Kit-of-parts and microbiological method for assessing folate status in serum and red blood cells

Abstract

Kits and methods for the microbiological determination of folate and folic acid in whole blood samples and the assessment of the folate status of an individual, comprising steps for the complete lysis of red blood cells (erythrocytes) and release of folate species, and for the release of lysosomal γ-glutamyl hydrolase from the cells contained in the whole blood sample and / or the addition of γ-glutamyl hydrolase and complete enzymatic hydrolysis of the γ-glutamyl chains of the follipolyglutamate species follitetraglutamate, follipentaglutamate and follihexaglutamate, followed by carrying out microbiological assays for comparative testing of growth and metabolism in the absence and presence of different amounts of treated whole blood sample and / or folate calibrators to assess the folate status of an individual in comparison to a folate reference.

Description

[Technical field]

[0001] The present invention relates to a microbiological method for assessing the folate status of a person (C12Q 1 / 02). [Background technology]

[0002] Vitamins are substances that the human body must ingest in the diet, and therefore specific deficiencies may occur in cases of disease, malnutrition, or malabsorption. Vitamin B is a class of water-soluble vitamins that share similar names (B1, B2, B3, etc.) but are chemically distinct. The B vitamins are referred to by their specific numbers, such as B1 for thiamine, B2 for riboflavin, and B3 for niacin. Some B vitamins are more commonly recognized by their names, niacin, pantothenic acid, biotin, and folate, than by their numbers. Folate occurs naturally in leafy vegetables and receives that name, while folic acid is the synthetic form of this vitamin. The vitamin activity of naturally occurring folates is less than that of folic acid, since not all naturally occurring folate compounds have vitamin activity. Folate is essential for cell growth and replication. Inadequate folate intake is associated with adverse health outcomes, and low maternal folate status may be associated with pre-eclampsia, spontaneous abortion, stillbirth, preterm delivery, low birth weight, autism, neural tube defects, and different congenital anomalies of the spine and brain (see review in WHO's Vitamin and Mineral Nutrition Information System-VMNIS 7,G 15.01 of 2015).

[0003] Although folate is primarily stored in the liver, a person's folate status can be assessed in serum, plasma, red blood cells, and urine. Serum folate levels are an indicator of only recent folate intake. Thus, a single folate measurement in serum cannot be used to distinguish between a temporary decrease in dietary folate intake and a chronic deficiency. Conversely, red blood cell folate concentrations respond slowly to changes in folate intake, since red blood cells have a life span of 120 days and accumulate folate during erythropoiesis. Thus, the folate content of whole blood samples could be used to assess a person's long-term folate status.

[0004] An individual's folate status further depends on a number of factors, including age, pregnancy, lactation, socio-economic status and access to dietary folate, as well as coexisting physiological factors such as homocysteine ​​and other vitamin levels. Genetics must also be considered, particularly polymorphisms in the methylenetetrahydrofolate reductase gene and mutations in the human glutamate carboxypeptidase gene II. As stated in the WHO report, pregnant women with low folate status are at higher risk of giving birth to children with neural tube defects and congenital heart disease. People with low folate status are generally at higher risk of metabolic disorders, cardiovascular diseases, colon cancer, and cognitive changes in older adults, including Alzheimer's disease in particular (US2004 / 0185487).

