Technical Note
Nephron 1992;62:104-107
Forschungslaboratorien der Boehringer-Mannheim GmbH, Mannheim, BRD
Key Words Inulin Inuiinase Inulin determination Inulin clearance
Fully Enzymatic Inulin Determination in Sm all Volum e Sam ples without Deproteinization
Abstract A fully enzymatic method for the determination of inulin in serum or plasma without deproteinization is described. The assay is carried out by means of a fructose determination after hydrolysis of inulin via inuiinase and simultaneous oxidation of the native glucose using glucose oxidase and H20 2. This time-saving procedure of high specificy, sensitivity and accuracy requires only small sample volumes. The usefulness of the method is shown by analysis of blood levels in humans and rabbits after an intravenous administration of Inutest®.
Introduction The classical compound for the determination of glom erular filtration rate (GFR) is inulin [1-4]. As this substance is largely pharmacologically inert, practically not bound to plasma protein and filtered exclusively via the glomeruli, it is extremely useful for GFR investigations. These advan tages have been compromised to a certain extent by the fact that the methods available for the determination of inulin are either difficult to use or inaccurate and nonspecific. To overcome these problems a fully enzymatic determination of inulin without deproteinization was developed. This procedure of high specifity, sensitivity and accuracy re quires only small serum volumes and can be carried out in any laboratory equipped with a photometer.
Inulin Determination Preliminary Comments To determine the pT .fructosan inulin this compound must be hydrolyzed into its fructose monomers by means o f inuiinase [5, 6]. Subsequently, the fructose moiety is enzymatically converted to glucose-6-Pby hexokinase (H K), which can be determined with the aid of
Accepted : October 31.1991
glucose-6-P-dehydrogenase (G-6-PDH). In order to avoid a separate determination o f glucose, which is always present in serum samples in varying concentrations, the native glucose is oxidized with glucose oxidase (GOD) and H20 2 during hydrolysis o f inulin. This is possible for serum glucose concentrations up to 700 mg/100 ml.
Glucose + H 2O 2
gluconate + H20
I C A O .) , ♦ n H:0 LI 1/
Fructose + ATP . . > fructose-6-P + ADP Mg-+ Fructose-6-P
PGI
glucose-6-P
G lucose-6-P+ NADP
G-6-PDH
gluconate-6-P+ NADPH2
Reagents (a) GOD (from Aspergillus niger. EC 1.1.3.4), purity grade I, lyophilized; (b) inuiinase (Novozym SP 230, Novo Industries, Cat. No. 811221, EC 3.2.1.7), activity: 3,000 U /g ; (c) H2O 2 (30%); (d) citric acid/sodium citrate; (e) test combination for glucose-fructose deter mination (Boehringer-Mannheim GmbH, Cat. No. 139106, food anal ysis).
Dr. H .F. Kuehnle Boehringer-M annheim G m bH Medizinische Forschung/Stoffwechsel S andhofcrStrasse 116, D-W-6800 Mannheim 31 (FRG)
© 1992 S. K arger AG, Basel 0028-2766/92/ 062I-0104S2.75/0
Downloaded by: Thomas Jefferson University Scott Library 147.140.20.32 - 1/24/2018 4:04:00 PM
H. F. Kuehnle K. V. Dahl F. H. Schmidt
Table 1. Precision of the inulin determi nation with inulinase in aqueous solution and in pooled rabbit serum
Preparation o f the Solutions Citrate buffer 0.2 M, pH 5.0: (a) 4.2 g citric acid (C6Hs0 7- H:0 ) are dissolved in distilled water up to 100 ml; (b) 5.88 g sodium citrate (C(,H?Na(0 7 - 2 H:0 ) are dissolved in water up to 100 ml: (c) 60 ml sodium citrate solution are mixed with 25 ml of the citric acid solution: the mixture is adjusted to pH 5.0. Test combination for the fructose determination: Bottle 1: TRA buffer, MgSOj, NADP, ATP are dis solved in 80 ml H:0 : bottles 2 + 3: H K /G -6-PD H /PG I are mixed in the ratio 1:1 and used undiluted. Incubation Media Medium I (without H20 2) Citrate buffer0.2 M. pH 5.0 GOD (about 250 U /m g) Novozym230 Medium II (with H20 2) Citrate buffer 0.2 M, pH 5.0 H:Oi (30%)
