WO2001025786A1

WO2001025786A1 - Method and kit for performing serum or plasma sialic acid assays

Info

Publication number: WO2001025786A1
Application number: PCT/EP2000/009106
Authority: WO
Inventors: Paulo Alberto Veronesi; Pablo Eduardo Andras Rodriguez
Original assignee: Therapicon Srl
Priority date: 1999-10-01
Filing date: 2000-09-14
Publication date: 2001-04-12
Also published as: AU7777300A; ITMI992056A0; ITMI992056A1; WO2001025786B1; IT1315232B1

Abstract

A chemical colorimetric testing method for the spectrophotometric assay of serum or plasma sialic acid for diagnostics and monitoring.

Description

METHODANDKITFORPERFORMING SERUMORPLASMA SIALICACIDASSAYS

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to serum sialic acid testing method, and more particularly to a test article comprising the reagents necessary for such an assay and methods which rely on the generation of a coloured or other visible signal from the test article.

Serum total sialic acid assays may be performed for diagnostic purposes, mainly the early detection of cancer processes of different origin or of other serious degenerative diseases. Moreover the variations of serum total sialic acid may be a significant and suitable indicator for the response of the patient to specific anti- cancer treatments. Unfortunately this assay is not commonly used in the medical practice mainly due to the high cost of the specific enzymatic reagents, which are also easily degradable, having a short shelf-life, and/or to the complexity of the involved steps, requiring long periods for the test execution.

Despite the above limitations, some quantitative estimation tests for sialic acid, either based on colorimetric or on enzymatic procedures, have been described in the literature and are presently in use. The colorimetric tests for which chemical reagents are used are not much precise and accurate, as required for such diagnostic purposes, and the results are not much reliable. One of the enzymatic method is the serum sialic acid enzymatic assay based on microtitre 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), which seems to be reliable, but unfortunately presents the same disadvantage of the high costs of the enzyme handling (refrigerated storage of the reagents and their limited _?

shelf-life) and the long execution caused by the complexity of the various involved steps.

Other methods have been also recently described, using monoclonal antibodies, but such methods present the same disadvantages and inconvenients of the enzymatic methods. Thus, the need of a convenient, practical and easy test for serum total sialic acid determinations is apparent, which shall combine also precision and relatively acceptable routine low costs, which seems to be achieved only by adopting a chemical test article.

2. Description of the Background Art

A great number of deaths following to cancer diseases could be avoided by an early detection of this pathology through a mass screening based on simple and low cost tests. Only few chemical methods have been found in the published literature related to the above subject, alike Warren L. "The Thiobarbituric Acid Assay of Sialic Acids", The Journal of Biological Chemistry, Vol. 234, N° . 8, pages 1971-1975, August 1959, Svennerhol L. "Quantitative determination of sialic acid", Biochimica et Biophysica Acta, Vol. 24, pages 604-611 and Miettinen T. and Takki- Luukkainen I.T., Acta Chem. Scand. , Short Communications "Use of Buthyl Acetate in Determination of Sialic Acid" , Vol. 13, N° . 4, 1959 and they are relatively old. One of the objectives of many institutions dedicated to cancer prevention and therapy is to find tests for the mass screening by stimulating research centres aiming to develop biochemical and/or immunological methods for the early detection of this serious disease based on the analysis of despholiated cells or of body fluids of the individual, apparently normal .

In fact it is already well known that the cancer cells show on their surface glycolipids and glycoproteins characterized by a modified composition of carbohydrates, which contributes to the known aberrant phenomena of cell- to-cell recognition, of adhesion, of antigenicity and invasivity observed between them.

These glycoconyugates may be released in the blood, as a consequence of an accelerated interchange of the constituents of the plasmatic membrane of the cancer cell, either by secretion or by release.

It also well known that sialic acid (Neu5Ac) is the most present component in the structure of the above glyco- compounds, as evidenced in the following Table 1 (Lindberg G., Rasta L., Gullberg B., Lundblad A., Nilsson -Ehle P. and Hanson B. - Serum concetrations of total sialic acid and sialoglycoproteins in relation to coronary heart disease risk markers - Atherosclerosis 103, pp. 123-129, 1993) :

TABLE 1 Most important proteins and lipids containing Neu5Ac

Globulin g Neu5Ac/100 ml serum

α i-Antitrypsin (α_x) 10.2

Orosomucoid (α 9.2

Haptoglobin (α₂) 6.

Hemopexin 6. α-macroglobulin (α₂) 5.

Transferrin ⁽β⁾ 4.

