DD270847A7 - METHOD FOR DETERMINING THE GLUCOSE CONTENT OF NON-ENZYMATICALLY GLUCOSYLATED PROTEINS AND REAGENT SET FOR CARRYING OUT THE PROCESS - Google Patents

Abstract

The invention relates to a method for determining the glucose content of non-enzymatically glucosylated proteins, characterized in that the solution in cells closed or water-insoluble proteins with an ionic or non-ionic detergent in solution, the glucose under pressure and using at least one N acid in the form of 5-hydroxymethyl-furfurol, deproteinized the reaction mixture, then the 5-hydroxymethyl-furfurol with an organic compound which forms a colored product with the aldehyde, condensed and finally determined the concentration of the product obtained spectrophotometrically. The invention further relates to a reagent kit for carrying out the above method in which the ratio of sodium dodecyl sulfate, oxalic acid, sulfosalicylic acid and barbituric acid is 0.02: 0.90: 2.0: 0.04, based on 1 unit of the protein to be determined.

Description

The invention relates to a method for the spectrophotometric determination of non-enzymatically glucosylated proteins and to a reagent set for carrying out this method.

Proteins may more or less bind glucose to their reactive groups (in particular to the N-terminal amino groups and the ε-amino groups of the lysine side chain) as a function of the glucose content of the surrounding medium. This process, which takes place in vivo and in vitro, is of great importance because certain properties of the glucosylated proteins (eg functional activity, immunological characteristics and stability) may change. The importance of the in vivo process is attributed above all to the increased, non-enzymatic glucosylation that has developed in diabetes mellitus, which plays an essential role in the development of later complications of this disease. The tracking of the in vitro process is important for the storage and reuse of various proteins, in particular of biological origin (eg, blood, milk, etc.).

In the management of patients suffering from diabetes mellitus and in the prevention of chronic complications, the control of the level of blood sugar control is of fundamental importance. The current blood sugar level measurement is in fact greatly influenced by the daily carbohydrate consumption, so that this measurement does not provide reliable information about carbohydrate bulking of the organism. Also, no information is provided on the location of the period preceding the experiment. For a few years, the determination of the amount of hemoglobin glucosylated at a specific site (N-terminal of the β-chain) (hemoglobin A _{! C} ; furthermore H jAic genanm), ie the amount of fast hemoglobin components, has been assumed as a suitable control method.

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The HbAic and the other glucosylated hemoglobins are formed in the course of postsynthetic glucosylation; their quantity is proportional to the glucose content of the blood. These glucosylated hemoglobins are constantly formed throughout the life of the erythrocytes so that their amount reflects the average value of the approximately three month carbohydrate metabolism. The latter statement represents the most important diagnostic value of the determination.

The above methods are mainly based on the determination of glucosylated Hämoglobinkompononten, namely the HbA _k component and for the determination of other glucosylated proteins unsuitable (LA Trivelli, HM Ranny, HT Lai. New England Jour, of Med ^1971, Volume 284 , Page 353). The majority of these methods are based on the chromatographic separation of the glucosylated hemoglobin; It is, therefore, indicated related value is not the total amount of glucose to hemoglobin gebundenon but of the glucosylated only at the N-terminal amino group of the side chain of hemoglobin (HbA _k). Commercially available reagent kits are suitable only for the specific purpose above, ie for the determination of HbA _tc , and are very sensitive to certain environmental factors (eg temperature of the laboratories / The colorimetric method developed by Flückiger and Winterhalter (FEBS Letters 71,356 (1976) The essence of this method is that the glucose bound to the amino groups of the proteins by a condensation reaction and submerged by an Arrwidori suspension is oxidized under the action of an acidic heat treatment in the form of 5 The method is suitable in this form only for the determination of glucose bound to water-soluble proteins.According to the original method, the determination was calibrated to the result of the chromatographic method and the method was therefore excl was based on the determination of HbAi ₀ . It has been found contrary to the above findings that this method can also be extended to cell-entrapped or water-insoluble proteins, foils previously brought into solution with a dilute ionic or nonionic detergent. The above finding allows the determination of the glucose content of other glucosylated proteins other than hemoglobin.

