WO2006096691A1

WO2006096691A1 - Determination of folate in samples of serum or plasma

Info

Publication number: WO2006096691A1
Application number: PCT/US2006/008033
Authority: WO
Inventors: Rita Skalevik; Per Magne Ueland
Original assignee: Bergen Teknologioverforing As
Priority date: 2005-03-04
Filing date: 2006-03-03
Publication date: 2006-09-14
Also published as: WO2006096691A9

Abstract

The invention relates to determination of folate levels in a biological sample, namely plasma or serum. The invention particularly relates to the determination of folate levels in fresh and stored serum and plasma samples as p- aminobenzoylglutamate (pABG). The invention has a wide spectrum of uses including use in the automated quantification of folate in clinical samples of plasma or serum obtained from human subjects.

Description

DETERMINATION OF FOLATE IN SAMPLES OF SERUM OR PLASMA

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/658,900, filed March 4, 2005, the contents of which are expressly incorporated herein by reference.

FIELD OF THE INVENTION

The invention generally relates to methods of determining the leevls or amount of folate in biological samples, e.g., serum or plasma, particularly those obtained from a human patient. The invention has a wide variety of applications including use as a diagnostic tool to measure the level of folate in human serum or plasma.

BACKGROUND OF THE INVENTION Folates are a group of B-vitamins supplied through vegetables and fruit. Folate deficiency is reported to be common. Additionally, folate has been implicated in the pathogenesis of chronic diseases, including colorectal cancer, neural tube defects and cardiovascular disease. Attempts have been made to address these diseases by increasing dietary folate intake in the US, Canada and other countries. See generally Stover PJ. Nutr Rev (2004); 62:S3-12; and Blakley, RX. (1969) in The Biochemistry of Folic Acid and Related Pteridines. NY, NY; and references cited therein.

See also Brody, T. et al., In Handbook of Vitamins (Machlin, L. J. Ed.), Marcel Dekker, Inc., New York, N. Y. (1984) pp. 459-496; Nussbaum, R. L. and Ledbetter, D. H., In The Metabolic Basis of Inherited Diseases (Scriver, C. R. et al., Eds.) McGraw Hill, New York, N. Y. (1989) pp. 327-341; and references cited therein.

Previous attempts to determine folate levels in various biological matrices have been unsuccessful or unreliable. For instance, folates have been measured for clinical diagnostic purposes in red blood cells (RBC), as well as serum and plasma. Unfortunately, there have been reported difficulties. For instance, measurement of RBC folate has largely been abandoned, because of methodological difficulties. These shortcomings have contributed to low precision, large inter-laboratory variability, and no external quality control (EQC), for instance.

Folate measurements in serum and plasma have also been problematic. See Gunter EW, et al. Clin Chem (1996);42:1689-94. In particular, several assays have been marketed, but each of these gives different results. Moreover, most clinical chemistry laboratories use different assays resulting in different reference ranges between regions and countries. Often there are changes is reference ranges related to altered assay calibrations by the producer or changes in the assays used.

There have been other reported shortcomings. For serum/plasma folate analyses, the EQC and assay calibrations between laboratories are difficult, mostly because folates are unstable. This drawback makes production, storage and distribution of quality control specimens impractical. Furthermore, instability alters folate content in serum/plasma during transportation to the analysis laboratory. Such instability creates great problems when carrying out large epidemiological studies based on stored samples. Assayable folate is decreased even in frozen samples, and is reduced to 80% after 1 year at -20 °C, and is essentially absent in frozen older samples. See Ocke MC, et al. J Clin Epidemiol 1995;48: 1077-85.

In humans, folic acid is metabolized to tetrahydrofolate and subsequently to 5- methyltetrahydrofolate. Different folate species exist that differ with respect to the oxidation states of the pteridine ring, substitutions at positions N5 and NlO (methyl, formyl, methenyl or methylene bridge between N5 and Nl 0) and number of glutamate residues connected via a gamma glutamyl bond (n= 1-6) (polyglutamates). The main species in fresh serum is 5-methyltetrahydrofolate. See eg. Ratanasthien K₅ et al. J Clin Pathol (191 '4) ,27:875-9.

It has been disclosed that folate catabolism is a major route of turnover that can involve production of breakdown products such as p-aminobenzoylglutamate (pABG), p-acetoamidobenzoylglutamate (apABG), and other compounds. See eg., Caudill, M. et al. J. Nutr. (2002) 132: 2613. In particular cleavage of the C9-N10 bond of folate to form pABG has been done to estimate the length of the polyglutamate chain. See Houlihan CM and Scott JM. Biochem Biophys Res Commun (1972);48:1675-81. Moreover, the pABG content in urine has been evaluated as an indicator of folate turnover. See Gregory JF, et al. JNutr (2000);130:2949-52. In whole blood, folate has been measured as para-aminobenzoic acid (pABA) after strong acid hydrolysis. See Dueker SR, et al. Anal Biochem (2000); 283:266- 75.

Many reported methods estimate the folate content by comparing activity to a standard curve generated using a single folate coenzyme as the standard in a separate assay. See Fleming, A. F. et al., Am. J. Clin. Nutr. (1971) 24:1257-1264; Home, D. W. and Patterson, D., Clin. Chem. (1988) 34:2357-2359; Tamura, T. In Folic Acid Metabolism in Health and Disease, (Picciano, M. F. et al., Eds.) Wiley-Liss, Inc., New York, N. Y., pp. 121-137).

