CN117969720A - Detection and pretreatment method for folic acid derivatives and related metabolic coenzymes in erythrocytes - Google Patents
Detection and pretreatment method for folic acid derivatives and related metabolic coenzymes in erythrocytes Download PDFInfo
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a detection and pretreatment method for folic acid derivatives and related metabolic coenzymes in erythrocytes for the first time, which solves the problems of complex detection steps and inaccurate detection results in the prior art; intensive researches on components of the red blood cell cleaning liquid find out the mechanism that glucose can maintain the stability of folic acid derivatives in red blood cells and can not influence the detection of folic acid related metabolic coenzymes; intensive researches on components of the erythrocyte lysate find that ascorbic acid and sodium dodecyl sulfate have the effect of protecting folic acid derivatives released by erythrocyte lysis and related metabolic coenzymes thereof, and the recovery rate of each detection index is improved; the novel deproteinized liquid is provided, so that on one hand, the recovery rate of each index can be improved, and on the other hand, the coprecipitation of folic acid related metabolic coenzyme and protein can be avoided; the separation gradient of the mobile phase and the liquid phase is improved, so that the quantitative limit of B2 and B6 can be simultaneously reached to the minimum, and the obtained chromatogram has good peak type and more accurate quantification.
Description
Technical Field
The invention belongs to the technical field of biological sample detection, and particularly relates to a detection and pretreatment method for folic acid derivatives and related metabolic coenzymes in erythrocytes.
Background
Folic acid (Folic Acid), also known as pteroylglutamic acid, is an important B-group water-soluble vitamin essential for synthesis of nucleic acid, thymidylate, neurotransmitter, phospholipid and hormone, and has an important role in protein synthesis and cell division and growth processes, and has a promoting effect on the formation of normal erythrocytes. Folate deficiency is associated with the occurrence of a variety of diseases, and accurate detection of folate levels in humans is the basis for assessment of folate status. For a long time, in clinical examination, the content of folic acid in human serum or blood plasma is generally measured to judge whether the human folic acid is deficient or not. In recent years, research proves that the folic acid content in red blood cells is 10-20 times of that in serum or blood plasma, and the blood plasma or blood serum folic acid represents the folic acid level in the in-vivo circulation state, but is easily influenced by factors such as diet and the like, and the concentration level fluctuation is larger. And the red blood cell folic acid is related to the red blood cell updating process and reflects the long-term change state of folic acid and the folic acid storage condition in the human body, so that the red blood cell folic acid measurement can more accurately reflect the tissue storage condition. The red blood cell folate concentration is considered to be an indicator that most reliably reflects folate status. Folic acid is not a single molecule, but consists of a series of derivatives. Folic acid, which is ingested by the human body on a diet in daily life, needs to be converted into 5-methyltetrahydrofolate through a series of metabolic processes to play a role. 5-methyltetrahydrofolate, also known as active folic acid, is metabolized in humans in the following general description: folic acid-dihydrofolic acid-tetrahydrofolic acid-5, 10-methylene tetrahydrofolic acid-5-methyl tetrahydrofolic acid-5, 10-methylene tetrahydrofolic acid, and important coenzymes involved in the metabolic process include folic acid related metabolic coenzymes VB2, folic acid related metabolic coenzymes VB3 and folic acid related metabolic coenzymes VB6.
There are three methods in the prior art to estimate the folate concentration of erythrocytes.
First, the vitamin distribution of the whole blood folic acid is quantitatively determined by adopting a liquid chromatography-tandem mass spectrometry method, and the folic acid concentration of the red blood cells is estimated according to the folic acid concentration of the whole blood. Specifically, a small portion of whole blood is first removed and the hematocrit is measured; taking out a small part of whole blood, and measuring the folic acid concentration of the whole blood; the whole blood folate concentration is then divided by the hematocrit to estimate the red blood folate concentration. The method has the defects that the contribution and the difference of the duty ratio of the plasma folic acid are ignored, the concentration of the red blood cell folic acid is overestimated, and the detection result is inaccurate.
Secondly, the main red cell folic acid derivatives of the subject are measured by using a stable isotope dilution liquid-tandem mass spectrometry, firstly, the red cell pressure and the whole blood folic acid concentration are measured, then the same tube blood is centrifuged to obtain blood plasma, the blood plasma folic acid concentration is measured, and then the red cell folic acid concentration is estimated according to the formula of red cell folic acid= [ whole blood folic acid-blood folic acid (1-red cell pressure accumulation) ]/red cell pressure accumulation.
Thirdly, the whole blood is centrifuged, the upper plasma is discarded, then the lower red blood cells are directly sucked to measure the folic acid concentration of the red blood cells, the plasma is separated from the red blood cells by a centrifugal layering technology, and the concentration of folic acid and 5-methyltetrahydrofluric acid in the red blood cells is detected.
There is no prior art solution for simultaneous determination of folic acid derivatives of erythrocytes and their related metabolic coenzymes.
Therefore, there is a need to develop a method for detecting and preprocessing folic acid derivatives and related metabolic coenzymes in erythrocytes, so as to improve the detection accuracy and reduce the detection error and cost.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a detection and pretreatment method for folic acid derivatives and related metabolic coenzymes in erythrocytes, wherein the folic acid derivatives and the related metabolic coenzymes comprise tetrahydrofolic acid (THF), 5-methyltetrahydrofolic acid (5-MTHF), 5, 10-methyltetrahydrofolic acid (5, 10-CH-THF), folic acid related metabolic coenzyme VB2 (VB 2 or vitamin B2), folic acid related metabolic coenzyme VB3 (VB 3 or vitamin B3) and folic acid related metabolic coenzyme VB6 (VB 6 or vitamin B6).
