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    • 专利摘要:
    Fluorometric method for the direct titration of nitrites/nitrates in serum and in biological tissue extracts. The titration of nitrites and nitrates is in increasing demand in both basic and clinical research laboratories. Some commercial fluorometric kits have the great drawback that they must be previously processed to eliminate interferences. The present invention describes a method for the titration of nitrites/nitrates for a concentration range of 0 to 1200 pmol of biological samples in a simple, direct way and avoiding the previous treatment of sample preparation to eliminate possible molecules that interfere in the result in other methods. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2785086A1
    申请号:ES202030312
    申请日:2020-04-17
    公开日:2020-10-05
    发明作者:González Juana Benedí；Alvarez Alba Garcimartín；Martín Jose Joaquín Merino；María Elvira López-Oliva；González Adrian Macho；González Pilar González
    申请人:Universidad Complutense de Madrid；
    IPC主号:
    专利说明:

    [0001] FLUORIMETRIC METHOD FOR THE DIRECT ASSESSMENT OF NITRITES / NITRATES IN SERUM AND IN EXTRACTS OF BIOLOGICAL TISSUES
    [0003] TECHNICAL SECTOR
    [0005] The present invention falls within the field of measuring parameters in serum and biological tissues in laboratories. More specifically, it refers to a method for determining nitrites and nitrates following a fluorometric method.
    [0007] BACKGROUND OF THE INVENTION
    [0009] In Biomedicine there is a growing interest in the measurement of nitric oxide (NO) in serum or plasma, since it can be indicative of various diseases such as psoriasis or cardiovascular diseases, among others. The measurement of NO is also of interest in cases of ischemia because high levels of NO protect against the sequelae induced by stroke. Research on the pathophysiology of chronic degenerative diseases currently very prevalent, such as Alzheimer's disease or metabolic syndrome, indicate that NO seems to play an important role in the low-grade inflammation present in all of them. Therefore, the analysis of the NO concentration in biological samples can be a useful tool to know the degree of evolution of these pathologies.
    [0011] The measurement of nitric oxide (NO) in biological samples presents the difficulty of the short period of the half-life of this molecule (between 6 and 10 seconds) that is transformed into nitrites and nitrates, nitrates being the most stable. For this reason, the titration of nitrites and nitrates is in increasing demand in both basic and clinical research laboratories.
    [0013] There are several nitrite titration methods that are based on colorimetric, fluorimetric, HPLC, and chemiluminescence techniques. Among them, the fluorimetric methods that use 2,3-diaminonaphthalene (DAN). The classical method is the one developed by Misko (Misko, TP et al. A fluorimetric assay for the measurement of nitrite in biological samples. Analytical Biochemistry (1993) 214, 11-16). There are companies on the market that use a methodology similar to Misko's (for example, the Abcam company kit). However, these methods have the fundamental drawback of the need to carry out a preliminary processing of the sample that requires expensive equipment (such as ultracentrifuges) or the purchase of commercial columns that also make the technique more expensive.
    [0015] For all the above, there is a need for a simple and direct method that avoids the previous step of sample preparation.
    [0017] EXPLANATION OF THE INVENTION
    [0019] The present invention describes a fluorometric method for the titration of nitrites / nitrates in biological samples that is based on the classic modified Misko method. The modifications introduced make it possible to eliminate the need for prior treatment of the sample and reduce the risk of ocular and respiratory toxicity of the DAN reagent.
    [0021] The method comprises the following stages:
    [0023] a) Prepare a 1mg / ml solution of 2,3-daminonaphthalene (DAN) in 0.6M HCl (from here on this solution will be called "concentrated DAN") and store in a freezer.
    [0024] b) At the time of each titration, prepare a solution by taking 10 µl of the previous solution (concentrated DAN) and 190 µl of 0.6M HCl (from here on, this solution would be referred to as "diluted DAN) c) Obtain a calibration curve for nitrite concentration in a range of 0 to 500 pmol. (Table 1) This range is sufficient although the nitrite curve could have a sensitivity up to at least 1200 pmol.