For example, a recognized method of folate analysis, i.e. the radioisotope competitive binding assay (RIDA), the folate content of a sample is calculated by comparing binding to a standard curve generated using folic acid or 5- methyltetrahydrofolate as an external standard. Patents relating to radioassays include U.S. Pat. Nos. 5,800,979; 4,276,280, 4,247,453, 4,091,087 and 3,989,812.

Another folate detection method involves use of a microbiological assay. Here, microorganisms requiring folates for growth are grown in the presence of a sample containing folates and compared to growth in the presence of a standard. In HPLC based methods, the identification of folates is based on synchronization of retention times, spiking of samples with markers or a combination of those strategies. See Varela-Moreiras, G. et al., J. Nutr. Biochem (1991) 2:44-53.

See also Owen WE, and Roberts WL. AmerJClin Pathol (2003);120:121-126. Various other assays to detect folate have been disclosed. See eg., USP 5,434,087 to Abbott Laboratories; 4,350,659 to Corning Glass Works; 4,028,465 to Bio-Rad Laboratories; and references cited therein.

It would be useful to have a more accurate, reliable, and reproducible method for assaying folate in biological samples, in particular fresh and stored human plasma and serum samples. BRIEF SUMMARY OF THE INVENTION

The instant invention is based, at least in part, on the discovery that treating serum or plasma in a way that maximizes production of p-aminobenzoylglutamate (pABG) from folate, allows for reliable measurement of the folate in the samples. That is, by chemically treating plasma and serum under conditions that convert essentially all folate therein to pABG, it is possible to measure the level of p ABG, which closely relates to the level of folate originally present in the sample. By converting the folate essentially entirely to pABG, it is possible to reduce and avoid problems that have hindered accurate and reproducible folate measurements using prior methods.

Accordingly, and in one aspect, the invention provides accurate, reliable and reproducible methods for measuring folate in a biological sample, typically blood serum or plasma. Without wishing to be bound to theory, it is believed that by subjecting the sample to oxidation and then to suitable "limited acid hydrolysis" conditions, it is possible to convert folate in the sample to pABG. Practice of the methods of the invention provide substantial benefits, for instance, by simplifying analysis of potentially heterogeneous folate species in some samples by providing a single detected species i.e., pABG. Prior folate instability problems are minimized and usually avoided. In addition, preferred invention methods are readily adapted to a variety of medical, veterinary, research, diagnostic and clinical platforms including those that currently rely on automated or semi-automated steps to measure folate in serum and plasma. The invention is thus flexible as it can be used to accurately and reliably measure folate in a variety of settings. In one embodiment of the foregoing method, the serum or plasma is combined with at least one acid, i.e., one or two acids, sufficient to deproteinize the serum or plasma. Preferred acids are known in the field and may be replaced with other known deproteinization methods provided such methods do not hinder production of the p ABG and do not cause the formation of p AB A. The methods may further include combining the deproteinized serum or plasma with at least one base sufficient to neutralize the acid, generally less than about 5 bases and usually one, two or three bases. Nearly any base or combination of bases is sufficient provided it can give the serum or plasma a basic pH (i.e., greater than pH 7.0). In most cases, the deproteinized serum or plasma will be treated with at least one oxidizing agent, i.e., one or two, under conditions that are sufficient to oxidize any folate species present in the treated serum or plasma. In some embodiments of the invention, the oxidizing agent is inactivated using a suitable compound. Examples of preferred folate species include, but are not limited to 5-methyltetrahydrofolate, 4α-hydroxy-5-methyltetrahydrofolate, folic acid and 5 -formyltetrahydro folate.

In one embodiment of the foregoing method, the serum or plasma is subjected to limited acid hydrolysis. By "limited acid hydrolysis" is generally meant chemical hydrolysis conditions that are sufficient to convert any oxidized folate species in the sample to essentially all pABG. It is an invention objective to control the hydrolysis so that essentially no pABA is formed. Such "limited acid hydrolysis" can be controlled by one or a combination of methods including selecting a suitable acid and/or concentration thereof in the invention methods. Alternatively, or in addition, the hydrolysis may be controlled by limiting the time of hydrolysis, the temperature of the reaction, etc. Nearly any suitable method can be used to test for "limited hydrolysis" in line with the invention including measuring pABG as discussed herein.

Typically, the pABG formed by the invention method is further detected and correlated to folate originally present, if any, in the sample. Particular methods for limiting acid hydrolysis in accord with the invention are discussed below including the Example section.

In another invention embodiment, the method further includes performing quantitative mass spectrometry (MS) to measure the pABG including, but not limited to use of MS systems that are operably linked to a liquid chromatography device (LC- MS/MS or LC-MS) or gas chromatography device (GC-MS or GC-MS/MS).

Additional advantages of the invention include the ability to detect degraded folate, for instance, in old serum or plasma samples.

Other embodiments of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the chemical structure of folates. Folates have common structural features including a pteridine residue, a p-aminobenzoate moiety and glutamyl residue.

Figure 2 depicts the oxidation pathway of folates following limited acid hydrolysis of 5-methyltetrahydrofolate to pABG.

