CA1078827A - Isotopically labeled derivatives of folic acid - Google Patents
Isotopically labeled derivatives of folic acidInfo
- Publication number
- CA1078827A CA1078827A CA245,371A CA245371A CA1078827A CA 1078827 A CA1078827 A CA 1078827A CA 245371 A CA245371 A CA 245371A CA 1078827 A CA1078827 A CA 1078827A
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- folic acid
- derivative
- accordance
- radioactive tracer
- assay
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D475/00—Heterocyclic compounds containing pteridine ring systems
- C07D475/02—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4
- C07D475/04—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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- Urology & Nephrology (AREA)
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- Immunology (AREA)
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- Biotechnology (AREA)
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- General Physics & Mathematics (AREA)
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Abstract
Abstract of the Disclosure .
Derivatives of folic acid comprising folic acid coupled through one or both of its carboxyl groups with the amino nitrogen of a hydroxyaryl-alkylamine such as tyramine or tyrosine or a tyrosine-containing peptide such as glycyl tyrorine. These derivatives can be iodinated with isotopic iodine to serve as tracers in a radioassay for serum folates.
Derivatives of folic acid comprising folic acid coupled through one or both of its carboxyl groups with the amino nitrogen of a hydroxyaryl-alkylamine such as tyramine or tyrosine or a tyrosine-containing peptide such as glycyl tyrorine. These derivatives can be iodinated with isotopic iodine to serve as tracers in a radioassay for serum folates.
Description
7~8~7 This inven~ion relates to derivatives o~ folic acid suitable for use as tracers in an assay for serum folates.
More particularly, the invention relates to folic acid derivatives which contain phenolic groups which can be substituted with a gamma emitting isotope such as isotopic iodine. The radioactive derivative is useful in a competitive protein binding procedure for determination of endogenous folates in serum.
One of the commonly performed clinical assays is the determination of serum folates. A desirable procedure for such a determination involves the principles of competitive protein binding. In such a procedure, a radioactively labeled tracer competes with endogenous serum folates for the binding sites of a folate-selective protein. The protein is added in such amounts that its binding capàcity is less than equivalent to the quantity of material present. Thus, the bound radioactivity is inversely proportional to the endogenous folate concentration and is evaluated with the use of àn empirically constructed standard curve.
i 20 Such a competitive binding folate assay is generallyknown in the art. However, previously described tracers are tritium labeled materials. These tracers require complex and expensive beta radiation (liquid scintillation) measuring instruments and special methodology to offset quenching and sample handling difficulties. Other non-radioassays such as the microbiological assay method which have been used for folate determinations are considerably more cumbersome to practice and ,:, ;' are therefore less desirable for routine use in a clinicaL
laboratory. The present invention improves upon this state of :,..... .
" . 1--~ bm~
., , : ~ .
,'~'- - .
` :
~10~8B~7 .
the art by providing a gamma emitting tracer which has been ~; found to be suitable for the determination of the mutually cross reacting natural folates in serum, It will be appreciated that a ~amma emitting tracer can be utilized with relatively simple instrumentation available in clinical laboratories.
`. More particularly, the present invention provides ~ derivatives of folic acid suitable for use as tracers in an : assay for serum folates comprising folic acid coupled through `~: 10 one or both of its carboxyl groups with the amino nitrogen of a member selected from . ., and ~ :
fHz fH2 ; HC - R~ Hf - C02R2 `~ NH2 NH
'` C=O
,,"
CHz .... I
. , ~H2 ,: .,.,.; .
` wherein R1 is selected from hydrogen, COOH and COOR3 in which R3 is lower alkyl (about 1 to 5 carbon atoms) and R2 is :~.; selected from hydrogen and lower alkyl (about 1 to 5 carbon :~ 1 ~ atoms).
J''" ' Folic acid has the following structural formula:
.,".~. .
