 and 10 min after addition of the acidic ammonium molybdate solution. Figure 2 shows that when plotting E7,,0 as a function of the molarity of xylitol, the experimental points of the sigmoidal part of the curves depicted in Fig. 1 were located on straightline sections. In this experiment it was possible to group the reaction times so that they were represented by two separate straight lines.
EFFECT
0
0.01
OF POLYOLS
0.02
ON
PHOSPHORUS
0 [PHOSPHATE]
151
ASSAY
0.1
a2
mM
FIG. 3. Effect of 25 mM xylitol, mannitol, and sorbitol on the phosphorus assays based on the Triton X-lOO-molybate methods. A: Effect on the turbidimetric method of Eibl and Lands (4). B: Effect on the calorimetric method of See and Fitt (5) (modified LowryLopez Assay II. as described by See and Fitt).
Table 1 shows the results from experiments similar to those described above, using a number of other polyols. The reactions were performed according to the standard Lowry-Lopez method and the results were, for practical reasons, thus given as extinctions of the mixtures following standing for various times at 22°C. Under the present conditions the polyols could be divided into two separate groups according to their ability to form complexes with molybdenum. Those forming rather strong chelates were perseitol, sorbitol, mannitol, and the two arabitols. Xylitol and ribitol formed weaker complexes, while glycerol and ethylene glycol did not obviously react at all. The behavior of i-erythritol was deviant: At low concentrations it increased the rate of the reaction between phosphate and molybdate, but the complexes which were formed at higher i-erythritol concentrations were as stable as those formed with xylitol or ribitol. Glucose, fructose, and sucrose did not interfere with the phosphorus assay at comparable concentrations.
The Triton methods of Eibl and Lands (4) and See and Fitt (5) were slightly affected by pentitols, hexitols, and perseitol. Figure 3 shows the effect of xylitol, mannitol, and sorbitol on these methods. Preliminary experiments showed that the malachite green method (3) was not affected by the polyols used. Preliminary experiments also showed that the formation of MO-containing complexes with heptitols, hexitols, and pentitols could be utilized for a quantitative determination of these polyols. Studies with xylitol indicated that the reaction mixture described could be used to determine 5 to 30 mM xylitol concentrations in aqueous solutions. Pentitols which formed stronger complexes and all tested hexitols and perseitol could be analyzed at still lower concentrations. This method presupposed, however, a prior removal of phosphorus from the sample, using, for example, the method of Lowry and Lopez (2). Addition of a constant amount of KH,PO, (as described in the Materials and Methods section) thus produced
152
KAUKO
K. MAKINEN
the required blue complex, the formation of which depended on the presence of the polyols mentioned. In the polyol assays the graphs shown in Fig. 2 were constructed. DISCUSSION
The interference of the polyols mentioned with the Lowry-Lopez phosphorus assay can be considered to result from the formation of complexes between the polyhydroxy compounds and Mo,Og;. The mechanism involved and the types of the complexes formed have been described in the literature. So, for example, ribitol and xylitol formed 2:1 complexes at low pH values with Na,MoO,, whereas arabitol and glycerol did not form chelates (6). Dimolybdenic o-mannitol or sorbitol complexes have been described by several authors (7- 13), although other types of complexes have also been reported. Also borate and boric acid form complexes with polyols (14,15,21). Other properties of the molybdate derivatives of carbohydrates have also been studied (14,16-20). In order to get a satisfactory fit to the present experimental results, the existence of 2: 1 molybdate-polyol complexes must be assumed in the case of polyols with four or more carbon atoms. The differences observed in the stability of the complexes between various polyols are in accordance with the proposal of Fedorov and Pavlinova (18), who showed that increasing the number of COOH groups in such molecules increases the stability of the molybdate complexes. This paper suggests that a similar effect can also be encountered when the number of OH groups increases in a polyol molecule. The stability of these complexes in general can be considered to be a result of the number of hydrogen bonds formed between the reacting species (12). The formation of binuclear complexes between molybdate and hexitols has been explained by a large number of chelating groups in the reagent molecule (10). This condition is not met, e.g., by glycerol and cyclic carbohydrate molecules which did
not interfere with the phosphorus assay. The interference of polyols with the phosphomolybdate techniques has been previously studied by other authors (22-24). The fact that the malachite green method was not affected by the polyols used may have resulted from the urea simultaneously present in high concentration. Urea has most likely prevented effective hydrogen bonding between polyols and molybdate. A suggestion concerning the possibility of determining MOO:- acidimetrically as a monoacidic base in a hexitol medium and hexitols as diacidic bases in a medium of MOO:- has been made (10). For example, 2 mg of sorbitol has been determined in a medium of tungstate and molybdate (10). The present study suggests that a similar method could be used for heptitols and several pentitols as well. However, as discussed previously (25), gas chromatography and enzymatic methods are most often convenient for the assay of polyols. REFERENCES I. Lohmar, R. L. (1962) in The Carbohydrates: Chemistry, Biochemistry, Physiology (Pigman. W., ed.). p. 241, Academic Press, New York. 2. Lowry, 0. H., and Lopez, J. A. (1957) in Methods in Enzymology (Colowick. S. P.. and Kaplan, N. 0.. eds.), Vol. III, p. 845, Academic Press, New York. 3. Kallner. A. (1975) C/in. Chim. Actu 59, 35-39. 4. Eibl, H.. and Lands, W. E. M. (1969) AM/. Biochem. 5.