    [0025] d) Take a volume between 5 and 30 µl of serum or biological sample extract
    [0026] e) Add distilled water to a total volume of 150 pl
    [0027] f) Add 10 µl of diluted DAN and keep the mixture for 15 minutes at room temperature and in the dark to allow the reaction between nitrites and DAN to take place.
    [0028] g) Add 50 µl of 3M NaOH and keep the mixture again for 15 minutes in the dark.
    [0029] h) Read the fluorescence at 340 nm exc. and 460 nm em. in a fluorimeter. i) Determine the nitrite concentration from the fluorescence read and the nitrite calibration curve.
    [0030] j) For the determination of nitrates, a step prior to the technique explained is necessary, in which nitrates are converted to nitrites by the enzyme nitrate reductase. Convert nitrates to nitrites and repeat the previous two steps to obtain the nitrite concentration plus nitrates. k) Determine the nitrate concentration by subtracting the nitrite value.
    [0032] The method allows knowing the nitrite / nitrate concentration for a concentration range of 0-1200 pmol (Figure 1) without the need to pretreat the sample to eliminate possible molecules that interfere with the result in other methods.
    [0034] BRIEF DESCRIPTION OF THE DRAWINGS
    [0036] To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description in which, with an illustrative and non-limiting nature, it has been represented following:
    [0038] Figure 1 shows the sensitivity of the nitrite standard curve in arbitrary fluorescence units (UAF).
    [0040] Figure 2 shows the effect of serum volume on the result of the fluorimetric titration of nitrites.
    [0042] Figure 3 shows the serum elution profile by passage of Sephadex G-25 Fine.
    [0043] Figure 4 shows the effect of the nitrite concentration corresponding to the mixture of fractions 21 to 31 of the Sephadex G25 Fine step, which would be the equivalent to the serum metabolites.
    [0045] PREFERRED EMBODIMENT OF THE INVENTION
    [0047] The present invention is illustrated by the following examples, which are not intended to be limiting of its scope.
    [0049] Example 1.
    [0051] This example refers to the preparation of the nitrite calibration curve.
    [0053] Prepare a 5 pM solution of nitrites in distilled water. From this solution, the pl indicated in Table 1 (pl NaNO 2 ) are taken to obtain a range of nitrite concentrations between 0 and 500 pmol. Distilled water is added to 150 µl and then a fixed volume of diluted DAN of 10 µl is added. Table 1 shows the mixes prepared.
    [0055] Table 1.
    [0060] Each prepared mixture is kept for 15 minutes at room temperature and in the dark, in order to allow the reaction between nitrites and DAN to take place:
    [0063] Once this time has elapsed, 50 µl of 3M NaOH is added and the mixture is left for another 15 minutes in the dark. Finally, the fluorescence is read at 340 nm exc and 460 nm em in a fluorimeter.
    [0065] Figure 1 represents the result obtained with a standard curve made with nitrite concentrations between 0 and 1200 pmol. It can be seen how the nitrite concentration is linear up to at least 1200 pmol of NaNO 2 as shown by the correlation coefficient of the curve (R = 0.9969).
    [0067] Example 2.
    [0069] This example refers to the influence of the degree of dilution of the serum in the mixture with water, DAN and NaOH on the titration of nitrites in serum. It is shown how the reaction is linear up to 30 µl of serum. Higher concentrations begin to have interference.
    [0071] Therefore, for the titration of nitrites in serum or in biological sample extract, different volumes of serum can be used, between 5 and 30 µl to which distilled water (H 2 O) is added to a total volume of 150 µl. 10 µl of diluted DAN and 50 µl of 3M NaOH are added in the same way as in Example 1.
    [0072] In Figure 2 the results obtained with different volumes of serum are shown. It can be seen that the relationship between the concentration of nitrites and the volume of serum in the mixture is linear in the range of 5 to 30 µl of serum according to the equation Y = 54.6 X 1.11 (R = 0.997). Higher amounts of serum volume decrease nitrite concentration.
    [0074] This indicates that there is some component in the serum (protein or metabolite) that at high concentrations (volume of serum above 40 µl in the mixture) decreases the fluorescence of the reaction.
    [0076] Example 3.