N N N
\ ~ \ / ~ / COOH
/ N/ ~ C -N - I -C--C -COOH
,', bH
:` -2-. ......................................................................... .
bm: p~
788;~7 From the formula it may be seen that there are two carboxyl groups, The present derivatives involve the coupling of the amino group nitro~en of the selected reactant with one or both of the carboxyl groups of folic acid. This coupling reaction between the amino group and the carboxyl group follows well-established organic reactions and employs conventional reagents for effecting the desired coupling. Suitable reagents and conditions are ~escribed in "Principles of Competitive Protein-Binding Assays", Odell, W.D., and Daughaday, W.H., eds., J.B, Lippincott Company 1971. Attention is particularly directed to page 25 thereof, where a table presents numerous alternatives for carrying out the reaction9 The derivative obtained is suitable for receiving isotopic iodine such as I~ 2 ~ ~ which can be introduced by substitution on the benzene ring containing the phenolic group.
A preferred method involves iodination with Chloramine-T
(p-toluenesulfonchloramide) and a sodium salt of I~2' or Il3l.
This method is widely used and is applicable to labeling a variety of proteins and peptides containing phenols, The reaction generally involves contact of the derivative with Chloramine-T in the presence of the radioactive sodium iodide for a short time at room temperature~
There are other known methods and reagents for accomplishing the desired iodination of the derivative including (a) iodine monochloride-(125) and ~131); (b) exchange labeling with sodium iodide-(125) and (131); (c) electrolytic iodination with I-(125) and I-(131); and (d) enzymatic iodination with lactoperoxidase catalyst. The following are selected references teaching these general methods:
, .
bm:ph .. . . .
- - . ; .. , ,. ~ ...
- . , ' ~ 7~3827 :
1, Liebster, J,, et al., Nature 183, 1474 (1959)
More particularly, the invention relates to folic acid derivatives which contain phenolic groups which can be substituted with a gamma emitting isotope such as isotopic iodine. The radioactive derivative is useful in a competitive protein binding procedure for determination of endogenous folates in serum.
One of the commonly performed clinical assays is the determination of serum folates. A desirable procedure for such a determination involves the principles of competitive protein binding. In such a procedure, a radioactively labeled tracer competes with endogenous serum folates for the binding sites of a folate-selective protein. The protein is added in such amounts that its binding capàcity is less than equivalent to the quantity of material present. Thus, the bound radioactivity is inversely proportional to the endogenous folate concentration and is evaluated with the use of àn empirically constructed standard curve.
i 20 Such a competitive binding folate assay is generallyknown in the art. However, previously described tracers are tritium labeled materials. These tracers require complex and expensive beta radiation (liquid scintillation) measuring instruments and special methodology to offset quenching and sample handling difficulties. Other non-radioassays such as the microbiological assay method which have been used for folate determinations are considerably more cumbersome to practice and ,:, ;' are therefore less desirable for routine use in a clinicaL
laboratory. The present invention improves upon this state of :,..... .
" . 1--~ bm~
., , : ~ .
,'~'- - .
` :
~10~8B~7 .
the art by providing a gamma emitting tracer which has been ~; found to be suitable for the determination of the mutually cross reacting natural folates in serum, It will be appreciated that a ~amma emitting tracer can be utilized with relatively simple instrumentation available in clinical laboratories.
`. More particularly, the present invention provides ~ derivatives of folic acid suitable for use as tracers in an : assay for serum folates comprising folic acid coupled through `~: 10 one or both of its carboxyl groups with the amino nitrogen of a member selected from . ., and ~ :
fHz fH2 ; HC - R~ Hf - C02R2 `~ NH2 NH
'` C=O
,,"
CHz .... I
. , ~H2 ,: .,.,.; .
` wherein R1 is selected from hydrogen, COOH and COOR3 in which R3 is lower alkyl (about 1 to 5 carbon atoms) and R2 is :~.; selected from hydrogen and lower alkyl (about 1 to 5 carbon :~ 1 ~ atoms).
J''" ' Folic acid has the following structural formula:
.,".~. .