Figure 3 graphically depicts the correlation of folate in serum determined by microbiological assay and as pABG equivalents. DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides methods for measuring folate in serum or plasma that involve detection of a single folate product. The method is reliable, relatively fast, and sensitive, especially when compared to many prior methods. In one embodiment, the method involves at least one and preferably more of the following steps:

(a) combining the serum or plasma with an acid sufficient to deproteinize the serum or plasma,

(b) combining the deproteinized serum or plasma with at least one base sufficient to neutralize the acid,

(c) incubating the deproteinized serum or plasma with an oxidizing agent under conditions sufficient to oxidize any folate species in the serum or plasma,

(d) inactivating the oxidizing agent, (e) subjecting the oxidized folate species in the serum or plasma to limited acid hydrolysis under conditions sufficient to form p-aminobenzoylglutamate (pABG), wherein the hydrolysis substantially reduces and preferably prevents formation of p AB A in the sample; and (f) correlating presence of the pABG so formed to the amount of folate in the blood serum or plasma sample.

The invention method is flexible and is not bound to any particular number or order of steps so long as intended results are achieved. As discussed, a particular invention objective is to control the hydrolysis step (e), for instance, so as to maximize production of pABG, while at the same time, minimize or preferably eliminate the production of p AB A.

As also discussed, prior methods of measuring folate are inaccurate, and have inter-assay as well as intra-assay variances. Existing methods include microbiological assays, radio-dilution methodology, and liquid chromatography- (tandem) mass spectrometry (LC-MS/(MS)) methods. For example, microbiological assays measure active folates supporting the growth of Lactobacillus casei. However, the microbiological assays experience interference from compounds inhibiting bacterial growth, including EDTA and some antibiotics. See eg., O'Broin S, and Kelleher B. J Clin Pathol (1992);45:344-7.

"Folate" as used herein refers to a genus of well defined B-vitamin compounds, including 5 -methyltetrahydro folate, 5-formyltetrahydrofolate, dihydrofolate, tetrahydrofolate, and folic acid. Folates exhibit similar vitamin activity within the body. Examples of suitable "folate oxidation products" and "oxidized folate species" as used herein include, but are not limited to, 5-methyldihydrofolate, 4α-hydroxy-5- methyltetrahydrofolate, and in some instances folic acid.

Other folate detection methods including those relying on radio-dilution methodology, a competitive binding or automated nonisotopic format have been used with varying success. These are typically associated with drawbacks. For instance, the radio-dilution assays use folate-binding proteins, but are unable to detect degraded or oxidized folate species. Another disadvantage is that the method does not exhibit an equimolar response to the various folate derivatives used as standards during the assay, thus producing inaccurate results. The same is true for the microbiological assay as well. Liquid chromatography- (tandem) mass spectrometry (LC-MS/(MS)) methods, which measure folate in serum/plasma, are only useful as reference techniques because of the technical complexity and expensive instrumentation necessary. See Pfeiffer CM, et al. Clin Chem (2004);50:423-32. Moreover, for past methods that have used LC-MS/(MS), such a method cannot be used successfully unless the folate species is known i.e., it is difficult or impossible to detect unknown folate species including degradation products.

In contrast, the present invention provides an accurate, efficient, and reliable method of measuring folate levels in a biological sample, namely serum and plasma, that can be practiced manually or can be adapted to one or a combination of automated or semi-automated clinical systems that are designed to measure folate routinely. There have been some attempts to measure certain folate derivatives, for instance, see PCT/GB03/00529 (WO 03/067263). However, the disclosed technique has many disadvantages including the use of relatively harsh acidic conditions used to treat samples. These and other drawbacks can contribute to the generation of multiple folate derivatives, thereby complicating analysis and leading to inaccurate measures of the folate levels. This shortcoming is still present even when binding partners disclosed by the PCT/GB03/00529 application are used in the analysis. As mentioned, the present invention avoids these and other problems by providing methods that produce a single and readily detectable species i.e., the pABG.

As discussed, the invention presents methods for measuring folate in serum or plasma. In one embodiment, the method includes deproteinizing the serum or plasma with an acid, combining the deproteinized serum or plasma with at least one base sufficient to neutralize the acid, incubating the deproteinized serum or plasma with an oxidizing agent under conditions sufficient to oxidize any folate species in the serum or plasma, inactivating the oxidizing agent, subjecting the oxidized folate species in the serum or plasma to limited acid hydrolysis under conditions sufficient to form pABG, and correlating presence of the pABG so formed to the amount of folate in the blood serum or plasma sample.

Typically, all steps are performed at temperatures between from about O⁰C to about room temperature (25⁰C). In one embodiment, the invention is preformed at room temperature, while in other embodiments, the methods described herein can be performed at various other temperatures including keeping reagents "on ice" for some or all of the method steps. Alternatively, a higher temperature than room temperature may be indicated for some embodiments, for instance, less than about 6O⁰C or less than about 5O⁰C. Nearly any temperature is suitable provided intended invention objectives are achieved. It is within the scope of the present invention to perform the method steps at one or a combination of different temperatures, for instance, to perform one or more steps at one temperature (e.g., "on ice") and to perform one or more other steps at a higher or lower temperatures. Choice of a particular temperature(s) to use will be guided by understood parameters including convenience. By "combining" is meant the mixing of sample, e.g., serum or plasma, and the reagent. The reagent, e.g., acid, base, or oxidizing agent, may be added to the serum or the serum may be added to the reagent.

For most invention embodiments, sufficient reagents used to deproteinize the serum or plasma include ample amounts of reagents to accomplish deproteinization. For example, sufficient reagents will lead to between about 90 to about 95% deprotenization of the sample. See Blanchard J. J Chromatogr (1981);226:455-60. Preferably sufficient reagents will lead to between about 95 to about 99% deproteinization, and more preferably about 99.9% deproteinization. In one embodiment of the folate detection method provided herein, the reagents is perchloric acid (HClO₄), although other reagents including, but not limited to, acetonitrile, trichloroacetic acid or sulfosalicylic acid may be used for some applications. In a related embodiment, more than one reagent is combined to the sample to achieve deproteinization, for instance, two, or three of same. Nearly any concentration orcombination of the reagents is suitable provided intended results are achieved.

