BG113543A - Method for electron paramagnetic resonance (epr) determination of serum albumin levels and hypoalbuminemia in patients presented with coronavirus (covid-19) infection - Google Patents

Abstract

The method is used in the study of changes in serum albumin levels and states of hypoalbuminemia, which are considered a distinctive marker of severe and fatal coronavirus infection or are a sign of complications in various diseases. The proposed protocol can be applied and introduced in medical diagnostics, as a method for determining serum albumin levels in biochemical laboratories, in diseases characterized by changes in serum albumin levels and hypoalbuminemia, incl. in patients with COVID-19. EPR spectroscopy in combination with 3-Maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-Maleimido-PROXYL, 5-MSL) is a highly specific and highly sensitive method applicable to the study of the protein structure of albumin, its dynamics , measuring the concentration of albumin in serum samples of patients with COVID-19 to make a diagnosis - hypoalbuminemia and includes five stages: Stage 1 / Performing a venipuncture according to a standard protocol - taking venous blood in a tube from the closed system (vacuum tube for serum /with a red cap); Stage 2/ Centrifugation of blood samples to separate serum; Stage 3/Separation of serum and its transfer to the Eppendorf system; Stage 4/ Incubation of the serum samples with a solution of the nitroxide radical 3- Maleimido-PROXYL; Stage 5/ Placing the test sample in the EPR-microcapillary and starting the EPR measurement, data processing and analysis.

Description

ELECTRON PARAMAGNETIC RESONANCE METHOD FOR DETERMINING THE LEVELS OF SERUM ALBUMIN AND HYPOALBUMINEMIA IN PATIENTS WITH CORONAVIRUS INFECTION (COVID-19)

DESCRIPTION

TECHNICAL AREA

This method refers to the use of the stable nitroxide radical 3Maleimido-2,2,5,5-tetramethyl-l-pyrrolidinyloxy (3-Maleimido-PROXYL, 5-MSL) and its analogs, in studying the rotational mobility of the radical, in the presence of albumin and aims to determine hypoalbuminemia in patients with SARS-Cov-2 infection (COVID-19) or other congenital and pathological changes characterized by serum albumin values outside the reference range 35-52 g/L. In particular, this invention consists of a spectroscopic method based on Electron Paramagnetic Resonance Spectroscopy (EPR/EPR) and the nitroxide spin detector 3-MaleimidoPROXYL.

By analyzing the EPR spectral parameters of nitroxide, information on serum albumin levels is provided:

* in COVID-19 patients, with low protein concentrations (hypoalbuminemia, albumin <35 g/L), free nitroxide rotation is reported;

* in healthy people, with serum albumin levels in the reference range of 35-52 g/L, difficult rotation of the spin-label with respect to the applied magnetic field is reported.

The method is aimed at studying the dynamics of 3-Maleimido-PROXYL in COVID-induced hypoalbuminemia or other congenital and pathological changes characterized by serum albumin values outside the reference range 35-52 g/L. The method subject to the inventions focuses on spin labeling of a cysteine residue (Cys-34) with paramagnetic synthetic pyrrolidine nitroxides, through which diamagnetic protein structures - albumin - are visualized.

PREVIOUS CONDITION

Human serum albumin is the most abundant transport protein in blood plasma, which is responsible for the reversible binding of numerous endogenous and exogenous compounds. It has a high overall binding capacity and is characterized as a major transporter of neutral lipophilic and acidic dosage forms, and maintains colloid-osmotic pressure (Raoufinia, R et al., 2016). At low albumin levels, osmosis is disrupted and fluid shifts from the intravascular to the interstitial space, which in turn activates baroreceptors and favors sodium and water retention (Arques, S. and Ambrosi, P., 2011).

Serum albumin levels below 35g/L usually result from various congenital (bisalbuminemia) (Chhabra, S et al., 2013) or acquired pathological conditions (hypoalbuminemia), including qualitative changes in albumin microheterogeneity, such as oxidized albumin and glycoalbumin (Watanabe A et al., 2004).

