ITMI20121636A1 - METHOD FOR THE DETERMINATION OF THE ACTIVITY OF ALFA-AMILASI - Google Patents

Description

Description of the patent for industrial invention entitled:

â € œMETHOD FOR DETERMINING THE ACTIVITY OF ALPHA-AMYLASEâ €

The present invention relates to a method for determining the activity of alpha-amylase in a sample of biological fluid by means of a colorimetric assay. The method of the invention allows an accurate evaluation of the catalytic activity of the alpha-amylase enzyme in the presence of hemoglobin in the biological fluid sample. The invention also relates to a kit and reagents for carrying out the assay.

Introduction

In laboratory diagnostics, hemolyzed venous and arterial blood samples cause significant clinical, analytical and organizational drawbacks due to biological, spectrophotometric and chemical interference caused by free plasma hemoglobin (1,2). Regarding the latter two types of interference, the increase in optical absorbance or the change in the baseline value in the measurement at wavelengths in which hemoglobin absorbs light most intensely, or attributable to pseudoperoxidase activity and to the spectral superposition with chemical reactions, it often causes the alteration of the results of multiple tests. The Î ± -amylase enzyme is mainly synthesized in the pancreas and salivary glands, but it can also be released to a lesser extent from other tissues such as the intestine (duodenum and ileum), ovary and testes. Although three specific isoenzymes of amylase occur in nature (Î ±, β and γ), Î ± -amylase is present only in humans, whose pancreas and salivary glands synthesize specific isoforms characterized by different mobility during focusing isoelectric. The main causes of serum or plasma - hyperamylasemia as well as macroamylasemia - include acute pancreatitis and other pancreatic disorders such as cancer, pancreatic obstruction and cystic fibrosis, breast, colon, lung and ovarian malignancies, trauma, burns, salivary and gastrointestinal disorders (i.e. ulcers, intestinal ischemia and perforation, appendicitis), liver dysfunction due to hepatitis and cirrhosis, various gynecological conditions including ectopic pregnancies and salpingitis, cholecystitis, peritonitis, the presence of gallstones and l chronic alcoholism. In most of these conditions, the evaluation of amylase in serum, plasma or even urine is an essential part of the diagnostic practice. The significance of quantifying amylase in biological fluids other than blood and urine has been proposed for the differential diagnosis in the cystic fluid of pancreatic lesions or for the early prediction of the severity of acute pancreatitis, as well as in pleural and peritoneal fluids for the differential diagnosis of peritonitis; and a variety of other disorders, including esophageal rupture, pancreatic pleural effusion, ectopic pregnancy, ovarian tumors, Meigs syndrome, alcoholic cirrhosis, lung, breast or kidney cancers.

Several studies have shown that the catalytic activity of amylase - measured with a variety of reagents and tests - can be substantially decreased in the presence of free hemoglobin in the sample (3-9), thus reducing the clinical usefulness of the evaluation of the € ™ alpha-amylase in serum or hemolyzed plasma, as well as in urine or other body fluids which often contain large numbers of intact or damaged red blood cells. This also applies to acute pancreatitis with complications from haemorrhagic shock, in which the plasma free hemoglobin concentration can be frequently increased due to hemolysis in vivo (10,11).

The most suitable approaches to overcome spectrophotometric and chemical interference from free hemoglobin in plasma in traditional clinical chemistry include changing reagent (s), measuring blank, and changing measurement wavelength or measurement times (12 ). All these methods carry specific disadvantages and limitations, such as disproportionate costs or excessive lengthening of the measurement time, applicability or not depending on the instrumentation and absorbance values that exceed the reading capabilities of the spectrophotometer.

Description of the invention

We have now found a method for determining the activity of alpha amylase which is significantly less affected than conventional tests by the interference produced by the hemoglobin possibly present in the biological sample. This result was obtained by subjecting the biological sample to a preliminary oxidation step of oxyhemoglobin to methhemoglobin or alternatively through the formation of a complex with carbon monoxide, thus preventing its interference with the spectrum of absorption during the execution of the colorimetric assay.

