CN115453017A - Method for detecting erythrocyte folic acid - Google Patents
Method for detecting erythrocyte folic acid Download PDFInfo
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- CN115453017A CN115453017A CN202110647553.6A CN202110647553A CN115453017A CN 115453017 A CN115453017 A CN 115453017A CN 202110647553 A CN202110647553 A CN 202110647553A CN 115453017 A CN115453017 A CN 115453017A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a method for detecting erythrocyte folic acid, which comprises the following steps: (1) Preparing erythrocyte suspension, detecting the hematocrit of the erythrocyte suspension, or preparing erythrocyte lysate, and detecting the hemoglobin concentration of the erythrocyte lysate; (2) Extracting and detecting folic acid, wherein the supernatant obtained by extracting folic acid is subjected to folic acid LC-MS/MS detection, and the concentration of folic acid in red blood cell suspension or red blood cell lysate is calculated according to the concentration of a standard substance; (3) Folate concentration calculation, wherein folate concentration is calculated according to the following formula: erythrocyte folate concentration = folate concentration in the erythrocyte suspension/hematocrit of the erythrocyte suspension; or erythrocyte folate concentration = folate concentration in the erythrocyte lysate/(erythrocyte lysate hemoglobin concentration 0.0029). The detection of the invention has higher accuracy and clinical value through verification.
Description
Technical Field
The invention relates to the field of folic acid detection, in particular to a method for detecting erythrocyte folic acid.
Background
Folate levels are closely related to reproductive health. The clinical detection of folic acid level comprises two types, namely serum (blood plasma) detection and erythrocyte detection.
The existing serum (plasma) detection technology is mature, and the detection methods comprise three methods: microbiological methods, protein binding methods, and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
The microbiological method utilizes the principle that the growth rate of lactobacilli is sensitive to folate, reflecting the concentration of added folate in the culture by measuring the turbidity of the bacteria after growth over a certain period of time (Grossowicz, n., et al, determination of folic acid in biological subjects and in nutrients with nutritional extracellular Exp Biol Med,1962.109 p.770-3..
Microbiological methods are recognized by the clinical Nutrition community as gold standards for folate detection and are often used for performance comparisons by new methods (Yetley, E.A., et al, biomarkers of folate status in NHANES: a roundable summary. Am J Clin Nutr,2011.94 (1): p.303S-312S). The main disadvantages of the microbiological method are that the detection takes long time, generally needs 3 days, has high requirements on aseptic operation, and is not beneficial to commercial popularization.
Commercial test kits based on the principle of protein binding have been widely used to this day since the last 70 s. The major international kit manufacturers include Roche
The detection of the Folate I and the Folate II of the Folate II is mainly based on the principle of competitive binding of endogenous folic acid and exogenously labeled folic acid to Folate binding protein, and utilizes a protein binding assay (protein binding assay) for generating a signal by chemiluminescence.
In recent years, detection methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) have also been increasingly widely used for serum folate detection and have shown very good specificity and sensitivity (Verstraete, J., et. Al., clinical determination of patients: recovery analytical protocols and strains. Anal Bioanal Chem., 2019.411 (19): p.4383-4399.).
The major drawback of serological tests is that they reflect a recent folate intake, are susceptible to transient dietary factors, are highly variable, and their concentrations are not widely accepted by academia for reference values in the avoidance of birth defects. WHO indicated in a nutritional guideline for the prevention of neural tube defects that serum or plasma folate thresholds are inadequate for preventing neural tube defects in women of child-bearing age; the concentration of erythrocyte folate must reach more than 906nmol/L to prevent the neural tube defect to the maximum (Cordero, A.M., et al, optimal serum and red blood cell concentration in the World of productive for the prevention of neural tube defects: world Health Organization guidelines. MMWR molar mineral Wkly Rep,2015.64 (15): p.421-3.).
Erythrocyte folate reflects long-term storage levels of folic acid in vivo, is not susceptible to recent folate uptake, and is considered by many scholars to be a better folate indicator (Farrell, C.J., S.H.Kirsch, and M.Hermann, red cell or serum folate: what to do in clinical practical Clin Chem Lab Med,2013.51 (3): p.555-69.).
However, the erythrocyte folate detection based on the protein binding method has the problem of low accuracy, for example, owen et al (2003) compares the detection data of 5 folate detection kits of different brands, and finds that the difference of She Suanzhi among different detection kits is as high as 250%, which is obviously higher than the difference of 40% of serum folate (Owen, W.E.and W.L.Roberts, comparison of five automated serum and white blood folate assays. Am J Clin Pathol,2003.120 (1): p.121-6.). The inaccuracy of the detection of the folic acid of the red blood cells seriously limits the wide application of the folic acid in clinic.
