Method, composition, and device for determining the specific gravity of a liquid

A method, composition, and device for determining the specific gravity of a liquid. In the method, a sample of the liquid to be tested is contacted with standardized quantities of substances which participate in a reaction that is affected by the level of solute in said liquid. The resulting effect of the solute on the reaction is a reproducible function of the concentration or amount of solute in the sample and relates to the specific gravity of the liquid tested. A unitized test composition is provided comprising at least one substance which participates in the standardized reaction. The standardized reaction is preferably chemical in nature and, in such a case, the test composition preferably includes an indicator responsive to a product of the chemical reaction.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to methods, compositions, and devices for 
determining the specific gravity of a liquid. In particular, this 
invention relates to means for determining the specific gravity of an 
aqueous liquid. The present invention finds application in the 
determination of the specific gravity of body fluids such as urine. 
Specific gravity is defined as the ratio of the density of a substance to 
that of a standard substance. Where the substance whose specific gravity 
is to be determined is in a liquid form, the standard substance generally 
is water. In the context of this disclosure, a liquid whose specific 
gravity is to be determined shall be defined as a mixture of substances 
whose aggregate is in a homogenous liquid state. The substance in the 
liquid that is present in the greatest amount shall be defined as the 
solvent or solvent component with the remaining substance or substances 
being defined as the solute. 
Specific gravity determinations are used in the fields of science, 
medicine, and industry for a wide variety of purposes. For example, it is 
often quite useful to ascertain the specific gravity of an unknown liquid 
to aid in identification thereof. The determination of the specific 
gravity of body fluids, particularly urine, is a part of nearly all 
routine urinalyses. Urine consists of water as solvent and various 
dissolved solids as solute. Only a minor portion, if any, of the solute 
component of urine consists of liquid substances. Thus, the specific 
gravity of urine indicates the relative proportions of dissolved solid 
components to the total volume of the specimen tested and therefore 
reflects the relative degree of concentration or dilution of the specimen. 
Under appropriate and standardized conditions of fluid restriction or 
increased uptake, the specific gravity of a urine specimen measures the 
concentrating and diluting abilities of the kidney. 
Normal urinary specific gravity ranges from 1.003 to 1.035, but usually 
remains between 1.010 and 1.025. Specific gravities below 1.010 can be 
indicative of diabetes insipidus, a disease caused by the absence of, or 
impairment to, the normal functioning of the antidiuretic hormone. Low 
specific gravity may also occur in patients with glomerulonephritis, 
pyelonephritis, and various renal anomalies. Specific gravity is high in 
patients with diabetes mellitus, adrenal insufficiency, hepatic disease, 
and congestive cardiac failure. Therefore, urinary specific gravity 
determinations are useful in routine urinalysis as a screening procedure 
for detecting potentially abnormal clinical conditions. 
2. Description of the Prior Art 
By its definition, the most straight forward method for determining the 
specific gravity of a liquid is to determine the ratio of the weight of a 
given volume of the liquid to the weight of the same volume of water under 
standard conditions. Such a method, however, requires precise volumetric 
and gravimetric techniques. More often, the specific gravity of liquids is 
determined using pycnometers or gravitometers. Such instruments have as 
their principle the fact that, if two manometers containing liquids of 
different densities are connected to a common source of suction, the 
heights of the liquids are inversely proportional to their densities, and 
hence their specific gravities. 
In the clinical laboratory, urinary specific gravity is determined in many 
ways. The specific gravity of urine is usually determined with a 
urinometer. A urinometer is a weighted bulb-shaped device having a 
cylindrical stem which contains a scale calibrated in units of specific 
gravity. The device is floated in a cylinder containing the urine. The 
depth to which the device sinks in the urine indicates the specific 
gravity of the urine and is read on the scale at the surface of the urine. 
The urinometer is sensitive to temperature requiring an adjustment of 
0.001 units for each 3.degree. C. difference between the calibrated 
temperature of the device and the temperature of the urine specimen. 
All of the methods and devices mentioned hereinabove for determining the 
specific gravity of a liquid require a relatively large sample volume in 
order to conduct the test. The need for a several milliliter volume of 
sample sometimes requires that an additional urine specimen must be 
obtained from the patient, thereby destroying the continuity of routine 
urinalysis. It is most desirable to obtain all of the routine urinalysis 
results from a single sample in order that the relationships between the 
test results may be properly analyzed. 
Through the use of a refractometer, urinary specific gravity determinations 
may be carried out using as little as one drop of urine. Since in urine 
the solute consists essentially of only dissolved solids, the refractive 
index of urine closely correlates with its specific gravity. Small hand 
refractometers are available specifically designed for determining urinary 
specific gravity. 
Many other diverse techniques are available for determining the specific 
gravity of a liquid. All of the known techniques require a bulky 
instrument or device of one sort or another which has to be consistently 
cleaned, maintained, and adjusted in order to produce reliable results. 
All such techniques also are based strictly on physical measurements such 
as measurements of volume, weight, height, and refractive index. 
Routine urinalysis, as practiced at the present time, involves three basic 
areas of investigation: a determination of the presence or absence of 
substances such as glucose, protein, occult blood, ketones, and so forth; 
a determination of specific gravity; and a microscopic examination of the 
urinary sediment. The first area of investigation usually involves the 
testing of the urine specimen with indicator papers or strips comprising 
reagent pads responsive to the urinary constituents to be determined. 
