ITMI992056A1 - DIAGNOSTIC SYSTEM FOR THE DETERMINATION OF SERIAL ACIDOSIALIC LEVELS AND ITS APPLICATION METHOD - Google Patents

Description

Description of the industrial invention entitled: "Diagnostic system for the determination of serum levels of sialic acid and its method of application" -

This invention generally relates to a method for determining the levels of sialic acid in human serum and more specifically to a diagnostic system comprising a set of reagents necessary to determine the titer and the procedures characterized by the production of a colored signal or other visible signals. Total serum sialic acid levels can be measured for diagnostic reasons, especially for the early detection of cancerous processes of various origins or other serious degenerative diseases. Furthermore, the serum variations of total sialic acid can constitute an important and reliable indicator of the patient's response to specific anticancer treatments. Unfortunately this test is not commonly used in medical practice mainly due to the high cost of specific enzymatic reagents, which are also easily degradable, having a very short half-life, and / or due to the complexity of the analytical steps required, which require long periods of execution. Although these limitations exist, some systems for the quantitative determination of sialic acid, based on both colorimetric and enzymatic procedures, have been described in the literature and are in use today. The colorimetric methods, for which chemical reagents are used, are not very precise and accurate, as required for the aforementioned diagnostic purposes and the consequent results are not very reliable. One of the enzymatic methods for serum sialic acid titer is based on microtiter plates (Simpson H. et al. "Serum sialic acid enzymatic assay based on microtitre plates: application for measuring capillary serum sialic acid concentrations", British Journal of Biomedical Science , 1993; 50: 164-167), and would seem to be reliable, but unfortunately it has the same disadvantages of very high costs, as in the case of the use of enzymes (refrigerated storage of reagents and their limited half-life) and the long execution due to the complexity of the various steps required. Other methods have recently been described, using monoclonal antibodies, but these methods have the same disadvantages and drawbacks as enzymatic methods. Therefore, the need is evident for a reliable, practical and simple diagnostic system for carrying out the determinations of total serum sialic acid, and which offers, in addition to precision, also a relatively low cost, elements that can be obtained by adopting a chemical diagnostic system. Furthermore, an early diagnosis of this pathology, with a mass monitoring based on simple low-cost tests, could avoid many deaths, due to late diagnosis. Only a few chemical methods related to this topic have been published in the literature, such as Warren L. "The Thiobarbituric Acid Assay of Sialic Acids", The Journal of Biological Chemistry, Vol. 234, N °. 8, pages 1971-1975, August 1959, Svennerholm L. "Quantitative determination of sialic acid", Biochimica et Biophysica Acta, Vol. 24, pages 604-61 1 and Miettinen Τ. and Takki-Luukkainen I.T., Acta Chem. Scand., Short Communications "Use of Buthyl Acetate in Determination of Sialic Acid", Vol. 13, N °. 4, 1959, but these are relatively old publications. One of the objectives of many institutions dedicated to cancer prevention and therapy is to find diagnostic systems useful for mass monitoring and to stimulate research centers to develop biochemical and / or immunological methods! for the early diagnosis of this serious disease, based on the simple analysis of desfoliated cells or body fluids of apparently healthy individuals. In fact, it is already well known that tumor cells present on their surface glycolipids and glycoproteins characterized by a modified composition of carbohydrates, which contribute to the aberrant phenomena of cell-cell recognition, adhesion, antigenicity and invasiveness. These glycoconjugates can be released into the blood, as a result of an accelerated exchange of the constituents of the piasmatic membrane of the tumor cell, either by excretion or by release. It is also well known that sialic acid (Neu5Ac) is the component most present in the structure of these glyco-compounds, as evidenced by the following Table 1 (Lindberg G., Rastam L, Gulberg B., Lundblad A., Nilsson- Ehle P. and Hanson B. - Serum concentrations of total sialic acid and sialoglycoproteins in relation to coronary heart disease risk markers - Atherosclerosis 103, pp. 1 23 - 1 29, 1 993):

TABLE 1

Consequently, the quantification of Neu5Ac levels in the serum of patients is of great interest as a non-specific indicator of serious degenerative diseases, more specifically of different types of tumor, although the specificity is relatively low since other processes, especially inflammatory, can cause increases in sialic acid levels. Below is an indicative list taken from the published literature (*) (°), in order to highlight the high correlation between the serum level of Neu5Ac and various diseases: ;; TABLE 2 ;; ; ;; {*) Shamberger R.J. - Evaluation of water soluble and lipid soluble sialic acid levels as tumor markers - Anticancer Res. 1986; 6; 717-720.

