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A new method for the determination of inulin in plasma and urine
470
CLINXCA CHIMICA ACTA
A NEW
METHOD
FOR THE IN PLASMA
ANTONIN
DETERMINATION
VOL. 1 (qj0)
OF l[NULIN
AND URINE
HEYROVSKY
Lahwatory of the Second. Medical Clinic, Charles University, Prague (Czechoslovakia)
The wide use of inulin for the measurement of glomerular filtration rate emphasizes the importance of a simple method for the determination of this substance. All the existing methods so far published for the determination of inulin are based upon estimation of fructose. This is accomplished by methods which make use of the reducing power of the fructose derived from the hydrolysis of inulinr~ 2, the reaction with diphenylamine that gives a bIue color+@ or the reaction with resorcinol that gives an orange-red col0rl0-~7. The color reaction of inulin with anthrone was aiso usedX8. The methods for the dete~ination of inulin by reduction and by formation of color with diphenyl~ine or anthrone suffer from the disadvantage that glucose is an interfering substance that must be removed. The removal of glucose is performed usually by means of the treatment with yeast. The procedure become long and often cumbersome because of the necessity of washing the yeast several times and detcrmining the volume of the cells in the yeast suspension by means of a hematocrit. This is absolutely necessary in order to determine the degree of dilution of the sample by the yeast suspension. The methods based on the reaction with resorcinol are sufficiently specific for fructose in the presence of the amounts of glucose found in normal blood and urine. These methods using resorcinol alone, resorcinol and thiourea, or resorcinol and ferric iron all suffer from the drawback that they require a very rigid temperature control during the final step of color development. Hence, in the routine practice of a clinical laboratory they are not as exact in most cases as they should be. During an investigation of the possibilities of determining carbohydrates by means of indole derivatives, it was found in the author’s laboratoryl* that /l-indolyiacetic acid is an excellent reagent for detection and for photometric determination of small amounts of fructose, with which it gives a bea~ltif~ll pur~Ie-blue color in the presence of concentrated hydrochloric acid. This reaction is of relatively high specificity and sensitivity. From a great number of compounds occurring in biologicaimaterials only fructose and fructose-containing saccharides (e.g. saccharose, inulin) give the reaction in comparable concentrations. The same color is given also by sorbose and tagatose but it is of different intensity from that given by equal amounts of fructose*v. Nitrites and nitrates in high concentrations not occurring in the material in question interfere by giving a somewhat similar color, which differs from the color obtained with fructose, especially in its diminished stability. The interference by gluIn view of these properties of the color, the reaccase is of the order of o.pr.o%. tion was used also for the determination of inulin during renal clearance studies. References p. 474
VOL.
1 (1g$)
INULIN
IN PLASMA AND URINE
471
EXPERIMENTAL
I. /3-indolylacetic acid. 0.5 g of the pure white preparation is dissolved in IOO ml of 46% ethanol. The solution is stable even at room temperature for many months. Only pure white substance should be used. Coloured commercial preparations must be recrystallized from hot dilute ethanol with the addition of charcoal. 2. Concentrated hy~ochloric acid (37% w /v ) was used throughout; grades “pro analysi” and “puriss.” were equally satisfactory. 3. Standard in&in solutions. A stock solution containing I mg of inulin (carefully dried in a vacuum desiccator prior to use) per I ml is diluted before use, I 150, 2 :50, 3 :50, 4 :50, 5 150, to obtain working standards containing 0.02; 0.04; 0.06; 0.08; 0.10 mg of inulin per 1 ml.
Procedwe
To I ml of plasma filtrate or diluted urine containing between 0.01 and 0.1 mg of inulin is added 0.2 ml of ,&indolyl acetic acid and 8 ml of concentrated hydrochloric acid. The contents of the tubes are mixed and left to stand at room temperature over-
Fig. 1. Spectrum
of
the color
given by
inulin and /Lindolylaceticacid in strong hydrochloricacid.