IgA (γ) 3.

IgG (γ) 2.

IgM (γ) 2.

Complement C3 1. c^-Antichymotrypsin ( _x) 1.

Ceruloplasmin (α₂) 0.

IgD (γ) 0.

Gangliosides - 0.

Consequently the quantification of Neu5Ac levels in serum of patients is of great interest as a non-specific marker for the diagnosis of serious degenerative pathologies, more specifically of different types of cancer, despite the specificity is relatively low due that other processes, basically inflammatory, may cause sialic acid increased levels .

An indicative list taken from published literature (*) (°) is reported hereby, in order to evidence the high correlation between Neu5Ac serum level and different diseases: TABLE 2

Disease Total Sialic Acid (μmol/ml)

Normal 2.12

Malignant cancers (general) 2.80

Gastrointestinal cancer 2.55

Rectal cancer 2.30

Breast cancer 2.40

Skin cancer (melanoma) 2.40

Pancreatic cancer and colecistis 2.90

Pulmonary cancer 3.90

Kidney cancer 2.84

Disseminated cancer (general) 4.59

Lynphogranulomatosis 3.17

Myeloid leukaemia 3.24

Lynphatic leukaemia 3.24

Mieloma 2.60

Macroglobulinemia 2.90

Pulmonitis 3.20

Bronquitis 2.35

Pulmonar fibrotic Tubercolosis 2.00

Pulmonar exudative Tubercolosis 3.00

Pulmonar active Tubercolosis 5.80

Inactive Tubercolosis 1.76

Miocardial infarction 3.06 Sub-acute bacteriological endocarditis 3.30

Hepathitis 2.30

Hepathic Cirrhosis 1.96

Cronic Hepathitis 2.69

Nephrosis 3.20

Nephroschlerosis 2.50

Kidney trasplant 2.50

Infectious diseases 2.80

Chronic and acute polyarthritis 2.74

Infantil A aurotic Dementia 2.80 Hemiplegia (from cardiovascular etiology) 3.00

Insulino-dependent Diabetes 1.90

No insulino-dependent Diabetes 2.50

Intestinal diseases 2.49

Hernia 2.25

Endocrinal diseases 2.30

Enteritis 2.90

Colitis ulcerosa 2.21

(*) 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 lung cancer: significance of total and lipid-bound sialic acid - Anticancer Res. 1991; 11; 2107 - 2110.

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

Schutter E.M., Visser J.J., Van Kamp G.J., Mensdorff- Pouilly S., Van Dijk 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 have been abstracted from different bibliographic references and desulted by using different method and therefore they are not homogeneous figures . From the above Table 2 it is clearly evidenced that the exact determination of serum level of Neu5Ac in many important diseases, alike cancer, may be useful for an early diagnosis and for monitoring the patient during the pharmacological treatment and as a marker of the remission of the disease. In fact it has been surprisingly noticed that the cancer remission is accompany to a decrease of levels of Neu5Ac to normal values.

Description of the invention

The present invention relates to a test article, which measures directly by chemical way the level of sialic acid (Neu5Ac) in the serum of patients with serious degenerative diseases, alike cancer.

The inventors have surprisingly noticed that the chemical colorimetric method of the instant invention allows to obtain specific and precise results as those obtained by the traditional enzymatic methods already well known in the art .

Moreover it has been discovered that the method of this invention can advantageously overcome the major problem arising with the known enzymatic methods, as long execution and high costs, which in the past substantially limited their use.

More specifically the invention relates to a chemical colorimetric method, which is carried out using the necessary reagents in a specific sequence, in order to produce from the seric or plasmatic Neu5Ac a chemical chromogen group, which permits to determine precisely and specifically by spectrophotometry Neu5Ac levels. In fact the preferred embodiment of the instant invention is a chemical method wherein the serum (or alternatively the plasma) of a patient, obtained with usual methods already well known, is firstly treated with H₂S0₄, which causes an hydrolysis of glycoproteins and glycolipids present in the serum or plasma, thus producing Neu5Ac . PHASE 1: The sample solution is placed in a test tube, preferably from plastic material, with stopper and heated at 80° C during 1 hour and finally cooled at room temperature. At this step trichloroacetic acid is added to the solution, which is then centrifugated at 3000 rpm during 15 minutes. After this latter operation the sample presents two different and well separated layers, the supranatant (Vt) and a lower proteic pellet

