US 2509140 A
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Patented May 23, 1950 Alfred H; "Free. Elkhai-tj 11141., assignor -to--Miles- Laboratories; :Inc., Elkhart, Ind., a. corporation of Indiana;-
No Drawing, ApplicationMarch 2,1948,
Serial No. 12,1699
The present invention relates to new-and inn proved diagnostiv compositions usefuliorr the."
qualitative detection and quantitativeestimation' of: ketonebo'dies in body fluids, particularly:
acetone bodies .inth'e urine.
More specifically, the invention-relates todie agnostic compositions. in dry-: form; preferably tabletted 511111) suitable; sized tablets; which com;-
positions can .readilybeaused even .byr-unskilled:
persons rapidly to .detect' the presence rof -ade'e tone" in .urine. without evolvement of ammonia;.
and without the use ofyequipment or apparatus:
other than some meansrfor'obtalning' a: drop. or-
droxy-butyric acid. Under normal circumstances no significant quantity of these ketone. substances appears in the urine: Howevewif.
there. is an excessive metabolism. offat; the-intermediate acetone bodies accumulate" inthe blood and are excretedrinthe urineinzvariable amounts. In diabetes mellitus; such an: excessive fat. metabolism occurs and.manyofsthe symp toms of the-disease can. be ascribed?tothe-toxic-.-
effects of the acetone bodies.- The medicalqpram titioner is well aware of the usef-ulnessiofxtests-r for acetonebodiesin theurinein diabetes-.; mel-.-.
litus.. Acetone bodies'also occurtin the urine in other Well. recognized disturbances of -metabolism, and in such cases also it is important to carry out tests for these substances.
In the past a varietyof reagents and techniques. have been used for the demonstration. of acetone'bodies in urine.
of asoluble .ni'troprussidev asa reactive ingredi ent or agent. Inonemodificati'o'n the nitr prusside reaction is carriedout'in the presence of ammonia in order to develop particular colorations. (See Patent'No. 2;186,902 to Fortune.) Careful study of availablereagents andctechniques for: detecting acetone indicates that theyaall are subject to onc or :more' of the :followin gr drawbacks or disadvantages:
1. A plurality of reagents arerequiredvfor most/rtests.
2. Most: testsrequire the skill of a: trained.-
3: Reagents :employed for most tests are unstable, poisonous and inconvenient to use;
A number of such" reagents. andv techniques have involved the use.
6 Claims: '(Cl. 252 -4403) 4. "Tests-accordingto" most techniques can only satisfactorily be performed in a laboratory.
5. Powder or dry reagent preparations-which contain an ammonia yielding ingredient are inherently unstable and-liberate ammonia and change" color .even when stored. in air-tight containe'rs.
6. Most dry. preparations evolvev noxious ammonia'duringthe performance of'the tests.
'7. Most 1 dry reagent preparations give a test with 'negativeurines which. maybe confusedwith a positive ,even' by a skilled operator."
The object of the present invention, generallystated; is theprovision. of diagnostic compositions: in stabledry 'io'rm; preferably tablets, which can be. used even-by an unskilled person conveniently and readily 'to give an accurate qualitative test for, anda quantitative estimation' of," the presence'of 'acetone bodies in .urine, which test clearly distinguishes between posi tive and negative specimens.
An. important object ofithe invention is the provision. of a "drwdiagnostic composition for the detection of acetonebodies "in urine-whichdoes not evolve ammonia either: in the dry state or "in-'the'wet'state; Certain otherobj'ects of the invention will, in-part,-be-obvious' and-wilt in part appear hereinafter;
Fora more complete understanding of thenature and scope of the invention. reference'may now be had to the followi'ngdetai leddescriptime thereofsettirig forth; by way of illustration; certain specific formulations which i constitute the presently' preferredembodiments of the in vention;
According to the present invention itnasbeen found that a solublenitro r-usside in. the ros ence of an aliphatic amino acid under alkaline: conditions; provides a diagnostid composition which is particularly adapted for the detection of acetone inurine: This diagnostic or zreagent composition: may beformulate'd in dry form; most suitabl lastablets.