night, The diluted standard solutions of inulin are treated in the same way. Plasma blank and urine blank are prepared from plasma and urine obtained before the administration of inulin. A reagent blank is prepared by substituting I ml of distilled water for r ml of the sample. Plasma may be deproteinized with CdSO,-NaOH, ZnSO,-NaOH or with trichloracetic acid. In most cases the dilution during deproteinization was maintained at I :5. For urine a dilution of I :IOO is satisfactory in the most experiments. Since proteins do not interfere, urine is not deproteinized even if proteinuria is present. When plasma was not deproteinized but only diluted I :5, errors were introduced since a light turbidity developed in the solution. Even with urines from patients with severe proteinuria no turbidity was observed in the solutions when the urines were diluted I : ICO. After standing overnight at room temperature the extinctions of the purpleK~ftmnces p. 474
A. HEYROVSKY
472
VOL. 1 (rc)$)
violet solutions are read with a photometer at the wave length 5300 A. Alternatively, the color may be developed in a waterbath at a constant temperature not exceeding 37’ C, the readings at 37’ C being performed after ho-80 minutes. From the standard solutions, which must be run simultaneously in each series of determinations, the inulin content of the samples is determined. After subtraction of the (in most cases El
10 Fig. z. Rate of formation
20
30 hours
of the color between inulin and /I-indolylacetic acid at room temperature.
acid in strong hydrochloric
very small) plasma blank and urine blank the inulin value is multiplied by the dilution used and by IOO to obtain the inulin content per IOO ml of the sample. DISCUSSION The purple-violet color given by fructose or fructose saccharides and P-indolylacetic acid in concentrated hydrochloric acid is in many ways similar to the color given under similar conditions by fructose and scatolen’. Nevertheless, it seems to be more sensitive. In addition, the reagent is easily available commercially in sufficient purity and is odorless in contradistinction to scatole, which is difficult to use in daily work owing to its very intense unpleasant odor. The color obtained in the new method is of excellent quality for photometric work. An absorption curve of the color is shown in Fig. I. Maximum absorption occurs at 5300 A. As can be seen in Fig. 3 the calibration graph is in good accord with the Lambert-Beer law. The intensity of the color depends on the concentration of hydrochloric acid, on the time of the development of the color and on the temperature. The best results were obtained with 8 ml of concentrated hydrochloric acid (37%) per I ml of sample*9; with ZOO/~ HCl only a faint color was formed at room temperature. At room temperature the intensity of the color increases slowly (Fig. 2) ; maximum intensity is attained after about 15-18 hours, the color then being stable for at least 20 hours. But it is not necessary to read the optical density of the solutions at maximum intensity, since the Lambert-Beer law holds even when the color is not at its maximum (see Fig. 3) and the standards are run simultaneously with each series of samples. At o0 C only a trace of color is formed during 24 hours, at 37’ C the maximum intensity of the color is attained after about 60-80 minutes, at 60” after 6 minutes, at Kefeercnces p. 474
VOL. 1 (1956) 100’
during the first minute.
INULIN IN PLASMA AND URINE
At higher temperatures
473
the colors fade rather rapidlyIs,
deviations from linearity are often observed and the interference from glucose and other sugars becomes serious. Therefore room temperature overnight or 37’ C for 60 minutes was adopted for the development of the coloP. At room temperature glucose gives about 0.57~ of the color intensity given by
0.1
0.2mg
Fig. 3. Calibration graphs for inulin. The color was formed at room temperature. 525 m/i were performed after (a) I hour; (b) 4 hours; (c) 7 hours; (d) 14 hours; (f) 26 hours.
