Reference blood filter paper for measuring the concentration of methionine in the blood

A reference blood filter paper for measuring the concentration of methionine in the blood, comprising a piece of blood filter paper, a blood material infiltered in the blood filter paper containing a known concentration of methionine and at least one water-soluble, sulfur-containing antioxidant represented by general formula: ##STR1## where n=1 or 2.

BACKGROUND OF THE INVENTION 
The present invention relates to a reference blood filter paper for 
diagnosis of homocystinuria, one kind of congenital disorder of amino acid 
metabolism, and more particularly to a reference blood filter paper of the 
kind mentioned above, which is used for the diagnosis of homocystinuria by 
measuring the concentration of methionine in the blood, and which can be 
preserved for a long period of time without any detectable deterioration. 
Congenital disorders of amino acid metabolism, such as phenylketonuria, 
histidinemia and homocystinuria, are fearful diseases leading to mental 
deficiency or hepatocirrhosis. These days, however, patients with such 
congenital disorders can be cured and their lives saved if the disorders 
are identified during early infancy and the patients are promptly and 
appropriately treated, for instance, by subjecting them to dietary 
treatment. 
In order to identify those disorders, it is necessary that the 
concentration of a particular amino acid in the blood of a new born baby 
be measured within a week after its birth, to determine whether or not 
that concentration is abnormally high. For this purpose, there is desired 
a simple and reliable screening assay. 
The assay which is in most general use for this determination of 
concentration at preset is Guthrie's Bacterial Inhibition Assay. The 
principle of that assay is as follows: 
When bacillus subtilis is cultured in an agar culture medium, if the agar 
culture medium contains a predetermined amount of a metabolism inhibitor 
which works on an amino acid which is indispensable to the growth of 
bacillus subtilis, the growth of bacillus subtilis will be inhibited by 
the action of the metabolism inhibitor. However, when a piece of filter 
paper into which sample blood has been infiltrated is placed on the 
above-mentioned agar culture medium containing the metabolism inhibitor, 
and bacillus subtilis is cultured there, bacillus subtilis can grow, 
utilizing the amino acid contained in the blood in the filter paper, so 
that a growth circle of bacillus subtilis, corresponding in size to the 
quantity of the amino acid contained in the blood, is formed. 
Likewise, bacillus subtilis is cultured on a piece of reference blood 
filter paper into which blood containing a known amount of the amino acid 
has been infiltrated, so that a reference growth circle of bacillus 
subtilis is obtained. 
By comparing the first mentioned growth circle with the second mentioned 
reference growth circle, the approximate concentration of the amino acid 
in the sample blood can be determined. 
The details of this procedure and measurement conditions for the Guthrie's 
Bacterial Inhibition Assay are described in Rinshobyori (Clinical 
Pathology) 24 (12) 962-973, 1976. 
In order to obtain highly stable and reproducible measurement results, it 
is indispensable that the amount of the amino acid contained in the 
reference blood filter paper not change with time, and the reference blood 
filter paper be preservable for a long period of time without any 
detectable deterioration. 
In a conventional reference blood filter for measuring the concentration of 
methionine in the blood, methionine contained in the reference blood 
filter is oxidized extremely easily during preservation and, accordingly, 
its properties also change during preservation. 
For instance, when it is preserved at a temperature of -20.degree. C., it 
is so deteriorated after 4 months that it cannot be used any longer. This 
is a significant shortcoming of the conventional reference blood filter 
paper. 
SUMMARY OF THE INVENTION 
It is therefore an object of the present invention to provide a novel 
reference blood filter paper for measuring the concentration of methionine 
in the blood, which reference blood filter paper is free from the 
shortcomings of the conventional blood filter paper and can be preserved 
for a long period of time without any detectable deterioration. 
The present invention is based on the discovery that an organic, 
water-soluble, sulfur-containing antioxidant with a sulfide structure, 
such as .beta.-thiodiglycol and .beta.-thiodipropanol, is capable of 
preventing methionine contained in the dried blood from being oxidized or 
changed in properties, without having any adverse effects on the growth of 
bacillus subtilis which serves as an indicator of the quantity of 
methionine contained in the blood. 