Indicator strips, usually in the form of single strips carrying multiple 
reagent pads responsive to the different urinary constituents to be 
determined, are dipped momentarily into the urine specimens, and the 
resulting color responses are compared to a color chart. Under present 
technology, separate analytical steps must be undertaken to determine 
urinary specific gravity and to microscopically examine the urinary 
sediment. 
SUMMARY OF THE INVENTION 
A new means for determining the specific gravity of a liquid has now been 
discovered. By the new means of the present invention specific gravity can 
be determined without requiring elaborate physical measurements or 
instrumental hardware. A method, test composition and device are provided 
which yield a response, preferably a colorimetric response, related to the 
specific gravity of a liquid contacted with the composition or device. One 
form of the device of the present invention is that of a reagent pad of 
the type used in the prior art for determining urinary constituents. Thus, 
such a reagent pad for determining urinary specific gravity may be readily 
included on an indicator strip which carries multiple reagent pads 
responsive to the different urinary constituents of interest. A multiple 
reagent strip therefore can be made which provides a means for determining 
urinary constituents and specific gravity simultaneously, thereby 
eliminating the additional analytical steps required in the prior art for 
determining urinary specific gravity. 
The present invention is based on the observation that specific 
standardized reactions which occur in the liquid whose specific gravity is 
to be determined, are affected by the level of solute present. 
Specifically, the present method comprises contacting the liquid to be 
tested with at least one substance capable of producing, on said contact, 
a predetermined reaction when the liquid contains a known amount of 
solute, and also capable of producing, on said contact, an aberration of 
the reaction when the liquid contains a different amount of solute. The 
specific gravity of the liquid may then be determined by the extent of any 
resulting aberration of the reaction.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The types of reactions contemplated by the present invention include those 
which involve a chemical or physical interaction which occurs, usually on 
a molecular level, in a liquid medium of the type whose specific gravity 
is to be determined and which is affected by the amount or concentration 
of solute in the liquid medium. Chemical reactions are preferred, since 
means for evaluating aberrations of chemical reactions are readily 
available. Usually, the liquid to be tested is contacted with the 
components of the standardized reaction and is additionally contacted with 
an indicator responsive to any aberration in the reaction which may occur 
due to the level of solute in the liquid. 
The indicator may be responsive to either physical or chemical aberrations 
and hence may function in a spectrophotometric, potentiometric, 
manometric, thermal, radiological, or turbidometric manner. Preferably the 
indicator consists primarily of one or more chemical reagents which 
produce a colorimetric response either to the presence or absence of the 
aberration in the standardized test reaction. In this way, the effect on 
the standardized test reaction of the presence of solute in the liquid to 
be tested may be determined either by a visual or instrumental observation 
of the indicator response. 
Normally, the results observed in a test run are compared to those observed 
in standard runs, and an aberration from standard results provides a 
measure of the specific gravity of the test liquid. The results of the 
standard runs are usually in the form of a standard graph plot or, where 
the preferred colorimetric indicators are used, a standard color chart. 
The comparison that is made between the aberration, if any, produced in 
the test run and the standard results can be accomplished automatically by 
instrument means, for example, where a single instrument observes the 
aberration and internally compares it to the standard, thereby yielding an 
output in units of specific gravity. Due to its convenient nature, the 
present invention is particularly suited to an uncomplicated test strip 
format. In one aspect, the present invention provides a "dip-and-read" 
type test device which yields a colorimetric response related to specific 
gravity within a less than two minute period of time after momentary 
immersion in the liquid to be tested. 
Of the chemical reactions which are preferably utilized according to the 
present invention, those whose rate is affected, either enhanced or 
inhibited by the solute, are particularly useful. Usually the rate of the 
chemical reaction is inhibited as the level of solute increases. Also, the 
chemical reaction preferably is one that produces a product. Any 
aberration in the reaction is readily determinable by monitoring the rate 
of disappearance of a reactant or the rate of appearance of a product. The 
rate of appearance of a product is usually more readily determinable 
particularly where an indicator for the product is present during the 
reaction. By choosing an indicator which yields a colorimetric response to 
the presence or absence of a product of the reaction, any effect that the 
presence of the test liquid has on the reaction results in an effect on 
the color change. When the aberration of the reaction due to the presence 
of solute in the test liquid is an effect on the rate of the reaction, 
test liquids having different specific gravities will produce different 
rates of color change. Thus, in such a case, the actual color change that 
occurs after a predetermined period of time is related to the specific 
gravity of the liquid tested. 
Numerous chemical reactions have been found to be affected by the level of 
solute in a liquid medium in which they occur. Various chemical reactions 
of this type are pointed out in the examples which follow. Particularly 
preferred are those chemical reactions which may be catalyzed by an 
enzyme. The specific activity of enzymes has been found to be especially 
sensitive to the level of environmental solute, particularly dissolved 
solids. Exemplary of enzymatic reactions which may be used in accordance 
with the present invention are those which involve the action of enzymes 
which are classified as transferases, hydrolases, lyases, isomerases, and 
ligases. Particularly useful are those reactions catalyzed by the 
oxidoreductases, especially those which catalyze the oxidation of a 
carbohydrate such as a hexose. 