On the other hand, the evaluation of the method according to the invention is not based on the chromatographic HbA ₁ C method, but only the extinction coefficient of the glucose derivative and the relationship between the formation and decomposition independent of the protein are taken into account.

The method is thereby extended to all proteins and is suitable for determining the total amount of glucose bound to proteins.

Another new feature in our method is that a higher acid concentration than the corresponding, usual in the known methods value of max. 0.6 M combined with a heat treatment carried out under overpressure. Acid hydrolysis may be accomplished with hats of a single acid or with a mixture of organic or inorganic acids, optionally in the presence of a catalyst. As acid can advantageously oxalic acid and acid mixture as a mixture of oxalic acid and acetic acid or oxalic acid .id sulfuric acid find utilization. As a catalyst can, for. B. HgCI ₂ can be used. On the one hand, this embodiment of the invention reduces the duration of the determination and, on the other hand, leads to well reproducible results in all cases.

It was further found during our experiments that the trichloroacetic acid exclusively used in the deproteinization can be advantageously replaced by sulfosalicylic acid. This reagent is much easier to treat and therefore offers technological advantages.

It has furthermore been found that thiobarbituric acid, which has hitherto been used exclusively in the colorimetric determinations, can be replaced by all aromatic compounds (otherwise) condensing with the aldehyde group of the 5-hydroxymethyl-furfurol formed during the acid decomposition and thereby forming an extended double bond system (delocalized n electrons) form a colored product. Barbituric acid has proven to be particularly advantageous for this purpose. It has also been found that in the case of thiobarbituric acid development, the accuracy of the measurement can be increased if the evaluation is carried out at 360 nm. The invention thus relates to a method for determining the glucose content of any non-enzymatically glucosylated proteins by formation of hydroxymethyl furfural by acid treatment, precipitation of the protein and color formation with the Hydroxymothylfurfural. According to the invention, the encapsulated or water-insoluble proteins are dissolved in solution with the aid of an ionic or nonionic detergent which cleaves glucose under excess pressure with at least 1 η acid, preferably in the presence of a catalyst in the form of 5-hydroxymethyl-furfurol. then deproteinating the reaction mixture, deproteinizing 5-hydroxymethyl furfural with an organic compound giving a colored product which condenses, finally determining the concentration of the product obtained spectrophotometrically.

C content is still p ^: h Reagenssatz with which the Serier.messung can always be performed with the same accuracy.

The reagent kit according to the invention consists, separately from each other, of 1-3 parts by weight of an ionic or nonionic detergent, 60-120 parts by weight of a hydrolyzing agent, 150-250 parts by weight of a protein precipitating agent and 2-6 parts by weight of a substance containing hydroxymethyl furfural Color reaction results. The reagent sets according to the invention preferably comprise sodium dodecyl sulfate as detergent, oxalic acid as hydrolysing agent, sulfosalicylic acid as protein precipitating agent, and substance which produces a color reaction with hvdroxymethylfurfural, preferably barbituric acid.

The composition of the reagent kit can be varied because the same chemical procedure can be performed with different reagents.

It was found that the most advantageous reagent kit for detergent determination was 2 ml of a 0.1% sodium dodecyl sulfate, 1 ml of 1N oxalic acid as a hydrolyzing agent, 1 ml of 2% sulfosalicylic acid as deprotein and 2 mg of hydroxymethylfurfural to form the colored product Each compound contains 0.5 ml of 0.05 molar barbituric acid. An advantageous composition of the reagent set for & U measurements is the following: 100 ml 0.1% sodium dodecylsulfate 50 ml 1N oxalic acid

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50ml 20% sulfosalicylic acid

25ml 0.05mg of barbituric acid.

In this reagent set of optimum composition, the ratio of reagents necessary to determine protein-bound glucose is as follows:

Sodium dodecylsulfate oxalic acid sulfosalicylic acid carburic acid = 0.02: 0.9: 2.00: 0.04.

