Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/04—Endocrine or metabolic disorders
  • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

• the present invention concerns oligosaccharide metabolites of glycosaminoglycans and their use in the diagnosis and treatment of diabetes, as well as the methods for detecting and titrating them.
• State of the art Pathologies with a chronic course are generally characterised by the appearance and development of physiological dismetabolites originating from alterations at both cellular and humoral level.
• the extent of the variation of the humoral parameters may be influenced by the general condition of the subject, so it does not always correctly reflect the state of evolution of the disease and may provide an incorrect indication of the extent of the damage produced and of the consequent risk of onset of related pathological complications.
• a correct assessment of the cellular damage would be desirable as it would be able to provide useful indication on the severity of the damage produced, on the speed of progression of the disease, on its regression following pharmacological treatment and on the potential onset of related pathologies.
• the humoral alterations in diabetes may be an expression, not only of a malfunction of the Islands of Langerhans, but also of a simultaneous endocrine disorder such as hyperpituitarism, hyperadrenocorticism, hyperthyroidism, etc.
• the aim of treating a diabetic patient is to maintain the blood glucose levels as close as possible to normal, also in order to limit the onset and progress of complications.
• the most frequent risks of complications in diabetes are microvascular disorders, retinopathy, nephropathy and neuropathy. On average these tend to appear about 10-15 years after the onset of the disease, but only the renal complications and disorders of the retina seem to be directly related to the severity of the hyperglycaemia, while this is not the case for atherogenic and neurological disorders. It must be stressed that some complications do not seem to regress significantly even when the glucose levels are reduced for 1-3 years.
• the form of monitoring most commonly used is to determine the glucose in order to highlight states of hyper- or hypoglycaemia.
• the basement membrane contains the proteoglycan of heparansulphate (also known as proteoheparansulphate) and traces of other proteoglycans.
• proteoglycans are structures formed by a central proteinaceous filament to which polysaccharides called glycosaminoglycans (hereinafter referred to as GAGs), composed of repetitive disaccharide units containing a derivative of an amino sugar, glucosamine or galactosamine are connected by acidic bonds.
• At least one of the disaccharide sugars possesses a carboxylate or sulphate group with negative charge.
• the presence of proteoheparansulphate, with its strong negative charge, can endow the basement membrane with the ability to block the passage of proteins from the blood to the urine; through its links with other structures of the membrane, it is also able to influence the processes involved in the inflammatory, atherosclerotic or thrombogenic pathogenesis, etc.
• oligosaccharide metabolites of the GAGs originating from the cellular membrane may be detected in the biological liquids in concentrations proportional to the possible onset of the diabetes or of its pathological complications. They may also be a therapeutic tool for the treatment of complications of diabetes.
• Figure 1 illustrates the electrophoretic bands of oligosaccharide metabolites of
• Figure 2 illustrates the electrophoretic bands of oligosaccharide metabolites of GAGs extracted from the urine of diabetic subjects, before and after treatment with chondroitinase A/C compared with standard GAGs (Sigma).
• Figure 3 illustrates the electrophoretic run obtained from the urine of a diabetic subject.
• Figure 4 illustrates the trend of the reading of the optic density in the affinity/specificity antigen/antibody test.
• Figures 5 and 6 illustrate the calibration lines of the affinity test for lectin from
• the present invention therefore refers to the use of oligosaccharide metabolites of GAGs as markers in biological fluids for assessing the state, severity, risk of onset of complications of diabetes, and for combating onset and progression thereof, and to the methods for revealing and titrating them.
• oligosaccharide metabolites of GAGs refers, more specifically, to heparansulphate, chondroitinsulphates A and C, dermatansulphate and their metabolic fragments. Particularly preferred for the purposes of this invention is the use of heparansulphate and of its metabolic fragments.
• the quantity of metabolites of the invention present in the biological liquids depends on the extent of the cellular damage caused by diabetes, but it is independent of the pharmacological control of hyperglycaemia. For this reason the determination of their quantity may be used in the control of the risk of onset of complications associated with diabetes, in monitoring the seriousness or regression of the pathology, in predicting the onset in subjects at risk. Since the concentration of metabolites of the invention is a value that does not depend on the variations of the humoral biochemistry, that is on the concentration of glucose in the subject's biological fluids, it may be considered as a real reference parameter of the state, severity, risk of onset, possibility of the genesis of complications, monitoring of the treatment of the diabetic disease.