Of the reactions which are catalyzed by an oxidoreductase, those involving 
the action of an oxidase have been found to yield particularly 
reproducible and distinct aberrations related to specific gravity. Oxidase 
reactions which yield a peroxide, particularly hydrogen peroxide, as a 
product are preferred, since indicators responsive to peroxide are well 
known in the art. Exemplary of oxidase reactions are those which involve 
the enzymatic oxidation of glycollate, malate, cholesterol, aryl alcohol, 
gulonolactone, pyranose, pyridoxin, alcohol, catechol, hydroxyacids, 
hypoxanthine, xanthine, glycine, L-amino acid, D-amino acid, uric acid, 
luciferin, aspartic acid, lactic acid, and various mono- and di-amines, 
and aliphatic and aromatic aldehydes. Particularly preferred are the 
hexose oxidase reactions such as those involving the oxidation of glucose, 
galactose, or sorbose. 
Indicators which are particularly suited to detecting the presence of 
peroxides, such as hydrogen peroxide, in liquids comprise a substance 
having peroxidative activity and a chromogen which is oxidized in the 
presence of peroxide and the substance having peroxidative activity to 
yield a spectrophotometric response, usually in the visible range. Such 
chromogens include those which are oxidation-reduction types having a 
potential appropriate to detecting the particular peroxide in the presence 
of the substance having peroxidative activity. Such chromogens thus 
include, water soluble iodide salts, o-tolidine, syringaldazine, 
vanillinazine, the combination of phenol and 4-aminoantipyrine, 
2,7-diaminofluorene, benzidine and derivatives thereof such as 
o-dianisidine. Substances having peroxidative activity comprise such 
naturally occurring peroxidases as horseradish peroxidase and potato 
peroxidase. Other substances having peroxidative activity include 
materials such as normal whole blood, red blood cells alone, lyophilized 
whole blood, urohemin, metalloporphyrins, and so forth. Certain inorganic 
compounds, such as the combination of iodide and molybdate salts, may also 
be used as an indicator. 
The basic first step of the method of the present invention comprises 
contacting the liquid to be tested with the substance or substances 
necessary for the reaction that is affected by the level of solute in said 
liquid. Thus, in one aspect, such first step involves the combination or 
intermixing of the test liquid with the necessary reaction constituents. 
It is important to standardize the reaction within certain parameter 
ranges in order to be sufficiently confident that any significant 
aberration of the reaction due to the presence of solute in the test 
liquid is a function essentially only of the level of solute present. It 
is usually sufficient to standardize the amounts or concentrations of the 
reaction constituents, i.e. reactants, catalysts, and so forth, and the 
environmental reaction conditions, such as temperature and pressure. The 
critical allowable ranges for such reaction parameters will of course vary 
from one type of reaction to another. The essence of the present invention 
in this regard is strictly empirical. 
Standardization of the environmental reaction conditions is usually a 
relatively simple matter, since, for the sake of convenience, the reaction 
is normally conducted under room conditions. The slight variations in 
temperature and pressure that may occur under room conditions usually have 
little, if any, effect on reactions selected for use in the present 
invention. The degree of control of the amounts or concentrations of the 
reaction constituents required for standardization purposes depends upon 
the sensitivity of the specific gravity-induced aberration produced in a 
particular reaction. For instance, where varying the amount of a 
particular reaction constituent does not significantly affect the specific 
gravity-induced aberration, precise control of the amount of such 
constituent used would not be required. Such a situation may exist where 
the chosen reaction is one whose rate is affected by specific gravity and 
which involves the action of a catalyst which may be present in widely 
varying amounts or concentrations without affecting the reaction rate. In 
such a situation, control of the quantity of catalyst present would not be 
critical. In other circumstances, the amount of catalyst present may 
affect reaction rate and would, therefore, be subject to critical 
limitations. 
Some of the reactions which are sensitive to specific gravity in the manner 
of the present invention involve reaction constituents which are normally 
present in relatively constant amounts or concentrations in the atmosphere 
or in the liquid to be tested. One substance which is present in the 
liquid to be tested at a relatively fixed concentration is the solvent 
portion thereof. It is contemplated that a particular reaction selected 
for use in the present invention could include the solvent as a reaction 
constituent. If the level of solvent is critical to the standardization of 
the reaction, the volume of sample tested should be predetermined, thereby 
standardizing the level of solvent present. 
Substances which are present in the atmosphere in relatively constant 
amounts and which may participate in a reaction selected in accordance 
with the present invention are the atmospheric gases, particularly oxygen. 
Many of the reactions which are contemplated by the present invention 
require atmospheric oxygen as a reaction constituent. This is particularly 
true of the most preferred reactions, namely, those involving the 
enzymatic oxidation of a carbohydrate. Where a reaction of this type is 
involved, such an atmospheric reaction constituent is automatically 
standardized by carrying out the reaction under ordinary room conditions. 
Therefore, the first basic step of the present method generally comprises 
contacting the liquid to be tested with standardized quantities of all of 
the substances which participate in the chosen specific gravity-affected 
reaction. Where one or more of the reaction constituents is present in the 
atmosphere, i.e., atmospheric reaction constituents, such a step may be 
accomplished by contacting the liquid to be tested with predetermined 
quantities of all of the non-atmospheric reaction constituents under 
standardized atmospheric conditions. 