In the rain pack, the hemolysis solution may also contain another detergent (eg Triton X100) instead of sodium dodecyl sulfate. The oxalic acid can be replaced by 2N acetic acid, sulfuric acid or another strong acid. Trichloroacetic acid can also be used to remove the proteins. In the color reaction, instead of the barbituric acid, thiobarbituric acid or another compound forming a colored product with hydroxymethylfurfurol may also be used. The use of the method will be further described by the following examples, without limiting the scope to these examples.

example 1 Determination of the glucose content of non-enzymatically glucosylated hemoglobin

a) 3 ml of heparinized blood are washed with 10 ml of physiological saline, after which 2 ml of 0.1% sodium dodecyl sulfate solution are added to 1 ml of washed erythrocytes. The hemoglobin concentration of the resulting

Hemolysate is determined; to 2 ml of which is added 1 ml of a 1N oxalic acid solution and the mixture is stirred at 112-115 ° C

and under a pressure of 1.6 atm. Subjected to a heat treatment for 2.5 hours. To the hydrolyzate, 1 ml of a 20% sulfosalicylic acid solution is added dropwise with vigorous shaking (temperature 4O ⁰ C). The resulting

The suspension is centrifuged and the light absorption of the supernatant fluid is read at 3S8nm (E, value). To 2ml

0.5 ml of a 0.05 molar barbituric acid solution is added to this liquid and the mixture is incubated at 40 ° C for 40 minutes. The mixture is cooled and the light absorption is read off for 10 minutes at 398 ηm against a blank sample without hemolysis (E ₂ value).

The glucose content (G) is calculated according to the following formula:

G. 2.40 ^E 2Q98) - ° - ^{3 E} 1Q98) '° · ²⁰ _{χ 1Q} -2 _mol

(F) glucose / 100 ml

hemoglobin

(F) = hemoglobin concentration (moles / l).

The constant contains the molar extinction coefficient of the thiobarbituric acid, the correction factor of the acid Decomposition occurred 5-hydroxymethyl-furfur J loss and dilution. If the hemoglobin concentration is determined by Drabkin's method, add 0.02 ml of hemolysate to 5 ml of reagent and

the light absorption bi 540nm measures (Em ₀ ), the formula is as follows:

G = 4.53, _'ol glucose / 100 ml

(E) hemoglobin

In the case of hemoglobin, the normal value is 7.5-12.0 moles glucose / 100 moles hemoglobin.

b) The procedure is as in Example a) with the difference that 0.05 molar thiobarbituric acid is used to determine the glucose and the reaction is monitored at 443 nm.

The following formulas were used for the evaluation:

₀ . _lf5 2 (^)! (<* ") - ° .l» _χ , ₀ -2., _ol

(P) glucose / 100 ml

hemoglobin

G = 2.83 ± iz ±> J _{:. : ol} glucose / 100 ml

() Hemoglobin

c) The procedure is as in Example a), with the difference that one follows the reaction at the more sensitive value of 360 nm. In this case, the following formulas are used:

G - 1.05 ^E 2 ^ 60) "° ' ^{8 E} l (3 &0)" ° ^{>] Λ} _{χ 10} -2. _:oil

(F) glucose / 100 moles

hemoglobin

or.

^E 2C * 60) - ° i8 Βι (χ £ .η) " ^{0) 1} ^ G = 1,98 ² _: moles of glucose / ICO

hemoglobin

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Considering that according to this method, the total amount of glucose bound to hemoglobin is determined, the result obtained is higher than the chromatographically determined or back-related, internationally recognized t used. The conversion factor between the two methods is G / HbA * = 1.8.