PHASE 2 : An accurately measured aliquot of supranatant (Va) , obtained from Phase 1, is then withdrawn and placed in a solution of concentrated HC1 containing Resorcinol, CuS0₄ and distilled water. The resulting solution is heated at 100° C during about 1 minute. After having cooled the solution at room temperature, a chromogen group is produced and then extracted with n-butyl acetate / n-butanol. Once having stirred until to have a complete homogeneity, the solution is centrifugated during 15 minutes at 3000 rpm. Finally the upper layer is withdrawn and the absorbance is measured by spectrophotometric route at 580 nm wavelength. The absorbance of the final colour of the reaction, is stable during 60 minutes at room temperature. Moreover it is particularly preferred to have a blank, constituted by distilled water and HC1 - Resorcinol - CuS0₄ solution, and a reference solution containing a known quantity of Neu5Ac in distilled water and HC1 - Resorcinol - CuS0₄ solution. The blank and the reference solution are processed as before described for the sample. The method of the invention shall be applied not only to fresh samples of serum and plasma, but also to old samples, provided they are preferably stored during 1 week at 4° C, 6 months at 0° C and 1 year at - 20° C.

The content of Neu5Ac in 1 ml of serum or plasma is calculated by using the following equation:

DOs x Vt x 0.0306 μ oles Neu5Ac/ml serum or plasma =

DOr x Va x Vs/p wherein:

DOs = Optical density of the sample Vt = Volume of the supranatant (ml) from Phase 1 DOr = Optical density of the reference solution Va = Volume of the supranatant (ml) from Phase 2 Vs/p = Volume of serum or plasma

0.0306 = μmoles Neu5Ac present in 20 μl of the reference solution used in Phase 2. This figure includes the correction factor of the reference solution, since this has not been processed with the acid treatment.

The test article of the invention may be performed manually by an operator or may be integrated into an automated equipment .

Another preferred embodiment of the instant invention is that the performance of the described method has been validated, as evidenced by the following statistic data:

Reproducibility: Treating at the same time different aliquots of the same sample the following results have been obtained:

Linearity: The reaction complies with the Lambert and Beer law and it is linear until 6.60 μmol Neu5Ac/ml. For higher values, it is necessary to dilute half quantity of the sample with an equal amount of distilled water. The absorbance of the obtained sample is measured and the calculation shall be corrected, multiplying the result by the diluting factor. The minimum detectable concentration of Neu5Ac is 0.007 μmol.

Recovery: Adding known quantities of Neu5Ac to different sera, a recovery between 98.7 % and 101.6 % is obtained for all tested levels.

In order that the invention may be more fully understood, the following Examples are given by way of illustration only.

EXAMPLE 1

Preparation of the reagents .

The reagents to be used for the test are prepared according to the following procedures:

H₂S0₄ 125 M:

Add in a 100 ml volumetric flask 0.7 ml of concentrated

H₂S0₄ (18 M / 36 N) and bring to volume with distilled water.

Trichloroacetic acid 5 %:

5 g of TCA are dissolved in 100 ml of distilled water.

CuS0₄ 0. 1 M (2. 5 g %) :

Dissolve 2.5 g of CuS0₄ in 100 ml distilled water.

HCl -Resorcinol - CuS0₄ reag-ent:

0.2 g of Resorcinol are dissolved in 10 ml of distilled water and added with 80 ml of concentrated HCl, 0.25 ml of 0.1 M CuS0₄ and distilled water to yield 100 ml volume. Mixture of n -butyl acetate / n -butanol : at the ration of 85:15(v/v).

Ref rence solution of sialic acid (Neu5Ac ) :

46.375 mg of Neu5Ac are exactly weighed and dissolved in 100 ml of distilled water. The obtained solution presents a concentration of 0.03 μmol/20 μl.

EXAMPLE 2

Quantification of the levels of Neu5Ac in the serum of 10 patients for an early diagnosis by using a test in-vitro.

Procedure :

Phase 1 :

Placet 50 μl of the serum of each patient (Vs/p) in a plastic test tube equipped with a stopper and add 200 μl of 125 mM of H₂S0₄. Incubate during 1 hour at 80° C and finally cool the sample at room temperature. Add to the sample 150 μl of 5 % TCA (w/v) , mix with a vortex until to obtain a perfect homogeneity and centrifugate at 3000 rpm during 15 minutes. The above operation permits to obtain in the sample two well separated layers, the supranatant (Vt=0.40 ml) and the precipitated proteic pellet.

Blank solution :

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

Reference solution :

Place in a test tube 230 μl of distilled water, 20 μl of Neu5Ac Reference solution (0.03 μmol) and 250 μl of HCl- Resorcinol reagent.