[0005] The concentration of folate in a sample can be determined by (i) chemical-physical methods, e.g., high pressure liquid chromatography coupled with different detectors such as mass spectrometry, (ii) immunological methods, (iii) non-related animal experiments, and (iv) microbiological methods. US 8,663,946 B2 (Fu Tzu-Fun) discloses a method and kit for detecting one or more folates, comprising the steps of (a) mixing a sample with an extraction buffer to form a mixture, boiling the mixture, then cooling to ambient temperature, and separating the supernatant from the mixture by centrifugation, (b) adding recombinant gamma-glutamyl hydrolase (GGH) and folate converting enzyme to the supernatant to drive the reaction, (c) stopping the reaction, and (d) analyzing the reaction mixture by high performance liquid chromatography to quantify one or more folates. Other chemical and chromatographic methods for detecting folate are disclosed by Doherty RF, Beecher RA A method for analysis of natural and synthetic folate in foods, J Agricultural and Food Chemistry, 2003, p.354-361, Chao Wang et al. A liquid chromatography-tandem mass spectrometric method for the quantitative determination of native 5-methyltetrahydrofolate and its polyglutamyl derivatives in raw vegetables, J Chromatography B, 2010, 2949-2958. EP1472545 (Axis-Shield ASA) describes a method for assaying folate in which a sample is subjected to hydrolysis to release para-aminobenzoic acid, para-aminobenzoylglutamic acid or a salt thereof, the released compound is contacted with a diazo binding partner, and the resulting binding partner is detected directly or indirectly.No. 6,329,162, assigned to AntiCancer Inc., patents a method for assessing folate levels in a biological sample, comprising providing to the sample glycine N-methyltransferase (GMT) and an excess of S-adenosylmethionine (SAM) and glycine, providing a folate-free control having amounts of the GMT and excess SAM and glycine comparable to those provided to the sample, and comparing the concentration of at least one product formed in the sample with the concentration of the product formed in the control, whereby the difference in the level of the product in the sample compared to the control is directly proportional to the level of folate in the sample.

[0006] Folate is a trace vitamin, and chemical-physical and immunological methods are generally less sensitive and useful due to the many biologically active and inactive folate species. Since genetically modified strains of Lactobacillus rhamnosus have become available for determination, the microbiological method for assessing folate status developed in the 1960s still forms the basis of current methods. This Lactobacillus strain is responsive to multiple folate forms while excluding species that do not have vitamin activity. However, a recent study compared the results of microbiological assays from three laboratories and observed that they gave substantially different results for the folate status of individuals. With regard to technical difficulties, microbiological assays typically require one or more dilution series of samples in assay buffer such that at the end of the incubation period the growth or turnover values ​​of the test microorganism fall within the measurement range of a parallel standard concentration series. A standard curve must be constructed for each assay. Furthermore, for reasons of safety and precision, each concentration level of the standard and sample series must be used at least three times. The folate content of the sample is determined by comparison with the known folate content of a parallel standard series. Generalized precision data are not possible, but the coefficient of variation should be about 10 percent or less. Furthermore, the inocula of the standard and sample series must be accurately added and have the desired sensitivity and specificity, which are sometimes uncertain.For further information see Gorin G et al. Determination of niacin...with lyophilized Lactobacillus arabinosus ATCC 8014, Appl Microbiol 1970, 20, 641-642; Conrad PB et al. Stabilization and preservation of Lactobacillus acidophilus in saccharide matrices, Cryobiology 2000, 41, 17-24; Kelleher BP et al. Microbiological assay for vitamin B12 performed in 96-well microtitre plates, J Clin Pathol 1991, 44(7), 592-595; Bui MH A microbiological assay on microtitre plates of thiamine in biological fluids and foods, J Vitam Nutr Res 1999, 69(5), 362-366.

[0007] Moloy AM et al.Microbiological assay for serum,plasma,and red cell folate using cryopreserved microtiter plate method,Methods Enzymol 1997,281,43-53 teaches that, despite being difficult to set up and difficult to maintain, the microbiological assay remains the "gold standard" and the best way to achieve the required results. However, routine analysis of serum and red cell folate remains a subject of debate, with only a few laboratories in the food sector specializing in this type of analysis, and even fewer clinical laboratories in human diagnostics. Although the microbiological method is labor intensive and requires a significant amount of laboratory organization, the technique has been improved and automated to such an extent that it is easier to perform, reliable to maintain, and fairly low cost than alternative methods, mainly involving a large number of samples. Three important advances in assay technology have contributed to this - the development of antibiotic-resistant organisms and genetically engineered strains of Lactobacillus rhamnosus. Secondly, the cryopreservation of the inoculum in multiple individual vials results in standardized growth curves that are reproducible for hundreds of assays. Finally, automated microtiter plate technology developed for enzyme-linked immunosorbent assay (ELISA) and its associated computer analysis packages are suitable for measuring the turbidity of microbiological growth. Although microbiological methods for determining the active folate content in human biological samples are available, the assessment of an individual's long-term folate status remains problematic and prone to error. The prior art represents a challenge. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 2004 / 0185487 [Patent Document 2] U.S. Pat. No. 8,663,946 [Patent Document 3] European Patent No. 1472545 [Patent Document 4] U.S. Patent No. 6,329,162