Readings at (e) 19 hours;
fructose; other sugars (except saccharides containing fructose, tagatose or sorbose) give only o-02% of the color given by an equal amount of fructose. Dextrans give at about 0.2% of the extinction given by an equal amount of inulin. Between 60” C and 100’ C sugars other than fructose interfere markedly; glucose, for example, yields 16-25% of the extinction of an equal amount of inulin or fructose. Experiments upon the recovery of known amounts of inulin added to urine or plasma showed that the new method is highly reliable for the determination of inulin in plasma and urine during clearance studies. The extinction values of the blanks for urine and plasma were in most cases negligible, differing only insignificantly from the blanks for reagents, thus showing that there is no error from normal constituents of these fluids in the procedure. Using the blank for urine in recovery experiments with urines containing 10% glucose or with plasma containing 1000 rng% glucose it was possible to determine the added amounts of inulin within ~-4%. In a number of recovery experiments the error of the method was about 1%. Using special microcuvettes and reducing the volume of the sample and of reagents it was possible to determine amounts of between o and 5 ,ug inulin, which corresponded to 0.02-0.1 ml of plasma or 0.0005-0.01 ml of urine, within an experimental error not exceeding 10%. SUMMARY
A new rapid and convenient method is described for the determination of inulin in plasma and urine. The determination is based on the formation of a purple-violet color by fructose with ,%indolylacetic acid in concentrated hydrochloric acid. Some advantages of the new method are discussed. References
p. 474
474
VOL. 1 (1g5h)
A. HEYROVSKY
L’auteur decrit une nouvelle methode rapide et commode pour l’inuline dans le plasma sanguin et dans l’urine. Le dosage est base sur d’une substance violet-pourpre qui resulte de la reaction du fructose P-indolyladtique en milieu d’acide chlorhydrique concentre. Quelques la nouvelle
methode
le dosage de la formation avec l’acidc avantages de
sont discutb. ZUSAMMENFASSUNG
Eine neue schnelle und bequeme Methode zur Bestimmung von Inulin in Plasma und Harn wird beschrieben. Die Bestimmung benatzt die Bildung eines purpurvioletten Farbstoffes der aus Fructose und /3-Indolylessigsaure in konzentrierter saure entsteht. Einige Vorteile der neuen Methode werden kurz besprochen.
Salz-
PE3K)ME AaHo 0nxaHxe ~0~0r0 6bIcTpoTo IIy~o6aoro MeToAa onpenenesllx HHyAxxzia~ nna3Me II Mo9e. OnpeaeheHTne ocHoBaH0 Ha noasheHuti nypnypH0 ~~owe~o~oT0 UBeTa AaaaeMoro COhsHOii KMCAOTe. +pyKTOBOfi C ,%IIH~OXU,-yKcyCHOi?i KIlCAOTOii B KOHgeHTpHpOBaHHOii 06cyx,aaIoTca HeKOTOpbIe KIpeHMy~CXTsa BTOI-0 HOBOrO MeTO,Z@.. KEFERESCES I J. A. SHANNON AND H. W. SMITH, J. Clin. Invest., 14 (1935) 393. 2 I?. MENNE, 0. WETTER, 0. CRAMER AND L. FISCHER, Klin. Wochschr., 30 (1952) 603. 3 A. S. ALVING, J. RUBIN AND B. F. MILLER, J. Biol. Chew, 127 (1939) 609. 4 H.C. CORCORAN AND I.H.PAGE, 1.BioZ.Chem.,~w (1939) bor. ; H. E. HARRISON, Procy Sac. Exptl.“BBiol. Med., 49 (1949) III. 6 D. ROLF. A. SURTSHIN AND H. L. WHITE, Proc. Sot. E-xptl. Biol. Med., 73 (1949) 3 jI 7 J. M. LITTLE, .I.Biol. Chem., 180 (1949) 747. 8 F. K. HERBERT,B&~~~. J., 32 (1938) 815. 9 E. BOJESEN, Acta Med. Stand., 142 (195~) suppl. 260, 275 IO K. STEINITZ, J.Biol.Chem.,rz6 (1938) 589. II R. S. HUBBARD AND T. A.LOOMIS, J.Biol. Chem.,145 (1942) 641. 12 P. KRUHOFER, Acta Physiol. Stand., II (1946) I. 13 J. H. ROE, J. H. EPSTEIN AND X. P. GOLDSTEIN, J. BioZ. Chem., 178 (1949) 839. 14 G. Ross AND R. MOKOTOFF, J. Biol. Chem., 190 (1951) 659. 15 A. HIGASHI AND L. PETERS, J.Lab.Clin.Med.,35 (1950) 475. 16 J. R. K. PREEDY,~. BioZ.Chem.,zro (1954) 651. 17 A. S. ALVING, J, FLOX , I. PITESKI AND F. B. MILLER, .f.Lab. Clin. Med., 27 (1941) IIj 18 R. P. WHITE AND F. E. SAMSON, J.Lab.Clin. Med., 43 (1954) 475. 19 A. HEYROVSKY, Chem. Listy, 50 (‘9.j4) (inpress).