Currently, there are various methods for determining serum albumin levels, which are part of routine clinical and biochemical laboratory analyzes (Soeters, R.V et al, 2019). By their nature, they are spectrophotometric methods and involve the use of specific dyes. Currently, methods for measuring serum albumin are based on measuring the absorbance of the protein-dye complex, at a certain wavelength, and undergo preliminary sample preparation. Standard spectrophotometric assays include the use of bromocresol purple, bromophenol blue, and bromocresol green indicator dyes. Known methods (see Patent EP0414935A1) require maintaining a relatively narrow pH range, which necessitates the use of additional reagents to maintain it. Another disadvantage of spectrophotometric methods for the analysis of albumin (Patent: G01N33/6839) is that the indicator dyes used are not highly specific in terms of binding to the protein, as they can bind to a number of other interfering proteins, for example globulins. This can lead to an inaccurate analysis due to insufficient specificity of the dye to albumin. In order to improve the determination of protein concentration (total protein analysis) in the test sample, some methods include a reagent system with two indicator dyes (e.g. bromophenol blue and methyl orange), without excluding the use of a suitable buffer (Patent: EP0361244A2), which makes analysis more expensive.

Commonly used dyes, such as bromocresol green (BCG) and bromocresol purple (BCP), are also characterized by a different analytical reading range of 38-53 g/L for BCG and 36-51 g/L for BCP. A disadvantage of these methods is that a large coefficient of variation is observed, which is due to the lack of a uniform standard for reagents and equipment in different laboratories. Also, in some diseases such as hypertension, diabetic nephropathy, liver cirrhosis, etc., these variations create the premise for inaccurate measurement of small deviations in serum albumin, which are particularly decisive in the pathophysiological assessment of the disease state and can delay the start of treatment. worsen the general condition of the body and lead to death. Another disadvantage is that concentration limits are also observed. Depending on the conventional method, some amount of albumin may be lost during sample pretreatment.

In clinical conditions characterized by high concentrations of serum globulins (> 39 g/L), a disadvantage of BCG methods is the occurrence of false positive results, due to the formation of a complex, not only between the dye and albumin, but also with the highly reactive α -2-macroglobulin. In case of nephrotic syndrome, BCG methods are not recommended. The presence of α and β globulins in the samples interferes with the measurement of albumin due to their easy binding to the dye and this leads to false positives and contributes to the poor specificity of the method.

Known patents so far proposed in connection with the study of physiologically important macromolecules (Patent: EP0714407B1), which involve the use of stable nitroxide radicals (incl. 3-Maleimido-PROXYL) as spin labels, are mainly aimed at labeling hemoglobin with nitroxide ( patent: WO1996029974A2). The in vivo active form of the radical allows application of this class of compounds in the development of blood substitutes with an oxygen detoxifying function mimicking that of red blood cells. The patents feature covalent attachment of 4-(2-Bromoacetamido)TEMPO and 3-Maleimido-PROXYL to cross-linked human hemoglobin and describe the synthesis of nitroxide-labeled physiologically compatible forms of hemoglobin. They also consider the influence of nitroxides on the course of redox reactions with the initiator superoxide anion radical. A disadvantage of these methods is that they involve additional processing in isolation of hemoglobin and are not used as a diagnostic assay in the study of low protein levels in viral infections and/or COVID-19.

In patients with COVID-19, hemoglobin levels are usually normal or mild anemia is observed (Hariyanto, T.I.; Kumiawan, A, 2020; Bergamaschi, G et al., 2021). A number of studies have reported a high rate of hypoalbuminemia, with physiologically low albumin levels being one of the main markers of severe disease course and are the basis for the development of a diagnostic method involving nitroxide radicals as spin detectors of COVID-induced hypoalbuminemia (Huang, J et al., 2020).