Plucinsky M.C. et al. - Total and lipid-associated serum sialic acid levels in cancer patients with different primary sites and differing degrees of metastatic involvement - Cancer 1986; 58; 2680-2685.

Verazin G., Riley W.M., Gregory J., Tautu C., Prorok J.J. et al. - Serum sialic acid and carcinoembryonic levels in the detection and monitoring of colonrectal cancer - Dis. Colo. Rectum. 1990; 33; 139-142.

Kakari S., Stringou E., Toumbis M., Ferderigos A.S., Poulaki I. et al. -Five tumor markers in! Ung cancer: significance of total and lipid-bound sialic acid - Anticancer Res. 1991; 11; 2107 - 2110.

Tautu C., Pee D., Dunsmore M., Prorok J.J., Alhadeff J.A. - Evaluation of serum sialic acid and carcinoembryonic antigen for the detection of earlystage colorectal cancer - J. Clin. Lab. Anal. 1991; 5; 247 - 254.

Schutter E.M., Visser J.J., Van Kamp G.J., Mensdorff-Pouilly S., Van Dij k W. et al. - The utility of lipid-associated sialic acid (LASA or LSA) as a serum marker for malignancy - Tumor Biol. 1992; 13; 121-132.

Narayanan S. - Sialic acid as a tumor marker - Annals of Clinical Laboratory Science 1994; 24; 376-384.

Vedralova E. et al. - Evaluation of serum sialic acid fractions as markers for malignant melanoma - Cancer Letters 1994; 78; 171-175.

(°) The values in Table 2 have been obtained from different bibliographic references and determined using different methods and therefore they are not completely homogeneous data.

From this Table 2 it is clear that the exact determination of the serum level of Neu5Ac in many important diseases, such as cancer, can be useful for early diagnosis and for monitoring the patient during drug treatment and as an indicator of disease remission. In fact, it has been surprisingly noticed that tumor remission is accompanied by a reduction of Neu5Ac levels to normal values.

This invention relates to a diagnostic system which directly chemically measures the level of sialic acid (Neu5Ac) in the serum of patients with severe degenerative diseases, such as cancer. The inventors have surprisingly discovered that the chemical colorimetric method of this invention allows to obtain specific and precise results, such as those obtained with the traditional enzymatic methods already well known in the art. Furthermore, it has been discovered that the method of this invention can advantageously overcome the major problem of the known enzymatic methods, ie the long execution and the high costs, which in the past have considerably limited their use. More particularly, the invention consists of a colorimetric chemical method, which is performed using the necessary reagents according to a specific sequence, in order to produce from! Serum or piasmatic Neu5Ac a chemical chromogenic group, which allows to determine precisely and specifically the levels of Neu5Ac by spectrophotometry. In fact the important aspect of this invention is a chemical method where the serum (or alternatively the plasma) of a patient, obtained with conventional methods already well known, is first treated with H2SO4, which causes the hydrolysis of the glycoproteins and glycolipids present. in serum or plasma, thus producing Neu5Ac. PHASE 1: The sample solution is placed in a test tube, preferably of plastic material, with a cap and heated at 80 ° C for 1 hour and then cooled to room temperature. At this stage, trichloroacetic acid is added to the solution, which is subsequently centrifuged at 3000 rpm for 15 minutes. After this last operation the sample has two different and well separated layers, the supernatant (Vt) and the protein precipitate on the bottom. STEP 2: An accurately measured aliquot of supernatant (Va) obtained in Step 1 is taken and placed in a concentrated HCl solution containing, Resorcinol, CUSO4 and distilled water. The resulting solution is heated at 100 ° C for 15 minutes. After cooling the solution to room temperature, a chromogenic group is generated, which is subsequently extracted with n-butyl acetate / n-butanol. After having stirred until a complete homogeneity is obtained, the solution is centrifuged for 1 minute at 3000 rpm. Finally, the uppermost layer is removed and the absorbance is determined by spectrophotometry at a wavelength of 580 nm. The absorbance of the final color of the reaction is stable for 60 minutes at room temperature. Furthermore, it is particularly important to prepare a blank, consisting of distilled water and a solution of HCl - Resorcinol - CUSO4 and a reference solution containing a known quantity of Neu5Ac in distilled water and a solution of HCl- Resorcinol -CUSO4. The blank and the reference solution are treated as described above for the sample. The method of the invention can not only be used on fresh serum or plasma samples, but also on old samples, previously taken, as long as they are stored at 4 ° C for a week, at 0 ° C for 6 months and at -20 ° C. for 1 year. The content of Neu5Ac in 1 ml of serum or plasma is calculated using the following equation:

where is it:

DOB = Optical density of the sample

Vt = Volume of the supernatant (in ml) of Phase 1

DOr = Optical density of the reference solution

= Volume of the supernatant (in ml) of Phase 2

Vs / p = Volume of serum or plasma

0.0306 = pmoles of Neu5Ac present in 20 μl of the reference solution used in Phase 2. This data includes the correction factor of the reference solution, since this has not been treated with acid.

The diagnostic system of the invention can be performed manually by an operator or can be integrated into an automated system. Another important aspect of this invention is that the reliability of the described method has been validated, as evidenced by the following statistical data:

Reproducibility: By treating different aliquots of the same sample at the same time, the following results were obtained:

Linearity: the reaction is in accordance with Lambert and Beer's law and is linear up to 6.60 pmoles Neu5Ac / ml. For higher values, it is necessary to dilute half the amount of the sample with an equal amount of distilled water. The absorbance determined on the diluted sample must be corrected by multiplying the result by the dilution factor. The minimum measurable concentration of Neu5Ac is 0.007 pine nuts.

Recovery: By adding known amounts of Neu5Ac to different sera, recovery between 98.7% and 101.6% is achieved for all levels tested.

In order to better illustrate the invention, the following examples are given for demonstration purposes only.

EXAMPLE 1

Preparation of reagents.

The reagents to be used in the test are prepared following these procedures:

H2SO4 125 mM:

Add 0.7 ml of concentrated H2SO4 (18 M / 36 N) to a 100 ml graduated container and make up to volume with distilled water.

5% trichloroacetic acid:

5 g of trichloroacetic acid are dissolved in 100 ml of distilled water.

CuSO4 0.1 M (2.5 g%):

Dissolve 2.5 grams of CUSO4 in 100 ml of distilled water.

Reagent HCl - Resorcinol - CuSO4:

0.2 g of Resorcinol are dissolved in 10 ml of distilled water and added with 8 0 ml of concentrated HCl, 0.25 ml of 0.1 M CUSO4 and distilled water until reaching the total volume of 100 ml.

Mixture of n-butyl acetate / n-butanol:

in the ratio of 85:15 (v / v).

Reference solution of sialic acid (Neu5Ac):

46,375 mg of Neu5Ac are weighed exactly and dissolved in 100 ml of distilled water. The solution obtained has a concentration of 0.03 pmoles / 2 0 μl

EXAMPLE 2

Quantitative determination of Neu5Ac levels in the serum of 10 patients for the early diagnosis of tumors using an "in vitro" diagnostic system.

Method

Phase 1:

Place 50 μl of each patient's serum (Vs / w) in a plastic tube with a cap and add 200 μl of H2SO4 at a concentration of 125 mM. Incubate the sample for 1 hour at 80 ° C and then cool it to room temperature. Add 150 μl of 5% (w / v) trichloroacetic acid to the sample, mix with a stirrer until perfect homogeneity is obtained and centrifuge at 3000 rpm for 15 minutes. This operation allows to obtain two well separated layers in the sample, the supernatant (Vt = 0.40 ml) and the protein precipitate.

White:

Place 250 μl of distilled water and 250 μl of HCl-Resorcinol reagent in a test tube.

