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Патент USA US3063827

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United States Patent 0
3,%3,8l2
C6
Patented Nov. 13, 1962
1
'2
3,063,812
DETERMINATEUN 0F ALBUMEW IN LEQUIDS
Galen F. (Iollins, Ellrhart, Ind, assignor to Miles Labora
tories, Inc., Eiiriiart, Ind, a corporation of lndiana
No Drawing. Filed Apr. 2, 1957, Ser. No. 650,068
17 (Ilaims. ((11. 23-230)
This invention relates to diagnostic compositions and,
smaller proportions of dye may be used satisfactorily
although with the smaller proportions the color is weak
ened rendering the test correspondingly less sensitive,
and with greater proportions the excess of dye is for the
most part wasted.
Inasmuch as the function of the cellulose in the com
positions is to adsorb albumin on the surface of the tablet
where it may be readily detected during the test, I prefer
more particularly, to new and improved compositions for
to
use cellulose in such an amount that it accounts for
the detection and quantitative estimation of albumin in
10 at least 35% of the weight of the total composition. In
aqueous solutions such as urine.
general, I prefer the cellulose component of my composi
it is an object of the invention to provide improved
diagnostic compositions in convenient tablet form which
may be used with con?dence, even by unskilled persons,
to detect and give a reliable quantitative estimation of
albumin in urine and in other aqueous solutions.
A further object is to provide diagnostic compositions
for the determination of albumin in aqueous liquids which
may be easily prepared from readily available materials
tions to comprise from about 45% to about 55% by
weight of the tablet; however, greater or lower percentages
of cellulose powder may be used if desired, compositions
having as little as about 25% and as much as about 75%
by weight of cellulose based on the weight of the total
composition giving satisfactory results. Four indicators
which I have found to work very well in my improved
compositions
and which are preferred are brom phenol
which is capable of detecting as little as about 5 mg.
blue and brom cresol green, tetra brom phenol blue and
percent (50 parts per million) of albumin in urine, and 20 tetrabromphenolphthalein
ethyl ester. Another indica
which may be stored over long periods of time without
losing its sensitivity in testing for albumin.
Other objects and advantages of the invention will be
apparent from the following description thereof.
The basis of the present invention is the phenomenon
of “protein error” exhibited by certain indicators where
tor which ‘will react as desired, but which gives a weak
color during the test, is m(p~anilinophenylazo) benzene
sulfonic acid sodium salt.
The function of the acid reacting substance is to lower
the pH of the urine below the acid-to-basic color change
point of the indicator, which for brom phenol blue is
pH 3.0, for brom cresol green is pH 3.8, for tetra brom
go their characteristic acid-to-base color change at a
phenol
blue is pH 3.0, and for tetrabromphenolphthalein
lower pH value than that at which they would change 30
ethyl ester is pH 4.0‘. When urine is the liquid tested,
color in the absence of protein. That is to say, an indica
by in solutions containing protein such indicators under
tor which exhibits “protein error” will, in solution contain
ing protein, under certain conditions show its basic color
although the pH of the solution may actually be well be
low that at which the color change normally occurs, and
the extent to which the color of the solution is changed is
the acid forms a buffer with the alkalinity of the urine
which assists in maintaining the pH of the liquid below
the color change point of the dye.
Many solid acid reacting substances, including a large
group of organic acids and a number of inorganic acid
rea‘cting salts’, may be employed for this purpose, those
having an ionization constant between about 10-5 and
The mechanism by which the presence of protein enables
about 10%, preferably about lO—3, and which are either
certain dyes to exhibit their basic color in solutions having
a pH below the acid-to-base color change point is not 4.0 slightly soluble or slowly soluble in' water being preferred.
Suitable acid reacting substances are citric, tartaric, giy
clearly understood but it is known that protein is limited
collie, malic, fuma'ri'c, phthalic, malonic, ma'ndeli'c, glu
in its range and capacity for bringing about this effect.
taric, aconitic, mal‘eic,‘ benzoic, adipic, salicylic, gallic and
Speci?cally, this color-change of the indicator, due to the
gentisic acids, and- aluminum sulfate and aluminum chlo
presence of protein, may be eifected only if the solution
ride. Of these I prefer to use salicylic, fumaric or
being tested has a ?nal pH not substantially lower than
phthalic acid.
about 1.25 pH units below the pH at which the normal
an indication of the amount of protein in the solution.
color change of the indicator occurs.
in accordance with the invention, a diagnostic compo
The acid used‘ must not be too strong, i.e., it must not
have an ionization constant greater than 10"2 since such
sition for determining albumin in liquids comprises a
an acid would tend to‘ rapidly reduce the pH of the al
which exhibits “protein error,” the mixture being dis
The cellulose powder used in my compositions may be
manufactured from‘ different source materials such as
mixture of a major proportion of a solid acid reacting 50 bumin causing a rapid disappearance of the basic color
of the dye characteristic of the‘ “positive” test.
