RU2034297C1 - Porphyrine metabolism disorder determination method - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: zinc-porphyrines are determined by intensity of luminescence duration. The property zinc-porphyrines demonstrate in specific conditions (buffer solution composition), that are optimized for given task. Method allows also simultaneously to determine value of hemoglobin in blood and calculate derivative index, ratio of zinc-porphyrine to gene, that also has high diagnostic value. Method is intended for wide use as initial screening to determine porphyrine metabolism among big groups of patients (clinical examination, professional diseases, risky groups examination). EFFECT: development of simple, highly resulting instrumental micromethod to determine porphyrine metabolism disorder. 7 cl, 4 dwg

Description

 The invention relates to methods for screening diagnostics of porphyrin metabolism disorders and can be used in medicine, environmental research, radiology, veterinary medicine, etc.

 Porphyrin metabolism disorders are caused by exposure to exogenous factors, in particular, acute and chronic poisoning by heavy metals (lead, cadmium, mercury, and others), chemicals (aromatic hydrocarbons, dioxin compounds, herbicides, etc.), low doses radiation. Violations are also manifested in a number of diseases: with muscle anemia, iron deficiency, cancer and several others. In most cases, there is a prolonged exposure of risk factors in small doses to large groups of the population, such as living in an environmentally unfavorable territory (the Chernobyl accident and environmental disasters), work in production with harmful technological cycles (lead, galvanic, metallurgical, and electrical , chemical and others). These effects are cumulative in nature and eventually lead to characteristic clinical manifestations and the need for therapeutic treatment.

 A characteristic biochemical manifestation for this group of disorders, and especially for acute and chronic lead poisoning, is, in particular, an increase in the levels of zinc protoporphyrin (CPP) in the blood. Currently, the great clinical significance of this indicator for the diagnosis of lead poisoning and other disorders of porphyrin metabolism has been proven.

 CPP is known to be a predominantly red blood cell component. Its levels are about 0.5 μmol / l normal. In cases of porphyrin metabolism disorders, CPP levels increase significantly to 5 μmol / L and higher. When determining CPP, whole blood or red blood cells are usually used. At the same time, the level of CPP in the blood alone is not reliable enough. More reliable and informative, as has been shown in many studies and on statistical material, is a derived indicator of the ratio of CPP / heme. This indicator mainly eliminates the influence of errors related to the irreproducibility of the blood sampling procedure, variations in the content of red blood cells and their integrity. Normal values for this parameter are of the order of 30 μmol / mol heme.

 Currently, a number of methods for determining CPP in biological fluids (blood, erythrocyte mass, tissue extracts, etc.) are described. Most methods used in clinical practice are highly specialized and make it possible to determine in the blood either only CPP or heme. The determination of CPP is usually based on the measurement of its fluorescence, and heme is determined by its absorption using appropriate instruments. The most reliable and accurate method for determining CPP is high performance liquid chromatography with fluorimetric detection. The determination is usually preceded by a pre-treatment step: dilution with an analytical lysis buffer or preliminary separation of a blood sample (extraction, or chromatography). The methods are usually expensive, time-consuming and have a relatively low productivity (about 5 analyzes per hour) and are not very suitable for use as a mass screening test. In this case, the determination of the CPP / heme ratio is possible only by combining two different techniques.

 The prototype of the proposed method for determining disorders of porphyrin metabolism, which is also based on the determination of CPP levels or the CPP / heme ratio in biological samples, is hematofluorimetry. The method has the appropriate instrumentation (hematofluorimeter, for example, Protofluor Z (USA) and is used in clinical practice for the diagnosis of lead poisoning and a number of other porphyrin metabolism disorders, (see above). It includes diluting a blood sample with a special solution that provides lysis red blood cells and transforms the heme into one spectral form, after which the sample is placed on a glass slide in a hematofluorimeter, which immediately measures the luminescence of the CPP (fluorescence) and heme absorption in a thin layer of solution sam ples.

 However, this method has a low productivity (no more than 10 samples per hour), is difficult to automate, and is not suitable for the analysis of large arrays of samples. It is also influenced by a number of factors (the optical properties of the blood and the fluorescent pigments that make up the blood, riboflavin and bilirubin), which are irreproducible and can affect the results of the analysis.

 The aim of the invention is the development of a simple, high-performance instrumental micromethod for determining disorders of porphyrin metabolism, based on the determination of zinc protoporphyrin levels, as well as the ratio of CPP / heme in biological objects, in particular in blood, erythrocyte mass, tissue extracts. The method is intended for widespread use as a primary screening for porphyrin metabolism disorders among large groups of patients (medical examination of occupational hazards and examination of risk groups, etc.).