30, 51-57.
See, Y. P., and Fitt. Biochem.
6.
P. S. (1972) Anul.
49,430-435.
Bayer, E.. and Voelter, W. (1966) Ann. 696,
Chem.
194-208.
7.
Posternak, Th.. Janjic. D., Lucken. E. A. C., and Szente. A. (1967) Helv. Chim. Artu 50,
8.
Shaw. E. H.. Jr., Daniels, D.. and Sawyer, L. D.
9.
Fedorov. A. A.. and Pavlinova. A. V. (1972) Zh.
1027-
1038.
(1%7) An&.
Proc~. S. Dak. Khim.
Acud.
Sci.
46, 174- 175.
27, 2409-2413.
IO.
Mikesova. M.. and BartuSek, M. (1978) Collec,f.
Il.
Hedman, B. (1974) Acru
Czech.
Chum.
Commun.
43, 1867-1877. Chem. Sand.
Ser.
A
28, 591-592. 12.
Chalmers, J. Inorg.
R. A., and Sinclair. NW/.
Chem.
A. G. (1967)
29, 2065-2080.
EFFECT 13. Brown,
D.
Inorg.
H..
Nucl.
and Chem.
OF
POLYOLS
Macpherson, 34,
1705-
J. (1972)
ON J.
1710.
14. Kankare. J. J. (1973) An&. Chem. 45, 2050-2056. 15. Ferrier, R. J. (1978) in Advances in Carbohydrate Chemistry and Biochemistry (Tipson, R. S.. and Horton, D., eds.), Vol. 35, p. 31, Academic Press, New York. 16. Voelter, W., Bayer, E.. Records, R., Bunnenberg. E., and Djerassi. C. (1969) Chem. Ber. 102, 1005-1019.
17. Lippke, 22, 18.
G., and Thaler.
Fedorov. Zh.
H. (1970)
Sfiirke
(Sturch)
344-351.
A. Vses.
A.. Khim.
and
Pavlinova.
Ohshchest.
A.
V.
17, 352-353.
(1972)
PHOSPHORUS
19. Fedorov, A. A. 1072-1075. 20.
153
ASSAY
Fedorov,
( 1975)
A. A. (1976)
21. BGseken,
J. (1949)
Zh.
Obshch,
Chem.
Absfr.
J. Advan.
Khim.
84,
Curbohyd.
45,
22792~. Chem.
4, 189. 22.
Vreman.
H.
B&hem.
23. Irving,
J., 17,
and Jiibsis, 118.
F. F. (1%6)
Anal.
10%
G. C. J., and Cosgrove, 36, 38 I-388.
D. J. (1970)Anul.
Biochem.
24. Ho,
C.
Biochem.
25. MLkinen,
H., 60,
and Pande, 413-416.
K. K. (1978)
S.
Experienria
V.
(1974) Suppl.
Anal.
30,
15.