The more important drawback of protein binding assays is that they detect total folate and cannot distinguish between various intermediate metabolites of folate in the body, which have different functions. In particular, 5-methyltetrahydrofolate (5-MTHF), one of the intermediate metabolites, acts as a methyl donor and is directly involved in homocysteine re-methylation, and is the main active state of folate in cells. The 5-MTHF concentration is directly influenced by the activity of methylenetetrahydrofolate reductase (MTHFR). The MTHFR677 site C- > T Polymorphism reduces the activity of the enzyme by 70%, significantly reducing the concentration of 5-MTHF, and thus increasing the risk of Neural Tube Defects (Yan, L., et al, association of the Material MTHFR C677T Polymorphism to Neural Tube Defects in Offspages: evolution from 25Case-Control Studies. PLoS ONE,2012.7 (10): p.e41689.). In contrast, total folate concentrations of erythrocytes detected by protein binding assay (Bio-Rad Quantaphase II radiology) exhibited an abnormal distribution of type TT > CT > CC among people of different genotypes of MTHFR677, in contradistinction to the results of the microbiological assay considered by the WHO as the gold standard for folate detection (Crider et al: MTHFR 677C → T genetic is associated with folate and homeostatic associations in a large, position-based, double-index tertiary of folate administration. The American Journal of Clinical Nutrition 2011,93 (6): 1365-1372).
The mass spectrum detection can accurately identify a single intermediate small molecule in the folate metabolism process by the specific chromatographic separation and the mass-to-charge ratio of primary and secondary target ions, so the detection is more accurate.
In recent years, there have been many reports on the use of LC-MS/MS technology to detect folic acid in whole blood, which results are closer to the results of gold-based microbiological methods for folic acid detection than chemiluminescence methods, and show higher accuracy (Verstraete, J., et al, clinical determinations of concerns: analytical protocols and strains, anal Bioanal Chem,2019.411 (19): p.4383-4399.). However, the application of mass spectrometry to the detection method of erythrocyte folic acid has not been reported at present.
Whole blood consists of blood cells (primarily red blood cells) and a liquid portion of plasma. Folate is present both in the cells and in the plasma, with plasma folate accounting for about 6% of whole blood folate and this ratio being subject to individual differences (Zhang, M., et al., manipulated RBC folate associations from cultured whole blood lipids, cultured hemoglobin, and expressed serum folate-cross-sectional data from the NHANES 1988-2010.The American Journal of Clinical nutrition,2020.111 (3): p.601-612.).
There are two methods in the literature to estimate the concentration of red blood cell folate. The first method is to estimate the concentration of folic acid in erythrocytes from the whole blood. Specifically, first, a small portion of whole blood is taken out, and the hematocrit is measured; an aliquot of whole blood was then removed and the whole blood folate concentration was measured. The whole blood folate concentration is then divided by the hematocrit to estimate the erythrocyte folate concentration (Smith, D.E., et al., quantitative determination of erythrocytic folate distribution by liquid chromatography. Clin Chem Lab. Med., 2006.44 (4): p.450-9 De Bruyn, E., B.gulbis, and F.Cotton, serum and red cell template testing for platelet determination: new tissue European Journal of Haematology,2014.92 (4): p.354-359.). This method results in an overestimation and inaccuracy of red blood cell folate concentrations due to neglect of plasma folate contribution and ratio variability. The second Method is to measure the red blood cell pressure and the whole blood folate concentration, centrifuge the same blood tube to obtain plasma, measure the plasma folate concentration, and estimate the red blood cell folate (Hoffbrand, a.v., b.f. a.newcomb, and d.l. mollin, method of assisted cell activity and the value of the assay a test for the assay specificity, 1966.19 (1): p.17-28, huang, Y., et al, quantification of key red blood cells from subjects with defined MTHFR 677C >. The method has the defect that the folic acid detection of the plasma and the whole blood needs to be carried out respectively, so that the detection error and the cost are increased.
In the whole blood folic acid extraction, the current conventional method is to add 1% ascorbic acid with 4-9 times volume of one part of EDTA whole blood, incubate at 37 ℃ or normal temperature for about 30-120 minutes, add the whole blood into a solid phase extraction column, wash the whole blood, and remove folic acid by using a solution containing methanol, acetonitrile, acetic acid and ascorbic acid. The folic acid thus purified and concentrated can be detected directly (Fazili, Z.and C.M.Pfeiffer, measurement of substrates in Serial and Collection preparation of Whole Blood samples: application of an Automated 96-Well Plate Isotope-Dilution library methods Chemistry,2004.50 (12): p.2378-2381.) or by liquid chromatography-Tandem Mass Spectrometry after drying with nitrogen and redissolving (Huang, Y.et., quantification of bound cell proteins from subjects with defined MTHFR 677C Tgenetic type analysis), see FIG. 12. See FIG. 3. 12. The present invention is not limited to methods of purification and concentration of folic acid. These extraction methods are complicated in procedure, i.e., increase cost, decrease efficiency, and may increase detection errors due to more steps.