In one aspect, the present invention provides a method for pretreatment of folic acid derivatives and their related metabolic coenzymes in erythrocytes, comprising the steps of:
s1, red blood cell cleaning: taking a red blood cell sample from which plasma is removed, adding a red blood cell cleaning solution, uniformly mixing, centrifuging, and removing an upper red blood cell cleaning solution;
S2 erythrocyte lysis: adding a red blood cell lysate into the red blood cell sample after the step S1 is completed, uniformly mixing, and carrying out enzymolysis;
S3, protein removal: adding deproteinized liquid into the red blood cell sample after finishing the step S2, uniformly mixing, centrifuging, and taking supernatant to be measured;
The erythrocyte lysate in the step S2 contains sodium dodecyl sulfate and ascorbic acid; the folic acid derivative and related metabolic coenzyme thereof comprise tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5, 10-methyltetrahydrofolic acid, folic acid related metabolic coenzyme VB2, folic acid related metabolic coenzyme VB3 and folic acid related metabolic coenzyme VB6.
The addition of ascorbic acid is beneficial to stabilizing several folic acid derivatives, while the recovery rate of VB3 is slightly reduced by more than 3 percent, and the recovery rate of VB6 is obviously reduced by more than 2 percent, so that the folic acid derivatives are not influenced by VB2, and probably because the folic acid derivatives are easy to oxidize after the erythrocyte is cracked, the folic acid is used as an antioxidant to protect the folic acid derivatives; the addition of low concentration sodium dodecyl sulfate can significantly improve the recovery rate of VB3, and as the concentration is further increased, the recovery rate of each index is reduced, probably due to the fact that too high concentration sodium dodecyl sulfate damages GGH enzyme and the structures of several folic acid related metabolic coenzymes.
Preferably, the erythrocyte lysate in step S2 is an aqueous solution containing 20U/ul GGH enzyme, 0.2% sodium dodecyl sulfate, 1% ascorbic acid.
Both ascorbic acid and sodium lauryl sulfate disrupt cell membranes. In the optimized final version of the invention, the content of ascorbic acid is 1% and the sodium dodecyl sulfate content is only 0.2%, and the component which plays the main role of destroying erythrocyte membranes is ascorbic acid. According to the principle of the method for measuring the labeling recovery rate, the standard substance is added after the red blood cells are cleaned and cannot be added into cell membranes. Whether the standard recovery rate reaches the standard is related to the stability of the compound in the pretreatment process and is not related to the rupture of cell membranes. Therefore, the principle of the invention of using the stable measurement index of sodium dodecyl sulfate is not the common function of the sodium dodecyl sulfate for destroying cell membranes, but the novel application of the sodium dodecyl sulfate for strengthening the stability of folic acid derivatives and related metabolic coenzymes is discovered.
Further, the red blood cell washing liquid in step S1 includes glucose, and the glucose concentration is greater than 5mM.
In the prior art, folic acid derivatives are proved to react with reducing sugar, particularly 5-methyltetrahydrofolate which is one of the detection indexes of the invention, has higher reactivity, the folic acid derivatives and the reducing sugar react at various temperatures to influence the stability of the folic acid derivatives, the reaction rate is high above 70 ℃, and the literature proves that the reaction exists at 37 ℃; glucose is a reducing sugar on the one hand, and reference is made to the literature on the other hand, which shows that folic acid derivatives react with dihydroxyacetone or dihydroxyacetone phosphate, which are metabolic intermediates of glucose, so that glucose theoretically reacts with folic acid derivatives, and the stability of folic acid derivatives is reduced. However, the invention finds that glucose can provide stability when detecting folic acid derivatives through the research of mechanisms, and specific results and mechanisms are as follows:
When no glucose was added to the red blood cell washing solution, the detection value of 5-MTHF was decreased by about 20% from the initial value, the detection value of THF was decreased by about 30% from the initial value, and the detection value of 5,10-CH-THF was increased by about 30% from the initial value after the washed red blood cells were stored at 4℃for 7 days. When glucose is added into the red blood cell cleaning liquid, the detection value and the initial value of each detection index have no obvious change, which indicates that the glucose provides stability for each folic acid derivative. According to the experiments of the present invention, the instability of folic acid derivatives may be due to the fact that these several folic acid derivatives lack glucose after plasma detachment and are converted to each other in erythrocytes. When sufficient glucose is added to the red blood cell washings, the glucose concentration in the red blood cells is far greater than the glucose concentration in the red blood cells (about 5 mM), glucose is absorbed by the red blood cells in a manner that aids in expansion, glucose provides continuous energy to the enzyme reaction, and the enzyme reaction reaches a steady state, and folic acid and its various derivative forms also reach a steady state similar to that in the human body. Based on the above mechanism, glucose provides an effect of stabilizing folic acid derivatives.
Further, the red blood cell washing liquid in step S1 contains 55mM glucose.
When the glucose concentration in the red blood cell cleaning liquid is 55mM, the stabilizing effect on the concentration of several folic acid derivatives is optimal, and the ratio of the detection value to the initial value of the three folic acid derivatives after 7 days is closest to 100%. In addition, the addition of 55mM glucose did not affect the detection of the relevant metabolic coenzymes VB2, VB3 and VB 6.
Further, the deproteinized solution in step S3 includes methanol, acetonitrile, and 25% aqueous trichloroacetic acid.
Preferably, the deproteinizing solution in step S3 is a mixture of methanol, acetonitrile and 25% aqueous solution of trichloroacetic acid, and the volume ratio of methanol, acetonitrile and 25% aqueous solution of trichloroacetic acid is 4:3:3.