 In addition, the aim of the invention is to develop a special composition necessary for the implementation of this method and which is its key link.

 The essence of the new approach in the determination of zincprotoporphyrin in biological fluids is to use the property of zinc porphyrin complexes to manifest, under certain conditions, prolonged luminescence (phosphorescence and delayed luminescence) in solutions at room temperature. This property underlies the selective detection and quantification of zinc porphyrins in blood samples based on measurements of the intensity of long-term luminescence. Although the intensity of long-term luminescence of the CPP is several times lower than the intensity of its fluorescence, the sensitivity of its detection when using a pulsed measurement mode with a millisecond time resolution is higher due to a radical decrease in background signals and the influence of various factors on the measurement results.

 The advantages of this approach are due to the fact that biological samples do not contain pigments and other components that would also have prolonged luminescence under the same conditions as zinc porphyrins and in the same spectral and temporal range. This fact makes it possible, using the luminescence measurement mode with millisecond time resolution, to exclude the influence of background glow (scattered light, fluorescence of fluorescent impurities, etc.) and selectively and with very high sensitivity to register only zinc porphyrins. Considering the fact that CPP dominates in biological samples from zinc porphyrins, which is a by-product of the heme biosynthesis from protoporphyrin IX, this approach allows only CPP to be determined in whole blood.

 The phenomenon of prolonged luminescence of organic compounds (labels) is now widespread in immunodiagnostics. Such labels, mainly europium fluorescent complexes, are used to label antibodies and provide high sensitivity and performance in solid-phase immunoassay (for example, the DELFIA method). The serial production of high-performance devices for this method, which record the long-term luminescence of europium complexes, is carried out, in particular, by Wallac (Finland). It is also known to use porphyrin dyes with long luminescence (water-soluble Pd-, Pt-, Zn-porphyrins) as highly sensitive labels. However, in the invention, the phenomenon of prolonged luminescence of metal complexes of porphyrins (in particular, zinc protoporphyrin) is used for a fundamentally different purpose for quantifying the substance itself, which is a biologically active component of living organisms.

 The main blood factor that can significantly affect the results of the analysis is the high hemoglobin content. Hemoglobin has intense absorption bands in the region of CPP excitation and can create an “internal filter effect” at high concentrations, i.e. distort the results of the determination of the CPP. Two ways can be used to eliminate the influence of this factor: repeated dilution of the sample (blood) during the analysis, or the preliminary stage of selective extraction of CPP from the sample with a suitable organic solvent, followed by determination of CPP in the extract by the proposed method.

 The measurements of the long-term luminescence of zincprotoporphyrin are carried out in a special buffer solution, which ensures the ignition of the long-term luminescence of the CPP in the solution and its high intensity. This buffer solution (composition) is designed specifically for the implementation of the described method for the determination of CPP in the blood. It consists of sulfite or bisulfite (sodium) and a nonionic surfactant. Sulfite is necessary for chemical deoxygenation of a solution (removal of dissolved oxygen), which quenches long-term luminescence. Triton X-100 additive is used as a lytic component to destroy red blood cells and obtain a true diluted blood solution, as well as to enhance the long-term luminescence of porphyrin metal complexes in aqueous solutions. It is known that additives of surfactants, mainly nonionic surfactants such as Triton, Twins, etc. at concentrations ensuring the solubilization of the dye molecules, they can increase the intensity of long-term luminescence several times. The buffer also standardizes pH measurement conditions.

 It was also found that the conditions under which the long-term luminescence of the CPP is measured are optimally also suitable for determining heme. When determining CPP in biological objects such as blood or erythrocyte mass, the components of the buffer solution perform a number of additional functions. A surfactant, in particular, also provides for the lysis of the cellular components of the blood, and obtaining a true diluted blood solution (action), and sulfite converts hemoglobin into one spectral form (restored). Under these conditions (i.e., a true solution containing only one form of heme), it is possible to determine the heme content by direct photometry of the diluted sample in the heme absorption region. With an appropriate selection of the dilution value, it is possible to determine both CPP (by its long-term luminescence) and heme (by its absorption) in the same diluted blood sample and as a result, calculate the value of the CPP / heme ratio.

 Thus, the second objective of the invention is the development and optimization of a special composition of a buffer solution, which is the determination of CPP, as well as heme. The composition of this composition includes the following main components: a chemical deoxygenator of aqueous solutions of sulfite or bisulfite (sodium), as well as a nonionic surfactant (such as Triton, Tween, etc.).