Specifically, the invention relates to a method for determining the activity of alpha-amylase in a sample of biological fluid containing hemoglobin, by means of a colorimetric assay comprising:

i) addition to the biological fluid sample of an oxidizing agent capable of oxidizing oxyhemoglobin to methemoglobin;

or alternatively

ii) addition of carbon monoxide to form the carbomonoxyhemoglobin complex;

And

iii) subsequent addition to the sample of an alpha amylase substrate bearing a chromophore group that is released by the enzymatic activity of the alpha amylase itself, generating a change in absorbance in a predetermined time interval, i.e. a time interval in which the reaction produces a linear absorbance change;

iv) determination of the absorbance generated by the sample in the predetermined time interval and therefore of the alpha amylase activity value by interpolating a standard reference curve previously obtained with known quantities of alpha amylase.

The hemoglobin present in the biological sample can be in free form, possibly conjugated with polyethylene glycol or treated with agents that form polymeric complexes, or it is contained within the erythrocytes.

To improve the amylase activity, potassium thiocyanate is added to the assay mix as a chaotropic agent to increase the solubility of apolar molecules and, therefore, the reaction rate.

The oxidizing agent is preferably selected from sodium nitrite and potassium ferricyanide. The first, more preferred, is generally added at a concentration of 10 mM and at pH 6. The concentration of sodium nitrite or potassium ferricyanide or other oxidizing reagent required depends on the incubation time before mixing with the chromophoric substrate of the alpha amylase. The pH of the solution can be varied with respect to pH 6, considering that the decrease in pH increases the speed of oxidation, and that the oxidation reaction is completed before mixing with the chromophoric substrate of the alpha amylase. Similarly, the oxidant concentration can vary according to the incubation time, considering that an increase in the concentration of the oxidant reagent increases the reaction speed.

Sodium nitrite is known to react with oxyhemoglobin to give methemoglobin and nitrate as final products. In this way the dioxygenase nitrite activity of oxyhemoglobin is exploited to obtain methemoglobin. With this approach, the oxidizing reagents that are present in commercial assays do not convert oxyhemoglobin to methemoglobin during the kinetic measurement and do not cause the concomitant absorbance changes, which are the source of free hemoglobin interference.

The sample is usually hemolyzed serum or plasma, urine or any biological fluid contaminated by the presence of intact or damaged erythrocytes.

Substrates of alpha-amylase bearing a chromophore group that is released by the enzymatic activity of the enzyme include 2-chloro-4-nitrophenylalpha-D-maltotrioside (CNPG3); 4,6-ethylidene (G7) -p-nitrophenyl (G1) -a, D-maltoheptaoside (EPS) or p-nitrophenyl phosphate (PNP).

Another aspect of the invention relates to a kit for carrying out the method described here, comprising, in separate containers, the oxidizing agent and the alpha-amylase substrate bearing the chromophore group and possibly buffer solutions.

The spectrum of potential clinical applications of the method of invention is wide. Firstly, it can be used to evaluate Î ± -amylase in the majority of samples in vivo or in hemolyzed samples, with important clinical, economic and organizational implications. Furthermore, thanks to its robustness against hemolysis (up to 75 g / L of hemoglobin), as well as its excellent analytical performance, it can be effectively used to measure the catalytic activity of amylase in urine and many other fluids. biologicals that are often contaminated by the presence of intact or damaged erythrocytes, without the need to process the sample with centrifugation or other complicated procedures to limit hemoglobin interference. Another application of the method of the invention concerns the determination of alphaamylase in patients undergoing the administration of blood substitutes based on hemoglobin, in particular hemoglobins conjugated with polyethylene glycol (PEG) or treated with agents that form polymeric hemoglobin complexes: these compounds are in fact known to produce negative interference on the assay similar to hemolysis. Finally, the method of the invention aimed at oxidizing hemoglobin to methemoglobin or producing a stable derivative of hemoglobin such as carbomonoxyhemoglobin can be applied in other clinical chemistry or immunochemistry assays to limit or even decrease the interference of hemoglobin for tests in which the origin of the interference is not biological, but rather chemical or spectrophotometric (for example, in the assays for cardiospecific troponin, uric acid, urea nitrogen, calcium, creatinine, bilirubin, lipase).