The addition of methanol in deproteinized liquid is favorable for the recovery of THF, the addition of acetonitrile is favorable for the recovery of 5,10-CH-THF, and the addition of trichloroacetic acid and perchloric acid is favorable for the recovery of VB 6. When only organic solvents are used, the recovery of VB6 is extremely low, probably due to the co-precipitation of VB6 with hemoglobin in the erythrocytes. In the invention, the deproteinized liquid comprises the following formula: methanol: acetonitrile: recovery of each index was optimized at 25% trichloroacetic acid=4:3:3 (volume ratio).
On the other hand, the invention provides a detection method of folic acid derivatives and related metabolic coenzymes in erythrocytes, which comprises the pretreatment method and also comprises liquid chromatography tandem mass spectrometry detection.
Further, mobile phase A detected by liquid chromatography tandem mass spectrometry is an aqueous solution containing 10mM ammonium acetate and 0.7% formic acid; the mobile phase B is a mixed solution of methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 8:2.
VB2 is difficult to detect when no ammonium acetate is added to mobile phase A. When the ammonium acetate addition is increased, the limit of the VB2 ration can be reduced, but the limit of the VB6 ration is increased; when the formic acid addition amount is increased, the limit of VB6 can be lowered, but the limit of VB2 can be raised. In the present invention, the mobile phase a additive formulation is: the quantitative limits of each index were optimized when an aqueous solution containing 10mM ammonium acetate and 0.7% formic acid was used.
Further, the liquid phase gradient of the liquid chromatography tandem mass spectrometry detection is:
When the time is 0 min, the volume percentage of the mobile phase B is 5%;
When the time is 0.5 min, the volume percentage of the mobile phase B is 30%;
When the time is 1 minute, the volume percentage of the mobile phase B is 30%;
when the time is 2 minutes, the volume percentage of the mobile phase B is 95 percent;
when the time is 3 minutes, the volume percentage of the mobile phase B is 95 percent;
When the time is 3.1 minutes, the volume percentage of the mobile phase B is 5%;
at 4.5 minutes, the volume percent of mobile phase B was 5%.
The folic acid derivative and the related metabolic coenzyme are difficult to separate in the chromatograph, tailing, peak cracking and the like are easy to occur, so that quantification is difficult to carry out.
In another aspect, the present invention provides a use of a red blood cell cleaning fluid comprising glucose, wherein the glucose concentration in the red blood cell cleaning fluid is higher than the glucose concentration in the red blood cell; the folic acid derivatives comprise tetrahydrofolic acid, 5-methyltetrahydrofolic acid and 5, 10-methenyl tetrahydrofolic acid.
The principle of the erythrocyte cleaning fluid for maintaining the stability of folic acid derivatives in erythrocytes is as follows: glucose provides sustained energy to the enzymatic reaction, allowing the enzymatic reaction to reach homeostasis, and folic acid and its various derivative forms to also reach homeostasis similar to that in humans.
Preferably, the red blood cell washing solution contains 55mM glucose.
In another aspect, the invention provides the use of glucose for the preparation of a reagent for maintaining the stability of a folic acid derivative in erythrocytes, said folic acid derivative comprising tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5, 10-methyltetrahydrofolic acid.
In another aspect, the present invention provides the use of a red blood cell lysate comprising sodium dodecyl sulfate, ascorbic acid for the preparation of a reagent for increasing the recovery of a leaf acid derivative and its associated metabolic coenzyme in red blood cells; the folic acid derivative and related metabolic coenzyme thereof comprise tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5, 10-methyltetrahydrofolic acid, folic acid related metabolic coenzyme VB2, folic acid related metabolic coenzyme VB3 and folic acid related metabolic coenzyme VB6.
Preferably, the erythrocyte lysate is an aqueous solution containing 20U/ul GGH enzyme, 0.2% sodium dodecyl sulfate, and 1% ascorbic acid.
In another aspect, the invention provides the use of sodium dodecyl sulfate for the preparation of a reagent for maintaining stability of folic acid derivatives and related metabolic coenzymes thereof, including tetrahydrofolate, 5-methyltetrahydrofolate, 5, 10-methyltetrahydrofolate, folic acid related metabolic coenzyme VB2, folic acid related metabolic coenzyme VB3, folic acid related metabolic coenzyme VB6.
In another aspect, the invention provides an application of deproteinized liquid in preparing a reagent for improving recovery rate of a leaf acid derivative and related metabolic coenzyme in erythrocytes, wherein the deproteinized liquid is a mixed liquid of methanol, acetonitrile and 25% trichloroacetic acid aqueous solution, and the volume ratio of the methanol, the acetonitrile and the 25% trichloroacetic acid aqueous solution is 4:3:3; the folic acid derivative and related metabolic coenzyme thereof comprise tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5, 10-methyltetrahydrofolic acid, folic acid related metabolic coenzyme VB2, folic acid related metabolic coenzyme VB3 and folic acid related metabolic coenzyme VB6.
In another aspect, the present invention provides the use of a mobile phase additive for the preparation of a medicament for simultaneously increasing the quantitative limits of the folate-associated metabolic coenzyme VB2 and the folate-associated metabolic coenzyme VB6, said mobile phase additive being 10mM ammonium acetate and 0.7% formic acid.