In certain embodiments, the volume of the acid and the serum or plasma is about equal. Alternately, the volume of acid is about 1/3 or about 1/4 the volume of the sample. The volume of acid to sample will depend, in part, on the strength of the acid, and the concentration of the sample, and the stoichiometry of the reaction. Of course, the invention is not bound to any particular acid volumes and other volumes may be just as suitable for other invention applications.

In a particular embodiment, the acid is perchloric acid having normality (N) of between from about 0.35 to about 2.0. The normality of the perchloric acid will depend in part on the concentration of the sample, and the stoichiometry of the reaction. In a preferred embodiment, the perchloric acid has a normality of about 1.6N.

The deproteinized serum or plasma, e.g., formed after combining the serum or plasma with an acid sufficient to deproteinize it, is combined with at least one base

(eg., two or three of same) sufficient to neutralize the acid. Suitable bases include, but are not limited to, potassium carbonate (KHCO₃ ), potassium hydroxide (KOH), NaOH, or Ca(OH)₂. In a related embodiment, combinations of bases suitable to neutralize the acid include KHCO₃ and KOH or NAOH and KHCO₃, and the like. Use of the invention is not bound to any particular base of combination thereof, provided intended results are achieved.

In a related embodiment, the base is sufficient to give the serum or plasma a basic pH. A basic pH is a pH from between about 7 and about 12 and preferably the base has a pH from between about 8 to about 11. In another related embodiment, the base has a molarity (M) of between from about IM to 2M. In certain embodiments, after neutralization, salt is precipitated from the solution. In one embodiment, the base is KHCO₃ and KOH and the pH is about 10. After the acid is neutralized, the sample is incubated with a suitable oxidizing agent under conditions sufficient to oxidize any folate species in the serum or plasma. In this context, "sufficient to oxidize any folate species in the serum or plasma," refers to an adequate amount of oxidizing, for example, to lead to about 95% pABG and about 5% pABA; preferably about 98% pABG and about 2% pABA; more preferably about 99% pABG and about 1% pABA; and still more preferably about 99.9% pABG and about 0.1% pABA; approaching about 100% pABG. The pABG and pABA can be measured and quantified by nearly any suitable method in the field including, but not limited to any of the techniques disclosed herein relying on mass spectrometry (MS).

As used herein, "inactivating the oxidizing agent," refers to stopping the reactivity of the oxidizing agent, for example, by precipitating the reagent or by adding H₂O₂ to the reaction to inactive the oxidizing agent. Useful concentrations OfH₂O₂ are between about 0.3% to about 4%. Preferably, the H₂O₂ concentration is about 3%. Of course, successful practice of the invention is not limited to the use OfH₂O₂ or particular concentration thereof so long as intended results are achieved.

In certain embodiments according to the invention, the oxidizing agent is potassium permanganate (KMnO₄). However, it may be helpful to use chromate ions (CrO₄ ^2"), dichromate ions (Cr₂O₇ ^2"), nitric acid (HNO₃), perchloric acid (HClO₄), and/or sulfuric acid (H₂SO₄) for certain applications. In a related embodiment, the concentration of the oxidizing agent (w/v) for use with the method is between from about 0.25% (w/v) to about 3% (w/v). In a preferred embodiment, the oxidizing agent is KMnO₄ having a concentration (w/v) of about 2%. The concentration of the oxidation agent may vary depending on the concentration of the sample, the stoichiometry of the reaction, presence of interfering agents in the sample, the strength and/or concentration of the oxidizing agent, and the like. Practice of the invention is not limited to the use of any particular oxidizing agent or concentration thereof such as those specified herein so long as intended invention results are achieved.

As used herein, the step of subjecting the oxidized folate species in the serum or plasma to limited acid hydrolysis under conditions sufficient to form pABG can be done in the stopped oxidation reaction solution, or the species may be isolated from the stopped oxidation reaction solution and then subjected to limited acid hydrolysis. The "limited acid hydrolysis" may vary, for example, in the concentration of the acid. With enough acid to get pABG but not more, the limited acid hydrolysis can proceed for at least 24 hours without formation of any essentially amount of pABA (eg., less than about 3 days, preferably less than about 1 or 2 days). However, in most invention embodiments, more rapid quantiation of the pABG will be useful. If desired, the reaction may be monitored to determine the optimal conditions, for example, by mass spectrometry, TLC, or HPLC. Optimal conditions refer to quantitative recovery of all folate species essentially as pABG, and formation of essentially no p AB A.

In a related embodiment, the limited acid hydrolysis is sufficient to convert essentially all of the oxidized folate species to the pABG. In this context, "essentially all" is intended to encompass about 95% pABG and about 5% pABA; about 98% pABG and about 2% pABA; about 99% pABG and about 1% pABA; about 99.9% p ABG and about 0.1% pABA; typically approaching about 100% pABG. Suitable methods for detecting the pABG and pABA (if present) are disclosed herein and include, but are not limited to, mass spectrometry. In one embodiment, the invention is used to attain pABG : pABA ratio > 98:2.