Disclosure of Invention
The present inventors have made extensive and intensive studies with respect to the deficiencies of the prior art, and have completed the present invention.
The invention aims to provide a novel method for detecting erythrocyte folic acid. The method mainly comprises a new method of red blood cell preparation, transportation, folic acid extraction and concentration calculation before mass spectrum detection, so that folic acid in red blood cells can be measured by an LC-MS/MS method.
In one aspect, the present invention provides a method for detecting erythrocyte folic acid, which comprises the following steps:
(1) Procedure for erythrocyte preparation
S1, placing a fasting venous blood sample into an anticoagulation tube, storing and transporting at 0-4 ℃, and performing the following operations within 72 hours at most: rinsing erythrocytes for at least 1 time by using isotonic non-toxic liquid at 0-4 ℃ to prepare erythrocyte suspension; detecting Hematocrit (HCT) of the red blood cell suspension; alternatively, the first and second liquid crystal display panels may be,
s1', placing a fasting venous blood sample in an anticoagulation tube, and storing or transporting the blood sample at 0-4 ℃, wherein the following operations are carried out within 72 hours at most: rinsing the red blood cells with isotonic non-toxic liquid at 0-4 deg.C for at least 1 time, centrifuging, collecting red blood cells, and freezing at-40-80 deg.C (preferably-80 deg.C or in dry ice) (for long-term storage and transportation); adding 1% ascorbic acid solution with the volume twice that of the red blood cells into the frozen red blood cells to thaw and suspend the red blood cells to prepare red blood cell lysate; detecting the hemoglobin concentration of the erythrocyte lysate;
(2) Folic acid extraction and detection
Respectively adding a folic acid extracting solution into the red blood cell suspension obtained in S1 or the red blood cell lysate obtained in S1' and the folic acid standard solution, and incubating for 30min-2h at 37 ℃; removing protein by using a chemical reagent capable of precipitating protein to obtain supernatant, directly carrying out folic acid LC-MS/MS detection on the supernatant, and calculating the concentration of folic acid in the erythrocyte suspension or erythrocyte lysate according to the concentration of a standard substance;
(3) Calculation of Folic acid concentration
The folate concentration was calculated according to the following equation
Erythrocyte folate concentration = folate concentration in the erythrocyte suspension/hematocrit of the erythrocyte suspension; or
Erythrocyte folate concentration = folate concentration in the erythrocyte lysate/(hemoglobin concentration in the erythrocyte lysate 0.0029).
In step (1), the isotonic non-toxic liquid is pH neutral isotonic non-toxic liquid which can be used in erythrocyte rinsing process. Preferably, the isotonic non-toxic liquid includes, but is not limited to, physiological saline, phosphate buffer, and the like.
In one embodiment, S1 comprises the steps of: placing the fasting venous blood sample into an anticoagulation tube (for example, an EDTA-K2 anticoagulation tube), temporarily storing at 0-4 ℃ in the dark, and transporting to a detection laboratory at 0-4 ℃ in the dark within 72 hours; after receiving the sample, the detection laboratory completes the following operations: separating plasma by conventional centrifugation (e.g., 2000g, 10min), discarding supernatant, and adding isotonic nontoxic liquid (preferably physiological saline); mixing, centrifuging (e.g., 2000g, 10min), discarding the supernatant, adding an isotonic non-toxic liquid (preferably physiological saline) with a volume twice that of the red blood cells, and making into an isotonic non-toxic liquid suspension of red blood cells.
In one embodiment, S1' comprises the steps of: placing the fasting venous blood sample in an anticoagulation tube (e.g., EDTA-K2 anticoagulation tube), storing in dark at 0 to 4 ℃, transporting in dark at 0 to 4 ℃ to a local sample disposal site within 72 hours: after the disposal site receives the sample, the following operations are completed: separating plasma by conventional centrifugation (e.g., 2000g, 10min), discarding supernatant, and adding isotonic nontoxic liquid (preferably physiological saline); mixing, centrifuging (e.g., 2000g, 10min), discarding supernatant, collecting red blood cells, and freezing at-40 to-80 deg.C (preferably at-80 deg.C or in dry ice); conveying the sample in dry ice to a detection laboratory; after receiving the sample, the detection laboratory completes the following operations: adding 1% ascorbic acid solution with the volume being two times that of the red blood cells into the frozen red blood cells to unfreeze and suspend the red blood cells to prepare a red blood cell lysate; and detecting the hemoglobin concentration of the erythrocyte lysate.
