Folic acid assay methods

Cells of Streptococcus fecalis (ATCC-8043) were grown in the AOAC folic acid assay medium [Lepper, Official and Tentative Methods of the Association of... 

J. M. Cooperman, Microbiological assay of folic acid activity in serum and whole blood. In Methods in Enzymology, XVIII, Part B (eds. D. G. McCormick and... 

The chemistry, metabolism, and clinical importance of folic acid have been the subject of many excellent reviews (A7, Gil, H14, H20, Rl). Folic acid deficiency leads to a macrocytic anemia and leucopenia. These symptoms are due to inadequate synthesis of nucleic acid. The synthesis of purine bases and of thymine, required for nucleic acid synthesis, is impaired in folic acid deficiency. Detection of folic acid activity in biologic fluids and tissues is of the utmost importance it distinguishes between the various anemias, e.g., those due to vitamin Bi2 or folic acid deficiency. Because morphology of the abnormal red cell does not help in diagnosing vitamin deficiency, one must rely on assay methods for differential diagnosis. Treatment of pernicious anemia with folic acid has led to subacute combined degeneration of the spinal cord despite... 

Chemical methods for folic acid detection are not useful for unfractionated biologic materials (H16). Reduction of folic acid in acid yields a methylpteridine and N-(p-aminobenzoyl) glutamic acid. The latter is estimated by a method for aromatic amines (B33). Another assay method of historical interest is the growth of chicks as a measure of the folic acid content of crude biological mixtures. 

Because of the multiplicity of folic acid factors reported in whole blood (Ul), the microbiologic assay for folic acid in whole blood and serum was regarded as valueless (C2, L8, W9) results based on S. faecalis methods (C2, C3) did not contradict this view. Streptococcus faecalis is inferior to L. casei in its utilization of the PGA polyglutamates... 

A modification of the above serum folic assay method was recently described (W4). The investigators confirmed the validity of this technique as a practical means of differentiating patients with folic acid from vitamin B12 megaloblastic anemias. Another modification of this method was also described (C4). These investigators reduced the over-all sensitivity by high serum dilutions and thus made the assay, as they used it, valueless as a diagnostic tool. 

Recently, tritiated folic acid became available, making possible a nonmicrobiological method for studying the metabolism of folic acid. It obviates the toxic effects of folic acid antagonists on microbial assay organisms. This technique was used to follow the uptake, metabolism, and excretory products of folic acid (A4, J2, J3). 

G12. Girdwood, R. H., Microbiological methods of assay in clinical medicine with particular reference to the investigation of deficiency of vitamin B12 and folic acid. Scot. Med. J. 5, 10-22 (1960). 

Deficiencies of folic acid and vitamin B1 are relatively common. Whenever macrocytic anemia is present, evaluation of these two vitamins is necessary 10 determine the cause of the condition, The standard method of measuring folic acid has been the microbiological assay (Bailey et al.. 19821. which can be used to measure folic acid in serum, blood, tissues, and foods. Improved high performance liquid chromatography (HPLC) methods have... 

An interesting example is die assay of clonidine hydrochloride in injections and tablets (British Pharmacopoeia, 1980). In 0.01 Mhydrochloric acid, clonidine exhibits two sharp maxima ne - 272 nm and 279 nm, which are not suitable for precise measurement. However, clonidine forms an ion pair with bromothymol blue, and this can be readily extracted into chloroform for subsequent measurement of the broad maximum near 420 nm. Because of the intiinsic variabihty of reagents used in such methods, a pharmacopoeial reference standard is employed for cahbration. A similar policy is adopted for assays involving chemical modification of the drug, as in the tetrazolium assay for corticosteroids, the assay for folic acid involving hydrolysis, diazotisation, and coupling with N-(l-naphthyl)ethylenediamine, and the reaction of penicillins with imidazole and mercuric salts. 