BIOCHEMISTRY
Interference
101,
148-153 (1980)
of Polyols with the Lowry-Lopez Phosphorus Reaction of Polyols with Mo,O& KAUKO
Assay:
K. MAKINEN
Department of Biochemistry, Institute of Dentistry, University of Turku, SF-20520 Turku 52, Finland Received May 3, 1979 Certain open-chain polyols were shown to interfere with the determination of phosphorus of the Lowry-Lopez method by forming a complex with Mo,O$;. The ability to interfere with the assay increased with increasing chain length of the polyols: Ethylene glycol and glycerol did not react at all; i-erythritol reacted to a small extent, but hexitols and perseitol formed stronger complexes. Depending on the polyol, interference occurred even at 0.2 mM (hexitols) or 2 mM (xylitol) concentrations. At these concentrations the polyols interfered only to a small extent with the phosphorus assays based on the use of Triton X-100 and molybdate. The comolex formation was exploited in the development of a calorimetric p0ly0l assay. -
It is a well-known fact that alditols form certain types of complexes with various inorganic polybasic acids or their salts and anhydrides in aqueous solutions (1). Consequently, complexes with boric, molybdic, tungstic, and other acids, as well as the oxides of antimony and arsenic, have been described. These complexes are believed to be true esters with one or more alditol molecules. A chelate type of structure may be involved in these complexes (1). As these complexes are often weak and form ideally under nonphysiological conditions involving either high salt concentrations or extreme pH values, they may have greater biological significance only in restricted cases. However, this reaction can be of significance in various chemical assays based on the formation of complexes between the above-mentioned inorganic acids, salts, or anhydrides, and the compounds to be analyzed if suitable alditols are simultaneously present. This communication describes a case in which polyols interfere with the determination of phosphorus with the Lowry-Lopez method which is based on 0003-2697/80/010148-06$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
148
the formation of a blue molybdenum-containing inorganic dye. MATERIALS
AND METHODS
Reagents. Phenazine methosulfate, nitro BT tetrazolium salt, and the polyols were obtained from Sigma Chemical Company (St. Louis, MO.),” except for xylitol which was a product of Xyrofin Ltd. (Baar, Switzerland). Hypoxanthine was obtained from E. Merck AG (Darmstadt, West Germany) and xanthine from Fluka AG (Buchs, Switzerland). Malachite green oxalate (C.I. 42000) and other reagents were from J. T. Baker Chemical Company (Phillipsburg, N. J.). Chemical methods. The effect of poly01s on the phosphorus assay was studied at 22°C in the original reaction mixture described by Lowry and Lopez (2), with small modifications. Consequently, the following reaction mixture (7.0 ml) was used: (a) 1.0 ml of a solution containing 1% L-ascorbic acid and 0.735 mM KH*PO,, (b) 4.0 ml of an aqueous polyol solution,
EFFECT
OF POLYOLS
ON PHOSPHORUS
1 =lO 2 = 20
3=30 4=40 5=60
10
1
MIN If
,, I' ,I
6 =120,
0
180,
240
20 [XYLITOL]
149
ASSAY
and
450MIN
30
40
mM
FIG. 1. Time dependence of the breakdown of molybdate-xylitol complexes in reaction mixtures of the Lowry-Lopez phosphorus assay method, plotted as the increase of E,,, versus xylitol concentration.
and (c) 2.0 ml of a solution containing 0.028 M acetate buffer, pH 4.0, 2.7 mM ammonium molybdate (VI) tetrahydrate 0.000167 N fW~,hMo,%-4&Ol, and
H,SO,. The reactions were started by adding (c) to a freshly made mixture of (a) and (b). The blue color which developed at different rates, depending on conditions,
0.1 a09 0.08
0.07 006 0.05
0.03 002 0.04
0
5
10
15 [XYLlTOq
20
25
30
35
mM
FIG. 2. Time dependence of the breakdown of molybdate-xylitol complexes. The data, obtained from Fig. I, were plotted on semilogarithmic paper. The tested reaction times fell into two categories which are represented by the two straight lines.