The invention will be further illustrated in the experimental part reported below and in the attached figures.

Brief description of the Figures

Figure 1: Bland-Altman plot of the new assay developed for Î ± -amylase versus the commercial Beckman Coulter assay for Î ± -amylase. The solid line drawn corresponds to the mean difference, while the dashed lines designate 95% of the confidence interval (95% CI).

Figure 2: Ratio of Î ± -amylase concentration in haemolyzed and non-haemolyzed samples, determined with the new assay developed with nitrite as oxidant (z) and with the commercial Beckman-Coulter assay ({). The thin lines designate the total error allowed.

Figure 3: Result of the study with the reagent developed with nitrite, compared with the same assay conducted with a pre-oxidation with 1.5 mM of potassium ferricyanide.

Figure 4: The carbon monoxide bond leads to a change in the absorption spectrum of hemoglobin, with a decrease in absorbance at 405-410 nm, the wavelength to which a series of colorimetric assays are conducted, with respect to to oxyhemoglobin. This decrease in absorbance also decreases the background contribution of hemoglobin at a given concentration, for measurements at 405-410 nm.

Figure 5: Measurement at 405 nm of the change in absorbance over time of a sample containing hemoglobin reacted with the commercial Beckman Coulter reagent () compared with the same reagent previously equilibrated in a carbon monoxide atmosphere (z) (concentration of carbon monoxide in the 1 mM solution).

Experimental part

Method description

The analytical performance of both the commercial Î ± -amylase assay and the newly developed assay were evaluated on the clinical chemistry platform AU5822 (Beckman Coulter Inc., Brea CA, USA), a fully automated instrumentation designed for general chemistry analysis , therapeutic monitoring, drug abuse, urinalysis, particular proteins and serological assays. A full description of the tool and its performance is available (13). The Beckman Coulter Î ± -amylase assay is a colorimetric kinetic test using 2-chloro-4-nitrophenyl-Î ± -D-maltotrioside (CNPG3) as a substrate. The substrate reacts directly with alpha-amylase and does not require the presence of auxiliary enzymes. The release of 2-chloro-4-nitrophenol (CNP) from the substrate and the resulting increase in absorbance at 410 nm is directly proportional to the enzymatic activity in the sample. According to the manufacturer, the test is linear within a range of enzymatic activity between 10 and-2000 U / L for serum and plasma, and has an inaccuracy of 0.8 to 2.0%.

The test is a colorimetric experimental method in kinetics for the quantitative determination of Î ± - amylase using 2-chloro-4-nitrophenyl-Î ± -D-maltotrioside (CNPG3) as substrate, with the formulation identical to that of the Beckman analogue reagent Coulter. Compared to this assay, the assay of the invention involves a step of pre-incubation of the biological sample, in which the acellular hemoglobin is completely oxidized. The mechanism for eliminating plasma free hemoglobin interference in the sample is attributed to this pre-oxidation step in which the molecule is converted to 10 mM sodium nitrite methhemoglobin, buffered in 50 mM MES, pH 6, before reacting with the chromophore substrate. Sodium nitrite is known to react with oxyhemoglobin to give methemoglobin and nitrate as final products. In this way the dioxygenase nitrite activity of oxyhemoglobin is exploited to obtain methemoglobin. With this approach, the oxidizing reagents that are present in commercial assays do not convert oxyhemoglobin to methemoglobin during the kinetic measurement and do not cause the concomitant absorbance changes, which are the source of free hemoglobin interference.