• the metabolites of the present invention may be detected in the biological liquids, preferably in serum and in urine, and even more preferably in urine.
• Said oligosaccharide metabolites are extracted from the biological liquids with specific methods, and characterised both by electrophoresis and by a specific reaction in response to a nitrosation process.
• the methods developed for determining these metabolites are of a ponderal, immonoenzymatic type, for example, competitive ELISA, immunoenzymatic-like, immunochromatographic-colorimetric, colorimetric and chromatographic, for example HPLC as described in the Journal of Chromatography, 212 (1981 ), 65- 73.
• the metabolites to which the present invention refers are detected with a competitive method, for example by means of the biotin/avidin system, by colorometric determination of glucuronic acid and of glucosamin; or by means of an immunoenzymatic-like method using lectins, for example from Triticum vulgaris.
• lectins to detect metabolites of glycosaminoglycans is particularly important for the purpose of this invention.
• Lectins have been used for some time in a chromatographic method for purifying sugars.
• lectins may also be applied to the recognition of glycosaminoglycans in immunoenzymatic-like systems.
• a further object of the present invention is therefore a kit for detecting glycosaminoglycans based on lectin adhering to a microplate or, alternatively, bonded to a detecting enzyme, for example peroxidase.
• the lectins particularly preferred for the purpose of the present invention are those derived from Triticum vulgaris.
• the kit based on lectins of the present invention works according to the so-called sandwich technique whereby the lectin or a suitable antibody is anchored to a solid support, made to react with the molecule that is to be detected. The latter is in turn reacted with, respectively, a suitable antibody or lectin, both marked with an enzyme, which then interacts with a chromogen in the presence of substratum for the final detection.
• sandwich technique whereby the lectin or a suitable antibody is anchored to a solid support, made to react with the molecule that is to be detected.
• the latter is in turn reacted with, respectively, a suitable antibody or lectin, both marked with an enzyme, which then interacts with a chromogen in the presence of substratum for the final detection.
• sandwich technique whereby the lectin or a suitable antibody is anchored to a solid support, made to react with the molecule that is to be detected.
• a suitable antibody or lectin both marked with an enzyme, which then interacts with
• the oligosaccharide metabolites of the present invention are also able to combat the onset and progression of complications of diabetes.
• metabolites taken, for example, from the urine of healthy subjects can compete in vivo for the enzymes produced in excess during the course of the diabetic pathology, for example ⁇ -N-acetyl-glucosaminidase and ⁇ -D-glucosidase.
• the administration to a diabetic subject of the metabolites of the invention constitutes an alternative substratum which prevents the above- mentioned enzymes from attacking the proteoglycans in the cell wall and producing the well known diabetic complications, that is, for example, microangiopathy, nephropathy, vasculopathy and angiopathy.
• the oligosaccharide metabolites of GAGs are therefore useful as therapeutic agents in nephropathy, retinopathy, vasculopathy, angiopathy and microangiopathy of diabetic origin.
• the metabolites of the present invention may be obtained by purifying the urine of healthy subjects using techniques well known to the expert in this field, for example by chromatographic purification procedures.
• the oligosaccharide metabolites to which of the present invention may be administered both orally and parenterally.
• the dose to be administered varies from 10 to 50 mg thrice a day.
• the parenteral administration the dose varies from 5 to 50 mg once or twice a day.
• Suitable pharmaceutical forms useful for the oral administration of the oligosaccharide metabolites of the present invention are, for example, tablets, capsules, sugar-coated pills, granulates, solutions and suspensions even of an extemporary nature, syrups.
• Suitable pharmaceutical forms for the parenteral administration of the oligosaccharide metabolites of the present invention are, for example, solutions for both intramuscular and intravenous injection, and also sublingual tablets, creams and ointments.
• the extraction, characterisation and quantitative detection of the oligosaccharide metabolites object of the present invention will now be illustrated by the following examples.