n Mean mg/ml SD SEM
cv
Rabbit serum
10 0.49 0.005 0.002 1.03
10 0.49 0.004
0.001 0.82
10.0 ml 5.0 mg 0.2 ml 5.0 ml 0.05 ml
Table 2. Results o f the inulin determina tion with inulinase in aqueous solution and pooled rabbit serum at various concentra tions (means o f 10 determinations each)
The incubation media 1 and II are stable at refrigerator tempera tures for at least I week. Procedure Sample Volumes. For determinations in man a uniform sample volume o f 0.1 ml serum or plasma can be used. With such a sample volume it is possible to determine the elimination kinetics o f inulin after intravenous administration o f 5 g/patient over a period up to 4 h. During this period the serum concentration in healthy subjects ranges approximately from 1.0 to 0.02 mg/ml. Due to the faster clearance compared to man a relatively high dose o f inulin should be used in small animal species. In orderto avoid exceeding the measuring range a 1:10 dilution of serum or plasma with saline is recommended if the expected value is above 1 mg/ml. Oxidation and Hydrolysis Reaction mixture
Spiked inulin mg/ml
Recovered aqueous solution
Rabbit serum
0.05 0.1 0.2 0.5 1.0
0.05 0.10 0.20 0.49 0.96
0.06 0.10 0.21 0.49 0.99
Animal samples: sample volume = 0.05 ml; cuvette volum e= 1.22 ml. AA(rua0M.x 1.183 = mg inulin/ml. With the diluted samples, the result must be multiplied by 10. Humans
Small animals
0.1
0.05
0.2
0.10
0.1
0.05
The reaction mixtures are mixed (e.g. Eppendorf mixer) and are incubated initially for 60 min at 37°C. Determination o f Fructose The determination is carried out at a wavelength of 365 nm. The following are pipetted into the incubation mixture: solution 1(bottle 1): 1-0 ml. This is well mixed and the entire mixture is transferred to a semi-micro cuvette. Read A ,: Addition o f the enzyme mixture (bottles 2 and 3) H K/G-6- P D H / PG1:0.02 ml. mix and wait for the completion o f reaction (about 10-20 min). Read A2: If the reagent blank cannot be ignored, then it has to be taken into account in the calculation:
A| —A?—AAj,|,n^■ A(nlctosc Calculation Human samples: sample voluine = 0.1 ml: cuvette volume = 1.42 ml. AA(ruc,osc x 0.688 = mg inulin/ml.
Results Precision. Table I shows the means and variations of 10 inulin determinations with inulinase from aqueous solution or pooled rabbit serum. The coefficients of variation were found to be between 0.8 and 1%. Recovery. Table 2 shows the results of inulin determina tions in various concentrations using inulinase in aqueous solution and pooled rabbit serum. The values documented show an excellent recovery rate in all concentration ranges. Examples. Figures 1and 2 show respectively the concen tration time curves of inulin in a healthy volunteer and in a patient with impaired renal clearance: 5 g of inulin was given as a rapid intravenous injection. Figure 3 shows the concentration time curve of inulin in a rabbit after a prime dose of Inutest® followed by a constant infusion of this compound.
105
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Sample, ml Incubation medium I, ml Incubation medium II, ml
Aqueous solution
Fig. 1. Concentration o f inulin in the serum o f a healthy volunteer after a rapid intravenous injection of Inutest (G F R = 118 m l/min).
Fig. 2. Concentration o f inulin in the serum o f a patient with impaired renal clearance after a rapid intravenous injection o f Inutest (GFR = 38 m l/m in).
Discussion
106
Fig. 3. Concentration o f inulin in the serum o f a rabbit after a prime dose o f 80 mg/kg Inutest followed by a constant infusion of 7 mg/min (G F R =9.I m l/min).
enzymatic determination of fructose. Using this method it is therefore possible to carry out a determination of inulin clearance with the aid of the single shot technique on only a few blood samples in an analogue manner to the radioac tive procedure.