Non-licensed documents

[0009]

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[0010] The subject of the present invention is a method for the microbiological determination of folate and folic acid in whole blood samples and for the assessment of the folate status of an individual, comprising: preparing one or more culture vessels for microbiological growth and metabolic measurements containing a predetermined number of viable Lactobacillus rhamnosus cells; obtaining a defined sample volume of whole blood from an individual whose folate status is to be determined; adding a volume of red blood cell lysis buffer to the sample of whole blood to lyse red blood cells for release of folate species; adding a detergent capable of permeabilizing an amount of γ-glutamyl hydrolase and / or lysosomes in order to release lysosomal γ-glutamyl hydrolase from cells contained in the whole blood sample; treating and incubating the lysed blood sample for a period of time at pH 5.5-7 to enzymatically hydrolyze the γ-glutamyl chains of the follipolyglutamate species follitetraglutamate, follipentaglutamate and follihexaglutamate; performing microbiological assays for comparative testing of growth and metabolism in the absence and presence of different amounts of the processed whole blood samples and / or folate calibrators to assess the folate status of the individual relative to a folate reference; The problem is solved by a method comprising the steps of:

[0011] In some embodiments, the red blood cell lysis buffer contains 1% ascorbate / ascorbic acid, pH 4.2-5.0, and a detergent to permeabilize lysosomes.

[0012] In some embodiments, the detergent for permeabilizing lysosomes is selected from sapogenins, steroid sapogenins, saponins, triterpene glycosides, terpenoids, alkylphenol-ethoxylates, (Triton® X-100), nonylphenol-ethoxylates, octylphenol-ethoxylates.

[0013] In some preferred embodiments of the present invention, the enzymatic hydrolysis of the γ-glutamyl chains of the follicle polyglutamate species, if not carried out in an ascorbic acid / ascorbate buffer at pH 4.2, is carried out in a phosphate buffer at pH 5.5-6.5 at ambient temperature to 37 degrees Celsius for 10-30 minutes.

[0014] In some embodiments, Lactobacillus rhamnosus is grown in assay medium buffered at pH 6 to counteract the inhibitory effects of produced lactic acid.

[0015] In some preferred embodiments of the invention, the Lactobacillus is chloramphenicol-resistant Lactobacillus rhamnosus (ATCC 7469).

[0016] In some most preferred embodiments, microbiological assays in microtiter plates.

[0017] Another aspect of the invention is a test kit for assessing the folate status of an individual by microbiological assay of folate and / or folic acid in a whole blood sample, comprising: A microtiter plate for microbiological growth and metabolism measurements, each of whose cavities contains a predetermined number of viable Lactobacillus rhamnosus cells and is rendered durable to shock-freezing and dry storage at ambient temperature by lyophilization; Ascorbic acid / ascorbate buffer system at pH 4.0-4.5 A test kit comprising:

[0018] Preferred embodiments of the invention can be understood from the detailed description and examples set forth below. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a graphical representation (standard calibration curve) of the growth of Lactobacillus rhamnosus in microtiter wells in dependence on folate concentration (OD measured at 630 nm - duplicate readings). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Folates, also called vitamin B9, differ in oxidation state, carbon substitution, and glutamate residue. Folic acid does not occur naturally, but only in individuals taking vitamin supplements or consuming fortified foods. Reduced folates are less stable than folic acid, and the stability depends on one-carbon substitution. Oxidation usually results in folate compounds that lack vitamin activity, although some can be converted to biologically active oxidized forms. The large number of folate derivatives, the instability of some, and the possibility that some of the folate derivatives may interconvert make the assessment of vitamin B9 or folate status chemically complex. Competitive protein binding-based assays are popular because they are available in commercial kit form. Some laboratories have also introduced mass spectrometry methods to measure individual folate one-carbon forms. Human plasma and serum naturally contain folate monoglutamates, mainly the 5-methyltetrahydrofolate form (5-methyl-THF), and red blood cells contain mainly polyglutamates of 5-methyl-THF. The assessment of folate status in whole blood samples is difficult because pteroylpolyglutamates cannot be metabolized by Lactobacillus rhamnosus. In contrast, such biological samples would be suitable to assess the long-term folate status of individuals. The above-mentioned genetic polymorphisms also cause a redistribution of folate forms between serum and red blood cells, so there is also growing interest in assaying folate one-carbon forms. Furthermore, the use of folate supplements has led to the appearance of free folic acid in blood samples. The excessive presence of synthetic forms of folate not only affects the assessment of folate status but may also have detrimental effects on the health of individuals.