Reference solution:

Place 230 μl of distilled water, 20 μl of Neu5Ac reference solution (0.03 pmoles) and 250 μl of HCl - Resorcinol reagent in a test tube. Phase 2:

Put 150 μl (Va) of supernatant (Vt) obtained in Step 1 into a test tube, add 100 μl of distilled water, and 250 μl of HCl - Resorcinol reagent. Heat the sample obtained, the blank and the reference solution at 100 ° C for 15 minutes and then cool to room temperature. Then add 1.5 ml of n-butyl acetate / n-butanol (85:15 v / v) to each of the three tubes. Mix the samples with a stirrer until complete homogeneity is obtained. Finally, centrifuge at 3000 rpm for 1 minute and measure the absorbance of the colored solution at the wavelength of 580 nm. Using the indicated volumes (Vt = 0.40 mi; Va = 0.15 mi; Vs / p = 0.05 mi) the equation is simplified as follows:

where is it:

DQs = Optical density of the sample

DOr = Optical density of the reference solution

Results:

In the following Table 3 it is clear and evident that each patient has different levels of sialic acid (Neu5Ac), calculated with precision and specifically with the chemical method of this invention. By comparing the results obtained '' in-vitro '' and the data already published in the literature (see Table 2), it is possible to formulate an early diagnosis for each patient based on the levels of Neu5Ac in serum or plasma. The results are summarized in Table 4.

FABLE 3

TABLE 4

Although the invention has been partially described with certain illustrations and examples, with the purpose of illustrating it with greater clarity and understanding, it is obvious that some changes and modifications may be made while remaining within the scope and scope of the claims themselves.

Claims (2)

CLAIMS: 1) A diagnostic system characterized by a colorimetric chemical method for the spectrophotometric measurement of the level of sialic acid (Neu5Ac) in serum or plasma taken from patients, which allows early detection of tumor processes or other serious degenerative diseases. 2) A diagnostic system as per Claim 1), characterized by the production of a chroniogenic group derived from Neu5Ac which can be examined by spectrophotometry, and which is produced by a precise sequence of chemical reactions and extractions in which the following reagents are used and solvents: a) Sulfuric acid (H2SO4) b) Trichloroacetic acid (TCA) c) Copper sulphate (CUSO4) d) Hydrochloric acid solution - Resorcinol e) Mixture of n-butyl acetate / n-butanol 3) A diagnostic system as per Claim 2), characterized by concentrations of the individual reagents and solvents included within the following ranges: a) Aqueous solution of H2SO4 from 75 mM to 175 mM; b) Aqueous solution of TCA from 2.5% to 10%; c) Aqueous solution of CUSO4 from 1 g% to 5 g%; d) Concentrated solution HCl - Resorcinol from 0.05 to 10%; e) Mixture of n-butyl acetate / n-butanol in a ratio of 60:40 v / v to 95: 5 v / v. 4) A diagnostic system according to Claim 3), characterized by concentrations of single reagents and solvents preferably consisting of: a2) Aqueous solution of H2SO4 125 mM; b2) 5% aqueous solution of TCA; c2) 2.5 g% aqueous solution of CUSO4; d2) Concentrated solution of HCl - Resorcinol of 0.2%; e2) Mixture of n-butyl acetate / n-butanol in the ratio 85:15 v / v. 5) A diagnostic system as per Claims 1) to 4), characterized by a chromogenic group obtained from serum or piasmatic Neu5Ac with the reagents and solvents of the following sequence of reactions and extractions: a3) treatment of serum or plasma with H2SO4 at 80 ° C for 1 hour to free NeuSAc from glycoproteins and glycolipids; b3) addition of TCA with subsequent centrifugation until the separation of 2 distinct layers; c3) taking an aliquot of supernatant (containing Neu5Ac) and adding the reagent HCl - Resorcinol - CUSO4 and heating at 100 ° C for 15 minutes until the development of the chromogenic group; d3) extraction of the chromogenic group with a mixture of n-butyl acetate / n-butanol and stirring until complete homogeneity; e3) centrifugation and measurement of the colorimetric signal of the chromogen group by spectrophotometry at 580 nm ± 5 nm. 6) A diagnostic system as per Claims 1) to 5), characterized to be particularly useful for determining the total level of Neu5Ac in the serum or plasma of patients, and can be used as a non-specific indicator for the early diagnosis of tumor processes or other serious degenerative diseases. 7) A diagnostic system referred to in Claims 1) to 5), characterized to be specific and suitable for the chemical determination of the total level of Neu5Ac in the serum or plasma of patients, which can be used to evaluate the efficacy and response of the patient to a specific immunological or chemotherapy treatment, acting as an indicator of disease remission.