Received
July
Ixth,
1956
CLINXCA CHIMICA ACTA
A NEW
METHOD
FOR THE IN PLASMA
ANTONIN
DETERMINATION
VOL. 1 (qj0)
OF l[NULIN
AND URINE
HEYROVSKY
Lahwatory of the Second. Medical Clinic, Charles University, Prague (Czechoslovakia)
The wide use of inulin for the measurement of glomerular filtration rate emphasizes the importance of a simple method for the determination of this substance. All the existing methods so far published for the determination of inulin are based upon estimation of fructose. This is accomplished by methods which make use of the reducing power of the fructose derived from the hydrolysis of inulinr~ 2, the reaction with diphenylamine that gives a bIue color+@ or the reaction with resorcinol that gives an orange-red col0rl0-~7. The color reaction of inulin with anthrone was aiso usedX8. The methods for the dete~ination of inulin by reduction and by formation of color with diphenyl~ine or anthrone suffer from the disadvantage that glucose is an interfering substance that must be removed. The removal of glucose is performed usually by means of the treatment with yeast. The procedure become long and often cumbersome because of the necessity of washing the yeast several times and detcrmining the volume of the cells in the yeast suspension by means of a hematocrit. This is absolutely necessary in order to determine the degree of dilution of the sample by the yeast suspension. The methods based on the reaction with resorcinol are sufficiently specific for fructose in the presence of the amounts of glucose found in normal blood and urine. These methods using resorcinol alone, resorcinol and thiourea, or resorcinol and ferric iron all suffer from the drawback that they require a very rigid temperature control during the final step of color development. Hence, in the routine practice of a clinical laboratory they are not as exact in most cases as they should be. During an investigation of the possibilities of determining carbohydrates by means of indole derivatives, it was found in the author’s laboratoryl* that /l-indolyiacetic acid is an excellent reagent for detection and for photometric determination of small amounts of fructose, with which it gives a bea~ltif~ll pur~Ie-blue color in the presence of concentrated hydrochloric acid. This reaction is of relatively high specificity and sensitivity. From a great number of compounds occurring in biologicaimaterials only fructose and fructose-containing saccharides (e.g. saccharose, inulin) give the reaction in comparable concentrations. The same color is given also by sorbose and tagatose but it is of different intensity from that given by equal amounts of fructose*v. Nitrites and nitrates in high concentrations not occurring in the material in question interfere by giving a somewhat similar color, which differs from the color obtained with fructose, especially in its diminished stability. The interference by gluIn view of these properties of the color, the reaccase is of the order of o.pr.o%. tion was used also for the determination of inulin during renal clearance studies. References p. 474
VOL.
1 (1g$)
INULIN
IN PLASMA AND URINE
471
EXPERIMENTAL
I. /3-indolylacetic acid. 0.5 g of the pure white preparation is dissolved in IOO ml of 46% ethanol. The solution is stable even at room temperature for many months. Only pure white substance should be used. Coloured commercial preparations must be recrystallized from hot dilute ethanol with the addition of charcoal. 2. Concentrated hy~ochloric acid (37% w /v ) was used throughout; grades “pro analysi” and “puriss.” were equally satisfactory. 3. Standard in&in solutions. A stock solution containing I mg of inulin (carefully dried in a vacuum desiccator prior to use) per I ml is diluted before use, I 150, 2 :50, 3 :50, 4 :50, 5 150, to obtain working standards containing 0.02; 0.04; 0.06; 0.08; 0.10 mg of inulin per 1 ml.
Procedwe
To I ml of plasma filtrate or diluted urine containing between 0.01 and 0.1 mg of inulin is added 0.2 ml of ,&indolyl acetic acid and 8 ml of concentrated hydrochloric acid. The contents of the tubes are mixed and left to stand at room temperature over-
Fig. 1. Spectrum
of
the color
given by
inulin and /Lindolylaceticacid in strong hydrochloricacid.
