FR2508646A1 - METHOD AND EQUIPMENT FOR THE DETERMINATION OF GLYCOSYLATED PROTEINS IN BIOLOGICAL FLUIDS - Google Patents

Abstract

METHOD AND EQUIPMENT FOR THE DETERMINATION OF GLYCOSYLATED PROTEINS IN BIOLOGICAL FLUIDS. THE PRESENCE AND QUANTITY OF NON-ENZYMATIC GLYCOSYLATED PROTEINS IN BIOLOGICAL FLUIDS ARE DETERMINED. THE PRESENCE OF FREE GLUCOSE IN THE TEST SAMPLE IS A DISTURBING FACTOR WHICH IS COMPENSATED EITHER BY PRECIPITATION OF ALL THE PROTEINS IN THE SAMPLE OR BY ENZYMATIC DIGESTION; AS A VARIANT, ACID HYDROLYSIS IS CARRIED OUT FOR A TIME NOT EXCEEDING 2 AND A HALF HOURS. IT IS STILL PROPOSED IN THE CASE WHERE THE PROTEINS ARE PRECIPITATED THAT THE TEST SAMPLE BE INCUBATED IN A WARM AND ACIDIC ENVIRONMENT IN ORDER TO DECREASE AND MAKE UNIFORM THE OPTICAL DENSITY OF THE CONTROL SAMPLES.

Description

Method and equipment for determining the

  glycosylated proteins in biological fluids.

  It is extremely important to keep the amount of glucose in the blood under close control in diabetics who, as we know, exhibit variations in

  blood sugar in a large number of cases.

  Since the evolution of secondary complications

  Progressive dies of diabetes is closely related to the frequency of Cu control of metabolic behavior of the patient, parameters have been investigated that may be able to give results independent of rapid changes in blood glucose as mentioned in the literature,

  and that are generated by a number of variables.

  One way to eliminate the state of glycemic control in these patients is to measure non-enzymatically glycosylated proteins (Yue, K et al, Diabetes, 29: 296, 1980). A reaction of this kind is common to a patient. certain number of proteins and is a process of blocking the

  glucose (Day, J F et al., J Biol Chem 254: 595, 1979).

  The control of this process provides important indications for establishing the degree of metabolic control in diabetics since it has been shown that an increase in blood glucose results in a persistent increase in

glycosylated.

  For measurement, glycosylated hemoglobin (Hb A 1) can be used as a long-term control of blood glucose since the life of this protein is very long (about 120 days). low utility if you want to track the progress of a treatment

  For the latter purpose, it may be more interesting

  to evaluate the degree of glycosylation of albumin since

  its life span is shorter than that of hemoglobin.

  glycosylated hemoglobin is isolated from total hemoglobin by ion exchange chromatography on a weakly resin

  acid, and is then measured colorimetrically.

  H 1 b A can also be determined by the reaction, by reaction with thiobarbituric acid (TBA), the amount of hydroxymethylfurfural (HMF) that is produced by acid treatment. In relation to the glycosylation of albumin, this parameter has has been evaluated with methods which provide for its Isolation by affinity chromatography on certain particular dyes, then the colorimetric determination after the reaction with TBA or in other ways by ion exchange chromatography and then by reading determination. the density

  optical fractions thus obtained.

  Once it has been shown that a very close correlation exists between the amount of glycosylated albumin and the amount of glycosylated proteinase, both of which are examined colorimetrically, a method of assaying them for the latter has been proposed, thereby removing the separation of albumin from serum with a considerable advantage both from the point of view of the time required for the test and the

simplicity of the operating mode.

  In a very regrettable way, it has been shown later in this assay that free glucose (and also other carbohydrates) is a disruptive factor and

  is absolutely necessary to eliminate it.

  To overcome this disadvantage, Kennedy et al (Diabetes, 29: 413 (1980) proposed a 15 hour dialysis at 4 C against a suitable saline solution. According to this method, the time required for the analysis is considerably prolonged.

  and the analytical method becomes more complicated.