[0021] [ka]

[0022] Folate, on the other hand, is essential for normal cell growth and replication, and folate and vitamin B 12Deficiency is recognized as the most common cause of macrocytosis (Kaferle J, Strzoda CE Evaluation of macrocytosis, Am Fam Physician. 2009, 79(3):203-8). Most importantly, poor maternal folate status can be associated with placental abruption, preeclampsia, spontaneous abortion, stillbirth, preterm labor, low birth weight, and severe congenital abnormalities of the brain and spine, such as neural tube defects (NTDs) (Molloy AM et al. Effects of folate and vitamin B 12 deficiencies during pregnancy on fetal, infant, and child development, Food Nutr Bull 2008;29(2):101-115, Hibbard BM et al.Folic acid and reproduction, Acta Obstet Gynecol Scand.1965,44(3):375-400).

[0023] Microbiological assays using Lactobacillus rhamnosa (also known as Lactobacillus casei) are recommended and used to determine the folate content of foods because they are responsive to multiple folate forms, excluding only those forms that have no vitamin activity (Anderson BB et al. Effect of light on the Lactobacillus casei microbiological assay, Am J Clin Pathol 1968,21:85-7; Yetley EA et al. Biomarkers of folate status in NHANES: a roundtable summary, Am J Clin Nutr. 2011;94(1):303S-312S. doi:10.3945 / ajcn.111.013011). Meanwhile, a recent study comparing microbiological assays from three laboratories showed that different results were obtained due to unknown issues not only with the species of folate calibrators but also with the genetics of the microorganisms (Pfeiffer CM et al. Comparison of serum and red blood cell folate microbiologic assays for national population surveys, J Nutr. 2011, 141(7): 1402-9. doi: 10.3945 / jn.111.141515). The inventors found that the different results were likely due to a lack of recovery of 5-methyltetrahydrofolate because, on the one hand, higher polyglutamate folate species in red blood cells were not degraded to usable folate forms with vitamin activity, and, on the other hand, precipitated or were trapped in cell membranes during cell lysis, or because the endogenous γ-glutamyl hydrolase of whole blood was not active or released from lysosomes. The present invention overcomes these challenges and provides kits and assay protocols that ensure complete release and solubilization of all folate species, as well as optimized enzymatic processing of pteroyl-polyglutamates by γ-glutamyl hydrolase, which is kept free in the reaction mixture, with specific permeabilization of lysosomes from blood cells.The method of the present invention is particularly useful for assessing folate status in the ranges commonly recognized as low and too low. Elimination of the possibility of under-recovery of 5-methyl-THF greatly contributes to the accurate assessment of an individual's folate status. It increases the reliability of clinical diagnosis and improves dosing with folate and vitamin supplements, especially for pregnant women.

[0024] Lysis of red blood cells is traditionally performed by the action of typically unbuffered 1% ascorbic acid, but ascorbic acid is known to be a strong reducing agent that can interfere with many blood chemistry tests, and its pKa of 4.17 and 11.6 was not known to lead to partial precipitation and trapping of the more water-soluble vitamin B9 in red blood cells, resulting in an inability to degrade vitamin B9 to pteroyl-monoglutamate. Thus, this percentage of vitamin B9 or active folate was removed from the microbiological assay of folate, leading to poor recovery of 5-methyl-THF. These challenges are overcome by using a buffer system of ascorbic acid and ascorbate at pH 4.2 for cell lysis of whole blood samples, and by adding a detergent to the whole blood sample to permeabilize the lysosomes to lysosomal γ-glutamate hydrolase. EXAMPLES