Example 2

Determination of non-enzymatically glucosylated proteins of the erythrocyte membrane

a) 10 ml of heparinized blood are washed three times with 50 ml each of a physiological saline solution, after which the precipitate is shaken with eight times the volume of an ice-cold hemolyzing solution and allowed to stand for 5 minutes. The suspension is then centrifuged, the supernatant liquid decanted and the remaining membrane fraction washed four times. The hemolyzing and washing solutions are based on the methods described in J. Physiol. 23 \ 5.551 (1973). The protein content of the hemoglobin-free membrane thus obtained is determined and the concentration of the membrane suspension adjusted to a value corresponding to 2.5 mg / ml protein. Thereafter, to a 2.5 ml membrane fraction, 2 ml of 0.1% sodium dodecyl sulfate and 1 ml of 1N oxalic acid are added, and the mixture is subjected to a heat treatment at 112-115 ° C under pressure for 2.5 hours. 1 ml of a 20% sulfosalicylic acid solution is added to the hydrolyzate (temperature 4O ⁰ C). The resulting suspension is centrifuged, the light absorption of the supematant fluid at 398nm read (E, value). To 2ml of which 0.5 ml of a 0,05molaren barbituric acid are added and the mixture is incubated for 40 minutes at 40 ⁰ C. The mixture is then cooled and the absorbance of light at 398 nm against a blank-prepared blank is read off within 10 minutes (E ₂ value). The glucose content (G) is calculated using the following formula:

G = 2.4-0 iU-PJ χ 10 ^d mol

(F) glucose / 100 rr.g

Protein ι

• ι

where (F) is the concentration of membrane protein (in mg / ml). The constant contains the molar extinction coefficient of the barbituric acid derivative, the correction factor of the 5-hydroxymethyl-furfurol loss which takes place during the acidic decomposition and also the dilution.

b) The preparation of the protein and the determination of glucose are carried out in the manner described in paragraph a), with the difference that 0.5 ml of a 0.05 molar thiobarbituric acid solution is used to determine the sugar derivative. The reaction is monitored at 360 and 443 nm, respectively.

To evaluate the measurement, the following formulas are used depending on the wavelength:

G = 1.05

(F) protein

or.

G = I ₁ SO - ^ ± JZ122 J 10 ^ moles of glucose / ICO ng

(F) protein

Example 3

Determination of glucosylation of plasma proteins

The plasma is separated from the blood. The protein content of blood plasma is determined and the protein concentration adjusted to 10 mg / ml. To 2 ml of 1 ml of 1N oxalic acid solution was added and subjected to 2.5 hours the mixture at 112-115 ⁰ C under pressure to heat treatment. 1 ml of a 0.6 molar aqueous trichloroacetic acid solution is added dropwise to the hydrolyzate, the resulting suspension is centrifuged and the light absorption of the supematant liquid is read off at 398 μm (Ei value). To 2 ml of this is a 0,05molaren barbituric acid is added 0.5 ml and the mixture incubated at 4O ⁰ C for 40 minutes. The mixture is cooled and the light absorbance read off for 10 minutes at 398 against a blank sample prepared without plasma protoin (E ₂ -WeH).

The glucose content (G) is calculated in the manner described in Example 1 a).

Also in the measurement of plasma proteins, the thiobarbituric acid reaction can be used to determine the cleaved glucose derivative. In this case, 0.5 ml of a 0.05 molar thiobarbituric acid solution is added to 2 ml of the supematant fluid. For evaluation, the absorbance values are read at 443 nm and the above formulas are used for evaluation.

Example 4

Determination of the glucose content of non-enzymic glycosylated albumin The plasma is separated from blood by centrifugation. The pH of the plasma is adjusted to 6.9 with a 2 molar hydrochloric acid solution. 15% Polyäth ·, imglykol in the form of a 50% aqueous solution are added dropwise with stirring to the mixture. The resulting suspension is stirred for 20 minutes, centrifuged and the pH of the supematant solution is adjusted to 4.6. After addition of 9% solid polyethylene glycol within one hour, the mixture is stirred for a further 30 minutes. The precipitate is removed by centrifuging and dissolved in a drop of distilled water. The solution is applied to a QAE Sephadex column (20 × 1.6 cm) brought into equilibrium with acetate buffer (pH 5.2). The polyethylene glycol is removed by washing and the albumin is eluted with an acetate buffer (pH 4.8). The glucose content of the albumin solution is determined and to 2 ml of which 1 ml of a 1N oxalic acid solution is added. The mixture is heat treated at 112-115 ° C under pressure for 2.5 hours. The hydrolyzate is 1 ml of a 20%