N N N
\ ~ \ / ~ / COOH
/ N/ ~ C -N - I -C--C -COOH
,', bH
:` -2-. ......................................................................... .
bm: p~
788;~7 From the formula it may be seen that there are two carboxyl groups, The present derivatives involve the coupling of the amino group nitro~en of the selected reactant with one or both of the carboxyl groups of folic acid. This coupling reaction between the amino group and the carboxyl group follows well-established organic reactions and employs conventional reagents for effecting the desired coupling. Suitable reagents and conditions are ~escribed in "Principles of Competitive Protein-Binding Assays", Odell, W.D., and Daughaday, W.H., eds., J.B, Lippincott Company 1971. Attention is particularly directed to page 25 thereof, where a table presents numerous alternatives for carrying out the reaction9 The derivative obtained is suitable for receiving isotopic iodine such as I~ 2 ~ ~ which can be introduced by substitution on the benzene ring containing the phenolic group.
A preferred method involves iodination with Chloramine-T
(p-toluenesulfonchloramide) and a sodium salt of I~2' or Il3l.
This method is widely used and is applicable to labeling a variety of proteins and peptides containing phenols, The reaction generally involves contact of the derivative with Chloramine-T in the presence of the radioactive sodium iodide for a short time at room temperature~
There are other known methods and reagents for accomplishing the desired iodination of the derivative including (a) iodine monochloride-(125) and ~131); (b) exchange labeling with sodium iodide-(125) and (131); (c) electrolytic iodination with I-(125) and I-(131); and (d) enzymatic iodination with lactoperoxidase catalyst. The following are selected references teaching these general methods:
, .
bm:ph .. . . .
- - . ; .. , ,. ~ ...
- . , ' ~ 7~3827 :
1, Liebster, J,, et al., Nature 183, 1474 (1959)
2. Koros, E., et al., Acta Chem, Acad Sci. Hung., 2~, 187 ~1961)
3. McFarlan, A.S., Nature 182 (1958)
4. Greenwood, F. C. and Hunter, W. M ., Nature, 194, 495 (1962)
5. Rosa, tJ., "Labeled Proteins in Tracer Studies", 17-28, 61-69, EUR 2950, Pisa, Italy ~1966) The following examples will illustrate preparation of derivatives of this invention and the introduction thereon of a gamma emitting isotope.
EXAMPLE I
Tyramine Derivative of Folic Acid Folic acid (0.441 g; lmM) in 0.05 M phosphate buffer, pH 7.4 (100 ml) was treated with tyramine hydrochloride (0.173 g; 1 mM) and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.382 g; 2mM). After three hours at ambient room temperature the material was centrifuged and the supernatant discarded. The resulting gel was washed twice with methanol and twice with acetone by suspending and centrifugation. The product was dried at room temperature, finely ground and used in the next Example without further purification.
-EXAMPLE II
-A. Iodination of Tyramine Folate Derivative The material prepared in Example I, 21.0 mg, was treated with 100 ~1 of concentrated aqueous ammonium hydroxide solution (approximately 29.4% NH9), for one minute (approximately), then agitated with 0.90 ml of distilled water --bm.p~ -.... , ... ... . . ... .. . . . ~ . .... . . . - . .. . . . :
~781 3~7 until ~issolv~d, and ~inally diluted wi~h additional 9.0 ml of distilled water, ~his solution was ~iltered through Whatman ~l filter paper, diluted l:lO with distilled water and stored frozen until required for the next step The solution prepared a~ove (50 ~1; approximately lO ~g o material) was mixed with 50 ~l of l M phosphate buffer, pH 7.4. Carrier free sodium iodide I~2' t4.3 mCi; approximately 50 ~l) was then added. The reaction was initiated by introduction of Chloramine-T dissolved in 0.05 M phosphate buffe~/ pH 7.4 ~50 ~l; 150 ~g) and stopped after one minute by ~he addition of 50 ~l of 1% aqueous solution of sodium metabisul~i~e. The incorporation of iodide into organic material estimated by electrophoresis or ion exchange chro~atography was typically in excess of 90%.