In the present invention, one of the above-mentioned antioxidants is used 
in the reference blood filter paper in order to attain the above-mentioned 
object of the present invention. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As the organic, water-soluble, sulfur-containing antioxidant having a 
sulfide structure that can be employed in the present invention, 
.beta.-thiodiglycol and .beta.-thiodipropanol can be employed. 
An embodiment of a piece of reference blood filter paper according to the 
present invention is prepared as follows: 
The above-mentioned antioxidant is dissolved in the waste blood of healthy 
persons so as to yield a mixture of the blood and the antioxidant, 
containing 30 mM to 120 mM of the antioxidant, preferably 35 mM to 60 mM 
of the antioxidant, therein. 
L-methionine is dissolved in the above-mentioned mixture to yield a mixture 
with a total concentration of 1 mg/dl of L-methionine. Likewise, six other 
mixtures with the total concentrations of L-methionine being 2 mg/dl, 4 
mg/dl, 8 mg/dl, 12 mg/dl, 16 mg/dl and 20 mg/dl are prepared. 
Each mixture is softly stirred and is then allowed to stand for 2 hours at 
room temperature so as to disperse the antioxidant and L-methionine 
homogeneously in the mixture. 
Each mixture is spread on a commercially available filter paper for 
diagnosis of phenylketonuria (PKU) with a diameter of 11 mm and is then 
air-dried, whereby a piece of reference blood filter paper according to 
the present invention is prepared. For use as a reference blood filter 
paper for Guthrie's Bacterial Inhibition Assay when the concentration of 
L-methionine in the blood of a patient is to be measured, a piece of the 
blood filter paper with a diameter of 3 mm is cut from the above-mentioned 
blood filter paper by use of a disc puncher. 
Unlike the conventional reference blood filter paper, the reference blood 
filter paper according to the present invention can be preserved without 
any detectable deterioration for more than one year at -20.degree. C. or 
more than 6 months at 4.degree. C., and, when preserved under the 
above-mentioned conditions, can provide a growth circle in accordance with 
each specified concentration of L-methionine, without having any adverse 
effects on the necessary factors for Guthrie's Bacterial Inhibition Assay, 
such as the growth of bacillus subtilis and the color of the blood. 
In Table 1, there are shown the results of comparisons between the blood 
filter paper according to the present invention and blood filter papers in 
which antioxidants other than the antioxidants according to the present 
invention are employed, in terms of the effects of the antioxidants on the 
blood when each blood filter paper is prepared and on the growth of 
bacillus subtilis in the Guthrie's Bacterial Inhibition Assay. 
TABLE 1 
__________________________________________________________________________ 
Suit- 
Antioxidant 
Effects on the Blood 
Effects on Guthrie's BIA* 
ability 
__________________________________________________________________________ 
.beta.-thiodiglycol 
In concentrations up to 120 mM, 
In concentrations up to 120 mM, 
Suit- 
no coagulation of the blood 
no formation of an inhibition 
able 
and no change in color of the 
circle and no difference in 
blood. growth of bacillus subtilis 
between the addition of this 
antioxidant and no addition 
thereof. 
.beta.-thiodipropanol 
In concentrations up to 120 mM, 
In concentrations up to 120 mM, 
Suit- 
no coagulation of the blood 
no formation of an inhibition 
able 
and no change in color of the 
circle and no difference in 
blood. growth of bacillus subtilis 
between the addition of this 
antioxidant and no addition 
thereof. 
Thioglycolic Acid 
Above 15 mM, immediate coagu- 
Excessive growth of bacillus 
Unsuit- 
lation of the blood, the color 
subtilis not corresponding to 
able 
thereof changing to dark 
the growth of amino acid. 
brown. 
.beta., .beta.'-thiodipropionic 
Above 10 mM, immediate coagu- 
-- Unsuit- 
acid lation of the blood, the color able 
thereof changing to dark 
brown. 