• Urine (10 I) of healthy human volunteers or subjects with confirmed diabetic pathogenesis was added with 0.1 M NaOH (40 g), stirred for 15 minutes and left to sediment for 4 hours, then filtered and the clear supernatant liquor was recovered.
• Triton X100 300 ml was added to the supernatant liquor and the pH brought to 6 with concentrated HCI.
• DEAE-Sephadex A-25 5 g was then added and the whole was kept under stirring for 20 minutes, then filtered. The recovered resin was loaded in a column, washed with 0.3M NaCI/Triton X100 3% (200 ml) and eluted with 1 M NaOH (20 ml), then 0.1 M (80 ml).
• Acetic acid (1.2 g) and absolute ethanol (400 ml) were added to the eluate and the mixture was kept for one night at 20°C.
• the formed precipitate was recovered by centrifugation at 1500xg for 10 minutes, washed 3 times with absolute ethanol and then dried at
• Table 1 shows the weight results of the recovery of glycosaminoglycans from human urine.
• This phase is useful for eliminating chondroitins A/C from the urine, then checking the behaviour of HS in the disease.
• the enzyme SIGMA C- 3667 was then added so as to have 0.1 ⁇ g/ml.
• the whole was then left to react for 24 hours, stirring slowly, protected from the light and at room temperature, then placed in a dialysis tube with cut 1000 against water.
• the resin was recovered and transferred to a column, washed with 0.3M NaCl / Triton X100 at 3% (20 ml/g of resin) and eluted with the smallest possible volume of 1 M NaOH.
• the eluate was brought to pH 5 using acetic acid, added with absolute ethanol, and kept in the freezer for one day.
• the precipitate was recovered by centrifugations at 1500xg for 10 minutes, washed 3 times with absolute ethanol and dried at 60°C.
• Table 2 shows the weight results of the recovery of glycosaminoglycans free from chondroitin A/C from human urine.
• Figure 1 illustrates the Sigma standard electrophoretic bands (a), metabolites of a healthy subject before treatment with chondroitinase A/C (b), and metabolites of a healthy subject after treatment with chondroitinase A/C (c).
• Figure 2 illustrates the Sigma standard electrophoretic bands (a), metabolites of a diabetic subject before treatment with chondroitinase A/C (b), and metabolites of a diabetic subject after treatment with chondroitinase A/C (c).
• Nitrosation is a characteristic reaction of the HS by which the stain that identifies it disappears from the electrophoretic run. This reaction was carried out using the method described by Cappelletti R. et al. , Anal. Biochem., 1980, 105, 430.
• Figure 3 illustrates the Sigma standard electrophoretic run (a), metabolites of a healthy subject before nitrosation treatment (b) and metabolites of a diabetic subject after nitrosation treatment (c). The immunological characterisation was carried out by the production of antibodies.
• BSA (45 mg) was dissolved in a physiological solution (20 ml), a 0.5M solution of dihydrazide of adipic acid was added (175 mg in 2 ml), at pH 5, and the volume was brought to 24 ml with physiological solution.
• Rabbits weighing about 1.5 kg were used. At time 0 (start of treatment) the animals were treated intradermically in 20 sites of the abdomen, with 0.1 ml/site of a suspension of PBS (1 ml) containing the immunogen GAGs-BSA (2 mg) obtained as described in example 3, and a complete Freund's adjuvant (1 ml, Sigma), in order to have the sensitisation reaction. After 21 days, the animals were treated intramuscularly with 1 ml of a suspension of PBS (1 ml) containing the immunogen GAGs-BSA obtained as described in example 3) and incomplete Freund's adjuvant (1 ml, Sigma), in order to have the priming reaction.
• GAGs-OVA prepared as described, respectively, in examples 3 and 4, in PBS.
• 100 ⁇ l of a solution containing GAGs-BSA were deposited in each well.
• the same operation was carried out with a solution of GAGs-OVA.
• the plates were placed in a thermostat at 37°C for 3 hours for the adhesion of the immunogen.
• 150 ⁇ l of a 2% casein solution (p/v) were added to each well for saturation of the non reacted sites.