 Thus, the proposed method and composition make it possible to measure the content of CPP (by long-term luminescence) and heme (by absorption) in a blood sample, and determine the ratio of CPP / heme. They provide high sensitivity and reliability of measurements. A feature of the new approach is that it is distinguished by exceptional methodological simplicity and high productivity. The method easily adapts to the existing instrument base and does not require any special equipment other than consumable utensils, automatic pipettes and simple solutions.

 The invention is as follows.

 PRI me R 1. The study of long-term luminescence of zincprotoporphyrin IX in solutions.

The standard preparation of zincprotoporphyrin IX was obtained by chemical synthesis based on protoporphyrin IX and ZnCl ₂ by a standard method. The purity of the CPP preparation was 96% according to HPLC.

The composition of the buffer solution for measuring the long-term luminescence of CPP was as follows: 10 mg / ml Na ₂ SO ₃ , 1% Triton X-100, 5 mg / ml KH ₂ PO ₄ , pH 7.4.

 The spectrum of continuous CPP luminescence (uncorrected) is shown in FIG. 1. It was recorded on an LS-50 luminescent spectrometer (Perkin Elmer, England) in the phosphorescence mode with the following measurement parameters: delay time after a lamp flash 0.4 ms; counting time 15.0 ms; integration time 5 s. The emission spectrum contains three bands with a maximum at 595 nm, 650 nm and 700 nm. The first two bands coincide with the fluorescence spectrum of CPP. They correspond to the spectrum of slow CPP fluorescence (uncorrected). The third band corresponds to the phosphorescence spectrum of CPP.

 For quantitative measurements, it is optimal to record in the short-wavelength region of the long-term luminescence of the CPP. The first band with a maximum around 595 nm is the most intense and is well detected by most photodetectors (PMTs). Luminescence excitation is most efficiently carried out in the region of the Soret absorption band (400-420 nm).

 The decay kinetics of long-term CPP luminescence is described by a simple single-exponential attenuation law, according to which the lifetime of long-term CPP luminescence was calculated. It is 12.5 ms in the above analytical buffer.

 In FIG. 2 shows the dilution curves (calibration curves) for determining the CPP. For curve A, measurements were performed on an LS-50 instrument in 0.5 ml plastic tubes, the counting conditions are similar to those described for FIG. 1. In the case of curve B, the CPP measurements were performed on a high-sensitivity Arcus-1231 instrument (Wallac, Finland) for DELFIA fluorescence immunoassay in 12-well plastic strips of collapsible microplates in 0.3 ml samples. Measurement conditions: excitation at 340 nm, registration at 613 nm; delay time 0.4 ms; account gate 2.4 ms; integration time 3 s.

 Despite the non-optimal conditions for measuring the luminescence of the CPP (both the excitation and recording wavelengths and the timing parameters of the count), which are associated with the design features of the Arcus-1231 instrument, the graph demonstrates the high sensitivity of the detection of CPP by this method and good linearity in a wide concentration range. The sensitivity of the method is about 0.1 nM CPP, which is about 3 orders of magnitude lower than the analytical range of the concentration of CPP in the blood.

 When the composition of the buffer solution was varied within 2–15 mg / ml for sulfite or bisulfite, 0.1–5% according to Triton-X-100, and for pH in the range 6.5–8.5, there was no noticeable change in the luminescence of CPP. Replacing Triton X-100 with Tween-20, Tween-40 or Bridge also did not significantly affect the determination results.

 In FIG. Figure 3 shows the effect of blood dilution on the results of the determination of CPP. As can be seen, with a dilution of 1: 500 and higher, the blood ceases to affect the results of the determination of CPP by prolonged luminescence.

 PRI me R 2. A direct method for determining CPP in the blood.

 Long-term luminescence measurements are carried out in the buffer described in Example 1. A finger blood sample (0.02-0.05 ml) is diluted 30 times in plastic tubes with a working buffer (0.01 ml of blood per 0.3 ml of buffer). 0.3 ml of working buffer was poured into the wells of a collapsible microplate (12 8-well strips, Nunc, England) and 0.01 ml of a diluted blood sample was added to each (see above, final dilution of blood 900 times). Some of the wells were used for standard CPP solutions (for example, 0.1; 0.03; 0.01; 0.005 micromol / L CPP) to construct a calibration curve.

 After that, the intensity of long-term luminescence in the wells is measured on an Arcus-1231 instrument under the conditions described in Example 1. Based on measurements according to the CPP standards, a calibration curve is constructed and the CPP content in the blood samples is determined from it (taking into account their dilution).