night, The diluted standard solutions of inulin are treated in the same way. Plasma blank and urine blank are prepared from plasma and urine obtained before the administration of inulin. A reagent blank is prepared by substituting I ml of distilled water for r ml of the sample. Plasma may be deproteinized with CdSO,-NaOH, ZnSO,-NaOH or with trichloracetic acid. In most cases the dilution during deproteinization was maintained at I :5. For urine a dilution of I :IOO is satisfactory in the most experiments. Since proteins do not interfere, urine is not deproteinized even if proteinuria is present. When plasma was not deproteinized but only diluted I :5, errors were introduced since a light turbidity developed in the solution. Even with urines from patients with severe proteinuria no turbidity was observed in the solutions when the urines were diluted I : ICO. After standing overnight at room temperature the extinctions of the purpleK~ftmnces p. 474
A. HEYROVSKY
472
VOL. 1 (rc)$)
violet solutions are read with a photometer at the wave length 5300 A. Alternatively, the color may be developed in a waterbath at a constant temperature not exceeding 37’ C, the readings at 37’ C being performed after ho-80 minutes. From the standard solutions, which must be run simultaneously in each series of determinations, the inulin content of the samples is determined. After subtraction of the (in most cases El
10 Fig. z. Rate of formation
20
30 hours
of the color between inulin and /I-indolylacetic acid at room temperature.
acid in strong hydrochloric
very small) plasma blank and urine blank the inulin value is multiplied by the dilution used and by IOO to obtain the inulin content per IOO ml of the sample. DISCUSSION The purple-violet color given by fructose or fructose saccharides and P-indolylacetic acid in concentrated hydrochloric acid is in many ways similar to the color given under similar conditions by fructose and scatolen’. Nevertheless, it seems to be more sensitive. In addition, the reagent is easily available commercially in sufficient purity and is odorless in contradistinction to scatole, which is difficult to use in daily work owing to its very intense unpleasant odor. The color obtained in the new method is of excellent quality for photometric work. An absorption curve of the color is shown in Fig. I. Maximum absorption occurs at 5300 A. As can be seen in Fig. 3 the calibration graph is in good accord with the Lambert-Beer law. The intensity of the color depends on the concentration of hydrochloric acid, on the time of the development of the color and on the temperature. The best results were obtained with 8 ml of concentrated hydrochloric acid (37%) per I ml of sample*9; with ZOO/~ HCl only a faint color was formed at room temperature. At room temperature the intensity of the color increases slowly (Fig. 2) ; maximum intensity is attained after about 15-18 hours, the color then being stable for at least 20 hours. But it is not necessary to read the optical density of the solutions at maximum intensity, since the Lambert-Beer law holds even when the color is not at its maximum (see Fig. 3) and the standards are run simultaneously with each series of samples. At o0 C only a trace of color is formed during 24 hours, at 37’ C the maximum intensity of the color is attained after about 60-80 minutes, at 60” after 6 minutes, at Kefeercnces p. 474
VOL. 1 (1956) 100’
during the first minute.
INULIN IN PLASMA AND URINE
At higher temperatures
473
the colors fade rather rapidlyIs,
deviations from linearity are often observed and the interference from glucose and other sugars becomes serious. Therefore room temperature overnight or 37’ C for 60 minutes was adopted for the development of the coloP. At room temperature glucose gives about 0.57~ of the color intensity given by
0.1
0.2mg
Fig. 3. Calibration graphs for inulin. The color was formed at room temperature. 525 m/i were performed after (a) I hour; (b) 4 hours; (c) 7 hours; (d) 14 hours; (f) 26 hours.