substance and a minor proportion of an indicator dye
persed throughout an inert carrier or matrix of ?nely
wood, cotton, etc’. I have found the‘ following speci?c
divided cellulose, and all of these ingredients being com
pressed into tablets of convenient size, e.g-., 7A5” in di 55 powdered cellulose products satisfactory for my purposes:
B quality coarse grade cellulose powder, B quality stand
ameter and weighing about .30 gram. As will be pointed
and‘ grade cellulose powder (both products manufactured
out in greater detail hereinafter, the test for albumin using
by W 82R BalstonLiLtd‘J‘, and alpha cellulose (a product
such a tablet requires only that a drop of the liquid speci
manufactured by Brown Company and sold under the
men to be tested, e.g.,~ urine, be placed on the tablet to
produce any color reaction due to the presence of albumin. 60 trade name of Solka Floc‘ BW-lOO')‘. The‘ products do
not necessarily need to be white'g‘howeven'the lighter the
If albumin is not present, the color of the tablet remains
the acid color of the indicator. However, if albumin is
present in the specimen it will be adsorbed by thecel
color, the‘ easier it' is to observe the color changes;
Example]
lulose and remain on the surface of the tablet where .it
contacts the dye which is present there and causes the 65
‘A, preferred
for
diagnostic compositions
dye to change color while the remaining liquid portion
comprises the following ingredients in the proportions
of the specimen is absorbed by the tablet.
indicated:
.
As would be expected, the proportion of dye in the
present compositions is very small, good results being
obtained using between about .02% and about .3%_ by 70
weight of dye based on the weight of tablet. This range
of proportions is not critical, however, and greater or
7
Parts by weight
Salicylic .acidug ____________________________ __ 600
Brom. phenol blue ___________________________ __
1
Powdered wood- cellulose ____________________ _'__ 500
Corn
starch_
' '
__
26
3,063,812
3
in liquids comprising a mixture of a major proportion of
a member of the group consisting of salicylic, fumaric and
phthalic acids and a minor proportion of an indicator
dye selected from the group consisting of brom phenol
blue, brom cresol green, tetra brom phenol blue, tetra
tablets of about 716” diameter, having a weight of about
.30 gram.
The cornstarch serves as a binder to assist in
tableting. If desired, other protein-free binders such as
sucrose and dextrose may be used instead of the corn
starch.
4
7. A diagnostic composition for determining albumin
The foregoing ingredients in the quantities given are
granulated, dried, intimately mixed and compressed into
bromphenolphthalein ethyl ester and m(p-anilinophenyl
,
azo) benzene sulfonic acid sodium salt, and a carrier for
Example 2
The following ingredients in the proportions given,
said mixture consisting essentially of ?nely divided cel
lulose, said ingredients being intimately mixed together
tableted as in Example 1, provide a modi?cation of the 10 and compressed in tablet form.
compositions of my invention:
8. A composition according to claim 7 wherein said
indicator is brom phenol blue.
9. A composition according to claim 7 wherein said
Parts by weight
Sodium citrate-2H2O ________________________ __
70
Citric acid anhydrous ________________________ .._ 180
‘Tetrabromphenolphthalein ethyl ester __________ _.. 0.1
15 indicator is brom cresol green.
10. A composition according to claim 7 wherein said
indicator is tetra brom phenol blue.
11. A composition according to claim 7 wherein said
Cellulose powder (whatman “B” quality) _______ __ 300
Sucrose
26
indicator is tetrabromophenolphthalein ethyl ester.
In testing a liquid specimen for albumin one drop of
12. A diagnostic composition for determining albumin
the specimen is placed on a tablet prepared as described 20
in liquids comprising a mixture of a major proportion of
above. After this drop is absorbed, two drops of Water
salicylic acid and a minor proportion of brom phenol
are placed on the tablet. If albumin is present in the
blue, and a carrier for said mixture consisting essentially
specimen it is adsorbed on the surface of the tablet while
of ?nely divided cellulose, said ingredients being inti
the liquid (e.g., urine) soaks into the mass of the tablet.
The two added drops of water wash all but the albumin 25 mately mixed together and compressed in tablet form.
13. A diagnostic composition for determining albumin
and reacted dye from the surface of the tablet so that
in liquids comprising about 600 parts by weight of
any color change due to the presence of albumin stands
salicylic acid, about one part by weight of brom phenol
out in detail. The presence of albumin is indicated by
blue, about 560 parts by Weight of cellulose powder and
the basic color of the indicator being observed on the
about 26 parts by weight of corn starch, said ingredients
surface of the tablet. In the absence of albumin, the
‘being intimately mixed together and compressed in tablet
color of the tablet does not change from the acidic color
form.
of the dye.