Dithiothreitol 
Above 45 mM, coagulation of 
Above 15 mM, conspicuous forma- 
Unsuit- 
the blood in 2 hrs., the color 
tion of an inhibition circle. 
able 
thereof changing to dark 
brown. 
2-mercaptoethyl 
Above 45 mM, coagulation of 
Above 15 mM, conspicuous forma- 
Unsuit- 
alcohol the blood in 2 hrs., the color 
tion of an inhibition circle. 
able 
thereof changing to dark 
brown. 
__________________________________________________________________________ 
Suit- 
Antioxidant 
Effects on the Blood 
Effects on Gurhrie's Assay 
ability 
__________________________________________________________________________ 
Sodium thiosulfate 
The color of the blood changes 
Above 30 mM, formation of an 
Unsuit- 
to that of fresh blood. 
inhibition circle is recognized. 
able 
Sodium hydrosulfite 
Above 10 mM, the color of the 
-- Unsuit- 
blood changes to dark purple. able 
Buthylated hydroxy- 
Practically insoluble in 
Forms the same growth circle 
Unsuit- 
anisole water and difficult to handle. 
regardless of the concentra- 
able 
tion of methionine. 
Buthylated hydroxy- 
Practically insoluble in 
Forms the same growth circle 
Unsuit- 
toluene water and difficult to handle. 
regardless of the concentra- 
able 
tion of methionine. 
EDTA.2Na Above 0.1 mM, inhibition of the 
Unsuit- 
growth of bacillus subtilis is 
able 
conspicuous. 
__________________________________________________________________________ 
Note:- 
*Bacterial Inhibition Assay 
TABLE 2 
__________________________________________________________________________ 
Antioxidant Duration of Stability Test 
Added Amount Added 
at 37.degree. C. (days) 
of Methionine 
Kind Amount 
2 7 14 22 37 
__________________________________________________________________________ 
L-methionine 
No addition 
0 3.6 mg/dl 
2.84 
2.64 
2.56 
2.44 
4 mg/dl for comparison 
(90%) (71) 
(66) 
(64) 
(61) 
.beta.-thiodiglycol 
50 mM 
3.96 mg/dl 
3.76 
3.60 
3.44 
3.32 
(99%) (94) 
(90) 
(86) 
(83) 
.beta.-thiodipro- 
50 mM 
3.97 mg/dl 
3.80 
3.62 
3.40 
3.29 
panol (99%) (97) 
(91) 
(85) 
(82) 
L-methionine 
No addition 
0 10.8 mg/dl 
9.6 
9.48 
9.36 
8.78 
12 mg/dl 
for comparison 
(90%) (80) 
(79) 
(78) 
(73) 
.beta.-thiodiglycol 
50 mM 
11.76 mg/dl 
11.52 
11.16 
10.92 
10.80 
(98%) (96) 
(93) 
(91) 
(90) 
.beta.-thiodipro- 
50 mM 
11.81 mg/dl 
11.59 
11.20 
10.88 
10.82 
panol (98%) (97) 
(93) 
(91) 
(90) 
__________________________________________________________________________ 
Note: 
Figures in the table indicate the found concentration of residual 
methionine. 
Figures in parentheses indicate the ratio of the residual methionine to 
the amount of methionine added. 
Referring to Table 1, the antioxidants which cause coagulation of the blood 
are not suitable for the object of the present invention since, if the 
coagulation takes place, the blood is not absorbed homogenously in the 
blood filter paper. Furthermore, the antioxidants which change the color 
of the blood placed on the reference blood filter paper to the extent that 
the color is quite different from the color of the blood to be tested, or 
the antioxidants which form inhibition circles inhibiting the growth of 
bacillus subtilis, are not suitable for the present invention since they 
may cause erroneous judgements in the comparative observations of the 
growth of bacillus subtilis in the Guthrie's Bacterial Inhibition Assay. 