Low albumin levels combined with massive fluid losses due to fever cause severe hypovolemia and shock, which are commonly seen in patients with COVID-19, especially in critical condition (Ramadori G, 2020). At the same time, hypoalbuminemia in COVID-19 represents a secondary disease state and creates a prerequisite for reducing the effectiveness of treatment and increasing the probability of a fatal outcome. In SARS-CoV-2 infection, timely administration of albumin is of utmost importance for adequate treatment, especially in critically ill patients who are diagnosed with low albumin levels. In the acute phase of the disease, albumin plays the role of an antioxidant. In a condition of cytokine storm and oxidative stress, the generation of abnormal levels of free radicals can lead to irreversible protein oxidation, a decrease in its serum concentration and subsequent damage to cells, tissues and organs. Clinical studies have shown that high levels of oxidative stress, combined with hypoalbuminemia, increase the risk of mortality in patients with COVID-19 (Belinskaia, D.A et al., 2021). Until now, there is no reported case in the literature of the application of 3-Melaimido-PROXYL and EPR spectroscopy in the study of albumin and hypoalbuminemia in patients with COVID-19. The method proposed by us serves for the diagnosis of the condition "hypoalbuminemia" in human serum samples, with Electron Paramagnetic Resonance Spectroscopy and the nitroxide radical 3-Melaimido-PROXYL, as this specific medical condition, at the moment, is determined by not-so-precise clinical- laboratory-diagnostic spectrophotometric methods.

Prior to the present patent proposal, the capabilities of the nitroxide radical 3-Melaimido-PROXYL and its application potential in medical diagnostics and biochemical screening, during and after coronavirus infection, have not been described and investigated. Scientific articles and patents available to date are not related to the development of a method for the analysis of hypoalbuminemia in patients with COVID-19.

Therefore, it is necessary to establish a new, unified, accurate, specific and standard method for the determination of serum albumin levels in patients with COVID-19, which involves the covalent binding of 3-Maleimido-PROXYL to albumin molecules and aims to overcome the shortcomings of the methods known to date (referred to in the patents cited above).

ESSENCE OF THE METHOD, APPARATUS AND TECHNICAL PARAMETERS

The spectroscopic method we developed, described in the present patent, is characterized by high accuracy, precision and reproducibility of the results, which, in the conditions of the covid pandemic, is of utmost importance when assessing the condition of patients with hypoalbuminemia, such as the intermediate-critical and critical cases.

To study albumin dynamics in COVID-induced hypoalbuminemia, the spin-label must have a stable paramagnetic moiety, include a functional group capable of selectively reacting with a specific amino acid without perturbing its structure, and be sensitive to albumin reorientation . Depending on the structure of the macromolecules and the properties of the local environment, the method should include the use of specific spin-labels meeting these conditions such as 3-Maleimido-2,2,5,5-tetramethyl-l-pyrrolidinyloxy (3-Maleimido-PROXYL , 5-MSL), 2,2,5,5-tetramethyl-l-oxyl-3-methyl methanethiosulfonate (MTSL), bifunctional derivatives, etc. The EPR spectroscopic analysis using the spin-label 3-MaleimidoPROXYL allows following conformational changes in the albumin molecule, by virtue of which the protein concentration is determined.

As a result of free rotation of 3-Maleimido-PROXYL in a solvent (Dimethyl Sulfoxide, DMSO), three sharp and closely spaced spectral lines are observed, which are of almost identical width and approximately the same intensity (control radical solvent, C1). In healthy people (control, C2), changes in the spectral parameters of the spin-label should be observed, which are the result of localization of the nitroxide in the protein molecule and lead to a decrease in its mobility with respect to the applied magnetic field.

At serum albumin levels above >35g/L, the Hmax and ΔΗ ⁰ values and the change in the EPR spectrum of the nitroxide, relative to that of 3-Maleimido-PROXYL/pa3TBopHTen, will be due to a high degree of specificity and binding of the radical to albumin . As a result of binding of the nitroxide to the protein, its rotation is hindered, which is reflected in a change in the spectral parameters compared to those of 3-MaleimidoPROXYL/solvent where no albumin is present. Conversely, at protein concentrations less than <35 g/L typical of patients with COVID-19, little change in Hmax and ΔΗ ⁰ values will be observed, with data comparable to that of the 3-Maleimido-PROXYL control /pa3TBopHTen. As a result of hypoalbuminemia in patients with COVID-19 and free rotation of the spin-label, the parameters Hmax and ΔΗ° will be the same in value as those of 3-Maleimido-PROXYL/pa3TBopHTen.