[0025] [Example 1] Hydrolysis and release of pteroyl-polyglutamates from erythrocytes. Theory: Folic acid (N-(4-{[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)-methyl]-amino}-benzoyl)-L-glutamic acid or pteroyl-L-glutamic acid) exists in erythrocytes (red blood cells) as polyglutamates (pteroyl-polyglutamates) and not as pteroyl-L-glutamic acid (monomer). See formula I above. The WHO recommends the measurement of folate levels in whole blood by a microbiological assay based on recombinant Lactobacillus rhamnosus ATCC 7469, which measures only short pteroyl-polyglutamates containing less than three glutamates. The present inventors have identified the need to maintain higher molecular weight pteroyl-polyglutamates in solution during cell lysis, as well as the need for hydrolysis and degradation to 5-methyl-THF by lysosomal γ-glutamase (glutamate conjugase) for accurate physiological assessment of an individual's folate status (vitamin B9 status). Complete release and solubilization of higher pteroyl-polyglutamates from red blood cells is particularly important when an individual is likely to suffer from insufficient folate uptake, since precipitated or trapped pteroyl-polyglutamates may constitute 30-50 percent of total red blood cell folate when cell lysis is induced by unbuffered 1% ascorbic acid solution (pH 2.5) as recommended in the prior art.

[0026] Test solutions and buffers: 96-well plates were prepared as described in EP 1 774 021, Examples 1 and 2. Assay medium (ASYMED) contained charcoal-treated pancreatic digested casein 10.0 g / L, dextrose 40.0 g / L, sodium acetate 40.0 g / L, potassium dihydrogen phosphate 1.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, DL-tryptophan 0.2 g / L, L-asparagine 0.6 g / L, L-cysteine ​​hydrochloride 0.5 g / L, adenine sulfate 10.0 mg / L, guanine hydrochloride 10.0 mg / L, uracil 10.0 mg / L, xanthine 20.0 mg / L, polysorbate-80 The storage medium was Difco™ Folic Acid Casein Medium containing 0.1 g / L, glutathione (reduced) 5.0 mg / L, magnesium sulfate 0.2 g / L, sodium chloride 20.0 mg / L, ferrous sulfate 20.0 mg / L, manganese sulfate 15.0 mg / L, riboflavin 1.0 mg / L, p-aminobenzoic acid 2.0 mg / L, pyridoxine hydrochloride 4.0 mg / L, thiamine hydrochloride 400.0 μg / L, calcium pantothenate 800 μg / L, nicotinic acid 800 μg / L, biotin 20 μg / L, and 0.05% ascorbic acid. The storage medium also contained 200 mmol / L trehalose, 10 mmol / L CaCl2. Folic acid standards were dissolved in 100 mmol / L potassium phosphate buffer, pH 6.1, 0.1% ascorbic acid.

[0027] Preparation of microtiter plates: A glycerol stock of Lactobacillus rhamnosus ATCC 7469 was inoculated into 10 ml of Lactobacillus medium and incubated. The culture was grown to log phase and the cells were harvested by centrifugation (2500G x 5 min). The cell pellet was washed three times with 0.85% NaCl solution, suspended in 10 ml of storage medium and diluted 1:10 in assay medium containing 200 mM / L trehalose. Dilutions were 10 per ml. 7Three microliters of the bacterial suspension was added to the bottom of each well of a microtiter plate, flash frozen at -80°C in a freezer, and lyophilized by applying a vacuum. Thus, each microtiter plate well contained exactly 3 x 10 viable bacteria of the same growth stage encapsulated in a trehalose / sugar / salt pellet attached to the bottom of the well. 4 The plates contained 100 live Lactobacillus rhamnosus bacteria. The adhesion of the pellets was further increased by adding small amounts of sucrose and dextrose to the freezing solution. The plates were packed sterile and protected from light with a desiccant (silica). The microtiter plates thus prepared are stable for long periods at room temperature without loss of microbial viability.

[0028] Lactobacillus rhamnosus prefers an assay medium with a pH between 5 and 7. It is preferable to buffer the system at about pH 6 during cultivation to keep the growth of Lactobacillus constant even in wells containing high concentrations of folate. Metabolism of folate leads to lactic acid, the acidity of which can have an inhibitory effect on the growth rate at higher concentrations. This can be achieved by using an assay medium (ASYMED) buffered with 50 mM KHPO4 / KH2PO4 buffer, pH 6.1.