14. A diagnostic composition for determining albumin
I claim:
in liquids comprising about 70 parts by weight of sodium
1. A diagnostic composition comprising a mixture of
a major proportion of a solid organic acid and a minor 35 citrate dihydrate, about 180 parts by weight of anhydrous
proportion of an indicator dye which exhibits “protein
error,” said mixture being distributed throughout a matrix
of powdered cellulose compressed in tablet form.
2. A diagnostic composition for determining albumin
in liquids comprising a mixture of a major proportion 40
of an acid-reacting substance selected from the group con
sisting of solid organic acids and acidic salts and a minor
proportion of an indicator dye which exhibits “protein
error,” and a carrier for said mixture consisting essentially
of ?nely divided cellulose, said ingredients being inti
lmately mixed together and compressed in tablet form.
3. A diagnostic composition in accordance with claim
2 including a protein-free binder for said ingredients se
lected from the group consisting of corn starch, sucrose
and dextrose.
'
r
citric acid, about 0.1 part by weight of tetrabromphenol
phthalein ethyl ester, about 300 parts by weight of cellu
lose powder and about 26 parts by weight of sucrose,
said ingredients being intimately mixed together and com
pressed in tablet ‘form.
l5. In a method of determining the presence of albu
min in a liquid wherein an acid reacting substance and
an indicator dye exhibiting “protein error” serve as the
determining media, the improvement which comprises
adsorbing the albumin contained in the liquid on cellulose
in the presence of said determining media, and separat
ing the non-albumin constituents of the liquid from the
adsorbed albumin by washing the latter.
16. The method of determining the presence of albu
50 min in a liquid comprising placing a drop of the liquid
4. A diagnostic composition for determining albumin
in liquids comprising a mixture of a major proportion of
'on a tablet containing in intimate admixture: a major
proportion of an acid-reacting substance selected from the
a solid organic acid having an ionization constant between
about 10*5 and about 10-2 and a minor proportion of
an indicator dye which exhibits “protein error,” and a
a minor proportion of an indicator dye which exhibits
:“protein error,” and a carrier for said acid-reacting sub
carrier for mixture consisting essentially of ?nely divided
cellulose, said ingredients being intimately mixed together
and compressed in tablet form.
5. A diagnostic composition comprising an intimate
mixture, compressed in tablet form, of a solid acid react
ing substance having an ionization constant between about
10-5 and about 10-2, an indicator dye which exhibits
group consisting of solid organic acids and acidic salts,
stance and dye consisting of ?nely divided cellulose; and,
'after said drop is absorbed in said tablet, placing thereon
two drops of water, the presence of albumin being indi
cated by the display of the basic color of the indicator
on the surface of said tablet.
17. The method of determining the presence of albu
min in a liquid comprising adsorbing the albumin from a
drop of said liquid on a quantity of compressed powdered
“protein error,” and powdered cellulose, the dye being
cellulose in the presence of a protein determining medium
present in an'arnount between about .02% and about 3%
by weight, said powdered cellulose being present in an 65 consisting essentially of (a) a major proportion of an
acid-reacting substance selected from the group con
amount between about 25% and about 75% by weight
sisting of solid organic acids and acidic salts, and (b) a
based on the weight of the total composition. >
minor proportion of an indicator dye which exhibits “pro
56. A diagnostic composition comprising an intimate
tein error,” and separating the non-albumin constituents
mixture compressed in tablet form, of a solid acid reacting
substance having an ionization constant between about 70 of the liquid from the adsorbed albumin by washing the
10-5 and about 10*, an indicator dye which exhibits
latter with two drops of water, the presence of albumin
“protein‘error” and powdered cellulose, the dye being
being indicated by the display of the basic color of the
present in an amount between about .02% and about 3%
indicator on the surface of the cellulose.
by weight, said powdered cellulose forming at least 25%
of the weight of said composition.
75 .,
(References on following page)
5
8,068,812
6
References Cited in the ?le of this patent
UNITED STATES PATENTS
2,171,961
2,178,550
2,281,758
2,314,336
2,331,573
2,387,244
2,854,317
2,912,309
OTHER REFERENCES
Abstracts of Papers, 131st Meeting Am. Chem. Soc.,
Fortune ______________ __ Sept. 5, 1939
March 25, 1957, pages 750 and 76C.
Acree ________________ __ Nov. 7, 1939
Fiegl: Microchim. Acta, v01. 2, pages 107-110, 1937.
Kolmer et aL: “Approved Lab Technic,” 5th Ed.
1951, page 142.
Galat ________________ __ May 5, 1942
Goodale _____________ __ Mar. 23,
Sheftel _______________ __ Oct. 12,
Compton ____________ __ Oct. 23,
Free et a1 _____________ __ Sept. 30,
1943
1943
1945
1958
Free ________________ __ Nov. 10, 11959
Free: “Gastroenterology,” v01. 24, No. 3, July 3, 1953,
pages 414 et seq.
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