Kuehnle/Dahl/Schmidt
Fully Enzymatic Inulin Determination
Downloaded by: Thomas Jefferson University Scott Library 147.140.20.32 - 1/24/2018 4:04:00 PM
Various procedures are known for the determination of inulin in biological samples. The methods using resorcinol [7], diphenylamine [8] and anthrone [9] are more or less nonspecific or inaccurate and can therefore be regarded as obsolete. Considerably better results are obtained by enzy matic determination of fructose with HK, PGI and G-6PDH after acid hydrolysis of the inulin. Glucose, which is always present in serum samples must, however, be taken into account and determined in a separate procedure. The first step in all the methods mentioned above is the acid hydrolysis of the polyfructosan inulin, which complicates the handling of samples considerably. As the hydrolysis is carried out at very low pH values, the sample must be deproteinized. Thereby small quantities of inulin may be precipitated. The fully enzymatic inulin determination method reported here does not show any of the disadvan tages of the procedures mentioned above. Both the glucose oxidation and the enzymatic hydrolysis of the inulin are carried out at the same time at 37°C at a mildly acid pH. This means that deproteinization can be omitted. Differen tial analysis is also avoided by oxidation of native glucose. This contributes considerably to the accuracy of the method as the concentration of native glucose can exceed the inulin concentration to be determined more than 100 times. An extremely high specificity is achieved by the subsequent
References 4
Price M, Schwartz R, Hoyt H: Evaluation and characteristics of currently available inulin. In vest Urol 1978;16:13-14. 5 Zittan L: Enzymatic hydrolysis of inulin - an alternative way to fructose production. Starch/ Stärke 1981:33:373-377. 6 Guiraud JP, Galzy P: Enzymatic hydrolysis of plant extracts containing inulin. Enzyme Microb Technol 1981;3:305-308.
7
8
9
Schreiner GF: Determination of inulin by means of resorcinol. Proc Soc Exp Biol Med 1950:74:117. Harrison HD: A modification of the diphenylamine method for determination of inulin. Proc Soc Exp Biol Med 1942:48:111. Davidson WD, Sackner MA: Simplification of the anthrone method for the determination of inulin in clearance studies. J Lab Clin Med 1963:62:351-356.
107
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1 Richards AN, Westfahl BB, Botl PA: Renal excretion of inulin, creatinine and xylose in nor mal dogs. Proc Soc Exp Biol 1934;32:73-75. 2 Shannon JA, Smith W: The excretion of inulin, xylose and urea by normal and phlorizinized man. J Clin Invest I935;I4:393. 3 Digregorio GJ: Agents used in liver, renal and cardiac diseases: in Dipalma JR (ed) Drill's Pharmacology in Medicine, ed. 4. New York. McGraw-Hill, 1971, pp 1851-1861.
Nephron 1992;62:104-107
Forschungslaboratorien der Boehringer-Mannheim GmbH, Mannheim, BRD
Key Words Inulin Inuiinase Inulin determination Inulin clearance
Fully Enzymatic Inulin Determination in Sm all Volum e Sam ples without Deproteinization
Abstract A fully enzymatic method for the determination of inulin in serum or plasma without deproteinization is described. The assay is carried out by means of a fructose determination after hydrolysis of inulin via inuiinase and simultaneous oxidation of the native glucose using glucose oxidase and H20 2. This time-saving procedure of high specificy, sensitivity and accuracy requires only small sample volumes. The usefulness of the method is shown by analysis of blood levels in humans and rabbits after an intravenous administration of Inutest®.
Introduction The classical compound for the determination of glom erular filtration rate (GFR) is inulin [1-4]. As this substance is largely pharmacologically inert, practically not bound to plasma protein and filtered exclusively via the glomeruli, it is extremely useful for GFR investigations. These advan tages have been compromised to a certain extent by the fact that the methods available for the determination of inulin are either difficult to use or inaccurate and nonspecific. To overcome these problems a fully enzymatic determination of inulin without deproteinization was developed. This procedure of high specifity, sensitivity and accuracy re quires only small serum volumes and can be carried out in any laboratory equipped with a photometer.
Inulin Determination Preliminary Comments To determine the pT .fructosan inulin this compound must be hydrolyzed into its fructose monomers by means o f inuiinase [5, 6]. Subsequently, the fructose moiety is enzymatically converted to glucose-6-Pby hexokinase (H K), which can be determined with the aid of
Accepted : October 31.1991
glucose-6-P-dehydrogenase (G-6-PDH). In order to avoid a separate determination o f glucose, which is always present in serum samples in varying concentrations, the native glucose is oxidized with glucose oxidase (GOD) and H20 2 during hydrolysis o f inulin. This is possible for serum glucose concentrations up to 700 mg/100 ml.