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Sulfosalicylsaurelösung (temperature 4O ⁰ C) was added, the resulting suspension is centrifuged and the light absorption of the supernatant fluid at 338nm read (Ε, value). To 2n-.i of which 0.5 ml of a 0.05 molar barbituric acid solution is added and the mixture is incubated at 40 ° C for 40 minutes. The mixture is cooled and the absorbance of light at 398 nm read off within 10 minutes against a blank sample prepared without albumin (E ₂ value). The glucose content (G) is calculated according to the following formula:

- 2 40 2Q98) '1Q98) - °: _ ² _{χ 10} -2

'Glucose / 100 mg

Albumin _f

where (A) means the albumin concentration in mg / ml.

The constant contains the molar extinction coefficient of the barbituric acid derivative, the correction factor of the

Acid decomposition 5-hydroxymethyl-furfurol loss also the dilution.

Example 5 Determination of glucosylation of immunoglobulins From clot-inhibited blood, the plasma is separated. The immunoglobulins are carried out after precipitation with rivanol Chromatography on OAE-Sephadex purified. To 2 ml of a 5% immunoglobulin solution is added 1 ml of an acid mixture (1N oxalic acid + 1N acetic acid, or

1N oxalic acid + 1N sulfuric acid). The hydrolysis is carried out for one hour at 100 ⁰ C and the protein precipitated by addition of 1 ml 0,6molarer sulfosalicylic acid. The resulting precipitate is centrifuged at 1500 rpm and to 2 ml of the supernatant fluid is added 0.5 ml of a 0.054 molar recorcinol, ₂ × 10 ^-3 molar CuSO ₄ solution. (Before addition of the reagents, the light absorption of the supernatant liquid is determined at 405 nm [Ei value]).

The reaction mixture is heated at 100 ^° C. for 15 minutes. After cooling, the light absorption at 405nm again

read (E ₂ value).

Glucose content (G) = 37.6 (E ₂ -E, -0.1) mol / 100 moles Examples Determination of glucosylation of hemoglobin To 1 ml of erythrocytes washed with physiological saline are added 2 ml of 0.1% Sodium dodecyl sulfate added. The concentration of the hemolysate is determined (Hb). To 2 ml of it is added 1 ml of one

1N oxalic acid 10 ~ ⁴ molar HgCl ₂ solution is added and the mixture is heated at 100 ⁰ C for one hour. The protein is precipitated with 1 ml of a 20% sulfosalicylic acid solution and removed by centrifugation. The light absorption of the supernatant fluid is determined at 442 nm (Ei). To 2 ml of it is added 0.5 ml of a 0.05 molar thiobarbituric acid egg. The mixture is incubated for 40 minutes at 4O C ⁰ and the light absorption is determined again (E ₂ value).

ρ-Ξ, X 0.8-0.1 ~

χ 10 "^ mol / 100 moles

Hb (mole / 1)

Example 7 Fast method for determining the glucose content of non-enzymatically glucosylated hemoglobin at low Tempbratur Blood anticoagulated with heparin is washed with a physiological saline solution until the supernatant Liquid becomes protein-free. 1 ml of washed erythrocytes are mixed with 2 μl of 0.1% sodium dodecyl sulfate solution

frozen twice and thawed. The concentration of the resulting: hemoglobin solution is determined to 2 ml of this

To the solution are then added 1 ml of 0.3N sulfuric acid and 0.1 ml of 10 " ⁴ M HgCl ₂ solution, and the mixture is allowed to stand for 30 minutes

allowed to stand at 40 ⁰ C. The protein content of the solution is precipitated with 1 ml of 0.6 molar sulfosalicylic acid, and the resulting

Precipitate by centrifuging for 10 minutes at 1500 rpm. The light absorbers of the clays

supernatant fluid is determined at 443nm (E ₁ -WeH). To 2 ml of the solution is added 0.5 ml of a 0.05 molar

Thiobarbituric acid solution added. The solution is allowed to stand at 40 ^J C for 40 minutes. After cooling, the Lichtabso. re-determined within 10 minutes at 443 nm (Ej value). Glucose content:

E - 3, x0, 8 - 0.06 _o

G = 1.38 × 10 -5 moles glucose / 100 moles Hb

Hb (Moie / 1)

Example 8 Determination of glucosylation of hemoglobin The erythrocytes were washed with physiological saline three times, then 3.5 ml of one was added to 2 ml of the sediment

0.1% sodium dodecyl sulfate solution. The hemoglobin concentration was determined and the hemoglobin concentration of the hemolysate (Hb%) was adjusted to a value between 9.0 and 11.0 g / 100 by dilution with concentrated water.