In step (2) of the present invention, the folic acid includes various intermediate forms in the folic acid metabolic pathway, including but not limited to folic acid (folic acid), 5-methyltetrahydrofolic acid (5-methyltetrahydrofolic acid), tetrahydrofolic acid (tetrahydrofolic acid), 5-formyltetrahydrofolic acid (5-formyltetrahydrofolic acid), 5,10-methylenetetrahydrofolic acid (5,10-methylhydrofolic acid), and the like.
The folic acid extract in step (2) of the present invention is an aqueous solution that provides an antioxidant environment and contains enzymes that hydrolyze polyglutamic acid folate to folic acid monoglutamate. In one embodiment, the folate extract comprises 1% ascorbic acid (or DTT, TCEP, β -mercaptoethanol), 1 μ g/ml GGH recombinase (or animal plasma or tissue extract containing GGH enzyme, or GGH enzyme purified from animal tissue), an isotopically labeled internal 5-methyltetrahydrofolate standard.
In step (2) of the present invention, the procedure for LC-MS/MS detection of folic acid in the supernatant is known to those skilled in the art. For example, it is done by (ultra) high performance liquid chromatography based on reverse phase chromatography columns, combined with triple quadrupole mass spectrometry detection. Specific chromatographic separation and mass spectrometric detection parameters are reported in various documents, with slightly different, but substantially similar, example parameter settings.
In one embodiment, the LC-MS/MS detection conditions are as follows:
the instrument comprises the following steps: SCIEX
6500+LC-MS/MS
Chromatographic conditions are as follows: the Column was separated using an ACQUITY UPLC BEH C18 Column,1.7 μm,2.1mm X50mm, mobile phase water (0.1% formic acid) as phase A, methanol (0.1% formic acid) as phase B, column temperature 40 deg.C; the sample injection amount is 5 mu L, the flow rate is 0.5ml/min, and gradient elution is carried out;
mass spectrum conditions: and performing mass spectrum scanning in a positive ion acquisition mode through a multi-reaction monitoring mode by adopting an electrospray ion source.
In step (2) of the present invention, the chemical agent capable of precipitating proteins is an organic or inorganic solvent capable of causing precipitation of proteins. Preferably, the chemical agent capable of precipitating proteins includes, but is not limited to, trichloroacetic acid (TCA), methanol, acetonitrile, zinc sulfide solution, chloroform-methanol mixture, and the like.
In one embodiment, step (2) comprises: adding folic acid extract (1% ascorbic acid, 1 μ g/ml GGH recombinase, isotope labeled 5-methyltetrahydrofolate internal standard) into the erythrocyte suspension obtained in S1 or erythrocyte lysate and folic acid standard solution obtained in S1', respectively, and incubating at 37 deg.C for 1h; a trichloroacetic acid (TCA) solution (e.g., 10% TCA solution) is added, allowed to stand (e.g., 30 minutes), centrifuged (e.g., 13000rpm,4 ℃,10 minutes), the supernatant is subjected to folate LC-MS/MS detection, and the concentration of folate in the erythrocyte suspension or erythrocyte lysate is calculated from the standard concentration.
The detection of the invention has higher accuracy and clinical value through verification.
The present invention has been described in detail hereinabove, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. Furthermore, there is no intention to be bound by any theory presented in the preceding prior art or the summary or the following examples.
Unless expressly stated otherwise, a range of values throughout this application includes any sub-range therein and any value incremented by the minimum sub-unit of a given value therein. Unless expressly stated otherwise, numerical values throughout this specification represent approximate measures or limitations to the extent that they include minor deviations from the given values, as well as embodiments having values about the mentioned as well as having the precise values mentioned. Other than in the operating examples provided at the end of the detailed description, all numbers expressing quantities or conditions of parameters (e.g., quantities or conditions) used in the specification (including the appended claims) are to be understood as being modified in all instances by the term "about" whether or not "about" actually appears before the number. "about" means that the numerical value so stated is allowed to be somewhat imprecise (with some approach to exactness in that value; about or reasonably close to that value; approximately). As used herein, "about" refers to at least variations that can be produced by ordinary methods of measuring and using such parameters, provided that the imprecision provided by "about" is not otherwise understood in the art with this ordinary meaning. For example, "about" can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%.
Drawings
Fig. 1 is a schematic flow chart of a detection method according to an embodiment of the present invention.
Fig. 2 is a schematic flow chart of a detection method according to another embodiment of the present invention.
FIG. 3 is a graph comparing the results of the assay of example 1 using the assay of the invention with the Beckman Coulter Access Folate assay for red blood cell Folate assay.