    [0078] In order to check whether proteins are the molecules responsible for the interference observed when a serum volume greater than 30 pL is used, the effect of proteins on fluorescence is measured.
    [0080] The effect of albumin (alb) concentrations in the range 160 to 640 pg on a 100 pmol sample of nitrites is determined. Table 2 shows the fluorescence result in arbitrary units (UAF) for various concentrations in this range. The standard error of the mean (SEM) and the probability value (p) are also indicated and compared with the control (100 pmoles of nitrites).
    [0082] Table 2.
    [0087] These results show that the presence of albumin, at concentrations similar to the proteins found in serum, does not appear to be responsible for the loss of fluorescence of the nitrite reaction at high serum concentrations.
    [0088] To reinforce this result, a nitrite titration is carried out in deproteinized serum by precipitation with ammonium sulfate [SO 4 (NH 4 ) 2 ] and in serum passed through a Sephadex G25 fine column and it is concluded again that the proteins do not appear to be responsible for the loss of fluorescence at high serum concentrations. These results are detailed in the following examples.
    [0090] Example 4.
    [0092] This example shows the nitrite titration in deproteinized serum with [SO 4 (NH 4 ) 2 ]
    [0094] Ammonium sulfate is used to precipitate most of the proteins present in serum due to its high solubility and the stabilization of proteins in this medium. For this, 100 mg of SO 4 (NH 4) 2 are added to 250 µl of serum. It is stirred and, subsequently, it is left to rest until the precipitation is complete. It is then centrifuged at 13000 rpm in Eppendorff for 15 minutes. After centrifugation, the supernatant (protein-free serum) is collected and nitrites are titrated.
    [0096] In Table 3 it can be seen that in serum samples from which most of the proteins have been eliminated, the amount of nitrites also decreases at higher sample volumes (samples containing serum, water, diluted DAN and NaOH). This supports the idea that proteins are not responsible for interference in nitrite titration. When assessing the ammonium sulfate concentrations in these samples, there is no effect on fluorescence due to nitrites.
    [0098] Table 3.
    [0101] Example 5.
    [0103] This example refers to the determination of nitrites in serum passed through a column of Sephadex G-25 fine.
    [0105] A column with dimensions 2 cm high 1 cm in diameter is used, equilibrated with 20 mM Tris-HCl buffer pH = 7.6. A 200 µl volume of serum is passed through the column eluting with the buffer. 96 fractions of 3 drops / fraction are collected on special Petri dishes to read at 260-280nm. The elution profile is read at 260 nm (nucletotide detection) and 280 nm (protein detection) in order to detect the protein fraction and metabolites. Higher molecular weight (MW) molecules come out first, then metabolites are eluted. This elution profile is shown in Figure 3.
    [0107] The fractions are joined as follows:
    [0108] - Mix 1: fractions 1 to 6
    [0109] - Mix 2: Fractions 7 to 15 (higher MW molecules, proteins and nucleic acids)
    [0110] - Mix 3: fractions 16 to 20
    [0111] - Mix 4: fractions 21 to 31 (metabolites)
    [0112] - Mix 5: fractions 32 to 49
    [0114] In mixture 4 (fractions 21-31), corresponding to metabolites, nitrites are assessed, the result being shown in Figure 4, where it is observed that the nitrite measurement of the fraction that contained only metabolites (mixture 4) is linear until the amount of 30 pl; Larger fractions decrease the amount of nitrites, which means less fluorescence of the sample. These results indicate that the molecule that lowers fluorescence at volumes greater than 30 pL of serum is a metabolite and not a protein.
    [0116] On the other hand, all these data indicate that the nitrite titration method of the present invention is valid for serum samples between 5 and 30 µl since in this interval there are no interferences from proteins or metabolites, so it is not necessary to eliminate them. previously, what the possibility of using the method directly, without the pre-processing of the sample carried out by the rest of the fluorimetric methods available on the market (see Figure 2).
    [0118] Example 6.
    [0120] This example shows titrations performed on diabetic sera.
    [0122] Table 4 presents measurements made with control sera and samples from diabetic patients, and it is observed that diabetic patients have a higher concentration of nitrites in serum.