B. Purification of Iodot~ramine Derivative of Folic Acid The reaction mixture obtained as above was streaked ~ut on a 500 micron thicX layer plate of Avicel~ and developed with the upper phase of n-butanol:2M acetic acid (l:l) system.
~ 20 The ~ain band was located by Polaroid~ autoradiography using - the method described in Prescott, K.M.~ and David~ G.S. t Analytical Biochemistry, 57, 232-239 (1974), scraped off and allo~ed to stand in 10 ml of dimethyl formamide overnight in -the col~, Following centrifugation, the supernatant solution of the tracer can be used in a folate radioassay.
EXAMPLE III
... ~
y osine ~ethyl Ester Derivative of Folic Acid Folic acid (0.441 9, lmM) in 0.05 M phosphate buffer, , .
' ~: p~
-^, ' ' ' ~ .
.~ . .
:. . ,' ' .~'~ , ~C~7~it8~7 pH 7.4 (100 ml) was treated with ty~osine methyl ester hydro-chloride (0,232 g, lmM) and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.382 g, 2mM)c After three hours at ambient room temperature, the material was centrifuged, washed, iodinated and purified exactly as in Examples I and IIo The displacement characteristic of this tracer is analogous to that obtained with material in Examples I and II, The foregoing examples are typical of the invention.
To obtain other derivatives and radioactive tracers within the scope of the ~resent invention, it is simply a matter of substituting the appropriate hydroxyaryl-alkylamine or tyrosine-containing peptide for the materials employed in the above examples, Thus, another preferred derivative can be obtained by substituting glycyl tyrosine for tyramine or tyrosine in the foregoing examples.
The efficacy of the radioactive tracers provided by this invention in a competitive protein binding assay of serum folate is demonstrated in two systems which employed commerically available assay procedures. The tracers of the present invention were substituted for the tracer provided in the commerical procedure, Otherwise, the work described below followed and used the commerical procedures and/or materials supplied by the manufacturer for use therewith. The procedures employed are summarized below in Flow Chart 1 and 2.
Flow Chart 1 represents the procedures and materials sold by Radio Immuno Assay Products, Inc., whereas Flow Chart 2 follows the folate assay sold by Diagnostic Biochemistry, Inc, As noted above, the directions of these manufactures were followed. However~ the tracers described in the examples were .
~6-; bm:p~
1~7~5l!3Z7 employed in these procedures, Figs, 1 and 2 show di~placement cUrves Using TEIFA
(N-methyltetrahydrofolic acid) and PGA (pteroyglutamic acid) as standards respectively~ and tyramine-I~ 2 ~ derivative described in Example II (B). Thus, a dose response, using either THFA or PG~ is demonstrated.
- bm: ~
! , . . :: ' ~.
,, ~' . ~ . ' .~ , :,, ; '` . - , ;
. '., ,: . ' :
~0~ 7 . . . .
.~
. ,R
n :
g ~ ~ O
I 1n Ul ~ .
('~'1 'UFw 0~ UF~
~il ~ ., ~
.~ I ~ O 0 0 O O ~ . ' .
. ~ O
'~` ~ ~ a ~ '~ a ~L ~ ~ 'I ,1 ' ~ o o O O O O O O ~ ~ ~}, O O ~ æ~
O ~ . .
. ~ i I I R
1~ ~ a -I ~ a a ~ '~
~ o oO oO oO O 0 ~0 ~
-,. ~ ~ ,~ a ~ ;~ $ ~
O ' ~i ~7 N ~ ~1 ~ I N N
~:p~
- . . , .: .
1. .,' ' , .. ~. , :. - . . . : ,~
.
` - . . ,. . . ~. .
,, .
O ~
~ $ g 8 o o 8 8 o ~s ~ U~ Lr) LO
~un~ o~ Uo~ UI) o~ ~ ,, ,, LO) ~1n U7 L'' ~ LO, o~ ~ ~ ~ " U
o o o o o o o o o o oooooo~oooo 1~ ~ ,o~
~ W
''~3 ~ O ~ 1 0 ~ ~ O .