More specifically, in the commercial amylase assay, the substrate analog CNGP3 (in 50 mM MES pH 6) is hydrolyzed by the enzyme itself, thus generating a chromophoric product which absorbs at 405-410 nm. To improve the amylase activity, potassium thiocyanate is present in the assay mixture as a chaotropic agent to increase the solubility of apolar molecules and, therefore, the reaction rate. It is known that, under acidic conditions, thiocyanate is oxidized to cyanide in the presence of oxyhemoglobin, but not in the presence of methemoglobin. These reactions are accompanied by significant changes in the absorption spectrum of hemoglobin in the range 400-450 nm, as well as up to about 700 nm. Since the substrate analogue of the amylase CNGP3 is hydrolyzed to generate a chromophore produced which absorbs at 405 nm, the spectral range and the kinetics of the two reactions (oxidation of hemoglobin by thiocyanate and formation of hydrolyzed compound) overlap and are the origin of the observed interference. To test this hypothesis, oxyhemoglobin was added to the commercial reagent for alpha-amylase, in the absence of alpha-amylase, and the spectral changes were monitored with a spectrophotometer. The final spectrum of the reaction mixture is very similar to that of cyanomet-hemoglobin. As reported, the pre-oxidation step of hemoglobin present in biological fluids converts it to a stable form that does not interfere with the detection of the color change developed in the amylase assay.

The interference caused by the oxidation of hemoglobin by the commercial assay solution is similarly removed or significantly reduced through the addition of carbon monoxide to the assay solution, which immediately gives rise to the formation of the complex stable with hemoglobin (carbomonoxyhemoglobin), which is much more stable to oxidation than oxyhemoglobin, and also shows a lower absorption at 405-410 nm, decreasing the contribution to the background signal.

Inaccuracy studies

The imprecision studies with the tests were performed using three serum samples with different concentrations of Î ± -amylase, low (45 U / L), intermediate (143 U / L) and high (270 U / L). The intra-test imprecision was evaluated in 20 sets of sequential measurements and the results were expressed as coefficient of variation (CV%).

Linearity

A routine serum sample with Î ± -amylase at a concentration of 282 U / L was serially diluted in fixed ratios (e.g., 1: 9, 2: 8; 3: 7; 4: 6; 5: 5 ; 6: 4; 07:03, 8: 2; 9: 1) with another routine serum sample with alpha-amylase concentration of 45 U / L. The dilutions were then evaluated with the new assay developed, in duplicate, and the theoretical values were calculated starting from the measurement results on the undiluted samples. Linearity was calculated by linear regression analysis and Spearman correlation coefficient (r).

Comparison study

The comparison study was carried out on 40 consecutive routine serum samples randomly selected from those indicated for stat test and display of a wide range of Î ± -amylase values. All samples were analyzed within 2 hours of arrival in the laboratory. The results of the new assay developed for alpha-amylase were compared with those obtained using the conventional Beckman Coulter test. The bias was evaluated by linear regression, Spearman's correlation coefficient (r), and Bland and Altman analyzes.

Hemolysis study

Free plasma hemoglobin interference on the alpha-amylase test was evaluated in both the new and commercial assays. The study was performed on 20 samples taken from healthy volunteers (10 females and 10 males, mean age 45 years, range 26-57 years). Venous blood was collected by a single experienced phlebotomist early in the morning in 6,0 ml vacuum tubes, siliconized, without separator gel and containing 18 U / L of lithium heparin (Vacuette, Greiner Bio-One GmbH, Frickenhausen, Germany), using a 20 gauge needle, 0.80x19 mm (Greiner Bio-One GmbH). The clotted blood was immediately divided into two aliquots of ~ 3 ml each. The first aliquot was immediately centrifuged at 1200 x g for 15 min at room temperature, while the second was subjected to mechanical hemolysis according to a validated method that has been thoroughly described elsewhere (14). Briefly, uncoagulated blood was passed 3 times through a small needle (30 gauge, 0.3x8 mm), a procedure that is known to produce mechanical injury to red blood cells as well as leukocytes and platelets (15). , and thus very precisely mimics hemolyzed samples present in clinical laboratories, rather than simply adding purified hemoglobin preparations to the samples (16). After this treatment, the sample was centrifuged at 1200 x g for 15 min at room temperature. Free plasma hemoglobin concentration was evaluated on all hemolyzed and non-hemolyzed samples by direct spectrophotometry on a Beckman Coulter DxC (Beckman Coulter, Inc.). The highly significant correlation between this method and the reference cyanomet-hemoglobin assay has already been assessed in a previous study (17). The statistical significance of the differences between rates was assessed by linear regression, Spearman's correlation coefficient (r), and the bias was correlated with the total permissible error derived from intra and inter-individual biological variation (18).