The method involves the use of a standard X-Band EPR spectrophotometer and is divided into five steps (see Scheme 1):

Stage 1: Performing a venipuncture according to a standard protocol - taking venous blood in a tube from the closed system (serum tube)

Step 2: Centrifugation of blood samples to separate serum

Step 3: Separation of serum and its transfer to an Eppendorf system

Step 4: Incubation of the serum samples with a solution of the nitroxide radical 3Maleimido-PROXYL

Step 5: Place the test sample in the EPR microcapillary and start the EPR measurement, data processing and analysis

Albumin

Sample Inversion (10k) | Incubate the sample (30 mln) |

HjC N CHj Ο*____________________

Albumin labeling with 3-Meleimido-PROXYL

Venipuncture using Clot Activator vacuum tubes for blood serum

Centrifuge for 15 mln at 3000 rpm

Transferred to a tan serum in an eppendorf cream

Samples were allowed to stand for 60 min before centrifugation Stage 5 |

Start the measurement

Processing and analysis of data from the EPR spectrum

STAGES OF SAMPLE PREPARATION AND ANALYSIS

Stage 1: Performing an injection according to a standard protocol - collecting venous blood in a closed system tube (vacuum tubes for serum with a red cap)

Step 2: Centrifugation of blood samples to separate serum

Stage 3: Separating the serum and transferring it to the Eppendorf system

Step 4: Incubation of the serum samples (P1) with a solution of the nitroxide radical 3-Maleimido-PROXYUDMSO (S1) 50 μΙ of solution S1 with a concentration of 200 µM and added to 450 µl test sample P1

Step 5: Place the test sample in the EPR-microcapillary and start the EPR measurement data processing and analysis

Scheme 1. Methodical and experimental protocol for the study of serum albumin levels and hypoalbiminemia.

DETERMINATION OF SERUM ALBUMIN LEVELS - GENERAL EPR PROTOCOL FOR SAMPLE PREPARATION AND ANALYSIS

STAGE 1:

A standard venous blood collection procedure is followed which includes a venipuncture protocol using Clot Activator vacuum blood serum tubes (PET vacutainer with red cap). 4 ml of venous blood is taken from each patient. Immediately after venipuncture, the tubes are carefully inverted (10x) to ensure proper mixing of the clot activator with the sample and to accelerate the clotting process. Samples were allowed to stand for 60 min before centrifugation.

STAGE 2 and 3:

The blood samples were centrifuged for 15 min and 3000 rpm, after which the blood serum was separated and transferred to a 2 ml eppendorf system.

STAGE 4:

4.1. A solution with a working concentration of 200 μΜ of 3-MaleimidoPROXYL in dimethyl sulfoxide (DMSO) solvent was prepared (S1).

4.2. Sample preparation for analysis: 50 μΐ of solution S1 is taken by variable pipette and added to 450 μΐ of test sample Pl, (P1, sample of patient with COVID-19 or other disease characterized by low albumin levels and hypoalbuminemia) .

4.3. Serum samples were incubated at temperature (20-24°C) with 3-MaleimidoPROXYL for 30 min, after which the assay was started. The step involves vortexing test samples for 5s, immediately after mixing S1 and P1 and immediately before starting the measurement.

STAGE 5:

The samples were incubated for 30 minutes at room temperature, after which the EPR measurement was started at parameters: Microwave Frequency: 9.846 GHz, Microwave Power: 1.290e+001 mW, Modulation Frequency: 100.000 kHz, Center Field: 3510.00 G; Sweep Width: 100.00 G; Sweep Time: 82.9 s; Time constant: 327.680 ms, Signal Phase: 180.0 deg, number of scans: 2. Sample analysis was performed with an X-band EMXmicro spectrometer (Brücker, Germany) equipped with a standard resonator. The test sample is placed in quartz capillaries, which are sealed and placed in a standard EPR quartz tube (i.d. 3 mm), which is fixed in the cavity of the EPR spectrometer. The measurement is started and the analysis of each sample is carried out in triplicate. Spectral processing was performed using Bruker WIN-EPR and SimFonia software programs.