FIG. 4 is a graph comparing the distribution of erythrocyte Folate measured by the method of the present invention in example 1 with the results of the assay using the commercial Kit Access Folate Kit [ A98032] (Beckman Coulter) and the microbiological method. Wherein, the left graph is the detection of the invention; the middle picture is detected by a Beckmann kit, and the right picture is detected by a microbiological method.
FIG. 5 is a graph showing the effect of the 4 ℃ standard transportation procedure in example 1 and the dry ice freeze transportation procedure in example 2 on red blood cell folate results.
Detailed Description
Hereinafter, the present invention will be described in detail by examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples
Example 1:
invention testMeasuring method
(1) Standard Red blood cell preparation procedure
Extracting 1 tube of fasting venous blood by using 2ml EDTA-K2 anticoagulation tube, turning slightly and reversing for 5 times, shaking up, placing in a refrigerator at 4 ℃ for temporary storage in dark, transporting to a treatment point at 4 ℃ within 72 hours to complete the preparation of the erythrocyte. After the blood sample is received at the treatment point, centrifuging for 10min at the normal temperature of 2000g, sucking away the blood plasma, adding 4ml of precooled physiological saline, fully shaking up, centrifuging for 10min at 2000g, and sucking away the supernatant. Then, precooled physiological saline of about twice the volume of the erythrocytes was added thereto, and the erythrocytes were suspended by gentle shaking to prepare a physiological saline suspension of erythrocytes (hereinafter referred to as an erythrocyte suspension). 500. Mu.l of the suspension of red blood cells was taken out for routine examination of blood or HCT (hematocrit) was measured by centrifugation. Then 100 mul red blood cell suspension is taken out for folic acid extraction, or frozen at minus 80 ℃ until folic acid extraction.
(2) Folic acid extraction and detection
Mu.l of folic acid extract (1% ascorbic acid, 1. Mu.g/ml GGH recombinase, isotope labeled 5-methyltetrahydrofolate internal standard) was added to 100. Mu.l of the erythrocyte suspension and the folic acid standard solution, respectively, and incubated at 37 ℃ for 1 hour. Then 500. Mu.l of 10% trichloroacetic acid (TCA) was added, and the mixture was left to stand for 30 minutes, centrifuged at 13000rpm and 4 ℃ for 10 minutes, and 100. Mu.l of the supernatant was taken for folic acid LC-MS/MS detection.
The LC-MS/MS detection conditions were as follows:
the instrument comprises the following steps: SCIEX
6500+LC-MS/MS
Chromatographic conditions are as follows: the Column was separated using an ACQUITY UPLC BEH C18 Column,1.7 μm,2.1mm X50mm, the mobile phase was water (0.1% formic acid) as phase A and methanol (0.1% formic acid) as phase B, the Column temperature was 40 ℃. The sample was taken in an amount of 5. Mu.L, and the flow rate was 0.5ml/min, followed by gradient elution.
Mass spectrum conditions: and performing mass spectrum scanning in a positive ion acquisition mode through a multi-reaction monitoring mode by adopting an electrospray ion source. Analyte ion pair information:
and calculating the concentration of folic acid in the erythrocyte suspension according to the concentration of the standard substance.
(3) Calculation of Folic acid concentration
Erythrocyte folate concentration = folate concentration in erythrocyte suspension/hematocrit of erythrocyte suspension
As a result:
(1) SCIEX after the above procedure for erythrocyte preparation
6500 LC-MS/MS mass spectrometer test result is compared with Beckman Coulter Access Folate assay erythrocyte Folate test result of Beijing Baozuo health institute:
the same EDTA blood is divided into 5 tubes with the same volume, each tube is 1ml, and the tube is sent to a certain woman and child health care institute in Beijing on the day of blood collection for Beckman Coulter Access erythrocyte folic acid detection. After the detection is finished, the red blood cells are sent to a treatment point at 4 ℃ within 48 hours, treated according to the red blood cell preparation program, and then processed by computer SCIEX
6500 LC-MS/MS mass spectrometer detection, and the folic acid concentration of the red blood cells is obtained by the calculation formula of folic acid in the above (3).
FIG. 3 is a graph comparing the results of the assay of example 1 using the assay of the invention with the Beckman Coulter Access Folate assay for red blood cell Folate assay.