In a related embodiment, the hydrolysis is controlled so that essentially no pAB A is formed. In another related embodiment, the limited hydrolysis is formation of less than about 2 % pABA. In yet another related embodiment, the limited hydrolysis is represented by a pABG : pABA ratio > 98:2. "Essentially no" pABA is intended to include about 5% pABA, about 4% pABA, about 3% pABA, preferably about 2% p AB A, more preferably about 1% pABA, and still more preferably <1% pABA in a sample upon completion of the limited acid hydrolysis. Methods for detecting these species are disclosed herein.

In one embodiment, the acid used in the limited hydrolysis is perchloric acid, TCA or HCl. Other useful acids will be apparent to one of skill in the art having the benefit of this disclosure.

As discussed, the invention can be used with one or a combination of suitable acids. In a preferred embodiment, about 30% (w/v) perchloric acid can be used. In such an embodiment, the limited acid hydrolysis is sufficient to provide the serum or plasma with a pH of between from about 0.5 to less than about 3. In a preferred embodiment, the wherein the pH of the serum or plasma is about 1.

Correlating presence of the pABG formed to the amount of folate in the blood serum or plasma sample may be done for example by one or more of the techniques including gas chromatography, liquid chromatography, mass spectrometry, TLC, or HPLC. The quantification of pABG in a sample is a measure of the folate content of that sample.

The correlation may take into account the amount of the pABG in the sample compared to a control amount of pABG (e.g., in normal subjects in whom folate deficiency is not detected). The correlation may take into account the presence or absence of p ABG as well as other factors known to those working in the field.

Mass Spectrometry (MS) Methods As discussed, successful practice of the invention can be achieved with one or a combination of methods that can detect, and preferably quantify the pABG. Mass spectrometry (MS) is a well known tool for analyzing chemical compounds. Thus in one embodiment, the correlation step of the present invention further includes performing quantitative MS to measure the pABG. In a related embodiment, the MS is operably/operationally linked to a liquid chromatography device (LC-MS/MS or LC-MS) or gas chromatography device (GC-MS or GC-MS/MS). Methods for performing MS are known in the field and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; USP 5,800,979 and references disclosed therein. For some invention uses, it may be helpful to purify the pABG made by the methods disclosed herein prior to subsequent analysis. Nearly any means known to the art for the purification and separation of small molecular weight substances, e.g., anion or cation exchange chromatography, gas chromatography, liquid chromatography or high pressure liquid chromatography may be used. Methods of selecting suitable separation and purification techniques and means of carrying them out are known in the art (see, e.g., Labadarious et. al., J. Chromatography (1984) 310:223-231, and references cited therein; and Shahrokhin and Gehrke, J. Chromatography (1968) 36:31-41, and Niessen J. Chromatography (1998) 794:407- 435). For invention embodiments in which reasonable accuracy is needed, it will often be useful to at least partially purified from samples before detection, using a combination of affinity chromatography, and anion or cation exchange, or reversed- phase or normal phase chromatography. Although not always necessary, it may be useful to modify the pABG for some invention applications, for instance, to facilitate purification and/or separation. This practice is well known in the art as derivatization, e.g., it may be desired to convert the target and reference compounds to analogs having improved solubility, increased volatility, different mass to charge ratio, etc. to facilitate purification and or separation and identification for analysis on the GC/MS (see, e.g., D. R. Knapp, Handbook of Analytical Derivatization Reactions, (1979) John Wiley & Sons, New York).

Capillary infusion is often useful to introduce the pABG to a desired MS implementation, for instance, because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a MS with other separation techniques including gas chromatography (GC) and liquid chromatography (LC). As discussed above, GC and LC can serve to separate a solution into its different components prior to mass analysis. Such techniques are readily combined with MS, for instance. One variation of the technique is that high performance liquid chromatography (HPLC) can now be directly coupled to mass spectrometer for integrated sample separation/and mass spectrometer analysis.

The invention can be practiced with one of several ionization techniques used in mass spectrometry including Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption/Ionization (MALDI). ESI is the production of highly charged droplets which are treated with dry gas or heat to facilitate evaporation leaving the ions in the gas phase. MALDI uses a laser to desorb sample molecules from a solid or liquid matrix containing a highly UV-absorbing substance. See e.g., K. Tang et al., at the May 1995 TOF-MS workshop, R. J. Cotter (Chairperson); K. Tang et al., Nucleic Acids Res. 23, 3126-3131, 1995.

Aside from MALDI, Electrospray Ionization Mass Spectrometry (ESI/MS) has been recognized as a significant tool used in the study of bio-molecules in general. ESI is a method of sample introduction for mass spectrometric analysis whereby ions are formed at atmospheric pressure and then introduced into a mass spectrometer using a special interface. Other well-known ionization methods may also be used. For example, electron ionization (also known as electron bombardment and electron impact), atmospheric pressure chemical ionization (APCI), fast atom Bombardment (FAB), or chemical ionization (CI). Quadrupole mass analyzers may also be employed as needed to practice the invention. Fourier-transform ion cyclotron resonance (FTMS) can also be used for some invention embodiments. It offers two distinct advantages, high resolution and the ability of tandem MS experiments. FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field. Coupled to ESI and MALDI, FTMS offers high accuracy with errors as low as .+-.0.001%.

Other MS embodiments are within the scope of the present invention such as use of a time-of-flight (TOF) analyzer and tandem mass spectrometry (MS/MS). An exemplary mass spectrometry instrument useful for practicing the instant invention is described in the Examples. A skilled artisan should readily understand that other similar instruments with equivalent function/specification, either commercially available or user modified, are suitable for practicing the instant invention.