Wneniac'etone positive specimens are tested" -withi this-diagnostic..compositiomia very markedi and definlte purple. reaction: or coloration-is prod'u-c'ed colorationqisxhighly characteristic and specific to acetone-positive specimens;
An important property-ref: the diagnostic compositions-isgthe-ifact that: acetone negative specie positions of the present invention should be made within one minute of the time that the powder or tablet is moistened with the specimen under examination.
The aliphatic amino acid of choice for the diagnostic compositions of the present invention is glycine (i. e. glycocol or amino acetic acid). This compound gives a. high quality coloration and has no blank color. Other aliphaticamino acids which give bright colors and which may be used in the diagnostic compositions are: alanine, glutamic acid, arginine, aspartic acid and lysine.
As mentioned above, alkaline conditions are required for testin specimens for acetone bodies in accordance with the invention. The a1- kaline material of choice for providing such alkalinity is disodium phosphate. With a dry powder or tablet preparation it is possible to use other alkaline solids such as carbonates, hy-
droxides, trisodium phosphate, dipotassium phos-. Instead of using a sepa-.
phate and the like. rate ingredient for imparting alkalinity, it is possible to use the alkali metal salts of the amino acids such as sodium or potassium glycinate. However, for practical purposes, disodium phosphate is the substance of choice since it is readily obtained as an anhydrous salt and its action in the test appears to be superior to that of a number of the other alkaline materials which can be used.
Any soluble nitroprusside appears to be useful in the reagent composition. Thus, any one of the alkali metal nitroprussides can be used. How-.
ever, sodium nitroprusside is the one of choice since it gives excellent results and is readily available on the chemical market.
It is possible, and often desirable, to use diluents along with the active constituents of the diagnostic compositions. Such diluents which have been satisfactorily used include starch, lactose, the borates, specifically sodium borate,
and a variety of other white or colorless com carry out a test since they are easily handled and may be conveniently disposed of.
However, the diagnostic compositions may, if desired, be used in powder or granular form and because of their stability in aqueous solution, the
compositions may even be used in liquid form.
The proportions of the amino acid, soluble nitroprusside and alkaline material, are not critical within broad limits, and these ingredients.
4 may be employed within a rather wide range of proportions. However, as the proportions of the ingredients in the diagnostic formulations are varied, it has been noted that the properties of the formulations are altered in the following respects:
1. The speed of reaction of the reagent with acetone positive urines.
2. The sensitivity of the reagent in detecting :small quantities of acetone bodies.
3. The presence or absence of a blank color with acetone negative urines. 4. The stability of the reagent when stored under adverse environmental conditions.
5. The quality and quantity of color obtained with urines having given concentrations of acei-tone bodies.
When sodium nitroprusside is used as the soluble nitroprusside in the diagnostic composition,
this ingredient should not comprise over approximately 5% by weight of the total bulk of the mixture and should represent at least 0.04% of the total bulk thereof. The preferred range of concentration is from 0.5% to 1%. Other soluble nitroprussides are preferably used in equivalent concentrations.
The relative proportions of disodium ph-os-' phate and glycine, or their equivalents, in the reagent compositions can vary quite widely and still the reagent will give satisfactory results with both acetone positive and acetone negative urines.
For instance, mixtures where the amount of' glycine was four times that of the sodium phos phate (i. e. circa glycine) gave clear cut tests. On the other hand, good tests were also obtained when the amount of glycine was only about 1% of that of the disodium phosphate. The optimum range of concentrations of the three preferred components is as follows in per cent by weight:
g Per cent Sodium nitroprusside 0.04- 5 Glycine 1.00-80 Disodium phosphate 20.00-99 When diluents are employed, they do not serve to'afiect the above preferred concentrations of the components except that the sodium nitroprusside or its equivalent should represent at least 0.04% of the total bulk. It will also be understood that equivalent components may be substituted in equivalent concentrations.
The two presently preferred embodiments of the invention are formulated as follows:
Example 1 50 grams of disodium phosphate, anhydrous,
50 grams of sodium borate, 2.5 gramsof glycine, and 0.8 gram of sodium nitroprusside are mixed together to give complete uniformity and this.-
mixture tabletted into tablets weighing approximately 0.3 gram each. Each of the tablets canbe used as a reagent in a test for acetone bodies in urine.
