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1. (WO1980000562) DERIVES DE L"ACIDE FOLIQUE POUR L"UTILISATION DANS DES ESSAIS DE RADIOIMMUNITE
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Description
Folic Acid Derivatives For Use In Radioimmunoassay

Technical Field
The present invention relates to folic acid derivatives useful in radioassaying folic acid and its metabolites in biological fluids such as blood serum. There are many methods known for the radioassay of folic acid and its derivatives in biological fluid. Competitive binding of labeled folic acid (compounds of the present invention) and test sample folic acid for the folate binding
protein (s) present in milk (Clinical Chem. 19, 1101, 1973) is a suitable method for determining folic acid.

Background Art
U. S. Patent 3,989,812 describes compounds of the formula:


wherein X is 125I or 131I substituted in the phenol ring having utility for determining folic acid in serum.
Belgium Patent 840,196 describes tyramine derivatives of folic acid labeled 125I or 131I.
The compounds of the present invention differ in that the labeled tryosine moiety is not in the terminal position and that the terminal position is occupied by glutamic acid. Labeled compounds of the present
invention more closely resemble folic acid in that both have glutamic acid in the terminal position.

Disclosure of Invention
The present invention encompasses compounds of the formula A and B:

mixtures of A and B and acid addition salts thereof wherein R is hydrogen or loweralkyl having 1-3 carbon atoms and the phenol moiety is labeled with 125I or 131I to provide radiolabeled folic acid derivatives useful for assaying for folic acid in biological fluids. Compounds of the present invention are prepared according to the following scheme:

These compounds are separated by conventional chroma tography techniques. The residual acid groups may be esterified by reaction with diazomethane or acid-catalyzed esterified with alcohols. Alternately, one of the
carboxylic acid groups of the glutamic acid portion of the starting folic acid can be esterified providing a single condensation product. Esters are converted to acids by conventional base catalyzed hydrolysis.
Radioactive iodine, 125I or 131I, is conveniently introduced by the phenol ring of tyrosine by the
chloramine-T method of Greenwood, et al, Biochem. J.,
89, 114 (1963).
Compounds of the present invention are most convenient ly used as the acid addition salt of organic and mineral acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric, benzoic, acetic, trichloroacetic, toluene-sulfonic acids and the like.

Best Mode For Carrying Out The Invention
Folic acid (485 mg, 1.1 mmole) is suspended in water (8.0 ml) and stirred magnetically, while a solution of L-tyrosyl-L-glutamic acid α,γ-dimethyl ester (396 mg, 1.17 mmole) in pyridine (8 ml) is added. To this reaction mixture is added l-ethyl 3 (3-dimethylaminopropyl)-carbodiimide hydrochloride (2.87 mg, 1.5 mmole). The mixture is stirred at 4°C overnight. 20 ml of 0.5% sodium bicarbonate solution is added to the reaction mixture and filtered. The filtrate is acidified with hydrochloride acid to pH 3.0. The resulting precipitate is filtered and washed with about 20 ml of cold water.
This precipitate of the folic acid-tyrosylglutamic acid dimethyl ester is suspended by stirring in 10 ml 0.2N sodium hydroxide, while nitrogen gas is bubbled through the reaction mixture. After 15-20 minutes of stirring at room temperature, the clear solution is acidified with 1N hydrochloric acid to pH 3.0. This precipitated material is filtered, washed. with cold water and dried in a vacuum desiccator to provide a mixture of Compound A and B where R is hydrogen. These compounds are separated by conventional techniques.
Iodination was performed by chloramine-T procedure, Biochem. J., 89, 114 (1963).

100μl of 0.5M sodium phosphate pH 7.5 is added to a glass test tube, followed by 10.0 mCi of Na I125. To this solution is added 25μl (8.3μg) of folic acid-tyrosylglutamic acid in sodium bicarbonate solution and 50μl of 0.4% chloramine-T solution in a 0.05M sodium phosphate buffer pH 7.5, respectively. After 60 seconds, mixing at room temperature, 50μl of 0.8% sodium metabi sulfite is added to the reaction mixture.
Incorporation of 125I into folic acid derivatives is in excess of 85%. The purification of the iodinated reaction mixture is achieved by ion exchange followed by cellulose column chromatography.

Industrial Applicability

Example 1
Binding Activity Of Folate Derivatives
The 125I folate compounds are tested for their
ability to bind to specific folate binders.
Partially purified bovine milk and goat's milk folate binders (J. Dairy Res., 36, 435 (1969)) are diluted serially with 0.05M borate buffer containing 0.1% dithio threitol and 0.05% gelatin pH 9.0. 125I folate (0.01 - 0.02μCi) is added to 0,5 ml of each respective binder dilution (final reaction volume is 0.6 ml). The mixture is vortexed and allowed to incubate 45 minutes at room temperature. At the end of the incubation period, 1 ml of a 0.7% dextran-coated charcoal suspension is added to each tube, vortexed and allowed to stand 5 minutes at room temperature. The tubes are centrifuged at l000g for 15 minutes . The supernatants are decanted into clean tubes and counted in a gamma scintillation counter.

Results
The 125I folate derivatives typically show a maximum binding ability of between 75-90%.

Example 2
Determination Of Folate Concentration in
Biological Fluids Preparation Of Samples
Serum is obtained, free of hemolysis and stored at
4°C for 24 hours. For longer storage periods samples are stored at -20°C.
Samples for red cell folate determinations are prepared by diluting one part of whole blood with 19 parts of a 0.2% ascorbic acid solution. Samples are allowed to stand at room temperature for 1-1/2 hours to allow for hydrolysis of folate polyglutamates. Samples are then stored at -20°C if not used immediately,

Example 3
Folate Assay
Pteroylglutamic acid (folic acid) is diluted seriall with 0,05M borate buffer containing 0.50% gelatin and
0.1% dithiothreitol; pH 9.0, The concentrations of folic acid used are 0, 2.5, 5, 10, 15 and 20 ng/ml,
Borate buffer (0.4 ml ) is added to a series of
polypropylene tubes, To this solution is added 0,05 ml of appropriate standard dilution or sample (serum or hemolysate).
The contents of the tubes are mixed and heated in a boiling water bath for 15 minutes, The tubes are loosely covered during the extraction.
The tubes are cooled to room temperature and 0.10 ml of 125I folate derivative in borate buffer (0.01-0.02μCi) is added to each tube followed by 0.01 ml of an appropriate dilution of folate milk binder in borate buffer to give about 50% binding. The tubes are vortexed and allowed to stand 5 minutes at room temperature. The tubes are then centrifuged at l000 xg for 15 minutes. The supernatants are decanted into clean tubes and counted in a suitable scintillation counter.

Results
The folate in the patient samples are determined by comparison with a logit-log plot of the standard curve. Red cell folates are corrected for dilution and hematocrit (Clin. Biochem., 6, 274 (1973)).