Statistics

Statistical evaluation was performed with Analyze-it for Microsoft Excel (Analyze-it Software Ltd, Leeds, UK). The study was carried out in accordance with the Declaration of Helsinki and under the terms of the relevant local legislation.

Results

The intra-analysis imprecision of the new test developed is between 1.3 and 2.2% (Table), and therefore comparable to that declared by the manufacturer for the commercial reagent. Linearity is also excellent, as can be seen from the linear regression equation found in theoretical concentrations (y = 1.01x - 5.6, r = 0.998, p <0.001). A very significant agreement was observed between trials (new assay Î ± -amylase = 1.06; Beckman Coulter Î ± -amylase -1; r = 1.00, p <0.001), the mean bias being -3.8% (95 % CI, -4.7% to - 3.0%) (Figure 1). The results of the hemolysis study are shown in Figure 2. The concentrations of free plasma hemoglobin and Î ± -amylase in the hemolyzed samples are between 7.5-75.0 g / L and 50-1150 U / L, respectively. Although a significant correlation between values in non-hemolyzed and hemolyzed samples was observed with both assays (commercial and new assay) (p <0.001), greater agreement was found with the new assay (r = 0.997 versus 0.818). It should be noted that with respect to the total permissible error derived from the intra- and inter-individual biological variation (i.e., ± 14.6%), no measure exceeds this threshold with the new assay, while the limit has been exceeded in most part of the samples using the commercial method (17/20, ie, 85%).

Table

Intra-assay imprecision on the newly developed assay tested on Beckman Coulter AU5822. The results are expressed as coefficient of variation (CV,%).

<Value>

(U / L) CV

Low serum 45 ± 1 1.3%

Intermediate serum 143 ± 3 1.7%

High serum 270 ± 6 2.2% Bibliography

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Claims (10)

CLAIMS 1. Method for the determination of alpha-amylase activity in a sample of biological fluid containing hemoglobin, by means of a colorimetric assay comprising: i) addition to the biological sample of an oxidizing agent capable of oxidizing oxyhemoglobin to methemoglobin; or alternatively ii) addition of carbon monoxide to form the carbomonoxyhemoglobin complex; And iii) subsequent addition to the sample of an alpha amylase substrate bearing a chromophore group which is released by the enzymatic activity of the alpha amylase itself, generating a change in absorbance in a predetermined time interval; iv) determination of the absorbance generated by the sample in the predetermined time interval, and therefore of the alpha amylase activity by interpolating a standard reference curve previously obtained with known quantities of alpha amylase. 2. Method according to claim 1, wherein the hemoglobin is in free, erythrocyte, conjugated form with polyethylene glycol or in the form of hemoglobin polymer. 3. Method according to claims 1-2, further comprising the addition of potassium thiocyanate in step iii). 4. Method according to claims 1-3, wherein said oxidizing agent is selected from sodium nitrite and potassium ferricyanide. The method according to claim 4, wherein sodium nitrite is present in the biological fluid sample at a concentration of 10 mM and at pH 6. 6. Method according to claims 1-5, wherein said biological fluid sample is selected from hemolyzed serum or plasma and urine. 7. Method according to claims 1-6, wherein said alpha amylase substrate carrying a chromophore group is selected from 2-chloro-4-nitrophenyl-alpha-D-maltotrioside (CNPG3); 4,6-ethylidene (G7) -p-nitrophenyl (G1) -a, D-maltoheptaoside (EPS) or p-nitrophenyl phosphate (PNP). 8. Kit for carrying out the method according to the preceding claims, comprising, in separate containers, the oxidizing agent and the alpha-amylase substrate bearing the chromophore group and possibly buffer solutions. 9. Use of (i) an oxidizing agent capable of oxidizing oxyhemoglobin to methhemoglobin or (ii) carbon monoxide capable of forming the carbomonoxyhemoglobin complex with hemoglobin, in a colorimetric assay of alpha amylase activity. 10. Use according to claim 9, wherein said oxidizing agent is selected from sodium nitrite and potassium ferricyanide. Milan, 1st October 2012