Multiple reactions may be monitored at the same time using this technique. In one embodiment, the method is performed in an automated or semi-automated format. This may be done, for example with an LC-MS/MS or LC-MS or a GC-MS or GC- MS/MS device in the multiple reaction monitoring mode. In a related embodiment, the method is performed in an automated format and the correlation step further comprises using a computer system to determine the amount of pABG present in the serum or plasma.

The invention is scaleable and readily adapted to automation, for instance, using methodologies employed for the automated quantification of folate from serum and blood samples. See Owen WE, and Roberts WL. Amer J Clin Pathol 2003;120:121-126. In one invention embodiment, the method further comprises outputting data from the computer system to a user of the method. This may be done, for example, by any algorithm or device useful for outputting data. The output maybe to a screen of a computer or to a printer.

In one embodiment, the oxidized folate species is 5-methyltdihydrofolate or 4α-hydroxy-5-methyltetrahydrofolate. In fresh samples, e.g., samples that were taken from a subject less than 2 days prior to the time of the assay, 5-methyltetrahydrofolate or 5-formyltetrahydrofolate and folic acid are the folate species detected. In one embodiment, the folate in the serum or plasma include 5- methyltetrahydrofolate, folic acid, 10-formyltetrahydro folate, or 5- formyltetrahydrofolate or oxidized species thereof. The methods described herein are intended to encompass unknown folate species that will be detectable in the herein described assays.

The methods described herein are typically used to measure folate in serum or plasma. If desired however, they can be used to assay other biological samples including urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus and amniotic fluid. , In particular embodiments, the body fluid is serum or plasma.

The invention also provides kits for assaying folate in a subject. The kits may include instructions for the assay, reagents, testing equipment (test tubes, reaction vessels, needles, syringes, etc.), standards for calibrating the assay, and/or equipment provided or used to conduct the assay. Reagents may include acids, bases, oxidizing agents, folate species, and folate oxidation product species, e.g., pABG and pABA.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et ah, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary? of Genetics, 5th Ed., R. Rieger et a (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

As used herein, "serum" refers to the fluid portion of the blood obtained after removal of the fibrin clot and blood cells, distinguished from the plasma in circulating blood. As used herein, "plasma" refers to the fluid, noncellular portion of the blood, distinguished from the serum obtained after coagulation. As used herein, "sample" refers to anything which may contain an analyte for which an analyte assay is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like. Biological tissues are aggregates of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).

As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from, e.g., infection or genetic defect, and characterized by identifiable symptoms. Unless otherwise specified, reference herein to a percentage (%) of a particular compound means (% w/v).

It should be appreciated that the invention should not be construed to be limited to the examples which are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.

For instance, in one variation, the pABG is detected using one or a combination of the binding partners disclosed according to the PCT/GB03/00529 (WO 03/067263) application. In one embodiment of the present invention, an antibody such as those disclosed by the PCT/GB03/00529 application are used to bind the pABG specifically i.e., to form a specific binding pair. One or more conventional immunological techniques can be used to detect and preferably quantitate the specific binding pair such as radioimmunoassay (RIA), ELISA, precipitation assay, sandwich assay and the like.

The following points illustrate the usefulness and advantages associated with preferred invention embodiments:

1) The prerequisites for a pABG method for folate determination in serum/plasma are established, i.e. there is no pABG or precursor of pABG other than folates in human serum/plasma.

2) Folate species in serum/plasma are quantitatively recovered as pABG by a procedure involving oxidation followed by limited acid hydrolysis under controlled conditions.

3) Concentrations of folate in fresh samples, determined as pABG equivalents, show a strong correlation with concentrations obtained by a reference method. 4) Folate are inactivated and degraded to unknown species upon prolonged storage, and these species are collectively determined as pABG equivalents by the procedure referenced under point 2. 5) The principles and procedures outlined above can form the basis for the construction of high-throughput folate assays for routine assessment of folate status in the clinical setting. The pABG assay can be adapted to commercial automated nonisotopic platforms by raising monoclonal antibodies against pABG that are used for pABG quantification. Such an assay is suitable for EQC and inter-laboratory calibration.

EXAMPLE 1 : Measurement of folate in serum/plasma as pABG

A key principle behind the pABG method is oxidation followed by limited hydrolysis of folate in serum/plasma, under conditions of quantitative conversion of all species to pABG. Quantification of pABG so formed will serve as a measure of folate status if there is no pABG or precursor of p AB G other than folates, in serum/plasma. Figure 2 depicts the pathway of oxidation followed by limited acid hydrolysis of 5- methyltetrahydrofolate to pABG.

Serum/plasma (100 μL) was deproteinized by adding 1.6 N perchloric acid (33 μL), the supernatant (90 μL) was neutralized by adding 1.2M KHCO3 and 1.44M KOH (34 μL). KC1O4 precipitated. To the supernatant (100 μL, pH 10), KMnO4 (2 %, 7 μL) was added to oxidize folate species, and after 15 minutes, the reaction was stopped by adding H2O2 (3%, 10 μL). Folates were then subjected to limited acid hydrolysis by adding perchloric acid (30 %, 15 μL) to this solution (80 μL), to obtain a final pH of 1.0. pABG was quantified as positive ions by LC-MS/MS in the multiple reaction monitoring (MRM) mode. The detection limit was 0.5 nmol/L serum, i.e. far below the concentration of folates in human serum. No detectable pABG (< 0.5 nmol/L) was found in human serum or plasma.