.~
~ a '~
ooooooOoOo ~ L~ u~ n Ln n In U~
~ O ~ 1 ~ H ~
~ ~ c) x .~
" , _9_ , : ' ~)7~ 7 The efficacy o the tracer of this invention was further d~monstrated b~ the spike recovery experiment summarized in Table I below. In this experiment, the assay was performed with the commercially available competitive proteln binding folate assay offered by Diagnostic Biochemistry, Inc., except that the tracer of this invention was utilized instead of the tracer commerically offered. In performing the experiment, a control serum having a very low folate level was spiked with PGA and THFA.
Table 1 Folates Ex- Folate No. of THFA PG~pected to Found Samples Added Added be Fo~nd m + S.D. % Recovery _ 0 23,24 2,87 + 0.29 88,6%
0 89.24 9.44 + 0.46 102.2%
2 03.24 2,49 + 0,33 76.8% ' ~ 10 8 09,24 7.92 + 0.43 85.7%
,, .
.', , ~ bm.p~
. '`:t"''' . '.
~! , , , `.. . .
' ~: . ' , ~'' '~; ' ' ' , ' ' ' ' ' : . . ' ' ' . , .
'.' ' . ~ " ' '' . " ' ~ . . . '' ' .. . . .
': ' ' ' ' ' ' ' ;~'
EXAMPLE I
Tyramine Derivative of Folic Acid Folic acid (0.441 g; lmM) in 0.05 M phosphate buffer, pH 7.4 (100 ml) was treated with tyramine hydrochloride (0.173 g; 1 mM) and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.382 g; 2mM). After three hours at ambient room temperature the material was centrifuged and the supernatant discarded. The resulting gel was washed twice with methanol and twice with acetone by suspending and centrifugation. The product was dried at room temperature, finely ground and used in the next Example without further purification.
-EXAMPLE II
-A. Iodination of Tyramine Folate Derivative The material prepared in Example I, 21.0 mg, was treated with 100 ~1 of concentrated aqueous ammonium hydroxide solution (approximately 29.4% NH9), for one minute (approximately), then agitated with 0.90 ml of distilled water --bm.p~ -.... , ... ... . . ... .. . . . ~ . .... . . . - . .. . . . :
~781 3~7 until ~issolv~d, and ~inally diluted wi~h additional 9.0 ml of distilled water, ~his solution was ~iltered through Whatman ~l filter paper, diluted l:lO with distilled water and stored frozen until required for the next step The solution prepared a~ove (50 ~1; approximately lO ~g o material) was mixed with 50 ~l of l M phosphate buffer, pH 7.4. Carrier free sodium iodide I~2' t4.3 mCi; approximately 50 ~l) was then added. The reaction was initiated by introduction of Chloramine-T dissolved in 0.05 M phosphate buffe~/ pH 7.4 ~50 ~l; 150 ~g) and stopped after one minute by ~he addition of 50 ~l of 1% aqueous solution of sodium metabisul~i~e. The incorporation of iodide into organic material estimated by electrophoresis or ion exchange chro~atography was typically in excess of 90%.
B. Purification of Iodot~ramine Derivative of Folic Acid The reaction mixture obtained as above was streaked ~ut on a 500 micron thicX layer plate of Avicel~ and developed with the upper phase of n-butanol:2M acetic acid (l:l) system.
~ 20 The ~ain band was located by Polaroid~ autoradiography using - the method described in Prescott, K.M.~ and David~ G.S. t Analytical Biochemistry, 57, 232-239 (1974), scraped off and allo~ed to stand in 10 ml of dimethyl formamide overnight in -the col~, Following centrifugation, the supernatant solution of the tracer can be used in a folate radioassay.
EXAMPLE III
... ~
y osine ~ethyl Ester Derivative of Folic Acid Folic acid (0.441 9, lmM) in 0.05 M phosphate buffer, , .