As a matter of course, the antioxidants which inhibit the growth of 
bacillus subtilis or which promote the growth of the same in such a manner 
as to be irrelevant to the concentration of methionine contained in the 
blood, cannot be used in practice, either. 
The comparative results shown in Table 1 show that the antioxidants 
suitable for the present invention are .beta.-thiodiglycol and 
.beta.-thiodipropanol. 
Referring to Table 2, there are shown the results of tests conducted as to 
the stability of L-methionine in the reference blood filter papers 
containing the above-mentioned antioxidants according to the present 
invention, which were conducted under severe conditions at 37.degree. C. 
In Table 2, the quantitative measurement of the remaining L-methionine was 
conducted by Bioassay employing lactobacillus. 
It is empirically known that reference blood filter paper which can hold 
therein more than 80% of L-methionine after severe testing for 14 days can 
be preserved for more than one year at -20.degree. C. and can then be used 
without any problems for the Guthrie's Bacterial Inhibition Assay. From 
this empirical knowledge, the results in Table 2 show that the reference 
blood filter paper produced by use of the blood to which 50 mM of 
.beta.-thiodiglycol or .beta.-thiodipropanol is added can be safely 
preserved for more than one year at -20.degree. C. 
Referring to Table 3, there are shown the results of tests for 
investigation of the relationship between the amount of each of 
.beta.-thiodiglycol and .beta.-thiodipropanol and the preservation effect 
thereof, which were conducted under the same conditions as the tests 
summarized in Table 2. 
TABLE 3 
______________________________________ 
Duration 
of Pres- 
Filter eruation Concentration of Antioxidant 
Paper at 37.degree. C. 
15 mM 30 60 90 120 
______________________________________ 
(1) Addition of .beta.-thiodiglycol 
With 14 days 3.16 mg/dl 
3.60 3.60 3.64 3.60 
methionine (79%) (90) (90) (91) (90) 
in concen- 
tration of 
28 days 3.12 3.36 3.40 3.44 3.44 
4 mg/dl (78) (84) (85) (86) (86) 
With 14 days 10.20 mg/dl 
11.28 
11.28 
11.40 11.40 
methionine (85%) (94) (94) (95) (95) 
in concen- 
tration of 
28 days 9.84 10.80 
10.80 
10.92 10.92 
12 mg/dl (82) (90) (90) (91) (91) 
(2) Addition of .beta.-thiodipropanol 
With 14 days 3.12 mg/dl 
3.44 3.68 3.72 3.68 
methionine (78%) (86) (92) (93) (92) 
in concen- 
tration of 
28 days 3.00 3.28 3.48 3.52 3.52 
4 mg/dl (75) (82) (87) (88) (88) 
With 14 days 9.96 mg/dl 
10.80 
11.28 
11.28 11.52 
methionine (83%) (90) (94) (94) (96) 
in concen- 
tration of 
28 days 9.72 10.44 
10.92 
10.82 11.16 
12 mg/dl (81) (87) (91) (90) (93) 
______________________________________ 
As can be seen from Table 3, when more than 30 mM of one of the 
antioxidants is added, the residual ratio of L-methionine in the blood 
filter paper is more than 80% and, therefore, by the addition of that 
amount of one of the antioxidants, the preservation effect for the object 
of the present invention can be sufficiently attained.

The present invention will now be explained more specifically by referring 
to the following examples of embodiments thereof. 
EXAMPLE 1 
6 l of the waste blood (with Hematocrit Value being 42%) of healthy persons 
was centrifuged at 2000 rpm for 20 minutes. From the centrifuged blood, 
402 ml of the supernatant solution, blood plasma, was removed by suction, 
in order to adjust the Hematocrit Value of the remaining blood solution to 
be 55%. 
25 ml of .beta.-thiodiglycol with a purity of 98% was placed in a 5-l 
volumetric flask. To the .beta.-thiodiglycol was added the above-mentioned 
blood solution to yield exactly 5 l of a mixture of the blood and 
.beta.-thiodiglycol. In the blood mixture, the concentration of 
.beta.-thiodiglycol was 49 mM/l. 