The beneficial effects of the invention are as follows:
1. the detection and pretreatment method for folic acid derivatives and related metabolic coenzymes in red blood cells solves the problems of complicated detection steps and inaccurate detection results in the prior art, has short reaction time, simple operation, accurate detection results and high sensitivity, and has wide application prospects;
2. The components of the red blood cell cleaning liquid are studied intensively, and through finding out the mechanism of glucose in maintaining the stability of folic acid derivatives in red blood cells, glucose is added to stabilize the folic acid derivatives, so that the detection accuracy and the reliability of the detection result are improved obviously, and the glucose does not influence the detection of folic acid related metabolic coenzymes;
3. Intensive researches on components of the erythrocyte lysate find that ascorbic acid and sodium dodecyl sulfate have the effect of protecting folic acid derivatives released by erythrocyte lysis and related metabolic coenzymes, so that the recovery rate of each detection index is improved, and the detection sensitivity and the quantitative accuracy are improved;
4. the novel deproteinized liquid is provided, so that on one hand, the recovery rate of each index can be improved, and on the other hand, the coprecipitation of folic acid related metabolic coenzyme and protein can be avoided, and the accuracy of quantification is improved;
5. The mobile phase is improved, and the fact that ammonium acetate or formic acid is added into the mobile phase can play a role in eliminating the quantitative limit of VB2 and VB6, so that the quantitative limit of VB2 and VB6 can be simultaneously minimized by adjusting the ratio of the two, and the detection sensitivity is improved.
6. The liquid phase separation gradient is improved, the complete separation of each detection index is realized, and the obtained chromatogram has good peak shape and more accurate quantification.
Drawings
Fig. 1: folic acid derivative in erythrocytes and detection method schematic diagram of related metabolic coenzyme thereof
Fig. 2: fitting function result of variation coefficient and average value of 5-MTHF
Fig. 3: statistical graph of concentration ratio of red blood cells stored at 4℃for 7 days at different glucose concentrations
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto. The reagents not specifically mentioned in this example are all known products and are obtained by purchasing commercially available products.
Example 1: method for detecting folic acid derivative and related metabolic coenzyme in erythrocytes
The flow of the detection method is shown in fig. 1, and specifically comprises the following steps:
1. reagent preparation
Red blood cell cleaning liquid: medical saline (0.9% aqueous sodium chloride solution) containing 55mM glucose.
Erythrocyte lysate: an aqueous solution containing 20U/ul GGH enzyme, 0.2% sodium dodecyl sulfate, and 1% ascorbic acid.
Deproteinized liquid: methanol-acetonitrile 25% aqueous trichloroacetic acid=4:3:3 (volume ratio).
Mixing standard curve solution: the 5-MTHF, THF, 5,10-CH-THF, VB2, VB3, VB6, FA (folic acid) standard were prepared into mixed working calibrator solutions by deionized water, and the solutions were continuously diluted with ionized water to a total of 7 concentration series of standard curve solutions (S1-S7), as shown in Table 1.
Table 1: preparation of mixed standard curve solution
Stable isotope internal standard solution: stable isotope internal standard of 5-MTHF, THF, 5,10-CH-THF, VB2, VB3, VB6 and FA (folic acid prototype) is respectively 5-MTHF-D4, THF-D4, 5,10-CH-THF-D4, VB2-D5, VB3-D4, VB6-D3 and FA-D4, and mixed stable isotope internal standard solution with concentration of 1000, 500, 200, 2000, 5000, 200 and 200 ng/mL is prepared by deionized water.
Mobile phase a was an aqueous solution containing 10mM ammonium acetate and 0.7% formic acid;
mobile phase B was methanol: acetonitrile=8:2 (volume ratio).
Red blood cell sample preparation: 10 parts of fresh human whole blood were collected using EDTA anticoagulant tubes, centrifuged (4 ℃ C., 2000g,10 min) within 2 hours after blood collection, and the upper plasma was removed to obtain a red blood cell sample.
2. Pretreatment method
(1) Erythrocyte washing
Adding 2 times volume of erythrocyte cleaning liquid into erythrocyte, centrifuging (4 ℃ C., 2000g,10 min), and removing supernatant to obtain a cleaned erythrocyte sample.
(2) Erythrocyte lysis
And (3) sucking the cleaned erythrocyte sample, adding a stable isotope internal standard solution and erythrocyte lysate, shaking and mixing uniformly, and incubating for 1 hour at 37 ℃.
(3) Protein removal
Adding deproteinized solution into sample solution after erythrocyte lysis is completed, shaking and mixing uniformly, centrifuging, and taking supernatant.
(Mixing standard Curve solution with sample pretreatment)
3. Liquid chromatography tandem mass spectrometry detection
A AB SCIEX TRIPLE QUAD 4500MD liquid phase-mass spectrometer is selected, a reverse chromatographic column (ACQUITY UPLC HSS T3.8 um 2.1×100 mm) is selected as the chromatographic column, the liquid phase gradient is shown in Table 2, the ion source parameters are shown in Table 3, and the ion pair parameters are shown in Table 4.
Table 2: gradient of liquid phase
Table 3: ion source parameters
Table 4: ion pair parameters
4. Analysis of results
(1) Linearity of
The peak areas of the 7 test compounds 5-MTHF, THF, 5,10-CH-THF, VB2, VB3, VB6, FA and their stable isotope internal standards can be determined by selecting the ion pairs detected by reaction monitoring and the corresponding retention times. Several compounds were separately quantified by stable isotope dilution: the linear regression equation of each compound was calculated by using the theoretical concentration of the standard curve as the independent variable x, the peak area of the compound corresponding to the concentration/the peak area of the stable isotope as the dependent variable y, and the result is shown in table 5.
Table 5: linear regression equation for each compound
Substituting the compound peak area/stable isotope peak area corresponding to the sample into a linear regression equation to obtain the compound concentration of the sample.