  To summarize and take into account the lay-up condition of serum glucose prior to assay, the method proposed by Kennedy et al can be summarized as follows: Reagents Test Assay Serum Specification Serum 1.0 Dialysis 15 hours Dialysis Serum 0.1 0.1 Hysiological solution (p H 7.4) 0.9 Physiological solution + Na BH 4 0.9 Oxalic acid 1-molar 0.5 0.5 Boiling bath for 5 hours Trichloroacetic acid 1, 0 1.0 Centrifugation Liquid supernatant 1.5 1.5 Thiobarbituric acid Maintained for 30 minutes at 400 C (TBA) 0.5 0.5 Optical density read at 443 nm (microns) The overall time required for the analysis is 21 to 22 hours (of which 15 are required for dialysis) and the various operating stages, even when they are not really difficult, at least in some cases

  who do not always agree with the customary routine.

  To a certain extent

  that the determination of non-glycosylated proteins

  Enzymatic is subject to analytical errors that are due to interference factors and that the only corrective action

  which has been proposed by the prior art is a preliminary dialysis

of the sample.

  The present invention provides two variants for the method indicated above, namely in one, the final stage of dialysis is replaced either by an enzymatic stage which removes the carbohydrates which are not bound on the proteins and in the other the test. is made in the presence of free carbohydrate but under such analytical conditions (eg controlled hydrolysis) that these carbohydrates can not interfere In the latter case, the acid hydrolysis is carried out in a time equal to 2 1/2 hours or shorter If the first of these two variants is chosen, a preliminary incubation of the sample to be examined is also proposed. In fact, a first object of the present invention is the provision of an improved method for assaying glycosylated proteins non-enzymatically, presented in biological fluids, inter alia in blood serum, said method providing for hydrolysis of the sample to be examined in an acidic medium and at a temperature above 80 C,

  hydrolysis possibly preceded by a preliminary incubation

  and provides for the elimination of carbohydrate and / or

  substances Detur: ries, the subsequent precipitation of

  the separation of the liquid phase and the addition thereto of a reagent capable of detecting the carbohydrates and / or their derivatives and the final reading of the optical density of both the sample and the blank test.

  The elimination of carbohydrates and

  Any event of different nature is carried out more

  in two directions, which will be described below.

after.

  According to the first route, all the proteins involved can

  It may be precipitated by the addition of particular precipitating agents which are selected from a number of reagents: these belong to the classes of organic compounds such as alcohols, polyols, carbonyl compounds, organic acids or their salts. mineral acid salts

can also be used.

  Among the above products p-toluenesulphonic acid,

  trichloroacetic acid, perchloric acid, ura-

  Nyl, sulphosalicylic acid, ammonium sulphate and sodium sulphate are preferred. The temperature is maintained at

  values that are middle or lower.

  The elimination of carbohydrates and / or other substances

  these disturbances may be effected enzymatically, possibly after adjusting the pH of the blood medium to an appropriate value. In this case, enzymes are added (either separately at different times or together with each other which have been selected in a particular manner). appropriate so

  to convert these substances into products that do not

terfere more.

  Do O Enzymes can be introduced: as is or

  properly immobilized, for example locked up in

  or covalently or otherwise bound to poly-substrates

natural (or synthetic) mothers.

  As noted above, operations related to

  the removal of the prurvary substances may be

  incubation of the sample in anticipation of removing potential interfering factors that

  were then released during the analytic cycle.

  Typical examples are preliminary treatments of the sample with appropriate enzymes (such as syalidase, aalactoxidase and others) or with acids at a temperature ranging from 800 C to 1000 C for a time not exceeding 30 minutes. . The acids to be used are preferably chosen from the group consisting of sulfuric, hydrochloric, sulphosalicylic and toluenesulphonic oxalic acids.

  mixtures of these acids with each other and with other acids.

  The process according to the present invention takes place

  then with a stage of hydrolysis in an acid medium during

  longer than 2½ hours and at a temperature of

  higher than 800 C The protein is precipitated

  The same precipitation agents and precipitators mentioned above can be used to achieve this. This stage is followed

  separation of the solid phase from the supernatant liquid

  In this case, the latter being in turn separated from the feedings at a portion of the liquid phase, a carbohydrate detector reagent (and / or a reagent for the detection of their derivatives, such as thiobarbituric acid) is added, while water is added to another part of the liquid phase in question

  to obtain a blank test if Na BH 4 is not used.