The concentration of L-methionine in the blood mixture was measured by an 
automatic amino acid detector to be 0.25 mg/dl. 
Seven L-methionine solutions with different concentrations of L-methionine 
were prepared and one ml of each solution was placed in each of seven 
100-ml volumetric flasks. Into each flask was placed the above-mentioned 
blood solution containing the antioxidant in such a manner as to yield 
exactly mixtures of 100 ml with the total concentrations of L-methionine 
being 1 mg/dl, 2 mg/dl, 4 mg/dl, 8 mg/dl, 12 mg/dl, 16 mg/dl and 20 mg/dl. 
Each of the mixtures was softly stirred and was then allowed to stand for 2 
hours at room temperature in order to disperse the L-methionine 
homogenously throughout the mixture. 
50 .mu.l of each of the mixture was dropped on pieces of the filter paper 
for PKU diagnosis manufactured by Toyo Filter Paper Co., Ltd., in such a 
manner that the dropped mixture spread in a circle with a diameter of 11 
mm. 
Each filter paper was dried at room temperature for about 2 hours and was 
then dried in a vacuum dryer and finally laminated. 
The thus prepared filter papers were preserved for 6 months and one year at 
-20.degree. C. and were compared with the conventional filter papers in 
terms of the growth circle of bacillus subtilis in the Guthrie's Bacterial 
Inhibition Assay. 
The results are shown in the following table: 
______________________________________ 
Con- Immediate- 
Preserva- 
Preserva- 
centration Antioxi- ly after tion for 
tion for 
of Methionine 
dant production 
6 months 
one year 
______________________________________ 
4 Conven- 0 25.2 mm 23.7 (+) 
20.5 (+) 
mg/dl tional 
product 
Product of 
.beta.-thiodi- 
25.5 mm 25.0 25.0 
the present 
glycol 
invention 49 mM 
12 Conven- 0 35.0 mm 32.5 30.0 (+) 
mg/dl tional 
product 
Product of 
.beta.-thiodi- 
35.0 mm 34.8 35.0 
the present 
glycol 
invention 49 mM 
______________________________________ 
(Note) 
(+) indicates the formation of an inhibition circle. 
EXAMPLE 2 
7.0 ml of .beta.-thiodipropanol was placed in a 1-l volumetric flask and 
the blood solution with the Hematocrit Value of 55%, prepared in Example 
1, was added to the .beta.-thiodipropanol to yield exactly 1 l of the 
mixture. The concentration of the antioxidant, .beta.-thiodipropanol, was 
50 mM in the mixture. Exactly in the same manner as in Example 1, a 
predetermined different amount of L-methionine was added to each blood 
solution to form blood mixtures with different concentrations of 
L-methionine and each mixture was dropped on each piece of the previously 
mentioned filter paper for PKU diagnosis. 
Each filter paper was dried to prepare a reference blood filter paper. 
The thus prepared filter papers were preserved for 6 months and one year at 
-20.degree. C. and were compared with the conventional filter papers in 
terms of the growth circle of bacillus subtilis in the Guthrie's Bacterial 
Inhibition Assay. 
The results are shown in the following table. 
______________________________________ 
Con- Immediate- 
Preserva- 
Preserva- 
centration Antioxi- ly after tion for 
tion for 
of Methionine 
dant production 
6 months 
one year 
______________________________________ 
4 Conven- 0 25.0 mm 23.7 mm 
21.0 mm 
mg/dl tional 
product 
Product of 
.beta.-thiodi- 
25.0 24.8 25.0 
the present 
propanol 
invention 50 mM 
12 Conven- 0 35.0 32.5 30.0 
mg/dl tional 
product 
Product of 
.beta.-thiodi- 
35.0 35.2 34.8 
the present 
propanol 
invention 50 mM 
______________________________________ 
(Note) 
Figures in the above table indicate the diameter of the growth circle of 
bacillus subtilis. subtillis.