The test composition provided by the present invention thus comprises at 
least one substance, and usually at least two substances, capable of 
producing, upon contacting the liquid to be tested, a predetermined 
reaction when the liquid contains a known amount of solute, and also 
capable of producing, upon said contact, an aberration of said reaction 
when the liquid contains a different amount of solute. Additionally, the 
test composition preferably comprises indicator means responsive to the 
aberration as described previously herein. Where the chosen reaction is 
catalyzed by an enzyme, the test composition preferably includes the 
enzyme. Where one or more of the reaction constituents is provided in a 
standardized quantity by the atmosphere, the test composition preferably 
comprises only the non-atmospheric reaction constituents. 
It should be noted that it is necessary to maintain the various reaction 
constituents in a non-reactive relationship at least until the time of 
contact thereof with the liquid to be tested. Usually the test composition 
is so constituted that the reaction constituents are brought into reactive 
relationship upon contact with the liquid to be tested. Normally the 
chosen reaction is one which occurs in a liquid of the type to be tested 
but which does not occur to any significant degree in a dry state or in 
certain other liquids. Thus, the test composition, prior to use, may be in 
a dry form, such as a powder, or may be in the form of a solution or 
suspension in which the reaction does not occur to a significant degree. 
The reaction constituents comprising the test composition may also be kept 
separated, to be combined at the time of use. 
To provide a useful test device according to the present invention, the 
test composition is usually incorporated with one or more carrier members. 
Suitable carrier members are usually constructed of material which is 
relatively inert with respect to the reaction constituents, porous and/or 
absorbent relative to the liquid to be tested. Such carrier members 
include bibulous paper; polymeric matrixes in the form of films, 
membranes, fleeces or the like, and so forth. The test composition or any 
portion thereof may be incorporated with a particular carrier member by 
impregnating the carrier member with a solution or dispersion of the 
composition, followed by drying; by coating the composition on the carrier 
member; by physically entrapping the composition within the carrier 
member; by chemically or physically bonding the composition to the carrier 
member; and so forth. A carrier member incorporated with the test 
composition or a portion thereof may be attached or otherwise associated 
with a base member or support as will be described more fully hereinafter. 
Useful base members or supports are usually in a strip form and may be 
constructed of plastic, paper, wood, metal foil, or the like. 
With reference to the drawing, FIG. 1 shows a test device 10 comprising an 
elongated rectangular strip forming a base member 11 to which member is 
attached a liquid absorbent carrier member 12. In one form of test device 
10, the base member is formed of transparent organoplastic material and 
the carrier member 12 is formed of bibulous paper incorporated with at 
least one, but less than all, of the non-atmospheric substances which 
participate in a reaction selected for use in the present invention. In 
use, the remaining non-atmospheric substances which participate in the 
selected reaction and not incorporated with carrier member 12 are added to 
a sample of the liquid to be tested, and the carrier member 12 is 
momentarily dipped into or otherwise contacted with the resulting mixture, 
allowing the selected reaction to take place. Any resulting aberration of 
the reaction is detected by indicator means incorporated with carrier 
member 12 or added to the liquid test sample either before or after 
contact of carrier member 12 therewith. Depending upon whether the 
indicator means is incorporated with carrier member 12 or is added to the 
test sample, the indicator response may be observed either on carrier 
member 12 or in the liquid test sample after removal of carrier member 12 
therefrom. Preferably, colorimetric indicator means are incorporated with 
carrier member 12 so that the resulting colorimetric response may be 
observed on or in carrier member 12 after removal from the test sample. 
In a second and preferred form of test device 10, all of the 
non-atmospheric reaction constituents, together with indicator means; are 
incorporated in carrier member 12. In use of this second form of test 
device 10, carrier member 12 is momentarily dipped into or otherwise 
contacted with a sample of the liquid to be tested, thereby initiating the 
reaction. Upon removal of carrier member 12 from the test sample and after 
any incubation or response-development period which may be required, any 
resulting aberration is detectable on carrier member 12. When a 
colorimetric indicator is used as the indicator means, the preferred form 
of test device 10 provides a convenient "dip-and-read" type test device 
for determining the specific gravity of a test liquid. Certain precautions 
must be taken in order to insure that the reaction constituents 
incorporated with carrier member 12 do not react with each other to any 
significant degree before contact with the test liquid. This may be 
accomplished by various methods. One method is to impregnate carrier 
member 12 with a solution or suspension of the reaction constituents which 
consists of a solvent or liquid in which the selected reaction will not 
occur. A second method is to impregnate carrier member 12 with a solution 
or suspension of the reaction constituents, at least one of which is 
dispersed or encapulated in a polymeric material which is soluble in the 
test liquid but which is insoluble in the solution used to impregnate 
carrier member 12. A third method is to impregnate carrier member 12 with 
a first solution containing at least one, but less than all, of the 
reaction constituents, drying carrier member 12, and then impregnating 
carrier member 12 with at least one additional solution which contains the 
remainder of the reaction constituents and in which the reaction is 
incapable of occurring to a significant degree. In each of the three cases 
described, carrier member 12 is subjected to a final drying step and 
attached to base member 11. 