As can be seen from FIG. 3, the CV values of both detection methods are equivalent (both < 10%), indicating that the present invention has excellent accuracy. However, the detection data of the invention is obviously different from the result of Beckman Coulter Access template detection (the mean value measured by a Beckman kit is 1586nmol/L, the mean value measured by the invention is 489nmol/L, the difference is 3.2 times, and p is less than 0.0001). In addition, the basic erythrocyte folic acid (without folic acid supplementation) of 75 women of childbearing age is detected by the method of the invention, and the median erythrocyte folic acid value is 500nmol/L (42-1403 nmol/L,2.5th-97.5th percent). Chen et al (2019) detected basic erythrocyte folate of 565 women of reproductive age by folate detection gold standard microbiological method, median value 533nmol/L (253-1244 nmol/L2.5 th-97.5th percent) (Chen, M.Y., et al, refining the plant folate conjugation with the red blot cell folate conjugation threshold for optimal neural tissue prediction: a ligation-based, random variant of nucleic acid supplementation. Am J Clin tr, 8978 zxNu8978 (5): p.1452-1461). Therefore, the result of the erythrocyte folic acid distribution measured by the invention is closer to the detection result of a microbiological method reported in the literature than the result of the Beckman Coulter Access Folate assay detection.
(2) The distribution of the red blood cell Folate detected by the detection method of the present invention is compared with the results of detection in the literature using a commercial Kit, accession Kit [ A98032] (Beckman counter) (Ni, J., et al, association between the MTHFR C677T polymorphism, blood and vitamin B12 discovery, and expressed serum total pathological in terms of chemical indexes in Yunnan protocol, chinese. Journal of the Chinese Medical Association,2017.80 (3): p.147-153.) and the results of detection by the microbiological method (Crater et al: HFR 677C → T genetic associated with cultured nucleic acid with nucleic acid, protein and protein group 3763, journal of molecular library, 1376. Journal of molecular library, 3).
The study examined basal erythrocyte folic acid (without folate supplementation) of 75 women of child bearing age and sequenced their MTHFR677 locus to obtain the erythrocyte folic acid distribution of the population with three genotypes of MTHFR 677C/C, C/T and T/T. Ni et al (2017.80 (3): p.147-153.) A commercial Kit for protein binding assay, A98032 (Beckman Coulter), analyzed 330 human red blood cell Folate and simultaneously detected polymorphisms at their MTHFR677 site, to determine the distribution of red blood cell Folate among the three genotypes. Crider et al (2011) have measured basal erythrocyte folate levels in 932 women of child-bearing age using microbiology and also polymorphisms at their MTHFR677 site.
FIG. 4 is a graph showing the distribution of the folic acid in erythrocytes detected by the detection method of the present invention in example 1, in comparison with the results of detection by the commercial Kit Access Folate Kit [ A98032] (Beckman Coulter) and the microbiological method. Wherein, the left graph is the detection of the method of the invention; the middle picture is the Beckmann kit detection, and the right picture is the microbiological method detection.
As can be seen from FIG. 4, the results of the assay according to the present invention show that the levels of 5-MTHF in the human being with folate metabolizing enzyme MTHFR 677T/T genotype are significantly lower and significantly different than those in the C/C and C/T populations. The difference of the erythrocyte folate 5-MTHF level of the population with the MTHFR 677T/T genotype relative to the population with the C/C genotype and the C/T genotype can be detected by a microbiological method, which shows that the MTHFR 677T/T has a reducing effect on the erythrocyte folate 5-MTHF level. The reduction in the level of 5-MTHF in erythrocytes coincides with the reduction in the enzymatic activity of MTHFR C677T mutation. The difference cannot be detected by the existing commercial kit. Since the decrease in 5-MTHF levels caused by MTHFR 677T/T is known to be associated with an increased risk of Neural Tube Defects (Yan, L., et al., association of the Material MTHFR C677T Polymorphism with surgery to Neural Tube Defects in offset weights: evaluation from 25Case-Control students. PLoS ONE,2012.7 (5310): p.e41689), the assay of the present invention has greater accuracy and clinical value.
Example 2:
the detection method of the invention
(1) Erythrocyte replacement preparation procedure
For remote areas without 4 ℃ cold chain transportation conditions in winter, an alternative procedure of dry ice refrigerated transportation may be employed. Extracting 1 tube of fasting venous blood with 2ml EDTA-K2 anticoagulation tube, reversing gently for 5 times, shaking up, placing in 4 deg.C refrigerator, keeping in dark place, centrifuging at 2000g at room temperature for 10min within 2 hr, removing blood plasma, adding 4ml pre-cooled physiological saline, shaking up, centrifuging at 2000g for 10min, and removing supernatant. The remaining erythrocytes were immediately frozen at-80 ℃ or in dry ice, which was transported to the treatment site. After the frozen blood sample was received at the processing site, 1% ascorbic acid approximately twice the volume of the red blood cells was added to the sample before thawing, and the mixture was shaken well to thaw and suspend the red blood cells, thereby preparing a red blood cell lysate. 100 μ l of the erythrocyte lysate was taken and hemoglobin was detected by absorptiometry. Then 100 mul of erythrocyte lysate is taken out for folic acid extraction, or frozen at minus 80 ℃ until folic acid extraction.