' ~: p~
-^, ' ' ' ~ .
.~ . .
:. . ,' ' .~'~ , ~C~7~it8~7 pH 7.4 (100 ml) was treated with ty~osine methyl ester hydro-chloride (0,232 g, lmM) and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.382 g, 2mM)c After three hours at ambient room temperature, the material was centrifuged, washed, iodinated and purified exactly as in Examples I and IIo The displacement characteristic of this tracer is analogous to that obtained with material in Examples I and II, The foregoing examples are typical of the invention.
To obtain other derivatives and radioactive tracers within the scope of the ~resent invention, it is simply a matter of substituting the appropriate hydroxyaryl-alkylamine or tyrosine-containing peptide for the materials employed in the above examples, Thus, another preferred derivative can be obtained by substituting glycyl tyrosine for tyramine or tyrosine in the foregoing examples.
The efficacy of the radioactive tracers provided by this invention in a competitive protein binding assay of serum folate is demonstrated in two systems which employed commerically available assay procedures. The tracers of the present invention were substituted for the tracer provided in the commerical procedure, Otherwise, the work described below followed and used the commerical procedures and/or materials supplied by the manufacturer for use therewith. The procedures employed are summarized below in Flow Chart 1 and 2.
Flow Chart 1 represents the procedures and materials sold by Radio Immuno Assay Products, Inc., whereas Flow Chart 2 follows the folate assay sold by Diagnostic Biochemistry, Inc, As noted above, the directions of these manufactures were followed. However~ the tracers described in the examples were .
~6-; bm:p~
1~7~5l!3Z7 employed in these procedures, Figs, 1 and 2 show di~placement cUrves Using TEIFA
(N-methyltetrahydrofolic acid) and PGA (pteroyglutamic acid) as standards respectively~ and tyramine-I~ 2 ~ derivative described in Example II (B). Thus, a dose response, using either THFA or PG~ is demonstrated.
- bm: ~
! , . . :: ' ~.
,, ~' . ~ . ' .~ , :,, ; '` . - , ;
. '., ,: . ' :
~0~ 7 . . . .
.~
. ,R
n :
g ~ ~ O
I 1n Ul ~ .
('~'1 'UFw 0~ UF~
~il ~ ., ~
.~ I ~ O 0 0 O O ~ . ' .
. ~ O
'~` ~ ~ a ~ '~ a ~L ~ ~ 'I ,1 ' ~ o o O O O O O O ~ ~ ~}, O O ~ æ~
O ~ . .
. ~ i I I R
1~ ~ a -I ~ a a ~ '~
~ o oO oO oO O 0 ~0 ~
-,. ~ ~ ,~ a ~ ;~ $ ~
O ' ~i ~7 N ~ ~1 ~ I N N
~:p~
- . . , .: .
1. .,' ' , .. ~. , :. - . . . : ,~
.
` - . . ,. . . ~. .
,, .
O ~
~ $ g 8 o o 8 8 o ~s ~ U~ Lr) LO
~un~ o~ Uo~ UI) o~ ~ ,, ,, LO) ~1n U7 L'' ~ LO, o~ ~ ~ ~ " U
o o o o o o o o o o oooooo~oooo 1~ ~ ,o~
~ W
''~3 ~ O ~ 1 0 ~ ~ O .
.~
~ a '~
ooooooOoOo ~ L~ u~ n Ln n In U~
~ O ~ 1 ~ H ~
~ ~ c) x .~
" , _9_ , : ' ~)7~ 7 The efficacy o the tracer of this invention was further d~monstrated b~ the spike recovery experiment summarized in Table I below. In this experiment, the assay was performed with the commercially available competitive proteln binding folate assay offered by Diagnostic Biochemistry, Inc., except that the tracer of this invention was utilized instead of the tracer commerically offered. In performing the experiment, a control serum having a very low folate level was spiked with PGA and THFA.