(2) Recovery rate
10 Samples were selected and tested as described above: after step 2 (1) was performed, the samples were divided into 2 groups, and standard substances having concentrations corresponding to the standard curve S4 were added to each of the experimental groups, the standard concentration X 0 was shown in table 1, the control group was not subjected to additional treatment, and all samples were continuously measured for concentration as in example 1. And finally, determining the concentration of a control group of each sample to be X 1, determining the concentration of an experimental group to be X 2, and averaging the labeling recovery rate of 10 samples to obtain the labeling recovery rate of the index, wherein the labeling recovery rate of the sample is Y= (X 2- X1)/ X0).
(3) Quantitative limit
10 Samples of low concentration level were selected and tested as described above: each sample was repeatedly tested 8 times per day, 4 times a day, and 4 times a afternoon, over 5 days. The precision of the quantitative limit is based on the repeated detection variation Coefficient (CV) of less than or equal to 10 percent. Using 5-MTHF as an example, raw data of 5-day detection concentration is shown in the following table (unit: ng/ml)
Table 6: raw data for 5 days of concentration detection
The mean value, standard Deviation (SD) and Coefficient of Variation (CV) of the detected concentration for each sample were calculated from the raw data, respectively, as shown in table 7.
Table 7: average value, standard deviation and variation coefficient of each sample detection concentration
From the above data, a scatter plot was drawn using an Excel table, the Coefficient of Variation (CV) was defined as independent variable (X), and the average value was defined as dependent variable (Y). The function is fitted using a power function model y=ax b: the fitting result is shown in FIG. 2, the fitting function equation is Y= 0.1931X -1.379, the precision target value CV=10% is taken as X into the equation, and the quantitative limit of the 5-MTHF of the method is found to be 4.62ng/ml. The quantitative limits of other folic acid derivatives and related metabolic coenzymes were also determined by reference to the methods described above.
Example 2: test of erythrocyte washing liquid
In the early-stage research of the invention, it is found that the folic acid derivative content in red blood cells changes with the increase of time, and if the folic acid derivative content changes in the time from blood sample collection to blood sample detection, the measurement result is inaccurate, so that the research on improving the stability of the folic acid derivative is needed; after 10 samples of erythrocytes were washed with normal physiological saline, the samples were stored at 4℃for 7 days, and it was found that the concentration of 5-MTHF was reduced by 23%, the concentration of THF was reduced by 32%, the concentration of 5,10-CH-THF was increased by 33%, the concentration of VB2 was reduced by 3%, the concentration of VB3 was reduced by 4%, and the concentration of VB6 was increased by 5%, and the folic acid derivatives were considered to be unstable due to their conversion to 5,10-CH-THF by referring to metabolic pathways of folic acid derivatives in vivo.
50 Parts of fresh human whole blood were collected using EDTA anticoagulant tubes using the procedure of example 1 and divided into 5 groups of 10 parts each, with the difference: the components of the red blood cell washing liquid were changed, different components were added to the physiological saline, the washed red blood cells were stored in a refrigerator at 4℃for 7 days, and were detected once before and after storage, and the concentration after storage was divided by the concentration before storage to obtain a concentration ratio, and the average value of the concentration ratio was calculated for 10 samples of each group, as shown in Table 8, and the results were shown in Table 8.
Table 8: concentration ratio of different red blood cell cleaning liquid to before and after preservation
The initial addition concentration (5%) of bovine serum albumin is the average concentration of protein in human plasma, and both 5% of bovine serum albumin and 25mM glucose can achieve a stable effect on the concentration of folic acid and derivatives thereof, while the addition of other monosaccharides or disaccharides similar to glucose has no obvious effect. It is possible that folic acid derivatives and related metabolic coenzymes exist inside erythrocytes during the washing of erythrocytes, and only glucose in the above saccharides can be absorbed and utilized by erythrocytes, and the remaining saccharides cannot function outside the cells.
Further, the concentration of bovine serum albumin was adjusted to obtain the results shown in Table 9.
Table 9: concentration ratio of bovine serum albumin of different concentrations to before and after storage
Further studies on the concentration of bovine serum albumin have found that altering the concentration of bovine serum albumin does not allow for a more stable concentration of folic acid and its derivatives.
To further determine at what concentration of glucose the stability of the indicator to be measured is optimal; using the procedure of example 1, 70 parts of fresh human whole blood were collected using EDTA anticoagulant tubes and divided into 7 groups of 10 parts each, with the difference: the red blood cells were washed with a physiological saline solution containing 0mM, 5mM, 25mM, 45mM, 55mM, 65mM and 85mM glucose, respectively, and the washed red blood cells were stored in a refrigerator at 4℃for 7 days, and were subjected to one-time detection before and after the storage, and the concentrations of folic acid derivatives and their related metabolic coenzymes before and after the storage were detected as shown in Table 10.
Table 10: folic acid derivatives and their related metabolic coenzyme concentrations before and after storage
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From the average concentration ratios in the above tables, a statistical chart is drawn, as shown in fig. 3, from the detection data of the compounds: when no glucose was added to the red blood cell washing solution, the detection value of 5-MTHF was decreased by about 20% from the initial value, the detection value of THF was decreased by about 30% from the initial value, and the detection value of 5,10-CH-THF was increased by about 30% from the initial value after the washed red blood cells were stored at 4℃for 7 days. It is particularly noted that when glucose is added in an amount equivalent to normal human plasma glucose concentration (5 mM), these folic acid derivatives are not significantly stabilized, probably because other compounds in human plasma are involved in the mass exchange of erythrocytes, helping the concentration of folic acid derivatives in erythrocytes to reach a steady state. However, when the amount of glucose added in the red blood cell washing liquid is increased, the concentration of the folic acid derivatives is still stable at 4 ℃ for 7 days after the red blood cells are separated from the plasma. As can be seen from the comparison chart: in the invention, when the glucose concentration in the red blood cell cleaning solution is 55mM, the stabilizing effect on the concentration of several folic acid derivatives is optimal, and the detection value/initial value of the three folic acid derivatives after 7 days is closest to 100%.