Thereafter, 1.5 ml of sulfuric acid-arsenate solution (0.01 mol of disodium hydrogen-arsenate in 0.3 N sulfuric acid solution) were added to 3.0 ml of the 1 -olysate, the mixture was shaken and incubated for one hour on the boiling water bath. Thereafter, the solution was cooled to 30-4O ⁰ C and 1.5ml of 40% trichloroacetic acid solution was added dropwise while the mixture was stirred with a vibrator. The solution was filtered, and 0.5 ml of 0.025 M thiobarbituric acid solution (solvent: 0.01 N sodium hydroxide solution) was added to 2.0 ml of the filtrate. The mixture was mixed, incubated for 40 minutes at 4O ⁰ C, then cooled to 20 ° C. The light absorption of the sample (E) was read off at 443nm within 30 minutes. The amount of glycosylated hemoglobin (HbAJ) was calculated from the measured values as follows:

HbA _{Ic <}

0.006 χ Hb, %

Claims (12)

1. A method for determining the glucose content of non-enzymatically glucosylated proteins by acidic heat treatment, by formation of hydroxymethyl furfural, precipitation of the protein, then color formation with the hydroxymethyl furfural, characterized in that the cell-enclosed or water-insoluble proteins with a ionic or nonionic detergent which releases glucose under pressure and using at least 1N acid, preferably in the presence of a catalyzer in the form of 5-hydroxymethyl-furfurol, deproteinizes the reaction mixture, then the 5-hydroxymethycfurfurol with an organic compound , which forms a colored product with it, condenses and finally determines the concentration of the product obtained spectrophotometrically. 2. The method according to claim 1, characterized in that one uses as a detergent sodium dodecyl sulfate. (Priority: October 1, 1982.) 3. The method according to claim 1 or 2, characterized in that the acidic hydrolysis under a pressure of 0.12-0.2at. - advantageously 0.15-0, i7at. - Performed with an oxalic acid solution. 4. The method according to claim 1 or 2, characterized in that one carries out the acidic hydrolysis in the presence of a catalyst, preferably mercury (II) chloride. 5. The method according to claim 1 or 2, characterized in that one carries out the acidic hydrolysis in the presence of a catalyst, preferably disodium hydrogen-arsenate. 6. The method according to claim 1 or 2, characterized in that one carries out the acidic hydrolysis with an acid mixture. 7. The method according to any one of claims 1-3, characterized in that one carries out the deproteinization with sulfosalicylic acid. 8. The method according to any one of claims 1-5, characterized in that is used as with the 5-Hydroxymeihyl-furfural a colored product forming organic compound barbituric acid. 9. The method according to claim 7, characterized in that one carries out the spectrophotometric determination at 398 nm. 10. Reagent set for the determination of the glucose content of non-enzymatically glycosylated proteins, characterized in that it separated from 1-3 parts by weight of ionic or nonionic detergent, 60-120 parts by weight of hydrolyzing agent, 150-250 parts by weight with hydroxymethyl furfural a color reaction resulting Substance exists. 11. Reagent set according to claim 10, characterized in that it contains as detergent sodium dodecyl sulfate, as a hydrolyzing agent oxalic acid, as a depottrating agent sulfosalicylic acid and as hydroxymethylfurfural with a color reaction resulting substance barbituric acid. 12. Reagenssatz for carrying out the method according to any one of claims 1-7, characterized in that it bestcnt from 2 parts by weight of sodium dodecyl sulfate, 90 parts by weight of oxalic acid, 200 parts by weight of sulfosalicylic acid and 4 parts by weight of barbituric acid.