Another form of a test device of the present invention is shown in FIG. 2. 
A test device 20 comprises an elongated strip base member 21 to which are 
attached carrier members 22 and 23 which are similar to carrier member 12 
in device 10. Base member 21 is formed with a transversely extending area 
of reduced cross-section 24 providing an integral flexible hinge allowing 
the portion of base member 21 bearing carrier member 23 to be bent or 
folded at the hinge portion 24 and relative to the remaining portion of 
base member 21 such that the major exposed, i.e. upper, surfaces of 
carrier members 22 and 23 may be brought into contact. In one form of test 
device 20, one of the carrier members 22 and 23 is incorporated with at 
least one, but not all, of the reaction constituents, the remainder of the 
reaction constituents being incorporated with the other of said carrier 
member. Either or both of carrier members 22 and 23 may also be 
incorporated with appropriate indicator means. In use, one or both of 
carrier members 22 and 23 are momentarily dipped into or otherwise 
contacted with the test liquid and thereafter are brought into contact 
with one another as previously described by folding at the hinge portion 
24 to allow the selected reaction to occur on such contact. In another 
form of the test device 20, carrier member 23 is incorporated with some or 
all of the reaction constituents in a manner described previously relative 
to test device 10, and a colorimetric indicator composition is 
incorporated with carrier member 22. Any test constituents not 
incorporated with carrier member 23 are either added to the test sample or 
incorporated with the carrier member 22. In use, carrier member 23 is 
momentarily dipped into or otherwise contacted with the test liquid, and 
thereafter carrier members 22 and 23 are brought into contact with one 
another by folding at the hinge portion 24 to provide an indicator 
response on either carrier member 22 or on both of carrier members 22 and 
23. 
FIG. 3 shows test device 30 comprising an elongated strip base member 31 
and two distinct carrier members 32 and 33 attached thereto and in 
edgewise abutting contact with one another. In one form of test device 30, 
one of the carrier members 31 and 32 is incorporated with some, but not 
all, of the reaction constituents, the remainder being incorporated with 
the other of said carrier members. In use, the end portion of carrier 
member 33 which is remote from carrier member 32 is dipped into or 
otherwise contacted with the test liquid. Carrier member 33 is formed of 
material of sufficient absorbency or capillarity to promote the movement 
of the test fluid longitudinally through carrier member 33 and into 
carrier member 32. The test fluid carries the reaction constituents from 
carrier member 33 into carrier member 32 for reaction with the 
constituents in carrier member 32. Either or both of carrier members 32 
and 33 may also be incorporated with an appropriate indicator means. In 
another form of test device 30, carrier member 32 is incorporated with all 
of the reaction constituents, and preferably also with an indicator 
composition in the manner described with respect to the test device 10. 
Carrier member 33 may be incorporated with an indicator composition and 
may comprise materials for removing interfering substances from the test 
liquid, either by chemical or physical means, as it flows through carrier 
member 33 toward carrier member 32. Some interfering materials, as will be 
discussed more fully below, may be eliminated or rendered harmless by mere 
exposure to the atmosphere as the test fluid flows through carrier member 
33, in which case carrier member 33 may comprise only absorbent material. 
A test device 40 is shown in FIG. 4 which comprises an elongated 
transparent strip base member 41 and carrier members 42 and 43 attached 
thereto in laminate relation by means of a thin transparent plastic sheet 
44 overlaying said carrier members and attached to base member 41 at 
opposite ends of the laminate structure comprised of carrier members 42 
and 43. Carrier members 42 and 43 are incorporated with the reaction 
constituents, and preferably an indicator means, in the same manner as 
carrier members 22 and 23 in test device 20. In use, carrier members 42 
and 43 are momentarily dipped into or otherwise contacted with the test 
liquid, and the indicator response is observed on carrier member 43 
through the sheet 44 or on carrier member 42 through base member 41. 
The present invention is particularly suited for the determination of 
specific gravity of liquids which have solutes consisting essentially of 
dissolved solids only. It has been found that for the purposes of the 
present invention the total dissolved solids content of a test liquid 
reproducibly affects a selected reaction of the present invention 
substantially independent of the proportion of the individual substances 
making up the total level of said dissolved solids. The test liquid is 
generally aqueous and usually comprises water as its solvent component. 
Thus, the present invention is particularly useful in determining urinary 
specific gravity. 
In testing the specific gravity of urine with test compositions and devices 
of the present invention, certain interfering reactions may affect the 
preferred colorimetric indicator compositions. Such reactions are usually 
due to abnormally high levels of substances which cause certain indicator 
compositions to yield an erroneous response. Such interfering reactions 
may be eliminated either by using an indicator composition which is not 
significantly affected by the presence of substances which cause erroneous 
results with other indicator compositions, or by pre-treating the urine 
sample so as to remove or render harmless such interfering substance. Such 
pre-treatment may be accomplished by adding appropriate reagents to the 
urine sample before contact with the test composition or by the use of a 
test device as shown in FIG. 3 incorporated with such appropriate 
reagents. Some of the preferred colorimetric indicator materials 
responsive to peroxide may be interfered with where the urine sample 
contains ascorbic acid levels exceeding about 5 mg./100 ml. Such ascorbate 
interference may be avoided by using an alternate indicator material that 
is not affected substantially by the presence of ascorbic acid, for 
example, a tribromophenol-aminoantipyrine indicator as disclosed in Clin. 