  Finally the optical densities are read (at an appropriate wavelength) for both the sample and the test.

  The process just described, as indicated above,

  above, can be applied to the determination of glycosylated proteins that are contained in biological liquors

of any kind and origin.

  The adoption of this process proved to be particularly

  advantageously for assaying the glycosylated proteins that are contained in blood serum samples and

  the authors of the present invention have found it useful to

  miter the examples given here to this specific case because of

  the extremely critical nature of such an application

  time, the use of the present method on samples

  from different origins does not cause any difficulty

  conqueet any extension of the method given as an example here will be a clearly routine operation entering

  in the context and spirit of the present invention.

  If the method of assaying glycosylated proteins is a first object of the present invention, a second important object is the provision of means to be used to carry out this method. These means consist of equipment which contains, Isolated in separate containers, or separate butreassembled in a single container, reagents selected from the group consisting of: a) medium acidifying agents b) protein precipitants c) ketone reducing agents d) hydrocarbonate detecting agents and / or

of their derivatives.

  More particularly, the equipment of diagnostic reagents according to the present invention is composed, taking into account the indications given above, of oxalic acid or acetic acid, trichloroacetic acid (TCA).

  of sodium borohydride and thiobarbituric acid (TBA).

  The previous operative details and other operational details

  will appear best using the examples

  given by way of illustration but not limited below.

EXAMPLE 1

  A portion of serum equivalent to 3.3 mg of protein is diluted after addition of traces of octyl alcohol, with 1 ml of 0.01 molar phosphate buffer, pH 7.4, containing 0.15 molar NaCl (PBS) and 0.85 molar sodium borohydride The sample is allowed to stand at room temperature for 1 hour and then for 18 hours at 4 ° C.

  This process is being considered to prepare the blank test.

  Another portion of the serum in the same amount of protein is diluted with 1 ml of the usual phosphate buffer, but without the reducing agent. Both samples are treated with 1 ml of 40% TCA and, after a rest period of 15 minutes. at room temperature, the samples are centrifuged for 15 minutes at 2500 g (2500 times the acceleration of gravity) The precipitate, resuspended in 1.5 ml of acetic acid 2 times normal, is maintained 24 hours at C While cooling, the suspension is treated with 0.5 ml of 40% TCA and centrifuged to recover the clear supernatant. 1.5 ml portions of the clear solution are reacted with 0.5 ml of TBA for 30 minutes. minutes at C The difference between optical densities (OD) taken at 443 nm from the blank and the sample is plotted on an appropriate graph, (a calibration chart established with 5-HMF (5-hydroxymethylfurfural) , so that the millimol The process can be summarized as follows: Reagents Sample Testing Operating Details White Serum 0.05 ml 0.05 ml PES 1 ml

  PBS + Na HBH 4 1 ml Leave 1 hour at the time

  ordinary temperature plus 18 hrs at 4 C 40% TCA 1 ml 1 ml Centrifuge Acetic acid 2 N 1.5 ml 1.5 ml Maintained 24 hours at 1 CO C TCA 0.5 ml 0.5 ml Centrifuge Liab 1.5 ml 1.5 ml TBA supernatant 0.5 ml 0.5 ml Lu at 443 nm after min at 40 ° C

EXAMPLE 2

  An operating model can be adopted that includes a preliminary treatment for 15 minutes at 100 C and then the

  Protein precipitation with TCA (40% concentrate).

  A portion of serum (0.4 ml) is diluted in a ratio of 1: 2 to 1:20 (1: 5 is preferred) with 0.6 normal oxalic acid directly into a test tube equipped with After being cooled to room temperature, 2 ml of 40% TCA is added. The sample is stirred, allowed to stand for 15 minutes at room temperature and then stirred for 15 minutes at 1000.degree. and then centrifuged at 2500 g for 10 minutes The supernatant liquid is discarded leaving the test dns /% inverted, resting for a few minutes on filter paper buffers 2.8 ml of oxalic acid O is then added to the residue, 6 and after having sealed the test tubes 1, they are kept on a water bath for 5 minutes.

  so that glucose bound to proteins is trans-

  formed in 5-hydroxymethylfurfural (5-HMF) The various test tubes are cooled to room temperature in a water bath for 10 minutes and 1 ml of 40% TCA is added to each tube After an additional 10 minutes at room temperature. The clear, supernatant liquid is collected and divided into two portions of 1.5 ml each at one serving.