[0029] Cell lysis: 4.5 mL lysis buffer (HSOL: 0.1% ascorbic acid / ascorbate, pH 4.2) is combined with permeabilizing detergent (PAF - 0.1 g / l sapogenin at final dilution). 25 μl of whole blood sample was mixed with 225 μl of HSOL / PAF (1:10) and incubated for 30 min at 37 °C. In this step, the ascorbic acid / ascorbate induces the lysis of all blood cells, mainly red blood cells (RBCs), and the γ-polyglutamates of folate (pteroylpolyglutamates - PteGlu). n) are released into the reaction solution and do not precipitate. Thus, the solubilized pteroylpolyglutamates can be enzymatically hydrolyzed by γ-glutamyl hydrolase released from the lysosomes of other blood cells, such as leukocytes and granulocytes. In this step, γ-glutamyl hydrolase can be added to the lysis buffer and the incubation time can be increased, but the γ-glutamyl hydrolase inherent in the lysosomes of whole blood cells is generally sufficient to dissolve all pteroyl-polyglutamates, provided that the pH is not below pH 4.2, which is the case when unbuffered 0.1% ascorbic acid is used for cell lysis. γ-glutamyl hydrolase is not active when the reaction buffer has such a low pH (pH 2.5 for unbuffered 0.1% ascorbic acid solution).

[0030] A higher pH is important during cell lysis because if the pH is too low, acidic pteroyl-polyglutamates may attach to proteins and cause aggregation. If the pH is too high or above pH 4.2, cell lysis may not be complete. Therefore, sapogenin is preferably added to ensure complete cell lysis and permeabilization of these cellular compartments to glutamase conjugase (γ-glutamyl hydrolase). During development, additional processing steps were tested and saponin was found to be optimal in combination with ascorbic acid / ascorbate buffer.

[0031] Following incubation, another sample dilution of 1:75 is performed and the samples and CTRL / STD are added to the microbiological assay in folate-deficient medium and incubated for 48 hours. The microbiological assay itself is performed as described above.

[0032] The kit contains the following components: 10 in each well 4 Microtiter plates pre-seeded with Lactobacillus rhamnosus cells.

[0033] SOL sample handling buffer 5 x 4.5 ml.

[0034] PAF-buffered folate in whole blood, lyophilized.

[0035] 5 x DIL water 4 x 30ml.

[0036] ASYMED Folate Assay Medium 4x.

[0037] STD folic acid standard, lyophilized 4x.

[0038] 4x FOL masking foil.

[0039] 1x FRA spare frame for repositioning microtiter strips.

[0040] ASYBUF Folate Media Treatment Buffer 4 x 1.5 ml.

[0041] CTRL 1 folate control 1, lyophilized 4x.

[0042] CTRL 2 folate control 2, lyophilized 4×.

[0043] [Example 2] Comparison of samples according to the invention with samples prepared according to the method published by Molloy et al.

[0044] Preparation of whole blood samples for erythrocyte folate analysis according to Molloy et al.: A whole blood sample obtained from an individual was thoroughly mixed in a 1.5 ml Eppendorf tube and 100 μl of it was added to 900 μl of 1% ascorbic acid solution freshly prepared by adding 1 g of ascorbic acid (not sodium ascorbate) to 100 ml of distilled water. The pH was not adjusted, resulting in a lysis mixture with an approximate pH of 2.3. The lysis mixture was then left to stand at room temperature for at least 30-40 minutes prior to folate assay to allow serum conjugates to convert folate polyglutamates released from erythrocytes to the assayable monoglutamate form. The erythrocyte / ascorbic acid lysate was then stored in a freezer below -20 degrees Celsius until assayed.

[0045] Preparation of samples according to the invention. In parallel, 25 μl of mixed whole blood sample was mixed with 225 μl of 0.1% ascorbic acid / ascorbate, adjusted to 5 with pH 4.2 (HSOL) containing 0.1 g / l sapogenin (PAF) for a final dilution of 1:10, and incubated for 30 minutes at 37° C. The red blood cell / ascorbic acid lysate was then also stored in a freezer below −20 degrees Celsius until assayed.

[0046] The results of the comparative measurements of 10 individuals are shown below in Table I. Figure 1 shows the standard calibration curve (OD at 630 nm) used for the 10 comparative measurements (in duplicate).