 Blood samples can be pre-collected in plastic tubes (moistened with heparin solution) and stored frozen for a long time. The determination of CPP after thawing samples is carried out similarly.

 The presence of other pigments, for example, free porphyrins, in concentrations up to 0.1 mmol / L, has practically no effect on the results of CPP measurements.

 PRI me R 3. The extraction method for the determination of CPP in the blood.

 A blood sample (0.02-0.05 ml) was extracted with 10 times the amount of dimethylformamide / methanol mixture (3: 7 v / v), shaking for 5 minutes, followed by centrifugation. 0.03 ml of the supernatant is added to the well of the microplate to 0.3 ml of the working buffer (final dilution of the sample 100 times), long-term luminescence is measured and the concentration of CPP is calculated in the same way as for the whole blood described above.

 The extraction method, despite a larger number of operations, allows working with less diluted blood samples and receiving higher signals when measuring luminescence, thereby increasing the reliability of the results.

 PRI me R 4. Optical properties of blood, diluted with the composition of the buffer solution and the definition of heme.

 The absorption spectrum of whole blood diluted 500 times with the working buffer is shown in FIG. 4 together with the absorption spectrum of CPP. The appearance of the spectrum indicates the presence of a single, restored form of heme. This is easily explained by the reducing effect of sulfite. The presence of one form of hemoglobin in the working buffer makes it extremely simple to quantify it by photometry using a standard heme solution. This can be done directly by photometric measurements of solutions in the heme absorption region (350-600 nm), for example, using an enzyme-linked immunosorbent reader with a 405 or 420 nm filter. Determination of heme by this method was carried out in polystyrene multi-well microplates (Nunc, Finland) in a volume of 0.2 ml on a photometric reader for enzyme-linked immunosorbent assay company Labsystems (Finland) at a wavelength of 410 nm. The working dilution of the blood sample in the buffer composition was 100-1000 times.

 PRI me R 5. Determination of the ratio of CPP / heme in the blood.

 The measurement of the content of CPP in the blood is carried out analogously to example 2. Then the same wells of the microplate photometrically on a reader at 410 nm. According to the results of absorption measurements, using the hemoglobin standard or the molar extinction value (see example 4), the heme content is determined. Based on the results of two measurements, the ratio of CPP / heme is calculated.

 The method was tested on blood samples of patients associated with work in lead production. For him, the adequacy of the results obtained and the ability to identify individuals with deviations of the CPP indicator and the ratio of CPP / heme from the norm (high levels) are shown.

 Thus, the new screening method for determining violations of porphyrin metabolism is distinguished by its methodological simplicity (only the stage of dilution of the sample), low cost (all reagents and consumables are easily accessible and inexpensive), high reliability and sensitivity, extremely high productivity (hundreds of analyzes per hour) and can be implemented on already manufactured equipment without significant modifications.

Claims (7)

 1. METHOD FOR DETERMINING PORPHYRININE IMPAIRMENT DISORDERS, including sampling of biological fluid, processing it with a buffer solution containing a lysing agent and a reducing agent, measuring the luminescence level of zincprotoporphyrin with subsequent calculation of its content and comparing with normal values, characterized in that the level of long-term luminescence of zincprotopin is measured, zincprotop moreover, the measurement is carried out under optimum conditions in a pulsed mode with a millisecond time resolution, not using a lysing agent ionic surfactants, and, as a reducing agent, sodium sulfite or bisulfite in concentrations providing respectively deoxygenation of the solution and the formation of a reduced form of hemoglobin, as well as lysis of the cellular and subcellular components of the sample and solubilization of zincprotoporphyrin.  2. The method according to claim 1, characterized in that the heme content is additionally measured by recording the absorption level, the zincprotoporphyrin / heme ratio is calculated, which determines the presence of porphyrin metabolism disorders.  3. The method according to claims 1 and 2, characterized in that the luminescence is measured at λ excitation 380 450 nm and l registration 550 700 nm.  4. The method according to claims 1, 2 and 3, characterized in that the content of heme and zincprotoporphyrin is carried out in one sample.  5. The method according to PP.2 to 4, characterized in that the absorption of the heme is measured at 380,650 nm.  6. The method according to PP.1 to 3, characterized in that before processing the blood sample with a buffer solution, it is subjected to extraction with an organic solvent and zincprotoporphyrin is determined in the extract.  7. The method according to PP. 1 6, characterized in that the buffer solution contains sodium sulfite (or bisulfite) sodium 2 mg / ml, Triton X-100 0.1 5.0% sodium phosphate 10 100 mn, pH 7.4.

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