  0.05 molar thiobarbituria acid, while the other

  Only water is added, this is the blank test.

  The dosage of the amount of 5-HMF that is contained in the sample is made by calculating the difference

  between the respective optical densities at 443 nm of the sample

  and the blank test, and comparing the difference thus found with a standard curve obtained with standard 5-HMF. The process according to this example can be summarized as follows Reagents Operative details Serum 0.4 ml Oxalic acid 0.6-nor 1.6 ml 15 min at 100 C 40% TCA 2 ml Centrifuge Oxalic acid 0.6-nor 2.8 ml Maintained for 5 hours at 1000 C TCA at 40% 1 LO ml Centrifuge

  SAMPLE SAMPLE SAMPLE TESTIMONIAL

Supernatant liquid 1.5 ml 1.5 ml

TBA 0.5 ml -

  Water 0.5 ml, D at 443 nm after min at 40 ° C

Claims (7)

  A method for determining the glycosylated proteins contained in biological fluids comprising treating the test sample in an acidic medium at a temperature above 80 ° C, preceded by non-pre-liminary incubation combined with the elimination of carbohydrates and / or other   disturbed substances Icek the subsequent precipitation of proteins   the separation of the liquid phase, the addition to it of a reagent detecting the presence of carbohydrates and / or their derivatives (sample), the addition of water or a suitable solution a portion of the liquid phase (control test) if the reducing agent, Na BH 4 and the final reading of the optical densities of the sample and the control test are not used. 2 Process for determining the glycosylated proteins contained in the biological fluids according to the preceding claim, characterized in that the acid treatment of the sample is the first operation and is carried out   for a period never exceeding two and a half hours.   Process for determining the glycosylated proteins contained in biological fluids according to claim 1, characterized in that it comprises the following stages: a) preliminary incubation of the sample; b) elimination of carbohydrates and / or disruptive substances c) acid treatment of the residue of the previous stage for a period of more than 2.5 hours d) precipitation of the proteins e) separation from the liquid phase with the precipitate   (f) the addition to a portion of the liquid phase of a reaction   tiff detecting the carbohydrates and / or their derivatives g) the addition of water or a suitable solution to a portion of the liquid phase in the same proportions as in the previous stage, and 6 h) the reading of the optical density of the sample and of the control test.   4 Method for determining glycosylated proteins   according to any one of the preceding claims, characterized   rised by the fact that stage b) is carried out by precipitating :: -   proteins contained in the sample.   Process according to the preceding claim, characterized in that the precipitation is carried out by adding to the sample a precipitating agent selected from the group consisting of organic compounds such as alcohols, polyols, acids, salts thereof. The process according to the preceding claim, characterized by the fact that the precipitation is carried out by adding   mineral salts to the sample.   7 Process according to claim 3, characterized in that stage b) is carried out by adding the specific carbohydrate enzymes to be eliminated, either alone   individually, either in mixture and / or at different times ent.   8 Process according to the preceding claim, characterized in that stage b) is carried out using immobilized enzymes. 9 Process according to claim 3, characterized in that the preliminary incubation stage is carried out   at a temperature between 80 C and 1000 C.   Method according to the preceding claim   in that the incubation is carried out in the presence of an acid selected from the group consisting of oxalic acids; acetic, sulfuric, hydrochloric, sulfosalicylic,   p.toluenesulfonic acid and mixtures thereof.   11 Process according to claim 3, characterized in that the protein precipitation operation of the stage   (d) is carried out by adding one of the preceeding agents   in claims 5 and 6.   12 Diagnostic reagent equipment for the determination of glycosylated proteins in biological fluids   only in a separate container or separate but   The reagents selected from the group consisting of: a) medium acidifying agents; b) protein precipitating agents; c) reagents for de-carbohydrates and / or their derivatives. Equipment according to the preceding claim, characterized in that the reagents are the acids   oxalic, trichloroacetic and thiobarbituric.