The preferred technique for testingspecimens for acetone using the diagnostic compositions of the present invention involves placing a tablet, or an equivalent pile of powder, on a flat surface and moistening the tablet or powder deposite with one drop of the urine specimen under examination. Within less than five seconds, acetone-containing urines cause the tablet or powder to acquire a characteristic type of purple or violet color, the intensity or shade of the color being proportional to the concentration of acetone bodies present. With negative urine samples the tablet or powder either does not undergo any color change or may assume a pale yellow or brown color which is easily diiferentiated from the characteristic purple color of the positive specimen.
Diagnostic compositions made in accordance with the present invention have been used clinically to study a series of acetone-containing urines from hospital patients as well as normal urines from healthy individuals. In all instances the urines were examined and checked by previously established methods as well as with the new reagent. The results of these clinical studies clearly showed that the diagnostic compositions of the present invention were particularly suited for use in detecting ketonuria and also for estimating the amount of ketone bodies in the urine. The clinical study emphasized the fact that the reagent formulations of the present invention were very reliable and that there was no difiiculty occasioned due to the confusion of the results of tests with negative urines and with positive urines.
The diagnostic compositions of the present invention have a number of advantages which increase their Value as reagents for testing specimens for the presence of acetone. Thus, the combination of disodium phosphate and glycine, or the equivalents thereof suggested above, comprises an extremely well buffered system that is unafiected by the reaction of the urine being tested. When the tests are made with the diagnostic compositions, n0 objectionable fumes of ammonia or other noxious gas are evolved as in the case with dry reagent compositions of the prior art. This is an important feature since in many instances, a technician will be testin a large number of specimens at the same time, often in a small room without adequate ventilation.
The combination of disodium phosphate and glycine, or their equivalents, is not reactive and is, accordingly, very stable. The anhydrous sodium phosphate is capable of taking up small amounts of water without deterioration so that the combination remains fully reactive even under adverse conditions of humidity,
Having thus fully described the invention and set forth formulations representing the preferred embodiments thereof, what is claimed as new is:
1. A diagnostic composition in solid dry form for detecting acetone bodies in urine without evolving ammonia, consisting essentially of, from 0.04-5.0% of sodium nitroprusside, from 1-80% of glycine, and from 20-99% of disodium phosphate.
2. A diagnostic composition in solid dry form for detecting acetone bodies in urine without evolving ammonia, consisting essentially of, approximately 5 parts of glycine, approximately 20 parts of disodium phosphate, and approximately 0.2 of a part of sodium nitroprusside.
3. A diagnostic composition in solid dry form for detecting acetone bodies in urine without evolving ammonia, consisting essentially of, approximately 2.5 parts of glycine, approximately parts of disodium phosphate, approximately 0.8 part of sodium nitroprusside, and approximately 50 parts of sodium borate.
4. Diagnostic tables tabletted from the composition called for in claim 3.
5. A diagnostic composition in solid dry form for detecting acetone bodies in urine without evolving ammonia, consisting essentially of from 0.04-5.0% of water soluble nitroprusside, from 1-80% of an aliphatic amino acid, and from 20-99% of an alkaline material.
6. A diagnostic composition in solid dry form for detecting acetone bodies in urine without evolving ammonia, consisting essentially of from 0.045.0% of an alkali metal nitroprusside, from 1-80% of an aliphatic amino acid, and from 20-99% of an alkaline alkali metal phosphate.
ALFRED H. FREE.
REFERENCES CITED The following references are of record in the file of this patent:
UNITED STATES PATENTS Number Name Date 2,171,962 Fortune Sept. 5, 1939 2,186,902 Fortune Jan. 9, 1940 2,283,262 Kamlet May 19, 1942 2,362,478 Galat Nov. 14, 1944 OTHER REFERENCES Gregory: Uses and Applications of Chemicals and Related Materials, vol. I, Reinhold Pub. 00., N. Y. C. (1939), page 112, column 2.
Kolthoff et al.: Acid-Base Indicators, The Mac- Millan 00., N. Y. (1937), pages 247-252 inclusive.