Chem. 19:522(1973). Alternatively, the urine sample may be pre-treated 
with a weak oxidizing agent in order to eliminate ascorbic acid 
interference. A test device as in FIG. 3 may be so constructed that prior 
to reaction, the urine sample must pass through the carrier member 33 
which is fabricated so as to prevent ascorbic acid from reaching the test 
composition in carrier member 32. This may be accomplished by forming 
carrier member 33 of a material which retards the capillary flow of 
ascorbic acid therethrough. For this purpose ion exchange papers may be 
utilized. Also, the carrier member 33 may be constructed of highly fibrous 
material such as filter paper to allow aerobic oxidation of ascorbic acid 
to occur as it travels through said carrier member. Materials which 
chemically alter ascorbic acid may also be incorporated in the carrier 
member 33. For example, the carrier member 33 may contain a weak oxidizing 
agent or an ascorbic acid metabolizing system, such as an ascorbic acid 
oxidase system. It is important to note at this point that interfering 
substances of the type discussed herein affect only the indicator means 
and not the selected specific gravity-affected reaction, and as such can 
be eliminated by properly coordinating the indicator means and the type of 
liquid to be tested. However, interference of the selected reaction caused 
by the presence of solute in the test liquid is indispensable to the 
present invention. 
It will be apparent from the foregoing that the present invention provides 
a new means for determining specific gravity which by virtue of its 
uncomplicated nature fulfills a long-felt need, particularly in the field 
of clinical medicine. 
The present invention will now be illustrated, but is not intended to be 
limited, by the following examples. 
EXAMPLE 1 
In this example a composition and device of the present invention was 
prepared and used in testing urine samples having different specific 
gravities. 
a. Preparation of test devices 
An aqueous solution having the following proportions of ingredients was 
prepared as follows: 
______________________________________ 
galactose oxidase 13,091 Worthington units.sup.1 
peroxidase (2500 units/mg.) 
16.4 mg. 
bovine albumin 1.09 g. 
o-tolidine . 2HCl 0.108 g. 
sodium phosphate, monobasic 
0.181 g. 
sodium phosphate, dibasic 
0.552 g. 
Elvanol 51-05.sup.2 
1.09 g. 
75% Aerosol OT.sup.3 
0.07 g. 
water 59.4 ml. 
______________________________________ 
.sup.1 Worthington Biochemical Corp., Freehold, New Jersey 
.sup.2 thickening agent from E.I. duPont de Nemours & Co., Wilmington, 
Delaware 
.sup.3 wetting agent from American Cyanamide Co., Wayne, New Jersey 
A sheet of SA2 ion exchange paper from Whatman, Inc., Cliffton, N.J. was 
immersed in the resultant solution and dried at 90.degree. C. for about 10 
minutes. Approximately 5 mm. square carrier sections or pads of the dry 
reagent-impregnated sheet were then attached to the surface of one end of 
5 mm. by 85 mm. plastic strips using double-faced adhesive tape, thereby 
providing test devices as shown in FIG. 1. 
b. Test method 
The reagent pad portions of the test devices so made yielded a green color 
when momentarily immersed in an aqueous solution containing galactose. The 
color of an unreacted pad after rehydration by immersion in distilled 
water was cream. A color chart was constructed consisting of four 
different colors to which were assigned values of 0, 4, 8, and 12 
respectively. The color having a value of 0 was that of a rehydrated, 
unreacted test strip pad. Green shades were assigned values 4, 8, and 12 
with the deeper shades having the higher values. 
Twenty-nine (29) random urine specimens having ascorbate levels below 5 
mg./100 ml. were collected and their specific gravities measured using a 
TS meter (B-5996) available from American Optical Co., Buffalo, N.Y. To 10 
ml. volumes of each of the specimens were added 0.7 ml. of a 10% w/v 
galactose solution. The reagent pad portions of twenty-nine (29) of the 
previously prepared test devices were each momentarily dipped into a 
separate one of the galactose-treated urine samples. The color response 
produced after one minute was assigned an integer value between 0 and 12 
based on a comparison with the color chart. The results appear in 
graphical form in FIG. 5 of the drawing. 
It was thus demonstrated that the present invention provides a convenient, 
useful method for distinguishing high (&gt;1.020), medium (1.010-1.020), and 
low (&lt;1.010) levels of specific gravity in urine. 
EXAMPLE 2 
Example 1 was repeated except that after 45 seconds from the time of 
contact between each urine sample and the respective test device pad the 
color response produced was measured in an Eyetone.RTM. reflectance meter 
available from Ames Company Division of Miles Laboratories, Inc., Elkhart, 
Indiana. This instrument has maximum activity at 580 nm. The results 
appear in graphical form in FIG. 6 of the drawing. 
It was thus demonstrated that the rate of color response produced by the 
standarized enzymatic galactose oxidation reaction is functionally related 
to the specific gravity of the test liquid. 
EXAMPLE 3 
In this example the effect of three major contributors to urinary specific 
gravity on a standardized chemical reaction was assessed. 