Glucose + H 2O 2
gluconate + H20
I C A O .) , ♦ n H:0 LI 1/
Fructose + ATP . . > fructose-6-P + ADP Mg-+ Fructose-6-P
PGI
glucose-6-P
G lucose-6-P+ NADP
G-6-PDH
gluconate-6-P+ NADPH2
Reagents (a) GOD (from Aspergillus niger. EC 1.1.3.4), purity grade I, lyophilized; (b) inuiinase (Novozym SP 230, Novo Industries, Cat. No. 811221, EC 3.2.1.7), activity: 3,000 U /g ; (c) H2O 2 (30%); (d) citric acid/sodium citrate; (e) test combination for glucose-fructose deter mination (Boehringer-Mannheim GmbH, Cat. No. 139106, food anal ysis).
Dr. H .F. Kuehnle Boehringer-M annheim G m bH Medizinische Forschung/Stoffwechsel S andhofcrStrasse 116, D-W-6800 Mannheim 31 (FRG)
© 1992 S. K arger AG, Basel 0028-2766/92/ 062I-0104S2.75/0
Downloaded by: Thomas Jefferson University Scott Library 147.140.20.32 - 1/24/2018 4:04:00 PM
H. F. Kuehnle K. V. Dahl F. H. Schmidt
Table 1. Precision of the inulin determi nation with inulinase in aqueous solution and in pooled rabbit serum
Preparation o f the Solutions Citrate buffer 0.2 M, pH 5.0: (a) 4.2 g citric acid (C6Hs0 7- H:0 ) are dissolved in distilled water up to 100 ml; (b) 5.88 g sodium citrate (C(,H?Na(0 7 - 2 H:0 ) are dissolved in water up to 100 ml: (c) 60 ml sodium citrate solution are mixed with 25 ml of the citric acid solution: the mixture is adjusted to pH 5.0. Test combination for the fructose determination: Bottle 1: TRA buffer, MgSOj, NADP, ATP are dis solved in 80 ml H:0 : bottles 2 + 3: H K /G -6-PD H /PG I are mixed in the ratio 1:1 and used undiluted. Incubation Media Medium I (without H20 2) Citrate buffer0.2 M. pH 5.0 GOD (about 250 U /m g) Novozym230 Medium II (with H20 2) Citrate buffer 0.2 M, pH 5.0 H:Oi (30%)
n Mean mg/ml SD SEM
cv
Rabbit serum
10 0.49 0.005 0.002 1.03
10 0.49 0.004
0.001 0.82
10.0 ml 5.0 mg 0.2 ml 5.0 ml 0.05 ml
Table 2. Results o f the inulin determina tion with inulinase in aqueous solution and pooled rabbit serum at various concentra tions (means o f 10 determinations each)
The incubation media 1 and II are stable at refrigerator tempera tures for at least I week. Procedure Sample Volumes. For determinations in man a uniform sample volume o f 0.1 ml serum or plasma can be used. With such a sample volume it is possible to determine the elimination kinetics o f inulin after intravenous administration o f 5 g/patient over a period up to 4 h. During this period the serum concentration in healthy subjects ranges approximately from 1.0 to 0.02 mg/ml. Due to the faster clearance compared to man a relatively high dose o f inulin should be used in small animal species. In orderto avoid exceeding the measuring range a 1:10 dilution of serum or plasma with saline is recommended if the expected value is above 1 mg/ml. Oxidation and Hydrolysis Reaction mixture
Spiked inulin mg/ml
Recovered aqueous solution
Rabbit serum
0.05 0.1 0.2 0.5 1.0
0.05 0.10 0.20 0.49 0.96
0.06 0.10 0.21 0.49 0.99
Animal samples: sample volume = 0.05 ml; cuvette volum e= 1.22 ml. AA(rua0M.x 1.183 = mg inulin/ml. With the diluted samples, the result must be multiplied by 10. Humans
Small animals
0.1
0.05
0.2
0.10
0.1
0.05
The reaction mixtures are mixed (e.g. Eppendorf mixer) and are incubated initially for 60 min at 37°C. Determination o f Fructose The determination is carried out at a wavelength of 365 nm. The following are pipetted into the incubation mixture: solution 1(bottle 1): 1-0 ml. This is well mixed and the entire mixture is transferred to a semi-micro cuvette. Read A ,: Addition o f the enzyme mixture (bottles 2 and 3) H K/G-6- P D H / PG1:0.02 ml. mix and wait for the completion o f reaction (about 10-20 min). Read A2: If the reagent blank cannot be ignored, then it has to be taken into account in the calculation:
A| —A?—AAj,|,n^■ A(nlctosc Calculation Human samples: sample voluine = 0.1 ml: cuvette volume = 1.42 ml. AA(ruc,osc x 0.688 = mg inulin/ml.