[0047] [Table 1]

[0048] As shown in Table I, sample preparation according to the method of the invention results in a test in which the lysis of cells (red blood cells) is complete and all released folate polyglutamates are enzymatically hydrolyzed to assayable forms. The high variability of the deviations in folate status also indicates that the degree of polymerization within folate polyglutamates was not constant among the individuals tested or followed a rule, but can be considered as a feature of the physiological state and folate supply of the individuals. Without wishing to be bound by any theory, the inventors believe that the low pH of the mixture, approximately 2.3 for the samples prepared by Molloy et al., causes the folate polyglutamate forms to be trapped or aggregated by cell membranes, so that they are only partially hydrolyzed by serum conjugases (=gamma-glutamyl hydrolases). The inventors found that complete enzymatic degradation of folate polyglutamate forms can only be achieved in buffers with a pH above 4.2, and that this pH shift is necessary due to the individual differences in the degree of glutamate polymerization. Therefore, the low pH of the lysed cell mixture used by Molloy et al. and by the prior art methods inevitably leads to insufficient recovery of folate and inaccurate evaluation of a person's folate status. The effect of pH and saponin on whole blood cell lysis in the context of erythrocyte folate analysis has been previously studied and reported by Wright AJA et al Erythrocyte Folate Analysis: Saponin Added During Lysis of Whole Blood Can Increase Apparent Folate Concentrations, Depending on Hemolysate pH, Clinical Chemistry (2000) 46: 12 1978-1986. However, they only investigated a single blood sample and therefore overlooked that the degree of polymerization within folate polyglutamates is variable and also that the hydrolysis of glutamate by intrinsic conjugase is highly pH-dependent and requires a pH shift. In contrast, Wright AJA et al. describe that a high pH above 4.5 is combined with incomplete cell lysis and inaccurate low evaluation of a person's folate status.

Claims

1. 1. A method for the microbiological measurement of folate and folic acid in a whole blood sample and for the assessment of the folate status of an individual, comprising: Preparing one or more culture vessels for microbiological growth and metabolic measurements containing a predetermined number of viable Lactobacillus rhamnosus cells; obtaining a defined sample volume of whole blood from an individual whose folate status is to be determined; adding a volume of red blood cell lysis buffer to the sample of whole blood to lyse red blood cells for release of folate species; adding a detergent capable of permeabilizing an amount of γ-glutamyl hydrolase and / or lysosomes in order to release lysosomal γ-glutamyl hydrolase from cells contained in said whole blood sample; treating and incubating the lysed blood sample for a period of time at pH 5.5-7 to enzymatically hydrolyze the γ-glutamyl chains of the follipolyglutamate species follitetraglutamate, follipentaglutamate and follihexaglutamate; performing microbiological assays for comparative testing of growth and metabolism in the absence and presence of different amounts of the processed whole blood samples and / or folate calibrators to assess the folate status of the individual relative to the folate reference; The method includes:

2. 2. The method of claim 1, wherein the red blood cell lysis buffer contains ascorbate / ascorbic acid at pH 4.2-5.0 and a detergent for permeabilizing lysosomes.

3. 3. The method according to claim 1 or 2, wherein the detergent for permeabilizing lysosomes is selected from sapogenins, steroid sapogenins, saponins, triterpene glycosides, terpenoids, alkylphenol-ethoxylates, (Triton® X-100), nonylphenol-ethoxylates, octylphenol-ethoxylates.

4. A method according to any of claims 1 to 3, wherein the enzymatic hydrolysis of γ-glutamyl chains of folipolyglutamate species is carried out in a phosphate buffer of pH 5.5 to 6.5 at ambient temperature to 37 degrees Celsius for 10 to 30 minutes.

5. The method according to any one of claims 1 to 4, wherein the Lactobacillus rhamnosus is grown in an assay medium buffered at pH 6 to counteract the inhibitory effect of the produced lactic acid.

6. The method according to any one of claims 1 to 5, wherein the Lactobacillus is chloramphenicol resistant Lactobacillus rhamnosus ATCC 7469.

7. The method according to any one of claims 1 to 6, wherein the culture vessel is a microtiter plate.

8. 1. A test kit for assessing the folate status of an individual by microbiological assay of folate and / or folic acid in a whole blood sample, comprising: A microtiter plate for microbiological growth and metabolism measurements, each of whose cavities contains a predetermined number of viable Lactobacillus rhamnosus cells and is made durable for dry storage at ambient temperature by shock freezing and lyophilization; Ascorbic acid / ascorbate buffer system at pH 4.0-4.5; A test kit comprising:

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