• the rabbit blood was taken from the marginal vein as described in the example and centrifuged at 3,000 rpm for 10 minutes.
• the resulting serum was diluted with PBS buffer solution in serial dilutions (1/20, 1/40, 1/80, 1/160, 1/320, 1/640, 1/1280).
• the quantity of immunoglobulins (antibodies) which bind with the immunogen adhering to the plate was measured by means of a rabbit anti-lgG antibody (Sigma) conjugated with the peroxidase enzyme (HRP) and diluted 1 :1000 with PBS. d) Performing the test
• phase a 100 ⁇ l of diluted anti-lgG-HRP conjugate were added to each well and the plates were put back in the thermostat at 37°C for 1 hour. They were washed again 2 times and dried in the same way as in phase a) and 100 ⁇ l of substratum for the enzyme (OPD - Sigma) were added to each well; the sample was again incubated at 37°C and finally 50 ⁇ l of stopper (Sigma) were added.
• the antibody formed in the rabbit was confirmed to be directed against the GAGs, since an overlapping reading was obtained for the two immunogens (HS-BSA and
• GAGs and their metabolic fragments compete with biotinilated GAGs added in standard concentrations against the anti-GAGs antibody adhering to a solid phase (microplate).
• concentration of biotinilated GAGs which binds with the antibody is inversely proportional to the concentration of GAGs and of its metabolic fragments in the sample. Detection is performed by titrating the biotin radical by a specific reaction with avidin-peroxidase. a) Preparation of the immunoglobulin IgG anti-GAGs and metabolites
• the proteins were precipitated by adding 50% ammonium sulphate; it was then centrifuged at 3,000 rpm for 15 minutes and the supernatant liquor was eliminated. The precipitate was taken up with PBS buffer solution (2 ml) and subjected to dialysis in a 10 kD tube, for 48 hours at 2-8°C. The protein yield was about 7/100 ml.
• the IgG were purified from the dialysed substance by low-pressure chromatography on a Tryacril column. On the purified IgG, proteinaceous titration was carried out using the Lowry micromethod (Sigma kit code 690.A). The yield in
• IgG was 1.8-2 g/ml of serum.
• the purified IgG were collected and brought to the final concentration of 12.9 mg/ml by adding PBS, then diluted 1 :100 with PBS for the subsequent preparation of the microplates.
• b) Preparation of the conjugate HS-biotin (biotinisation of HS) or of GAGs-biotin Conjugation was carried out on a 50% mixture of HS and metabolites extracted anhd purified from normal subjects (HSN) and from diabetic subjects at different stages of the disease, more or less affected by complications (HSP), in order to have competition with the largest possible panel of metabolites.
• HSN and HSP Two initial solutions of HSN and HSP were prepared (10 mg/ml in water). 230 ⁇ l of each were placed in a bath of ice. A separate solution of 0.1 M NalO 4 was prepared in 0.1 M acetate buffer, pH 5.5, and this too was cooled in a bath of ice. 3.52 ml of this solution of NalO 4 were then added to the solutions of HSN and HSP and the reaction was allowed to proceed for 20 minutes in a bath of ice. 150 ⁇ l of 1 M ethylenglycol were then added and allowed to react for 30 minutes at room temperature. The whole was placed in a Centricon 3,000 and centrifuged at 1 ,000 x g for 30 minutes (the operation was repeated 5 times).
• PBS buffer solutions were prepared, containing, in a volumetric ratio 1 :1 , a fixed concentration of 100 ⁇ g/ml biotinilate GAGs and scalar concentrations of standard GAGs starting from a maximum concentration of 100 ⁇ g/ml (1 st point) and diluting in scale 1 :2 for the subsequent points.
• 100 ⁇ l of the various solutions were placed in each well, except one to which only the buffer was added (blank). The microplates were incubated at 37°C for 1 hour, then the wells were emptied, washed and dried as described above.
• serum it was obtained from venous blood taken in the absence of anticoagulant and centrifuged at 3,000 rpm for 15 minutes. The volume of serum used was 100 ⁇ l. The same volume was used for the urine, after filtration, if it did not appear perfectly clear. The indicated sample volume was added in a volumetric ratio of 1 :1 to the solution of biotinilated GAGs (amounting to 100 mg/ml). 100 ⁇ l of this mixture were placed in a well in the microplate sensitized with anti-GAGs antibody, then left to incubate for 1 hour at 37°C.