Results Precision. Table I shows the means and variations of 10 inulin determinations with inulinase from aqueous solution or pooled rabbit serum. The coefficients of variation were found to be between 0.8 and 1%. Recovery. Table 2 shows the results of inulin determina tions in various concentrations using inulinase in aqueous solution and pooled rabbit serum. The values documented show an excellent recovery rate in all concentration ranges. Examples. Figures 1and 2 show respectively the concen tration time curves of inulin in a healthy volunteer and in a patient with impaired renal clearance: 5 g of inulin was given as a rapid intravenous injection. Figure 3 shows the concentration time curve of inulin in a rabbit after a prime dose of Inutest® followed by a constant infusion of this compound.
105
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Sample, ml Incubation medium I, ml Incubation medium II, ml
Aqueous solution
Fig. 1. Concentration o f inulin in the serum o f a healthy volunteer after a rapid intravenous injection of Inutest (G F R = 118 m l/min).
Fig. 2. Concentration o f inulin in the serum o f a patient with impaired renal clearance after a rapid intravenous injection o f Inutest (GFR = 38 m l/m in).
Discussion
106
Fig. 3. Concentration o f inulin in the serum o f a rabbit after a prime dose o f 80 mg/kg Inutest followed by a constant infusion of 7 mg/min (G F R =9.I m l/min).
enzymatic determination of fructose. Using this method it is therefore possible to carry out a determination of inulin clearance with the aid of the single shot technique on only a few blood samples in an analogue manner to the radioac tive procedure.
Kuehnle/Dahl/Schmidt
Fully Enzymatic Inulin Determination
Downloaded by: Thomas Jefferson University Scott Library 147.140.20.32 - 1/24/2018 4:04:00 PM
Various procedures are known for the determination of inulin in biological samples. The methods using resorcinol [7], diphenylamine [8] and anthrone [9] are more or less nonspecific or inaccurate and can therefore be regarded as obsolete. Considerably better results are obtained by enzy matic determination of fructose with HK, PGI and G-6PDH after acid hydrolysis of the inulin. Glucose, which is always present in serum samples must, however, be taken into account and determined in a separate procedure. The first step in all the methods mentioned above is the acid hydrolysis of the polyfructosan inulin, which complicates the handling of samples considerably. As the hydrolysis is carried out at very low pH values, the sample must be deproteinized. Thereby small quantities of inulin may be precipitated. The fully enzymatic inulin determination method reported here does not show any of the disadvan tages of the procedures mentioned above. Both the glucose oxidation and the enzymatic hydrolysis of the inulin are carried out at the same time at 37°C at a mildly acid pH. This means that deproteinization can be omitted. Differen tial analysis is also avoided by oxidation of native glucose. This contributes considerably to the accuracy of the method as the concentration of native glucose can exceed the inulin concentration to be determined more than 100 times. An extremely high specificity is achieved by the subsequent
References 4
Price M, Schwartz R, Hoyt H: Evaluation and characteristics of currently available inulin. In vest Urol 1978;16:13-14. 5 Zittan L: Enzymatic hydrolysis of inulin - an alternative way to fructose production. Starch/ Stärke 1981:33:373-377. 6 Guiraud JP, Galzy P: Enzymatic hydrolysis of plant extracts containing inulin. Enzyme Microb Technol 1981;3:305-308.
7
8
9
Schreiner GF: Determination of inulin by means of resorcinol. Proc Soc Exp Biol Med 1950:74:117. Harrison HD: A modification of the diphenylamine method for determination of inulin. Proc Soc Exp Biol Med 1942:48:111. Davidson WD, Sackner MA: Simplification of the anthrone method for the determination of inulin in clearance studies. J Lab Clin Med 1963:62:351-356.
107
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1 Richards AN, Westfahl BB, Botl PA: Renal excretion of inulin, creatinine and xylose in nor mal dogs. Proc Soc Exp Biol 1934;32:73-75. 2 Shannon JA, Smith W: The excretion of inulin, xylose and urea by normal and phlorizinized man. J Clin Invest I935;I4:393. 3 Digregorio GJ: Agents used in liver, renal and cardiac diseases: in Dipalma JR (ed) Drill's Pharmacology in Medicine, ed. 4. New York. McGraw-Hill, 1971, pp 1851-1861.
![[Amniotic fluid volume determination using inulin].](/assets/img/hold.png)
