US 3542649 A
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Nov. 24, 1970 I s cY 3,542,649
COLOR DEVELOPING COMPOSITION 'CONSISTING OF AN, ENZYME, A SALICYLATE AND A HYPOCHLORI'IE DONOR Filed. April 7; 1967 2 Sheets-Sheet 1 SAMPLE o a I PLATE 0 o TUBING GLASS COIL ML/MIN 91A RECORDER \5 MM F/c FLOWCELL COLORIMETER FIG. I
Nov. 24, 1970 R. L. SEARCY 3,542,649
COLOR DEVELOPING COMPOSITION CONSISTING OF AN ENZYME, A
SALICYLATE AND A HYPOCHLORITE DONOR Filed April .7, 1967 2 Sheets-Sheet. 2
0 'I I I I I I I 0 20 40 so 80 I00 I20 I40 I60 UREA NITROGEN CONCENTRATION (mg./lO0mI.)
United States Patent Ofice Patented Nov. 24, 1970 US. Cl. 195-1035 3 Claims ABSTRACT OF THE DISCLOSURE The present invention relates to an automated system for the an alytical determination of urea nitrogen in blood serum, plasma or in urine and to a color developing composition useful in the system consisting of an enzyme, a salicylate, preferably an alkali metal salicylate, and a hypochlorite donor, preferably sodium or potassium dichloroisocyanurate.
BACKGROUND OF THE INVENTION The determination of urea in blood serum, plasma or urine has been accomplished by several techniques. In one such prior art technique, serum, plasma or diluted urine is incubated with buffered urease for conversion of urea to ammonia. The ammonia is then measured by colorimetry which is based upon the reaction of the ammonia and reagents such as sodium phenate and hydrochlorite to form a colored complex. The intensity of the color developed is proportional to the ammonia concentration. This procedure has been proposed for use in automated analyzers.
Other methods which are routinely employed for the determination of urea in biological fluids are also available. One of the more commonly used methods involves hydrolizing urea to ammonia carbonate by means of the enzyme urease in the presence of a buffered solution. Ammonia is liberated from the carbonate salt by the addition of sodium borate and then distilled into 0.05 N hydrochloric acid. The amount of nitrogen present is then determined colorimetrically after nesslerization. This procedure is described in detail in the Manual of Clinical Laboratory Methods, Fourth Edition by Opal Hepler, Publisher, Charles C. Thomas, Springfield, Ill.
Another commonly employed method relies upon the amount of yellow pigment formed by condensing diacetylmonoxime with urea in acid solution in a filtrate of the fluid which is protein free. This procedureis currently utilized in automated analytical techniques. A description of diacetylmonoxime methodology appears in Marsh et a1, Amer. J. Clin. Path 28, 681, 1957.
More recently, it has been found that the determination of urea in biological fluids can be manually accomplished utilizing a color developing composition comprising sodium salicylate, sodium nitroprusside and sodium dichloroisocyanurate.
All of the prior art techniques suflered from certain defects. For example, the last-mentioned method while suitable for manual procedures did not develop color of suitable intensity to admit of its use in automated analyzers. Additionally, sodium nitroprusside is sensitive to atmosphere and could conceivably form toxic HCN. Other of the techniques are concentration dependent and require a large sample size. Furthermore, time is also a critical factor in the use of some of such prior art procedures and hence, extreme caution is required by the user if results are to be meaningful. Also, certain of the reagents utilized in prior art techniques were found to be unstable, further limiting the usefulness of the procedure which employs same.
It is an object of this invention to provide an automated system for determining urea nitrogen in biological fluids and materials usable in the system which do not suffer from any such defects.
BRIEF SUMMARY The present invention relates to a reagent combination for the automatic analysis of urea nitrogen in a specimen of a biological fluid which consists essentially of mixing the specimen with an amount of enzyme which reacts with the urea in the biological fluid to liberate ammonia, dialyzing the so-formed solution against a salt of a salicylate and developing color by adding a hypichlorite donating agent, to the recipient stream of the dialysis.
BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a schematic flow diagram illustrating a novel automated system for automatically analyzing urea nitrogen in biological fluids utilizing the novel color developing combination of the prsent invention.
FIG. 2 is a recording of the photometric response obtained when utilizing the automated system of FIG. 1.
FIG. 3 is a plot in terms of optical density of the photometric responses illustrated in FIG. 2.