Table 1 Folates Ex- Folate No. of THFA PG~pected to Found Samples Added Added be Fo~nd m + S.D. % Recovery _ 0 23,24 2,87 + 0.29 88,6%
0 89.24 9.44 + 0.46 102.2%
2 03.24 2,49 + 0,33 76.8% ' ~ 10 8 09,24 7.92 + 0.43 85.7%
,, .
.', , ~ bm.p~
. '`:t"''' . '.
~! , , , `.. . .
' ~: . ' , ~'' '~; ' ' ' , ' ' ' ' ' : . . ' ' ' . , .
'.' ' . ~ " ' '' . " ' ~ . . . '' ' .. . . .
': ' ' ' ' ' ' ' ;~'
Claims (11)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A derivative of folic acid suitable for use as a tracer in an assay for serum folates comprising the main band obtained by thin layer chromatographic separation of the reaction product of folic acid and a compound selected from and wherein R1 is selected from hydrogen, COOH and COOR3 in which R3 is lower alkyl and R2 is selected from hydrogen and lower alkyl, the benzene ring of the selected compound being substituted with isotopic iodine.
2. A derivative of folic acid in accordance with claim 1 wherein said isotopic iodine is I125.
3. A derivative of folic acid in accordance with claim 1 wherein said member selected is glycyl tyrosine.
4. A derivative of folic acid in accordance with claim 1 wherein said member selected is tyramine.
5. A derivative of folic acid in accordance with claim 1 wherein said member selected is tyrosine.
6. A derivative of folic acid in accordance with claim 1 wherein said member selected is the methyl ester of tyrosine.
7. In the assay for serum folates by the com-petitive protein binding method using a selective protein as a binding agent in the presence of a radioactive tracer, the improvement wherein said radioactive tracer is a folic acid derivative in accordance with claim 1.
8. In the assay for serum folates by the competitive protein binding method using a selective protein as a binding agent in the presence of a radioactive tracer, the improvement wherein said radioactive tracer is a folic acid derivative in accordance with claim 3.
9. In the assay for serum folates by the competitive protein binding method using a selective protein as a binding agent in the presence of a radioactive tracer, the improvement wherein said radioactive tracer is a folic acid derivative in accordance with claim 4.
10. In the assay for serum folates by the competitive protein binding method using a selective protein as a binding agent in the presence of a radioactive tracer, the improvement wherein said radioactive tracer is a folic acid derivative in accordance with claim 5.
11. In the assay for serum folates by the competitive protein binding method using a selective protein as a binding agent in the presence of a radioactive tracer, the improvement wherein said radioactive tracer is a folic acid derivative in accordance with claim 6.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US55146275A | 1975-02-20 | 1975-02-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1078827A true CA1078827A (en) | 1980-06-03 |
Family
ID=24201371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA245,371A Expired CA1078827A (en) | 1975-02-20 | 1976-02-10 | Isotopically labeled derivatives of folic acid |
Country Status (4)
| Country | Link |
|---|---|
| CA (1) | CA1078827A (en) |
| DE (1) | DE2605387A1 (en) |
| FR (1) | FR2301525A1 (en) |
| GB (1) | GB1504263A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4298735A (en) * | 1979-04-30 | 1981-11-03 | Union Carbide Corporation | Folic acid derivatives |
| GB2056459B (en) * | 1979-07-04 | 1983-03-09 | Daiichi Radioisotope Lab | Pterin derivatives and an assay method for determining pterins |
-
1976
- 1976-02-10 CA CA245,371A patent/CA1078827A/en not_active Expired
- 1976-02-11 DE DE19762605387 patent/DE2605387A1/en active Pending
- 1976-02-13 FR FR7604034A patent/FR2301525A1/en active Granted
- 1976-02-19 GB GB662876A patent/GB1504263A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GB1504263A (en) | 1978-03-15 |
| FR2301525A1 (en) | 1976-09-17 |
| DE2605387A1 (en) | 1976-09-02 |
| FR2301525B1 (en) | 1979-08-31 |
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