    [0124] Table 4.
    [0129] Example 7.
    [0131] This example shows titrations performed on isolated cell and tissue extracts.
    [0133] Extracts with tissues from liver, bladder, endothelial cells and polymononuclear cells (PMBC) are prepared using 20mM Tris-HCl buffer pH = 7.6 in a ratio of 1/10 (100 mg of tissue per ml of buffer 20 mM Tris- HCl pH 7.6).
    [0135] The results of the titration of these extracts are shown in Table 5.
    [0136] Table 5.
    [0139] Example 8.
    [0141] This example shows the titration of nitrates in serum and in extracts of biological samples.
    [0143] To carry out the titration of nitrates, first they are passed to nitrites, the total amount of nitrites is valued, then the difference is calculated. To convert nitrates into nitrites, the enzyme nitrate reductase is used, which catalyzes the following reaction:
    [0145] Nitrate reductase
    [0146] Nitrates NADPH ------------------------- * Nitrites NADP H20
    [0148] To perform the titration, 20 µl of serum are mixed with 5 µl of NADPH (2mg / ml), 23 µl of 20 mM Tris-HCl buffer pH = 7.6 and 2 µl of nitrate reductase (20 mU), in a total volume 50 µl, and the mixture is incubated at 37 ° C for 15 minutes. Then 100 µl of water are added, leaving a total volume of 150 µl and the nitrite is titrated by adding 10 µl of diluted DAN and 50 µl of NaOH. A standard curve is obtained in the same way as is shown in Example 1 for the case of nitrites, but adding to each sample NADPH (2mg / ml) and 23 μl of 20 mM Tris-HCl buffer pH = 7.6 to compensate for possible fluorescence due to NDAPH and / or Tris-HCl buffer. Table 6 shows the standard curve data.
    [0150] Table 6.
    [0155] With this calibration curve, the nitrites plus nitrates (transformed into nitrites) of the serum sample or biological extract (nitrites totals). The nitrate value would be obtained by subtracting the result of the free nitrite titration that is done simultaneously as indicated in the nitrite titration section.
    权利要求:
    Claims (4)
    [1]
    1. Fluorimetric method for the direct titration of nitrites / nitrates in serum and in biological tissue extracts, characterized in that it does not require prior treatment to eliminate proteins or metabolites and that comprises the following steps:
    a) Prepare a 1mg / ml solution of 2,3-daminonaphthalene (DAN) in 0.6M HCl and store in a freezer.
    b) At the time of each titration, prepare a solution by taking 10 µl of the previous solution (concentrated DAN) and 190 µl of 0.6M HCl.
    c) Obtain a calibration curve for nitrite concentration in a range from 0 to 500 pmol.
    d) Take a volume between 5 and 30 µl of serum or biological sample extract
    e) Add distilled water to a total volume of 150 pl
    f) Add 10 µl of diluted DAN and keep the mixture for 15 minutes at room temperature and in the dark to allow the reaction between nitrites and DAN to take place.
    g) Add 50 µl of 3M NaOH and keep the mixture again for 15 minutes in the dark.
    h) Read the fluorescence at 340 nm exc and 460 nm in in a fluorimeter. i) Determine the nitrite concentration from the fluorescence read and the nitrite calibration curve.
    j) Convert nitrates to nitrites and repeat the two previous steps to obtain the concentration of nitrites plus nitrates.
    k) Determine the nitrate concentration by subtracting the nitrite value.
    [2]
    2. Fluorimetric method for the direct titration of nitrites / nitrates, according to claim 1, where the concentration of nitrites / nitrates in the serum sample or biological extract is comprised in the range of 0 to 1200 pmol.
    [3]
    3. Fluorimetric method for the direct titration of nitrites / nitrates, according to any of claims 1 and 2, wherein the volume of the serum sample or biological sample extract is between 5 and 30 µl.
    [4]
    4. Fluorimetric method for the direct titration of nitrites / nitrates, according to any of claims 1 to 3, where the biological extract comes from liver, bladder or endothelial and polymorphonuclear cells tissues.
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