AU steps have been performed in room temperature. They have also been performed on ice, and experiments conducted at both temperatures showed the same results. A Sciex API 4000 MS/MS was used to measure pABG.

This result has been verified in more than 100 samples. For example, serum samples were spiked with various folate species (5-methyltetrahydrofolate, 5- formyltetrahydrofolate, folic acid and 4α-hydroxy-5-methyltetrahydrofolate), and a conversion of these species to pABG in samples subjected to oxidation and hydrolysis was obtained as detailed above. Figure 3 graphically depicts the results of an exemplary correlation of folate in serum determined by microbiological assay and as pABG equivalents.

The pABG assay was verified by comparison of folate concentrations in fresh serum samples determined by the microbiological assay and the concentrations measured as pABG equivalents. A strong correlation was observed (Pearson r of 0.97), as depicted in figure 3. The pABG assay was also validated by determining by both methods the folate concentrations in serum samples stored at —20 ⁰C for about 25 years. In these samples, 8 out of 10 samples had folate concentration far below the normal reference limits as determined by the microbiological assay. In contrast, all samples had essentially normal folate concentration when determined by the pABG method (Table 1).

The use of pABA as a marker of folate in serum/plasma was investigated because others have reported that the high folate content in erythrocytes is quantitative converted to pABA by strong acid hydrolysis. See Dueker, SR, supra. The invention provides that pABA is not a reliable marker of the low concentrations (in the nanomolar range) of folate in serum/plasma because a substantial fraction of healthy subjects (about 25 %) has material in serum/plasma other than folates that was converted to pABA.

Table L Determination of folate in old serum samples Method

Sample no. PABG (nmoJ/L) Microbiological (runαl/L)

1 8.3 2.9

2 7.4 2.8

3 19.8 8,9

4 17,2 9,4

5 7.6 2,9

6 83 2.8

7 7.9 2.1

S 8.4 2.7

9 13 2.8

10 9.2 1.8

Mean 10.7 3,9 Incorporation by Reference

The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the instant invention and the following claims.

Claims

What is claimed is:

1. A method for measuring folate in serum or plasma, the method comprising the following steps: (a) combining the serum or plasma with a reagent sufficient to deproteinize the serum or plasma,

(b) combining the deproteinized serum or plasma with at least one base sufficient to neutralize the sample,

(d) inactivating the oxidizing agent,

(e) subjecting the oxidized folate species in the serum or plasma to limited acid hydrolysis under conditions sufficient to form p-aminobenzoylglutamate (pABG), and

(f) correlating presence of the pABG so formed to the amount of folate in the blood serum or plasma sample.

2. The method of claim 1, wherein the hydrolysis is controlled so that essentially no paraaminobenzoic acid (pABA) is formed.

3. The method of claims 1-2, wherein the precipitant is perchloric acid (HClO₄), acetonitrile, trichloroacetic acid or sulfosalicylic acid.

4. The method of claims 1-3, wherein the volume of the reagent and the serum or plasma is about equal.

5. The method of claims 1-4, wherein the reagent is perchloric acid having a normality (N) of between from about 0.35 to about 2.0.

6. The method of claims 1-5, wherein the perchloric acid has a normality of about 0.8-1.6N.

7. The method of claims 1-6, wherein the base is potassium carbonate (KHCO₃ ), potassium hydroxide (KOH), NaOH, or Ca(OH)₂.

8. The method of claims 1-7, wherein the base is sufficient to give the serum or plasma a basic pH.

9. The method of claims 1-8, wherein the base has a molarity (M) of between from about 1.2M to 1.5M.

10. The method of claims 1-9, wherein the base has a molarity (M) of between from about IM to 2M.

11. The method of claims 1-10, wherein the pH is between from about 8 to about 11.

12. The method of claims 1-11, wherein the base is KHCO₃ and KOH and the pH is about 10.

13. The method of claims 1-12, wherein the oxidizing agent is potassium permanganate (KMnO₄), chromate ions (CrO₄ ^2"), dichromate ions (Cr₂O₇ ^2"), nitric acid (HNO₃), perchloric acid (HClO₄), or sulfuric acid (H₂SO₄).

14. The method of claims 1-13, wherein the concentration of the oxidizing agent (w/v) is between from about 0.25% (w/v) to about 3% (w/v).

15. The method of claims 1-14, wherein the oxidizing agent is KMnO₄ having a concentration (w/v) of about 2%.

16. The method of claims 1-15, wherein the folate species being oxidized are 5- methyltetrahydrofolate, folic acid, 4α-hydroxy-5-methyltetrahydrofolate or 5- formyltetrahydrofolate.

17. The method of claims 1-16, wherein the limited acid hydrolysis is sufficient to convert essentially all of the oxidized folate species to the pABG.

18. The method of claims 1-17, wherein the limited hydrolysis is formation of less than 2 % pABA.

19. The method of claims 1-18, wherein the limited hydrolysis is represented by a pABG: pABA ratio > 98:2.

20. The method of claims 1-19, wherein the acid used in the limited hydrolysis is perchloric acid, trichloroacetic acid (TCA) or hydrochloric acid (HCl).

21. The method of claims 1-20, wherein the concentration of the acid (w/v) is between from about 5% to about 10%.

22. The method of claims 1 -21 , wherein the limited acid hydrolysis is sufficient to serum or plasma to a pH of between from about 0.5 to about 1.5.