According to the test results, the concentration ratio before and after preservation can be influenced by the components of different red blood cell cleaning solutions, wherein the concentration ratio before and after preservation can be more approximate to 100% by the red blood cell cleaning solution with glucose only, namely the stability of the folic acid derivative is improved; the change in the measured concentration of folic acid derivatives may be due to the fact that these folic acid derivatives lack glucose after removal from plasma and are converted into each other in erythrocytes, and when sufficient glucose is added to the erythrocyte washing solution, the glucose concentration outside the erythrocytes is far greater than that in erythrocytes, glucose is absorbed by erythrocytes in a manner that assists in expansion, glucose provides continuous energy for the enzyme reaction, and the enzyme reaction reaches a steady state, and folic acid and its various derivative forms also reach a steady state similar to that in humans.
The results of the comparative experiments in which bovine serum albumin was added to a wash containing 55mM glucose are shown in Table 11.
Table 11: comparative test of adding bovine serum albumin to glucose-containing washing solution
According to the test results, there was no more remarkable gain effect by adding 5% bovine serum albumin to the red blood cell washing solution containing 55mM glucose.
If the stability of the three folic acid derivatives is increased in other ways, a method of inhibiting the enzyme activity by adding inhibitors or the like can be adopted to inhibit the conversion between the folic acid derivatives, but the structure of relevant metabolic coenzymes VB2, VB3 and VB6 can be destroyed in such a way; while 55mM glucose addition did not affect the detection of the relevant metabolic coenzymes VB2, VB3 and VB 6.
Example 3: testing of erythrocyte lysate
After the red blood cells are cleaned, interference of folic acid derivatives and the like contained in plasma on detection is eliminated, and the next step is to lyse the red blood cells and release substances in the red blood cells into a solution for measurement; however, in the cracking process and the steps after the cracking, various detection indexes are easy to degrade in the solution to be detected, and the early test shows that the recovery rate of THF and 5-MTHF is lower than 75%, and the recovery rate of 5,10-CH-THF and VB3 is lower than 70%, so that the accuracy of quantification is seriously affected, and therefore, certain treatment is needed in the cracking step to reduce the degradation rate and improve the accuracy of quantification.
The procedure of example 1 was used, with the difference that: the labeling recovery rates of the respective detection indexes were examined using the following red blood cell lysates of several formulations, and the results are shown in Table 12.
Table 12: labeling recovery rate of different erythrocyte lysate and each detection index
According to the test results, the GGH enzyme is singly used for erythrocyte lysis, and the recovery rate of three folic acid derivatives and folic acid related metabolic coenzyme VB3 is low, which indicates that the three folic acid derivatives can not be successfully extracted in the lysis process or the compounds can be degraded in the lysis process; the effect of using sodium dodecyl sulfate or sodium dodecyl benzene sulfonate alone is poor, and the extraction effect is hardly achieved; when GGH enzyme and antioxidant are used cooperatively, the recovery rate can be improved to a certain extent, but the GGH enzyme has no obvious effect on folic acid related metabolic coenzyme VB 3; when GGH enzyme and sodium dodecyl sulfate are used cooperatively, the recovery rate of folic acid related metabolic coenzyme VB3 can be improved to a certain extent, but the effect is poor, and the recovery rate is still lower; when GGH enzyme is used cooperatively with EDTA or ammonium chloride, the recovery rate of each index has no obvious improvement effect; when GGH enzyme, sodium dodecyl sulfate and ascorbic acid are cooperatively used, the recovery rate of each index is close to 100%, which shows that the three can cooperatively produce effects, and simultaneously, the extraction effect and the stabilizing effect on detection indexes are improved.
Further, the ratio of GGH enzyme, sodium dodecyl sulfate and ascorbic acid was tested, the GGH enzyme was immobilized at 20U/ul, the ratio of sodium dodecyl sulfate to ascorbic acid was adjusted, and the standard recovery rate of the red blood cell lysate at different ratios was measured, and the results are shown in Table 13.
Table 13: labeling recovery rate under erythrocyte lysate of different proportions
The addition of ascorbic acid is beneficial to stabilizing several folic acid derivatives, while the recovery rate of VB3 is slightly reduced by more than 3 percent, and the recovery rate of VB6 is obviously reduced by more than 2 percent, so that the folic acid derivatives are not influenced by VB2, and probably because the folic acid derivatives are easy to oxidize after the erythrocyte is cracked, the folic acid is used as an antioxidant to protect the folic acid derivatives; the addition of sodium dodecyl sulfate at low concentration can significantly improve the recovery rate of VB3 because VB3 is rapidly degraded after red blood cells are ruptured, the addition of SDS at low concentration can inhibit the activity of VB3 degrading enzyme to a certain extent, the structure of VB3 in the pretreatment process is maintained, but has no effect on folic acid derivatives, and the recovery rate of 5-MTHF is reduced to a certain extent, and as the concentration of sodium dodecyl sulfate is further increased, the recovery rate of each index is reduced, which is probably caused by the fact that GGH enzyme is damaged by sodium dodecyl sulfate at too high concentration and the structures of several folic acid related metabolic coenzymes.