Test devices were prepared as in procedure a. of Example 1. Four (4) sodium 
biphosphate, four (4) sodium chloride, and six (6) urea aqueous solutions 
having various specific gravities were prepared. To 10 ml. volume samples 
of the prepared solutions were added 0.1 ml. of a 10% w/v galactose 
solution. A separate one of the reagent pad portions of the previously 
prepared test devices was momentarily dipped into each of the 
galactose-treated solutions. The sample-inoculated pads were placed in an 
Eyetone.RTM. reflectance meter, and the time (in seconds) required for the 
color reaction to reach a reading of 100 units was recorded. The results 
appear in tabular form below and in graphical form in FIG. 7 of the 
drawing. 
______________________________________ 
specific grams added 
specific time (seconds) 
gravity to 100 ml. gravity to reach 
contributor 
water (T.S. meter) 
100 reading 
______________________________________ 
phosphate 
1 1.003 19 19 
(NaH.sub.2 PO.sub.4) 
2 1.007 38 39 
3 1.011 76 80 
4 1.015 150 143 
chloride 1 1.004 17 16 
(NaCl) 
2 1.009 28 29 
3 1.014 45 45 
4 1.018 85 95 
urea 1 1.003 13 14 
2 1.007 21 21 
3 1.011 30 30 
4 1.015 44 45 
5 1.019 58 55 
6 1.023 95 85 
______________________________________ 
It was thus demonstrated that major contributors to urinary specific 
gravity affect the standardized chemical reaction in a very similar 
manner, the effects produced by the two predominant contributors, chloride 
ion and urea, being essentially the same. 
EXAMPLE 4 
In this example test devices as in FIG. 1 were prepared and used in testing 
liquid samples having different specific gravities. 
The test devices were prepared as in procedure a. of Example 1 except that 
the dry reagent-impregnated sheet of ion exchange paper was immersed in a 
0.5% w/v solution of galactose in pyridine and air dried for about 10 
minutes, after which it was cut into squares which were attached to 
plastic strips. The resultant test devices yielded color responses related 
to specific gravity upon contact with aqueous test samples which did not 
contain galactose. The test samples comprised two containing aqueous 
solutions of sodium biphosphate at 3% and 6% w/w concentrations 
respectively, two containing sodium chloride at 3% and 6% w/w 
concentrations respectively, and two containing urea at 3% and 6% w/w 
concentrations respectively. When contacted with the reagent pad portions 
of test devices as prepared in this example, each of the phosphate, 
chloride, and urea solutions produced a color response in the reagent pad 
portion contacted therewith. It was observed that in each case the 3% w/w 
solution produced a color response more rapidly than the 6% w/w solution. 
EXAMPLE 5 
In this example test devices as in FIG. 3 were prepared and used in testing 
the specific gravity of urine having a high ascorbate level. 
Reagent-impregnated pads about 5 mm. square were prepared as in procedure 
a. of Example 1 and attached to plastic base members as the pad 32 in FIG. 
3. Strips of E&D 204 paper available from Eaton-Dikeman Co., Mount Holly 
Springs, Pa. were immersed in an aqueous solution containing 500 mg./100 
ml. galactose and allowed to dry. The dry galactose-impregnated strips 
were cut into approximately 5 mm. by 20 mm. pads and attached to the 
aforementioned base members as the pad 33 shown in FIG. 3. 
These test devices were used in testing the specific gravity of urines 
having ascorbate levels above 5 mg./100 ml. and specific gravities of 
1.006, 1.012 and 1.022 respectively. In testing the urine, the outer end 
portion of the pad 33 of each test device was dipped into the urine. After 
a short time, the sample flowed through the pad 33 and into the pad 32 of 
the test device whereupon a color response was produced in the pad 32. One 
minute after contacting a urine sample having a specific gravity of 1.022 
the reagent pad portion 32 yielded a greenish color, whereas a dark 
blue-green color was produced in pad 32 when testing a urine sample having 
a specific gravity of 1.012. 
One minute after contacting the pad 33 of a test device with a urine sample 
treated with galactose as in Example 1, having a specific gravity of 1.006 
and containing 15 mg./100 ml. of ascorbate, the reagent pad portion 32 
yielded a blue-green color. When the same urine sample treated with 
galactose was contacted by the pad 12 of the test device prepared as in 
Example 1, no color response was produced because of ascorbate 
interference. 
It was thus demonstrated that the form of the invention shown in FIG. 3 
permits the determination of specific gravity without interference from 
high ascorbate levels in urine samples tested therewith. 
EXAMPLE 6 
In this example test devices as in FIG. 4 were prepared and used in testing 
the specific gravity of urine. 
A dry reagent-impregnated sheet of SA2 ion exchange paper was prepared 
according to the procedure a. of Example 1. Strips of E&D 204 paper were 
immersed in an aqueous solution containing 500 mg/100 ml. galactose and 
allowed to dry. Both the reagent-impregnated sheet and the 
galactose-impregnated strips were cut into approximately 5 mm. by 5 mm. 
sections or pads. Test devices were prepared by placing one pad of each 
type in a laminate fashion onto the surface of one end of a 5 mm. by 85 
mm. plastic base strip. The laminate structure of FIG. 4 was produced by 
overlaying the superimposed pads with a piece of transparent plastic 
adhesive tape which was pressed onto the base strip as shown in FIG. 4. 