23. The method of claims 1-22, wherein the acid is about 30% (w/v) perchloric acid.

24. The method of claims 1 -23, wherein the pH of the serum or plasma is about 1.

25. The method of claims 1-24, wherein the folate in the serum or plasma is 5- methyltetrahydrofolate, folic acid, 10-formyltetrahydro folate, or 5- formylterrahydrofolate or oxidized species thereof.

26. The method of claims 1-25, wherein the correlation step further comprises performing quantitative mass spectrometry (MS) to measure the pABG.

27. The method of claims 1-26, wherein the MS is operably/operationally linked to a liquid chromatography device or gas chromatography device.

28. The method of claims 1-27, wherein the method is performed in an automated or semi-automated format.

29. The method of claims 1-28, wherein the method is performed in an automated format and the correlation step further comprises using a computer system to determine the amount of pABG present in the serum or plasma.

30. The method of claims 1-29, wherein the method further comprises outputting data from the computer system to a user of the method.

31. A kit for measuring folate in serum or plasma comprising instructions for use and pABG.

32. The kit of claim 31, further comprising perchloric acid, potassium carbonate, and potassium permanganate.

33. A method for measuring folate in serum or plasma, the method comprising the following steps: contacting the serum or plasma with a reagent sufficient to deproteinize the serum or plasma; neutralizing the sample by the addition of a base; oxidizing the sample by the addition of an oxidizing agent; subjecting the oxidized folate species in the serum or plasma to limited acid hydrolysis by the addition of an acid under conditions sufficient to form p- aminobenzoylglutamate (pABG); and correlating presence of the pABG in the sample to the amount of folate in the sample.

34. The method of claim 33, further comprising inactivating the oxidizing agent prior the performing the limited hydrolysis.

35. The method of claim 33, wherein the hydrolysis is controlled so that essentially no paraaminobenzoic acid (pABA) is formed.

36. The method of claim 33, wherein the serum or plasma is deproteinized by perchloric acid (HClO₄), acetonitrile, trichloroacetic acid or sulfosalicylic acid.

37. The method of claim 33, wherein the volume of the reagent and the serum or plasma is about equal.

38. The method of claim 36, wherein the reagent is perchloric acid having a normality (N) of between from about 0.35 to about 2.0.

39. The method of claim 38, wherein the perchloric acid has a normality of about 0.8-1.6N.

40. The method of claim 33, wherein the base is potassium carbonate (KHCO₃ ), potassium hydroxide (KOH), NaOH, or Ca(OH)₂.

41. The method of claims 33 or 40, wherein the base is sufficient to give the serum or plasma a basic pH.

42. The method of claims 33, 40 or 41, wherein the base has a molarity (M) of between from about IM to 2M.

43. The method of claim 42, wherein the base has a molarity (M) of between from about 1.2M to 1.5M.

44. The method of claim 33, wherein the pH is between from about 8 to about 11.

45. The method of claim 44, wherein the base is KHCO₃ and KOH and the pH is about 10.

46. The method of claim 33, wherein the oxidizing agent is potassium permanganate (KMnO₄), chromate ions (CrO₄ ^2"), dichromate ions (Cr₂O₇ ^2"), nitric acid (HNO₃), perchloric acid (HClO₄), or sulfuric acid (H₂SO₄).

47. The method of claim 46, wherein the concentration of the oxidizing agent (w/v) is between from about 0.25% (w/v) to about 3% (w/v).

48. The method of claim 47, wherein the oxidizing agent is KMnO₄ having a concentration (w/v) of about 2%.

49. The method of claim 48, wherein the folate species being oxidized are 5- methyltetrahydrofolate, folic acid, 4α-hydroxy-5-methyltetrahydrofolate or 5- formyltetrahydrofolate.

50. The method of claim 33, wherein the limited acid hydrolysis is sufficient to convert essentially all of the oxidized folate species to the pABG.

51. The method of claim 35 , wherein the limited hydrolysis is formation of less than 2 % pABA.

52. The method of claim 33 or 51, wherein the limited hydrolysis is represented by a pABG: pABA ratio > 98:2.

53. The method of claim 33, wherein the acid used in the limited hydrolysis is perchloric acid, trichloroacetic acid (TCA) or hydrochloric acid (HCl).

54. The method of claim 33, wherein the concentration of the acid (w/v) is between from about 5% to about 10%.

55. The method of claim 33, wherein the limited acid hydrolysis is sufficient to bring the serum or plasma to a pH of between from about 0.5 to about 1.5.

56. The method of claim 33, wherein the acid is about 30% (w/v) perchloric acid.

57. The method of claim 55, wherein the pH of the serum or plasma is about 1.

58. The method of claim 33, wherein the folate in the serum or plasma is 5- methyltetrahydrofolate, folic acid, 10-formyltetrahydro folate, or 5- formyltetrahydrofolate or oxidized species thereof.

59. The method of claim 33, wherein the correlation step further comprises performing quantitative mass spectrometry (MS) to measure the pABG.

60. The method of claim 59, wherein the MS is linked to a liquid chromatography device or gas chromatography device.

61. The method of claims 33-60, wherein the method is performed in an automated or semi-automated format.

62. The method of claims 33-60, wherein the method is performed in an automated format and the correlation step further comprises using a computer system to determine the amount of p AB G present in the serum or plasma.

63. The method of claims 33-62, wherein the method further comprises outputting data from the computer system to a user of the method.

64. A kit for measuring folate in serum or plasma comprising instructions for use and pABG.

65. The kit of claim 64, further comprising perchloric acid, potassium carbonate, and potassium permanganate.