Readings at (e) 19 hours;
fructose; other sugars (except saccharides containing fructose, tagatose or sorbose) give only o-02% of the color given by an equal amount of fructose. Dextrans give at about 0.2% of the extinction given by an equal amount of inulin. Between 60” C and 100’ C sugars other than fructose interfere markedly; glucose, for example, yields 16-25% of the extinction of an equal amount of inulin or fructose. Experiments upon the recovery of known amounts of inulin added to urine or plasma showed that the new method is highly reliable for the determination of inulin in plasma and urine during clearance studies. The extinction values of the blanks for urine and plasma were in most cases negligible, differing only insignificantly from the blanks for reagents, thus showing that there is no error from normal constituents of these fluids in the procedure. Using the blank for urine in recovery experiments with urines containing 10% glucose or with plasma containing 1000 rng% glucose it was possible to determine the added amounts of inulin within ~-4%. In a number of recovery experiments the error of the method was about 1%. Using special microcuvettes and reducing the volume of the sample and of reagents it was possible to determine amounts of between o and 5 ,ug inulin, which corresponded to 0.02-0.1 ml of plasma or 0.0005-0.01 ml of urine, within an experimental error not exceeding 10%. SUMMARY
A new rapid and convenient method is described for the determination of inulin in plasma and urine. The determination is based on the formation of a purple-violet color by fructose with ,%indolylacetic acid in concentrated hydrochloric acid. Some advantages of the new method are discussed. References
p. 474
474
VOL. 1 (1g5h)
A. HEYROVSKY
L’auteur decrit une nouvelle methode rapide et commode pour l’inuline dans le plasma sanguin et dans l’urine. Le dosage est base sur d’une substance violet-pourpre qui resulte de la reaction du fructose P-indolyladtique en milieu d’acide chlorhydrique concentre. Quelques la nouvelle
methode
le dosage de la formation avec l’acidc avantages de
sont discutb. ZUSAMMENFASSUNG
Eine neue schnelle und bequeme Methode zur Bestimmung von Inulin in Plasma und Harn wird beschrieben. Die Bestimmung benatzt die Bildung eines purpurvioletten Farbstoffes der aus Fructose und /3-Indolylessigsaure in konzentrierter saure entsteht. Einige Vorteile der neuen Methode werden kurz besprochen.
Salz-
PE3K)ME AaHo 0nxaHxe ~0~0r0 6bIcTpoTo IIy~o6aoro MeToAa onpenenesllx HHyAxxzia~ nna3Me II Mo9e. OnpeaeheHTne ocHoBaH0 Ha noasheHuti nypnypH0 ~~owe~o~oT0 UBeTa AaaaeMoro COhsHOii KMCAOTe. +pyKTOBOfi C ,%IIH~OXU,-yKcyCHOi?i KIlCAOTOii B KOHgeHTpHpOBaHHOii 06cyx,aaIoTca HeKOTOpbIe KIpeHMy~CXTsa BTOI-0 HOBOrO MeTO,Z@.. KEFERESCES I J. A. SHANNON AND H. W. SMITH, J. Clin. Invest., 14 (1935) 393. 2 I?. MENNE, 0. WETTER, 0. CRAMER AND L. FISCHER, Klin. Wochschr., 30 (1952) 603. 3 A. S. ALVING, J. RUBIN AND B. F. MILLER, J. Biol. Chew, 127 (1939) 609. 4 H.C. CORCORAN AND I.H.PAGE, 1.BioZ.Chem.,~w (1939) bor. ; H. E. HARRISON, Procy Sac. Exptl.“BBiol. Med., 49 (1949) III. 6 D. ROLF. A. SURTSHIN AND H. L. WHITE, Proc. Sot. E-xptl. Biol. Med., 73 (1949) 3 jI 7 J. M. LITTLE, .I.Biol. Chem., 180 (1949) 747. 8 F. K. HERBERT,B&~~~. J., 32 (1938) 815. 9 E. BOJESEN, Acta Med. Stand., 142 (195~) suppl. 260, 275 IO K. STEINITZ, J.Biol.Chem.,rz6 (1938) 589. II R. S. HUBBARD AND T. A.LOOMIS, J.Biol. Chem.,145 (1942) 641. 12 P. KRUHOFER, Acta Physiol. Stand., II (1946) I. 13 J. H. ROE, J. H. EPSTEIN AND X. P. GOLDSTEIN, J. BioZ. Chem., 178 (1949) 839. 14 G. Ross AND R. MOKOTOFF, J. Biol. Chem., 190 (1951) 659. 15 A. HIGASHI AND L. PETERS, J.Lab.Clin.Med.,35 (1950) 475. 16 J. R. K. PREEDY,~. BioZ.Chem.,zro (1954) 651. 17 A. S. ALVING, J, FLOX , I. PITESKI AND F. B. MILLER, .f.Lab. Clin. Med., 27 (1941) IIj 18 R. P. WHITE AND F. E. SAMSON, J.Lab.Clin. Med., 43 (1954) 475. 19 A. HEYROVSKY, Chem. Listy, 50 (‘9.j4) (inpress).
Received
July
Ixth,
1956