(2) Folic acid extraction and detection
Mu.l of folic acid extract (1% ascorbic acid, 1. Mu.g/ml GGH recombinase, isotope labeled 5-methyltetrahydrofolate internal standard) was added to 100. Mu.l of erythrocyte lysate and folic acid standard solution, respectively, and incubated at 37 ℃ for 1h. Then, 500. Mu.l of 10% trichloroacetic acid (TCA) was added thereto, and the mixture was allowed to stand for 30 minutes, centrifuged at 13000rpm for 10 minutes at 4 ℃ and 100. Mu.l of the supernatant was subjected to LC-MS/MS detection of folic acid (the LC-MS/MS detection method was the same as in example 1). And calculating the concentration of folic acid in the erythrocyte suspension according to the concentration of the standard substance.
(3) Calculation of Folic acid concentration
Erythrocyte folate concentration = folate concentration in erythrocyte lysate/(hemoglobin concentration in erythrocyte lysate 0.0029)
Note: 0.0029 is the conversion factor of hematocrit to hemoglobin, i.e., HCT = Hg 0.0029.
As a result:
the effect of the 4 ℃ standard transportation procedure in example 1 and the dry ice freeze transportation procedure in example 2 on the results of the detection of erythrocyte folate was investigated
The inventors collected blood from 9 persons, each sample was divided into two aliquots, one of which was shipped at 4 ℃ as in example 1, and the other of which was rinsed as in example 2 and then shipped frozen on dry ice. And (4) extracting folic acid according to respective programs after receiving the sample, and performing LC-MS/MS detection and calculation.
FIG. 5 is a graph showing the effect of the 4 ℃ standard transportation procedure in example 1 and the dry ice freeze transportation procedure in example 2 on red blood cell folate results.
As can be seen from fig. 5, it was shown that the results of the two detections are highly linearly related (r 2=0.95, p-t-s 0.0001). The mean error of the erythrocyte folate measured by the dry ice freeze transportation procedure was 9.2% compared to the standard transportation procedure at 4 ℃.
Claims (11)
1. A method for detecting folic acid of red blood cells comprises the following steps:
(1) Procedure for erythrocyte preparation
S1, placing a fasting venous blood sample into an anticoagulation tube, storing and transporting at 0-4 ℃, and performing the following operations within 72 hours at most: using isotonic non-toxic liquid with the temperature of 0 to 4 ℃ to rinse the red blood cells for at least 1 time and preparing red blood cell suspension; detecting the hematocrit of the red blood cell suspension; alternatively, the first and second electrodes may be,
s1', placing a fasting venous blood sample into an anticoagulation tube, and storing or transporting at 0-4 ℃, wherein the following operations are carried out within 72 hours at most: rinsing erythrocytes with isotonic nontoxic liquid at 0-4 deg.C for at least 1 time, centrifuging, collecting erythrocytes, and freezing at-40-80 deg.C; adding 1% ascorbic acid solution with the volume twice that of the red blood cells into the frozen red blood cells to thaw and suspend the red blood cells to prepare red blood cell lysate; detecting the hemoglobin concentration of the erythrocyte lysate;
(2) Folic acid extraction and detection
Respectively adding a folic acid extracting solution into the red blood cell suspension obtained in S1 or the red blood cell lysate obtained in S1' and the folic acid standard solution, and incubating for 30min-2h at 37 ℃; removing protein by using a chemical reagent capable of precipitating protein to obtain supernatant, directly carrying out folic acid LC-MS/MS detection on the supernatant, and calculating the concentration of folic acid in the erythrocyte suspension or erythrocyte lysate according to the concentration of a standard substance;
(3) Calculation of Folic acid concentration
The folate concentration was calculated according to the following equation
Erythrocyte folate concentration = folate concentration in the erythrocyte suspension/hematocrit of the erythrocyte suspension; or
Erythrocyte folate concentration = folate concentration in the erythrocyte lysate/(erythrocyte lysate hemoglobin concentration 0.0029).
2. The method for detecting erythrocyte folic acid according to claim 1, wherein, in the step (1), the isotonic non-toxic liquid is pH-neutral isotonic non-toxic liquid which can be used in erythrocyte rinsing process.
3. The method for detecting erythrocyte folic acid according to claim 2, wherein the isotonic non-toxic liquid is physiological saline or phosphate buffer.