Phase 2 :

Place 150 μl (Va) of the supranatant (Vt) obtained from

Phase 1 in a test tube, add 100 μl of distilled water and

250 μl of HCl-Resorcinol reagent.

Heat the obtained sample, the blank and the reference solution at 100° C during 15 minutes and after cool them at room temperature . Then add to each of the three tubes 1.5 ml of n- butylacetate/n-butanol (85:15 v/v) . Mix the samples by a vortex until to obtain a complete homogeneity. Finally centrifugate at 3000 rpm during about 1 minute and measure the absorbance of the coloured solution at 580 nm wavelength.

By using the indicated volumes (Vt=0.40 ml; Va=0.15 ml; Vs/p=0.05 ml) the equation is simplified in the following way:

DOs x 1.632 μmol Neu5Ac/ml serum =

DOr

DOs = Optical density of the sample

DOr = Optical density of the reference solution

Resul s:

From the following Table 3 it is clearly evidenced that each patient show different levels of sialic acid (NeuδAc) , calculated precisely and exactly with the chemical method of the instant invention. Comparing the obtained results "in vitro" with the data already published in the literature (see Table 2) it is possible to formulate for each patient an early diagnosis based on the serum or plasma levels of Neu5Ac . The results are summarized in Table 4.

TABLE 3

TABLE 4

Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practised within the scope of the appended claims.

Claims

CLAIMS :

1) A test article comprising a chemical colorimetric testing method for the spectrophotometric determination of sialic acid (Neu5Ac) level in serum or plasma withdrawn from patients for the early detection of cancer processes or of other serious degenerative diseases.

2) A test article as in claim 1) , where the chromogen group produced from Neu5Ac to be tested by spectrophotometry is induced by a sequence of chemical reactions and extractions in which are involved the following reagents and solvents: a) Sulphuric acid (H₂S0₄) b) Trichloroacetic acid (TCA) c) Cupper sulphate (CuS0₄) d) Resorcinol hydrochloric solution e) Mixture of n-butylacetate / n-butanol

3) A test article as in claim 2), where the concentration of the reagents and solvents is comprised within the following ranges: a_±) H₂S0₄ aqueous solution from 75 πiM to 175 mM; b TCA aqueous solution from 2.5 % to 10 %; c_x) CuS0₄ aqueous solution from l g % to 5 g %; d ) Resorcinol concentrated hydrochloric solution from 0.05 to 10 %; e_x) Mixture of n-butylacetate / n-butanol in ratio from 60:40 v/v to 95:5 v/v.

4) A test article as in claim 3), where the reagents and solvents are preferably selected at the following concentrations : a₂) H₂S0₄ aqueous solution at 125 mM; b₂) TCA aqueous solution at 5 %; c₂) CuS0₄ aqueous solution at 2.5 g %; d₂) Resorcinol concentrated hydrochloric solution of 0.2 %; e₂) Mixture of n-butyacetate / n-butanol in ratio of 85:15 v/v. 5) A test article as in claim from 1) to 4) , where the chromogen group is produced from Neu5Ac with the following reactions and extractions sequence: a₃) treatment of serum or plasma with H₂S0₄ at 80° C during

1 hour to release Neu5Ac from glycoproteins and glycolipids; b₃) addition of TCA with subsequent centrifugation until separation of 2 layers; c₃) withdrawal of an aliquot of the supranatant (containing Neu5Ac) and addition of HCl-Resorcinol -CuS0₄ reagent and heating to 100° C during 15 minutes until development of the chromogen group; d₃) extraction of the chromogen group with n-butylacetate / n- butanol and stirring to complete homogeneity; e₃) centrifugation and detection of the colorimetric signal of the chromogen group by spectrophotometry at 580 nm ± 5 run.

6) A test article as in claims from 1) to 5) , suitable for the determination of Neu5Ac levels in serum or plasma of patients as a non-specific marker for the early detection of cancer processes or of other serious degenerative diseases .

7) A test article as in claims from 1) to 5) , suitable for the chemical determination of Neu5Ac levels in serum or plasma of patients to evaluate the response of the patient during immunological or chemiotherapeutic treatment as a marker of the remission of the disease.

8) A method of testing comprising a spectrophotometric determination of the sialic acid level in serum or plasma as defined in any one of the preceding claims.

9) A diagnostic or monitoring test kit comprising reagents for performing a method of testing according to claim 8.

10) An article, method or diagnostic or monitoring kit as claimed in any preceding claim substantially as hereinbefore described.