• the measurement was carried out in the same way as described for calibration in point d), reading at 490 nm against the blank.
• the optic density value (D.O.) extrapolated on the line of calibration and multiplied by the dilution factor supplies the concentration of GAGs and of its oligosaccharide fragments in the sample.
• Table 3 shows the values obtained from the urine of diabetic subjects compared with that of healthy subjects.
• the concentration of GAGs in the urine of sick subjects is dramatically higher than that in the urine of healthy subjects.
• the concentration of HS and of its metabolites, following treatment with chondroitinase A/C, in the urine of sick subjects is dramatically higher than that in the urine of healthy subjects.
• Triticum vulgaris Lectin-Peroxidase Labeled Triticum vulgaris was used (Sigma L.3892) diluted before use 1 mg in 500 ml of PBS buffer. c-1 ) Principle of the method
• Triticum vulgaris lectin has affinity for N-acetyl- ⁇ -D-glucosamine residues and for
• N-acetyl- ⁇ -D-glucosamine oligomers are components of HS and of its structural analogs.
• HS, the analogs and their metabolic fragments in the sample of biological fluid bind with the specific antibody adhering to the wall of the microplate. They are detected by adding lectin conjugate of Triticum vulgaris- peroxidase and subsequent spectrophotometric determination of the reaction that develops between the enzyme and the specific substratum
• S,, S 2 , S 3 , S 4 e S 5 standards, composed of a mixture of GAGs or of HS from normal and diabetic subjects in a PBS buffer solution with the following scalar concentrations in ⁇ g/ml (respectively 200, 50, 12.5, 3.125, 0.781 ).
• C ⁇ C 2 , C 3 , and C 4 samples (urine).
• the plates are placed to incubate in a thermostat at 37°C for 1 hour. They were then washed 3 times consecutively by adding 300 ⁇ l of PBS buffer solution containing 0.2% Tween in each well, and the wells were then dried perfectly by shaking. 100 ⁇ l of lectin-HRP conjugate, prediluted as described above in point b- 1 ), were added to each well. The plate was then put back in the thermostat at 37°C for 1 hour and 3 washes were carried out as described above, after which each well was added with 100 ⁇ l of chromogen obtained by dissolving, at the time of use, one 2 mg tablet of OPD (Sigma) in 5 ml of PBS buffer solution and adding 10 ⁇ l of H 2 O 2 at 15%. The plate was put back in the thermostat at 37°C for 30 minutes, then 50 ⁇ l of 2N H 2 SO 4 were added to each well.
• the optical density was read at 492 nm within 30 seconds, resetting against the blank. e-1 ) Calibration line and determination of the sample
• optical densities read for the samples prepared according to the methods described are marked on the calibration line, allowing extrapolation of the content in metabolic derivatives of the sample expressed in ⁇ g/ml.
• Table 5 shows the total values of GAGs and HS and the respective metabolic fragments from the same samples from normal and pathological subjects, obtained with the sandwich method and detection with lectin from Triticum vulgaris conjugated with peroxidase (HRP).
• HRP peroxidase
• Triticum vulgaris adhering to the solid phase are detected by adding conjugate between the specific IgG antibody directed against them and the peroxidase enzyme, and subsequent spectrophotometric determination of the colouring developed by the reaction between the blocked enzyme and the specific substratum.
• a-2) Preparation of the microplate A solution of lectin of Triticum vulgaris (Sigma) in buffer 0.1 M CO3 2 7HCO3 ⁇ pH 9.5 was deposited in the wells of a microplate in doses of 100 ⁇ l/well, and the plate was incubated at 37°C for 2 hours. After the washing procedure already described above, 150 ⁇ l of a 2% casein solution in PBS buffer were added to each well. The microplate was left for one night at 4°C, then washed 3 times following the procedures described above.
• Conjugation was carried out between IgG obtained by purification of the anti- GAGs antiserum and the HRP enzyme.
