Note: Descriptions are shown in the official language in which they were submitted.
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:
, .
-3-
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 --
-4-
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,
, .
-5-
' ~: 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
- 8
~: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%
,, .
.', ,
--10--
~ bm.p~
. '`:t"''' . '.
~! , , , `.. . .
' ~: . ' , ~'' '~; ' ' ' , ' ' '
' ' : . . ' ' ' . , .
'.' ' . ~ " ' '' . " ' ~ . . . '' '
.. . . .
': ' ' ' ' ' ' ' ;~'