DETAILED DESCRIPTION The present invention in detail relates to a procedure for the chemical determination of urea nitrogen in bio logical fluids such as blood serum, plasma or urine.
In FIG. 1, an automated testing system is shown schematically wherein a specimen sample to be tested which may be oxalated blood or urine is drawn up in sequence from separate depressions in the sample plate which rotates at a constant speed to provide the system with 60 specimen samples per hour. A sample, so drawn, is mixed with an enzyme, preferably urease, which reacts with urea in biological fluids to liberate ammonia. The mixture is passed through a glass incubation coil of conventional design maintained at 37 C. After the mixture is passed through the glass incubation coil, it is next pumped into a dialyzer modual that is provided at its entrance with a small cellophane membrane or the like for dialysis on the opposite side of the membrane against the salicylate color reagent, preferably, an alkali metal salicylate, most advantageously, sodium salicylate. The residual, non-diffusable portion of the sample-urease mixture is discarded. The hypochlorite donating reagent, (preferably, an alkaline solution of an alkali salt of dichloroisocyanurate, most preferably sodium dichloroisocyanurate) is added to the recipent stream of dialysate. After mixing the resulting cbmbination in a suitable mixer, color developed at 37 during transit through an incubation coil. Photometric measurements are then performed at 660 mg. in a 15 mm. floWcell colorimeter, i.e. the absorbance of the solution to be tested is measured at 660 millimicrons in a Klett-Summerson colorimeter using a red filter (No. 660) against a reagent blank. The results obtained are recorded on a conventional recording mechanism.
The system illustrated in FIG. 1 aspirates at a rate of 60 specimens/hour. The rate of flow in ml./min. of the materials entering the system according to a preferred technique is illustrated in FIG. 1. The materials entering the system are pumped into it by any suitable pumping means adjusted to maintain the rate of flow illustrated in FIG. 1. The mechanism for the system of the present invention can be conveniently provided by a modification in accordance with the system illustrated in FIG. 1 of the manifold of the Technicon Auto Analyzer which uses the diacetylmonoxime procedure for determining urea nitrogen in biological fluids and the system disclosed in US. Pat. No. 2,879,141 issued Mar. 24, 1959.
FIG. 2 is a representative recording of the results obtained with the automated system depicted in FIG. 1 utilizing sample solutions containing urea nitrogen ranging in concentrations of from 5 to 150 mg. per 100 ml.
When these results are plotted in terms of optical density, they exhibit a linear relationship to urea nitrogen levels at the concentrations examined as shown in FIG. 3.
In an alternate procedure, aliquots of ammonia-free distilled water are placed between each specimen on the sample plate illustrated in FIG. 1 to preclude cross contamination.
Any enzyme which is capable of reacting with urea to liberate ammonia is suitable for the purposes of the present invention. Most preferred, however, is urease. Urease preparations suitablt'for the purposes of the present invention are readily available commercially in states of greater or lesser purity. The preferred urease preparations to be employed with the automated technique which constitutes the presitnt invention (as illustrated in FIG. 1) have an activity of from about 0.8 to about 1.0 Sumner units per mg. as measured at 30 C. When employed in the method of the invention, the enzyme urease is put in solution. The solution is buffered to a pH which favors enzyme activity and stability utilizing conventional buffers such as monopotassium dihydrogen phosphate, disodium monohydrogen phosphate and ethylenediamine tetraacetic acid and salts thereof. Particularly advantageous results are realized when a chelating agent such as an alkali metal salt of ethylene diamine tetraacetic acid, preferably, disodium ethylene diamine tetraacetate, is employed as the buffer.
A particular preferred buffered urease solution suitable for the purposes of the present invention is obtained by dissolving grams of disodium ethylene diaminetetraacetate and 200 mg. of urease (activity equal to 0.8 modified Sumner units per mg.) in about 750 ml. of ammoniafree distilled water. The resulting mixture is adjusted to pH 6.5 with a small amount of 2.5 N sodium hydroxide and the volume brought to 1 liter with additional deionized water. The enzyme solution is ready for use in the automated system illustrated in FIG. 1. The so-prepared enzyme solution, if desired, may be stored indefi nitely at C.