The analysis of the EPR spectra is carried out after labeling the cysteine residues by analyzing the hyperfine splitting constants Htach measured from the external peaks a and c and ΔΗ ⁰ - central resonance line (b) in the EPR spectrum of the nitroxide, which indicate the degree of inversion of the radical and are a marker of its mobility. At physiologically compatible albumin levels, such as those observed in healthy humans, the rotational mobility of 3-Maleimido-PROXYL is restricted (Figure 2B), whereas at serum albumin lower than 35 g/L, rotation of the spin-label is free (Figure 2C), and the measured parameters were identical to the radical/solvent control (Figure 2A).

Values of ΔΗ°= 1.66 G and H _max = 30 G are characteristic of the control 3-MaleimidoPROXYL in DMSO solvent (albumin-free control sample).

In samples from non-hypoalbuminemic patients with normal serum albumin levels (35-52 g/L), reported values of ΔΗ°= 3.6 G and H _max = 60-62G.

In patients with COVID-19-induced hypoalbuminemia, the values of ΔΗ°= 1.66^-1.75 G and Hmax= 30±32 G were comparable to those of the control 3-Maleimido-PROXYL in the solvent DMSO in which albumin was not present. .

The results (Table 1) of the EPR spectral analysis for the evidence of hypoalbuminemia in patients with COVID-19 were compared and validated by comparing data from a standard conventional colorimetric method. Conventional analysis was performed with a Cobas Integra® 400 Plus automated clinical laboratory analyzer (Roshe DIAGNOSTICS).

EPR method for determining serum albumin levels EPR parameters 3-Maleimido-PROXYL/DMSO control sample without serum Healthy patients without hypoalbuminemia Patients with COVID-19 and hypoalbuminemia ΔΗ, G 1.66 ±0.0 3.03-3.75 ±0.05 1.66-1.75 ±0.05 Nstach, G 30 ±0.00 60-62 ± 0.00 30-32 ± 0.00 A conventional test for determining serum albumin levels - Healthy patients without hypoalbuminemia Patients with COVID-19 and hypoalbuminemia Albumin — serum Reference range 35-52 g/L - 35-52 g/L <35 g/L

Table 1. The EPR spectral analysis for proving hypoalbuminemia in patients with COVID-19 and a conventional colorimetric method for determining serum albumin levels in COVID-19-induced hypoalbuminemia. All tested samples are measured at 20-24°C, and the results are determined from the arithmetic mean value of three measurements.

EXAMPLES OF APPLICATION OF THE METHOD

When examining serum albumin levels and states of hypoalbuminemia, which is considered a distinctive marker of severe and fatal coronavirus infection. The EPR spectroscopic examination of serum albumin levels with 3-Maleimido-PROXYL represents an adequate analytical method for albumin monitoring and is characterized as a rapid, specific and inexpensive assay.

The method is applicable for disease states accompanied by changes in serum albumin levels, incl. those associated with disruption of its protein structure or genetically determined conditions (alloalbuminemia or bisalbuminemia, familial disalbuminemic hyperthyroxinemia and hypertriiodothyroninemia), leading to changes in the conformation and concentration of albumin. These conditions are of particular clinical importance, as affected individuals are at risk of inappropriate treatment or may experience unwanted side effects of drug therapies (Caridi G et al., 2022).

In the acute phase of diseases, which are characterized by high levels of globulins (over 39 g/L), in which conventional spectrophotometric clinical laboratory methods are inapplicable due to the formation of complexes between α- and β-globulin with the fluorophore. EPR spectroscopy in combination with 3-Maleimido-PROXYL represents a powerful, accurate and specific method for studying the protein structure of albumin, its dynamics, provides the necessary sensitivity through a good signal/noise ratio, including at protein concentrations less than 1 nmol /L protein, which allows resolution at the protein molecule level, i.e. beyond the possibilities of the conventional spectrophotometric methods currently used in the biochemical monitoring of albumin. The assay is performed at physiological pH and at room temperature.

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12. Patent: "Method and composition for the assay of albumin", (EP-0414935-Al).

13. Patent: "Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green, (G01N33/6839).

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15. Patent: "Composition and methods using nitroxides to avoid oxygen toxicity", (EP-0714407-B1).

16. Patent: "Compositions and methods utilizing nitroxides in combination with biocompatible macromolecules", (WO-1996029974-A2).