The resnlts of folic acid measnrements may vary depending on the assay method nsed. Decreased serum folic acid levels indicate a folate deficiency megaloblastic anemia that may coexist with a vitamin B12 deficiency anemia. An erythrocyte folic acid level is less volatile than sernm levels, as it is slow to decrease in an acnte process such as drug-induced folic acid deficiency, and slow to increase with oral folic acid replacement. However, the clinical ntflity of determining the erythrocyte folic acid level is qnestionable, and the procedure should be reserved for cases in which the clinician snspects folic acid depletion and the serum folic acid may be falsely elevated or depleted. 

Several B vitamins, including folic acid, niacin, pyridoxine, and pantothenic acid, are routinely determined using microbiological assays, details of which can be found in the AOAC Official Methods of Analysis. Standard methods for thiamine determination using fluorimetric detection are also detailed in the AOAC methods in addition, LC techniques are now being used routinely for thiamine and other B vitamins, e.g., riboflavin. 

Several advantageous features can be noted for our lAC assay for folic acid determination compared with conventional chromatographic or MBA methods, including ... 

For meat samples, SIDA required affinity clean-up prior to LC-MS/MS as the folate levels in this matrix are known to be low. In analogy to other foods, 5-formyl-H4folate and folic acid could not be detected by LC-FD, whereas the more selective SIDA confirmed the presence of both compounds although the latter were below limit of quantitation. The total folate values calculated as folic acid for both methods were quite close to another. This comparison demonstrated that the LC-FD assay only produces reliable results if the matrix is not too demanding. 

Nutritional studies with Lactobacillus casei and Streptococcus faecalis R resulted in the discovery of new growth factors given the names norite eluate factor and folic acid, respectively. Assay methods using these two organisms were employed in the isolation of folic acid and related compounds from natural extracts. However, a number of previously reported biological responses to natural extracts may now be attributed to the folic acid group. 

Albumin activated with glutaraldehyde has been coupled with catalase and the product used in a thermometric enzyme-linked immunosorbent assay. Folic acid has been used to modify albumin in a carbodi-imide-mediated reaction. The folate-derivative was then coupled to agarose and used for affinity chromatography. Albumin alkylated with l-[ C]iodoacetamide has been used in the development of a new method for preparing affinity chromatography matrices. ... 

A pressing need is simplification of the Tetrahymena and other assay methods for folic acid so that an enzymologist, needing to know whether a likely folic acid cofactor is absent or tightly bound, can buy from reliable... 

Mild heat extraction was applied by Bohlman and Nau (56) in the analysis of rat plasma folates. Plasma was mixed with 0.1 M acetate buffer (pH 5.5) containing ascorbic acid (1%) and 2-mercaptoethanol (0.2M), after flushing with argon, the mixture was heated for 5 min at 80°C. Kelly et al. (45) described an assay for serum folate involving HPLC fractionation of deproteinized serum prior to Lactobacillus rhamnosus assay of the collected peaks. The method was validated particularly for the determination of unmetabolized folic acid in human plasma. The limit of detection for folic acid using L. rhamnosus microtitration plate assay was 1 ng/mL serum. 

Flynn, L.M., Microbiological assays of folic acid activity in foods changes in results with variation in experimental conditions and methods, J. Assoc. Offic. Anal Chem., 48, 1230-1235, 1965. 

Minerals are usually analyzed by the traditional method of atomic absorption spectroscopy or with the newer induced coupled plasma analyzers. Most vitamins are analyzed by HPLC systems or colorimetric assays. Many laboratories still quantify folic acid with the use of a microbiological assay. The analysis of dietary and detergent (neutral and acid) fibers are critically important for foods and feeds, respectively. Raw materials and feeds should be examined for rodent contamination, pathogenic bacteria, molds and mycotoxins, and undesirable toxicants such as PCB, insecticides, herbicides, and heavy metals. The proper selection of ingredients will ensure the production of high-quality foods that satisfy sensory properties required for humans, and feeds that are palatable and can meet sensory properties required for domestic animals (Chapter 18). 

Clinically important drugs, such as methotrexate 4-amino-lO-methyl folic acid MTX - which has considerable significance as an antileukaemic agent can be assayed using chemiluminescence immunoassay methods, coupling the drug with firefly luciferase. 

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