When ascorbic acid and sodium dodecyl sulfate are combined with GGH enzyme at the same time, too high concentration of ascorbic acid reduces the recovery rate of VB6, while too high concentration of sodium dodecyl sulfate reduces the recovery rate of 5-MTHF and VB3, in the invention, the recovery rate of each index is optimal only when the adding amount of sodium dodecyl sulfate in the erythrocyte lysate is 0.2% and the adding amount of ascorbic acid is 1%.
Both ascorbic acid and sodium lauryl sulfate disrupt cell membranes. In the optimized final version of the invention, the content of ascorbic acid is 1% and the sodium dodecyl sulfate content is only 0.2%, and the component which plays the main role of destroying erythrocyte membranes is ascorbic acid. According to the principle of the method for measuring the labeling recovery rate, the standard substance is added after the red blood cells are cleaned and cannot be added into cell membranes. Whether the standard recovery rate reaches the standard is related to the stability of the compound in the pretreatment process and is not related to the rupture of cell membranes. The principle of the invention of using a stable measure of sodium dodecyl sulfate is therefore not its common role for destroying cell membranes, but rather its effect of enhancing the stability of each compound is found, which is a new use of sodium dodecyl sulfate found in the invention.
Example 4: assay of deproteinized fluids
The step of removing the protein is to remove the protein in the solution to be detected, eliminate the interaction between the protein and substances such as folic acid derivatives and the interference on detection caused by the chromatographic effect, and in the step of removing the protein, trichloroacetic acid is often selected, and can form insoluble salts with the protein under the acidic condition, or denature the protein to aggregate and settle; however, in the detection of folic acid derivatives and related metabolic coenzymes in erythrocytes according to the present invention, trichloroacetic acid was used as deproteinizing solution, and the recovery rates of THF and 5,10-CH-THF were lower than 60%, indicating that trichloroacetic acid and its effect on proteins severely affected the detection of the above-mentioned indicators.
To find a suitable deproteinized solution, this example was subjected to a plurality of experiments by referring to the method of example 1, except that the components of the deproteinized solution were changed, and the recovery rates of the respective indexes under the different deproteinized solutions were examined, and the results are shown in Table 14.
Table 14: recovery of deproteinized liquid fraction and index
The recovery rate data of each index can be seen: the addition of methanol in deproteinized liquid is favorable for the recovery of THF, the addition of acetonitrile is favorable for the recovery of 5,10-CH-THF, and the addition of trichloroacetic acid and perchloric acid is favorable for the recovery of VB 6. When only organic solvents are used, the recovery of VB6 is extremely low, probably due to the co-precipitation of VB6 with hemoglobin in the erythrocytes. In the invention, the deproteinized liquid comprises the following formula: methanol: acetonitrile: 25% trichloroacetic acid = 4:3:3 (volume ratio), the recovery rate of each index is optimized.
Example 5: testing of mobile phase additives
Early experiments showed that in conventional organic solvent-water mobile phase systems, the relevant metabolic coenzymes were difficult to detect, where VB2 could not be detected, and VB6 was limited to a quantitative limit of up to 20ng/ml, possibly due to the microscopic molecular structure and biochemical nature of the relevant metabolic coenzymes, and this example attempted to alter the composition of the mobile phase from the mobile phase additive point of view to achieve simultaneous high sensitivity detection of VB2 and VB 6.
Multiple replicates were performed as described in example 1, except that the composition of mobile phase a was varied and the quantitative limits of each index for different mobile phases a were examined and the results are shown in table 15.
Table 15: quantitative limits of mobile phase additives and various indices
From the quantitative limit data for each index, VB2 was difficult to detect when no ammonium acetate was added to mobile phase A. When the ammonium acetate addition is increased, the limit of the VB2 ration can be reduced, but the limit of the VB6 ration is increased; when the formic acid addition amount is increased, the limit of VB6 can be lowered, but the limit of VB2 can be raised. In the present invention, the mobile phase a additive formulation is: the quantitative limits of each index were optimized when an aqueous solution containing 10mM ammonium acetate and 0.7% formic acid was used.
Example 6: testing of liquid phase separation gradients
In the previous test, the peak shape of each detection index in the chromatogram was found to be poor, and the repeated test was performed according to the method of example 1, except that the liquid phase separation gradient was changed, the liquid phase gradients 1 to 3 are shown in tables 16 to 18, and the peak shape of each index under the different liquid phase separation gradients is shown in table 19.
Table 16: liquid phase gradient 1
Table 17: liquid phase gradient 2
Table 18: liquid phase gradient 3
Table 19: peak shape of different liquid phase separation gradients and various indexes
Therefore, the liquid phase gradient 3 is used, so that the peak shape of each index is good, and the separation degree among each index is optimal.
Example 7: comparison with the detection results of the prior art
10 Parts of fresh human whole blood were collected using EDTA anticoagulant tubes, centrifuged (4 ℃ C., 2000g,10 min) within 2 hours after blood collection, and the upper plasma was removed to obtain a red blood cell sample. All erythrocyte samples were divided into three groups, each treatment condition was as follows:
Control group: adding erythrocyte cleaning solution with volume 2 times of that of erythrocyte, centrifuging (4 ℃ C., 2000g,10 min), and removing supernatant to obtain cleaned erythrocyte sample. (mode for detection of the application)
Experiment group 1: and directly carrying out the next operation without processing. (corresponding to the prior art, in particular to the detection mode of patent CN 116087373B)
Experiment group 2: adding red blood cell cleaning solution with volume 2 times of that of red blood cells, centrifuging (4 ℃ C., 2000g,10 min), and removing supernatant to obtain a cleaned red blood cell sample; adding red blood cell cleaning solution containing mixed standard substance with a certain concentration (the final concentration is equivalent to the standard curve S3) in an amount which is 2 times that of red blood cells again, centrifuging (4 ℃ C., 2000g,10 min), and removing supernatant to obtain a cleaned red blood cell sample. (corresponding to the detection mode based on the application, the standard substance is additionally added, and the measured concentration is higher)
The other steps of the above three groups were the same as in example 1 except for the above operation steps, and the detection results are shown in table 20.