When the laminated pad structure of test devices thus prepared were 
momentarily dipped into urine samples having a specific gravity of 1.020, 
a green color response was observed in the laminated pads whereas a dark 
blue-green color was produced in said pads were urine samples having a 
specific gravity of 1.005 were tested therewith. 
EXAMPLE 7 
In this example test devices as in FIG. 1 were prepared and used in testing 
urine samples having different specific gravities, the selected 
standardized chemical reaction involved being the enzymatic oxidation of 
glucose. 
Test devices comprising reagent pad portions 12 as in FIG. 1 were 
impregnated with glucose oxidase, peroxidase, and a chromogen 
substantially as described in U.S. Pat. No. 3,453,180. Four (4) test 
solutions were prepared as follows. The first test solution was water 
containing 0.1% w/v glucose. The other three test solutions were urine 
samples which has specific gravities of 1.003, 1.011, and 1.027 
respectively and which were adjusted to contain 0.1% w/v glucose. The 
reagent pad portions of four (4) of the test devices were each momentarily 
dipped into a separate one of the prepared test solutions. The color 
responses produced after 10 seconds were as follows: 
______________________________________ 
specific gravity color 
of test solution response 
______________________________________ 
1.000 deep purple 
1.003 deep purple 
1.011 purple 
1.027 pale reddish-purple 
______________________________________ 
It was thus demonstrated that various specific gravity levels in an aqueous 
liquid can be distinguished using test devices in accordance with the 
present invention in which the selected standarized reaction involves the 
enzymatic oxidation of glucose. 
EXAMPLE 8 
In this example test devices as in FIG. 1 were prepared and used in testing 
urine samples having different specific gravities, the selected 
standardized chemical reaction involved being non-enzymatic, color-forming 
reaction sensitive to nitrite. 
Solutions a. and b. were prepared as follows: 
______________________________________ 
Solution a. 
p-arsanilic acid 0.13 g. 
Gantrez AN.sup.1 0.5 g. 
sodium lauroyl sarcosinate 
0.25 g. 
methanol 75 ml. 
water 25 ml. 
______________________________________ 
.sup.1 an equimolar copolymer of methylvinyl ether and maleic anhydride 
available from General Aniline and Film Corp., New York, New York 
______________________________________ 
Solution b. 
N-(1-naphthyl)ethylene- 
diamine . 2HCl 0.1 g. 
d-tartaric acid 1.0 g. 
polyvinylpyrrolidone 6.2 ml. 
Renex 698.sup.2 0.1 g. 
chloroform 75 ml. 
methanol 18.8 ml. 
______________________________________ 
.sup.2 an ethoxylated nonyl phenol-wetting agent available from Atlas 
Chemical Industries, Wilmington, Delaware 
A sheet of Whatman No. 17 filter paper from W. R. Balston, Ltd, Maidstone, 
Kent, England, was immersed in Solution a. and dried at 110.degree. C. for 
about 10 minutes. The filter paper sheet was then immersed in Solution b. 
and dried at 75.degree. C. for about 10 minutes. Approximately 5 mm. 
square sections or pads of the dry reagent-impregnated sheet were attached 
to the surface of one end of 5 mm. by 85 mm. plastic strips using 
double-faced adhesive tape, thereby providing test devices as shown in 
FIG. 1. 
Three (3) urine samples were obtained which had different specific 
gravities and ascorbate levels below 5 mg./100 ml. and which produced no 
observable color change upon contact with the reagent pads of the test 
devices thus prepared. Sodium nitrite was then added to each of the urine 
samples to a concentration of 0.5 mg/100 ml. The reagent pad portions of 
three (3) of the thus prepared test devices were each momentarily dipped 
into a separate one of the nitrite-treated samples. The color responses 
produced after one minute were as follows: 
______________________________________ 
specific gravity color 
of test solution response 
______________________________________ 
1.005 pink 
1.014 pale pink 
1.026 buff 
______________________________________ 
It was thus demonstrated that various specific gravity levels in an aqueous 
solution can be distinguished using test devices in accordance with the 
present invention in which the selected standardized reaction is 
non-enzymatic. 
EXAMPLE 9 
In this example test compositions were used in testing urine samples having 
different specific gravities, the selected standardized chemical reaction 
involved being a non-enzymatic, color-forming reaction sensitive to 
reducing sugars. 
Test tablets comprising cupric sulfate, sodium hydroxide, and an 
effervescent couple were prepared substantially as described in U.S. Pat. 
No. 2,387,244. Three (3) urine samples having different specific gravities 
were obtained and the reducing sugar content of each was adjusted to a 
concentration of 0.35 g./100 ml. Five (5) drops of each sample were placed 
in a separate test tube. Ten (10) drops of water and one tablet thus 
prepared were added to each test tube. The following color responses were 
observed 15 seconds after the foaming caused by the effervescent couple 
had ceased: 
______________________________________ 
specific gravity color 
of test solution response 
______________________________________ 
1.003 yellowish-green 
1.011 pea green 
1.027 green 
______________________________________ 
It was thus demonstrated that various specific gravity levels in an aqueous 
solution can be distinguished using test compositions in accordance with 
the present invention in which the selected standardized reaction is 
non-enzymatic.