4. The method for detecting erythrocyte folate of claim 1, wherein S1 comprises the following steps: placing the fasting venous blood sample in an anticoagulation tube, placing the anticoagulation tube at 0-4 ℃ for temporary storage in a dark place, and transporting the anticoagulation tube to a detection laboratory in a dark place at 0-4 ℃ within 72 hours; after receiving the sample, the detection laboratory completes the following operations: centrifugally separating plasma, discarding supernatant, and adding isotonic nontoxic liquid; mixing, centrifuging, removing supernatant, adding isotonic nontoxic liquid with twice volume of erythrocyte, and making into suspension.
5. The method for detecting erythrocyte folate of claim 1, wherein S1' comprises the following steps: placing the fasting venous blood sample in an anticoagulation tube, temporarily storing at 0-4 ℃ in a dark place, and transporting to a local sample disposal site at 0-4 ℃ in a dark place within 72 hours: after the treatment site receives the sample, the following operations are completed: centrifugally separating plasma, discarding supernatant, and adding isotonic nontoxic liquid; mixing, centrifuging, removing supernatant, collecting erythrocyte, and freezing at-40 deg.C to-80 deg.C; transporting the sample in dry ice to a detection laboratory; after receiving the sample, the detection laboratory completes the following operations: adding 1% ascorbic acid solution with the volume being two times that of the red blood cells into the frozen red blood cells to unfreeze and suspend the red blood cells to prepare a red blood cell lysate; and detecting the hemoglobin concentration of the erythrocyte lysate.
6. The method for detecting erythrocyte folic acid according to claim 1, wherein, in the step (2), the folic acid extract is an aqueous solution which can provide an antioxidant environment and contains an enzyme capable of hydrolyzing folic acid polyglutamate into folic acid monoglutamate.
7. The method for detecting erythrocyte folic acid according to claim 1, wherein the folic acid extracting solution comprises: 1% ascorbic acid or DTT or TCEP or β -mercaptoethanol; 1 microgram/ml of GGH recombinant enzyme, animal plasma or tissue extract containing GGH enzyme, or GGH enzyme purified from animal tissue; an isotopically labeled 5-methyltetrahydrofolate internal standard.
8. The method for detecting erythrocyte folic acid according to claim 1, wherein in the step (2), the detection conditions of the folic acid LC-MS/MS detection on the supernatant are as follows:
the instrument comprises: SCIEX
6500+LC-MS/MS
Chromatographic conditions are as follows: the Column was separated using an ACQUITY UPLC BEH C18 Column,1.7 μm,2.1mm X50mm, the mobile phase was water (0.1% formic acid) as phase A, methanol (0.1% formic acid) as phase B, column temperature 40 ℃; the sample injection amount is 5 mu L, the flow rate is 0.5ml/min, and gradient elution is carried out;
mass spectrum conditions: and performing mass spectrum scanning in a positive ion acquisition mode through a multi-reaction monitoring mode by adopting an electrospray ion source.
9. The method for detecting erythrocyte folic acid according to claim 1, wherein in the step (2), the chemical reagent capable of precipitating protein is an organic or inorganic solvent capable of causing protein precipitation.
10.The method for detecting erythrocyte folic acid of claim 1, wherein the chemical reagent capable of precipitating protein comprises trichloroacetic acid, methanol, acetonitrile, zinc sulfide solution and chloroform-methanol mixed solution.
11. The method for detecting folic acid of red blood cells according to claim 1, wherein the step (2) comprises: respectively adding a folic acid extracting solution into the red blood cell suspension obtained in the S1 or the red blood cell lysate obtained in the S1' and the folic acid standard solution, and incubating for 1h at 37 ℃; and adding trichloroacetic acid solution, standing, centrifuging, taking supernate, performing folic acid LC-MS/MS detection, and calculating the folic acid concentration in the erythrocyte suspension or erythrocyte lysate according to the standard substance concentration.
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| PCT/CN2022/097535 WO2022257950A1 (en) | 2021-06-09 | 2022-06-08 | Red blood cell folate test method |
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| CN116087373A (en) * | 2023-03-27 | 2023-05-09 | 北京和合医学诊断技术股份有限公司 | Detection method and pretreatment method for folic acid and 5-methyltetrahydrofolate in erythrocytes |
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| CN100543462C (en) * | 2007-07-06 | 2009-09-23 | 山东优生医疗科技有限公司 | Method for measuring human body red blood cell acidum folicum content |
| CN112083108B (en) * | 2020-09-23 | 2021-04-27 | 辽宁润生康泰医学检验实验室有限公司 | Accurate detection method and kit for folic acid in blood |
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| CN116087373A (en) * | 2023-03-27 | 2023-05-09 | 北京和合医学诊断技术股份有限公司 | Detection method and pretreatment method for folic acid and 5-methyltetrahydrofolate in erythrocytes |
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