• the purification of the antiserum IgG was carried out as described in the example 7, a), while the conjugate was prepared as follows.
• the resulting enzyme solution was chromatographed on a Sephadex column (eluent: 0.1 M CO3 2 7HCO3 _ buffer, pH 9.5).
• IgG (10.2 mg) was added to the fractions containing activated HRP and they were left to react for 2 hours while stirring at room temperature, protected from the light. 510 ⁇ l of NaBH 4 (4 mg/ml of water) were added and the reaction allowed to continue for a further 2 hours while stirring at room temperature, protected from the light. The mixture was dialysed in a 100kD tube against PBS buffer. To perform the test, the resulting solution was diluted in 0.1 M PBS , pH 7.4 in a ratio of 1 :900.
• Figure 6 shows an example of a calibration line applying the same system as in point e-1 ) above.
• Table 6 shows the values of the contents of the biological liquids obtained operating on the same samples and with the same procedures.
• the GAGs metabolites and their fragments are blocked on a chromatographic column which may be composed of a matrix such as, for example, IgG anti-GAGs or anti-HS adhering to an inert support, or lectin of Triticum vulgaris immobilised on Sepharose 6MB (Sigma L.6257).
• the eluent may be 0.1 N HCI or 0.5-1.5M N- acetyl- ⁇ -D-glucosamine.
• concentration of GAGs or HS and their metabolic fragments is determined by detection of the content of uronic acids (glucuronic) or of glusosamine, carried out with spectrophotometric methods.
• IgG anti-GAGs 200 mg were bonded to Sigma G4643 beads (1 g equal to
• test tubes containing 0.0125M sodium tetraborate (1.2 ml) in H 2 SO 4 at 96%, and 0.2 ml of a sample, or 0.2 ml of standard or 0.2 ml of blank (PBS) were placed in a bath of ice and stirred gently, then for 5 minutes in bain-marie ar 100°C and then cooled.
• m-Hydroxy-diphenyl 0.15% in NaOH at 0.5% (40 ⁇ l) was added and after 5 minutes the reading of the optical density was taken at 520 nm resetting against the blank. 3.
• Chromatographic columns (0 0.6x10 cm) were loaded with glass beads with the immobilised anti-GAGs antibody, suspended again in PBS buffer solution. The chromatographic bed was washed with the same buffer solution, eluted with 30 ml of sample urine and washed again with the same buffer. It was then eluted with a solution of 0.1 M N-acetyl- ⁇ -D-glucosamine (5 ml) and the eluate was subjected to spectrophotometric determination of the concentrations of uronic acids in the urinary GAGs and their metabolites. The sample concentrations were defined from the ratio of the titre determined with reference to the respective standards.
• Table 7 shows the values of urinary GAGs and their fragments isolated from the sample.
• the size of the ⁇ % values is a clear indication of how the concentration of GAGs in the urine is in proportion to the severity of the diabetic pathology and to the onset of complications.
• the fragments of sulphate GAGs present in the sample are bonded to the specific antibody adhering to the membrane. They are detected by adding lectin of
• IgG anti-HS (30 mg/ml in PBS) was deposited on a membrane in a dose of 3 ⁇ l.
• the membrane was dried and then immersed in 3% casein in PBS. After 15 minutes it was drained on paper and placed in a thermostat at 37°C for 30 minutes, then placed on the dedicated support.
• the lectin-HRP conjugate of marked Triticum vulgaris (Sigma L.3882) (1000 ⁇ l) was diluted with 10 ml of PBS 3% casein, and the mixture was stirred where protected from the light. 3. Performing the test
• the wells were subjected to a cycle of 3 washes with 300 ⁇ l of PBS buffer solution containing 0.2% Tween 20, then 5 ⁇ l of lectin-HRP of marked Triticum vulgaris (Sigma L.3882), diluted in PBS, were added. After 10 minutes the wells were washed again as above and 10 ⁇ l of tetramethylbenziline (TMB) in a buffer solution (chromogen substratum) were added. The results were visually assessed, comparing the intensity of colouring of the wells containing the sample with the positive control wells.
• TMB tetramethylbenziline

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