Table 20: comparison of detection results
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From the detection data of each compound, it can be seen that: except for folic acid related metabolic coenzyme VB3, the detection concentration of each compound shows the trend that the experimental group 1 is higher than the control group; wherein, the folic acid prototype FA can not be detected in the control group, but is detected in the experimental group 1; the concentrations of all compounds tested showed a trend for experimental group 2 to be higher than the control group.
The technology adopted in the experiment group 1 is that red blood cells are not cleaned and are directly detected, so that the concentration of folic acid derivatives, related metabolic coenzymes and other multiple compounds is higher, the root cause is that the concentration of each compound in blood plasma in which the red blood cells are dispersed is detected simultaneously, the cleaned red blood cells are added with cleaning liquid containing each compound standard substance in the experiment group 2 and are cleaned again, so that the red blood cells are dispersed in the cleaning liquid containing the standard substance, the effect similar to that of the experiment group 1 is achieved, and the fact that the concentration measured by the detection mode in the prior art is higher is shown. The folic acid related metabolic coenzyme VB3 in the experimental group 1 is not significantly increased compared with the control group because: VB3 is metabolized to other forms in the plasma, and residual plasma does not affect the detection of VB 3. The comparative group could not be detected for the prototype FA, but for experimental group 1, the prototype FA could be detected, probably because: the folate prototype is metabolized rapidly after entering tissue cells.
The invention has been described in great detail, but the foregoing embodiments are illustrative, and the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments, as will be apparent to those skilled in the art, in view of the teachings herein. Accordingly, such modifications, adaptations, substitutions, or alternatives can be made without departing from the spirit of the invention and are intended to be within the scope of the invention as set forth in the following claims.
Claims (10)
1. A method for pretreatment of folic acid derivatives and their related metabolic coenzymes in erythrocytes, characterized in that the pretreatment method comprises the steps of:
s1, red blood cell cleaning: taking a red blood cell sample from which plasma is removed, adding a red blood cell cleaning solution, uniformly mixing, centrifuging, and removing an upper red blood cell cleaning solution;
S2 erythrocyte lysis: adding a red blood cell lysate into the red blood cell sample after the step S1 is completed, uniformly mixing, and carrying out enzymolysis;
S3, protein removal: adding deproteinized liquid into the red blood cell sample after finishing the step S2, uniformly mixing, centrifuging, and taking supernatant to be measured;
The erythrocyte lysate in the step S2 contains sodium dodecyl sulfate and ascorbic acid; the folic acid derivative and related metabolic coenzyme thereof comprise tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5, 10-methyltetrahydrofolic acid, folic acid related metabolic coenzyme VB2, folic acid related metabolic coenzyme VB3 and folic acid related metabolic coenzyme VB6.
2. The method according to claim 1, wherein the erythrocyte lysate in step S2 is an aqueous solution containing 20U/ul GGH enzyme, 0.2% sodium dodecyl sulfate, 1% ascorbic acid.
3. The method of claim 1, wherein the red blood cell washing fluid in step S1 comprises glucose at a concentration of greater than 5mM.
4. The method of claim 3, wherein the red blood cell washing fluid in step S1 comprises 55mM glucose.
5. The method of claim 3, wherein the deproteinized solution in step S3 comprises methanol, acetonitrile, 25% aqueous trichloroacetic acid.
6. The method according to claim 5, wherein the deproteinized solution in step S3 is a mixture of methanol, acetonitrile and 25% aqueous trichloroacetic acid solution, and the volume ratio of methanol, acetonitrile and 25% aqueous trichloroacetic acid solution is 4:3:3.
7. A method for detecting folic acid derivatives and related metabolic coenzymes in erythrocytes, comprising the pretreatment method according to claim 5, and further comprising liquid chromatography tandem mass spectrometry detection; mobile phase a, detected by liquid chromatography tandem mass spectrometry, was an aqueous solution containing 10mM ammonium acetate and 0.7% formic acid; the mobile phase B is a mixed solution of methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 8:2.
8. The method of claim 7, wherein the liquid phase gradient detected by liquid chromatography tandem mass spectrometry is:
When the time is 0 min, the volume percentage of the mobile phase B is 5%;
When the time is 0.5 min, the volume percentage of the mobile phase B is 30%;
When the time is 1 minute, the volume percentage of the mobile phase B is 30%;
when the time is 2 minutes, the volume percentage of the mobile phase B is 95 percent;
when the time is 3 minutes, the volume percentage of the mobile phase B is 95 percent;
When the time is 3.1 minutes, the volume percentage of the mobile phase B is 5%;
at 4.5 minutes, the volume percent of mobile phase B was 5%.
9. Use of a red blood cell lysate for the preparation of a reagent for increasing the recovery of a leaf acid derivative and its associated metabolic coenzyme in red blood cells, characterized in that the red blood cell lysate comprises sodium dodecyl sulfate, ascorbic acid; the folic acid derivative and related metabolic coenzyme thereof comprise tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5, 10-methyltetrahydrofolic acid, folic acid related metabolic coenzyme VB2, folic acid related metabolic coenzyme VB3 and folic acid related metabolic coenzyme VB6.
10. Use of glucose for the preparation of a reagent for maintaining the stability of a folic acid derivative in erythrocytes, characterized in that said folic acid derivative comprises tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5, 10-methyltetrahydrofolic acid.
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