150
KAUKO
K. MAKINEN
TABLE
1
RELATIVE ABILITY OF CERTAIN POLYOLSTO FORM COMPLEXES WITH Mo,O$;” Time after mixing (min) Polyol
10
20
30
40
60
140
Perseitol Galactitol D-Mannitol D-Sorbitol L-ArabitoP Ribitol Xylitol i-Erythritol Glycerol Ethylene glycol Water
0.013 0.014 0.021 0.038 0.039 0.144 0.170 0.345 0.165 0.191 0.168
0.015 0.016 0.023 0.059 0.062 0.263 0.291 0.418 0.294 0.334 0.296
0.018 0.017 0.025 0.081 0.087 0.382 0.383 0.422 0.419 0.449 0.425
0.020 0.018 0.028 0.105 0.114 0.445 0.427 0.425 0.458 0.479 0.472
0.021 0.020 0.032 0.136 0.155 0.462 0.441 0.424 0.480 0.497 0.500
0.024 0.023 0.043 0.183 0.222 0.468 0.443 0.426 0.541 0.558 0.568
o The experiments were carried out as described in the Materials and Methods section. The concentration of each polyol was 4 mg in the 7.0-ml reaction mixture which in principle corresponded to the original method of Lowry and Lopez (2). The results are given as extinctions (700 nm) of the reaction mixtures as a function of time following mixing of the reagents. The values given for water show the development of the blue color in a normal mixture in which the reaction between phosphate and Mo,O$; proceeded freely. The lower values shown for the polyols indicate competition between phosphate and polyols for Mo,O%. * D-Arabitol yielded essentially similar results.
was determined at 700 nm with a Beckman Model 25 spectrophotometer equipped with a sipper system and a Beckman Model 3 115 printer. Other phosphorus assays were carried out according to Kallner (3) (malachite green method), Eibl and Lands (4) (Triton method), and See and Fitt (5) (calorimetric method). RESULTS
Preliminary studies showed that xylitol, at concentrations above approximately 2 mM, interfered with the Lowry-Lopez phosphorus assay. The interference appeared as a delay in the formation of the characteristic blue color. At very high xylitol concentrations the final color practically did not develop at all during the working day. More detailed experiments showed that the time dependence of the development of absorption at 700 nm followed the pattern shown in Fig. 1. The reaction was considered to have resulted from the formation of a complex between the Mo,O& aggregate and xylitol. The complex formed was not,
however, very stable under the conditions described (in 0.1 M acetate buffer, pH 4.0). The time dependence of its breakdown was linear only during a short period after mixing of the reagents, and particularly at low xylitol concentrations. During prolonged standing (longer than 20 min), the breakdown curve assumed a sigmoidal shape and thus the longer reaction times indicated in Fig. 1 resulted in virtually similar curves. Consequently, at a xylitol concentration of 25 mM, for example, the phosphorus assay according to the standard method was impossible. The standard method presupposes the measurement of the extinction 5 and 10 min after addition of the acidic ammonium molybdate solution. Figure 2 shows that when plotting E7,,0 as a function of the molarity of xylitol, the experimental points of the sigmoidal part of the curves depicted in Fig. 1 were located on straightline sections. In this experiment it was possible to group the reaction times so that they were represented by two separate straight lines.
EFFECT
0
0.01
OF POLYOLS
0.02
ON
PHOSPHORUS
0 [PHOSPHATE]
151
ASSAY
0.1
a2
mM
FIG. 3. Effect of 25 mM xylitol, mannitol, and sorbitol on the phosphorus assays based on the Triton X-lOO-molybate methods. A: Effect on the turbidimetric method of Eibl and Lands (4). B: Effect on the calorimetric method of See and Fitt (5) (modified LowryLopez Assay II. as described by See and Fitt).
Table 1 shows the results from experiments similar to those described above, using a number of other polyols. The reactions were performed according to the standard Lowry-Lopez method and the results were, for practical reasons, thus given as extinctions of the mixtures following standing for various times at 22°C. Under the present conditions the polyols could be divided into two separate groups according to their ability to form complexes with molybdenum. Those forming rather strong chelates were perseitol, sorbitol, mannitol, and the two arabitols. Xylitol and ribitol formed weaker complexes, while glycerol and ethylene glycol did not obviously react at all. The behavior of i-erythritol was deviant: At low concentrations it increased the rate of the reaction between phosphate and molybdate, but the complexes which were formed at higher i-erythritol concentrations were as stable as those formed with xylitol or ribitol. Glucose, fructose, and sucrose did not interfere with the phosphorus assay at comparable concentrations.
The Triton methods of Eibl and Lands (4) and See and Fitt (5) were slightly affected by pentitols, hexitols, and perseitol. Figure 3 shows the effect of xylitol, mannitol, and sorbitol on these methods. Preliminary experiments showed that the malachite green method (3) was not affected by the polyols used. Preliminary experiments also showed that the formation of MO-containing complexes with heptitols, hexitols, and pentitols could be utilized for a quantitative determination of these polyols. Studies with xylitol indicated that the reaction mixture described could be used to determine 5 to 30 mM xylitol concentrations in aqueous solutions. Pentitols which formed stronger complexes and all tested hexitols and perseitol could be analyzed at still lower concentrations. This method presupposed, however, a prior removal of phosphorus from the sample, using, for example, the method of Lowry and Lopez (2). Addition of a constant amount of KH,PO, (as described in the Materials and Methods section) thus produced
152
KAUKO
K. MAKINEN
the required blue complex, the formation of which depended on the presence of the polyols mentioned. In the polyol assays the graphs shown in Fig. 2 were constructed. DISCUSSION
The interference of the polyols mentioned with the Lowry-Lopez phosphorus assay can be considered to result from the formation of complexes between the polyhydroxy compounds and Mo,Og;. The mechanism involved and the types of the complexes formed have been described in the literature. So, for example, ribitol and xylitol formed 2:1 complexes at low pH values with Na,MoO,, whereas arabitol and glycerol did not form chelates (6). Dimolybdenic o-mannitol or sorbitol complexes have been described by several authors (7- 13), although other types of complexes have also been reported. Also borate and boric acid form complexes with polyols (14,15,21). Other properties of the molybdate derivatives of carbohydrates have also been studied (14,16-20). In order to get a satisfactory fit to the present experimental results, the existence of 2: 1 molybdate-polyol complexes must be assumed in the case of polyols with four or more carbon atoms. The differences observed in the stability of the complexes between various polyols are in accordance with the proposal of Fedorov and Pavlinova (18), who showed that increasing the number of COOH groups in such molecules increases the stability of the molybdate complexes. This paper suggests that a similar effect can also be encountered when the number of OH groups increases in a polyol molecule. The stability of these complexes in general can be considered to be a result of the number of hydrogen bonds formed between the reacting species (12). The formation of binuclear complexes between molybdate and hexitols has been explained by a large number of chelating groups in the reagent molecule (10). This condition is not met, e.g., by glycerol and cyclic carbohydrate molecules which did
not interfere with the phosphorus assay. The interference of polyols with the phosphomolybdate techniques has been previously studied by other authors (22-24). The fact that the malachite green method was not affected by the polyols used may have resulted from the urea simultaneously present in high concentration. Urea has most likely prevented effective hydrogen bonding between polyols and molybdate. A suggestion concerning the possibility of determining MOO:- acidimetrically as a monoacidic base in a hexitol medium and hexitols as diacidic bases in a medium of MOO:- has been made (10). For example, 2 mg of sorbitol has been determined in a medium of tungstate and molybdate (10). The present study suggests that a similar method could be used for heptitols and several pentitols as well. However, as discussed previously (25), gas chromatography and enzymatic methods are most often convenient for the assay of polyols. REFERENCES I. Lohmar, R. L. (1962) in The Carbohydrates: Chemistry, Biochemistry, Physiology (Pigman. W., ed.). p. 241, Academic Press, New York. 2. Lowry, 0. H., and Lopez, J. A. (1957) in Methods in Enzymology (Colowick. S. P.. and Kaplan, N. 0.. eds.), Vol. III, p. 845, Academic Press, New York. 3. Kallner. A. (1975) C/in. Chim. Actu 59, 35-39. 4. Eibl, H.. and Lands, W. E. M. (1969) AM/. Biochem. 5.
30, 51-57.
See, Y. P., and Fitt. Biochem.
6.
P. S. (1972) Anul.
49,430-435.
Bayer, E.. and Voelter, W. (1966) Ann. 696,
Chem.
194-208.
7.
Posternak, Th.. Janjic. D., Lucken. E. A. C., and Szente. A. (1967) Helv. Chim. Artu 50,
8.
Shaw. E. H.. Jr., Daniels, D.. and Sawyer, L. D.
9.
Fedorov. A. A.. and Pavlinova. A. V. (1972) Zh.
1027-
1038.
(1%7) An&.
Proc~. S. Dak. Khim.
Acud.
Sci.
46, 174- 175.
27, 2409-2413.
IO.
Mikesova. M.. and BartuSek, M. (1978) Collec,f.
Il.
Hedman, B. (1974) Acru
Czech.
Chum.
Commun.
43, 1867-1877. Chem. Sand.
Ser.
A
28, 591-592. 12.
Chalmers, J. Inorg.
R. A., and Sinclair. NW/.
Chem.
A. G. (1967)
29, 2065-2080.
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