A preferred salicylate color reagent utilized in the mechanism depicted schematically in FIG. 1 is prepared by dissolving 170 grams of sodium salicylate in 1 liter of ammonia-free distilled water. This reagent can be used immediately in the system illustrated in FIG. 1. Alternatively, it can be stored for later use. It is found stable for many months when stored in glass at room temperature.
While sodium salicylate is indicated above as being the most advantageous salicylate salt for the purpose of the present invention, the use of other salicylate salts capable of effecting the desired end with equal efiicacy are contemplated. Thus, for the purposes of the present invention, there can be utilized 4-amino salicylate, phenyl salicylate, calcium salicylate, zinc salicylate, strontium salicylate, copper salicylate, lithium salicylate, magnesium salicylate, ammonium salicylate and salicylaldoxime. With the exception of phenyl salicylate and salicylaldoxime, a solution containing any of the above, can be prepared by the same procedure described above in connection with preparation of the most advantageous salicylate color reagent containing sodium salicylate. However, since phenyl salicylate and salicylaldoxime are not particularly soluble in water, when such are utilized, they are dissolved in 1 liter of 1.0 N sodium hydroxide, rather than 1 liter of ammonia-free distilled water as described above in connection with the preparation of the sodium salicylate color reagent.
The preferred hypochlorite donor utilized in the system schematically illustrated in FIG. 1 is prepared by dissolving 5.0 gm. of sodium dichloroisocyanurate in 1 liter of 0.6 N sodium hydroxide. The so-prepared alkaline donor medium can be used immediately or alternatively, it can be stored for use at some subsequent time. The donor medium resists deterioration when stored in glass at refrigerator temperature, i.e., 5 C.
Representative of other salts of dichloro isocyanurates equally efficacious for the purposes of the present invention is the potassium salt of dichloroisocyanurate.
In alkaline solutions, the salt of dichloroisocyanurate dissociates to produce hypochlorite ions and, is therefore, functioning as the hypochlorite donor. The developed color is due to the propensity of ammonia to react with the hypochlorite ions to form an intermediate which, when a salicylate is present, forms a chromatic substance. The chromatic substance obtained when utilizing sodium salicylate in the system illustrated schematically in FIG. 1 takes a brilliant emerald green color.
Utilizing other salicylates, chromogenic responses obtained are as follows:
REACTIVITY OF VARIOUS SALICYLATES WITH AMMONIA Compound: Chromogenic response:
Amino salicylate green Phenyl salicylate blue Calcium salicylate green Zinc salicylate green Strontium salicylate green Copper salicylate green Lithium salicylate green Magnesium salicylate blue green Ammonium salicylate green salicylaldoxime brownish green In testing for the presence of urea nitrogen, the invention described herein will be employed in the following manner. First, the enzyme, e.g. the buffered urease solution prepared as described above and the sample are pumped into the system illustrated schematically in FIG. 1 whereby a mixture of the two is obtained. The resultant mixture is then incubated, after which it flows into the salicylate color reagent entering the system. The so-prepared combination then flows along the system to a point whereat the hypochlorite donating agent meets therewith. An immediate color formation occurs. The optical density developed by the so-formed mixture is measured by the use of an accurate measuring instrument such as spectrophotometer or colorimeter which measures the transmission of light or other suitable radiation through the resultant medium. By the application of Beers law, the concentration of the color compound present can be determined. From a comparison of this concentration to a standard solution, the original concentration of urea present is calculated and recorded. A particular salient feature of the use of the salicylate and hypochlorite donating agent exclusively as described above is the rapidity in which the color formation occurs. Thus, by the novel color forming composition of the present invention, a technique is provided which is eminently well-suited for an automated system and yet avoids the problems inherent in prior art automated systems designed to efifect this end.
The reaction with urea nitrogen according to the present invention is highly sensitive, specific and reproducible.
What is invented is a particularly efiicacious method for measuring urea nitrogen automatically in biological fluids by the simple technique disclosed. In its use, there is avoided materials which may be caustic to equipment and hence, cause corrosion problems. Furthermore, the use of nitroprusside is avoided. Under certain conditions, this substance can form HCN, a gas which is highly toxic and dangerous. Thus, the use of a substance which could conceivably effect the health and well-being of the technicians utilizing the mechanism illustrated in FIG. 1 is avoided. Furthermore, if nitroprusside were to be utilized in the automated technique schematically illustrated in FIG. 1, the system is no longer suited for testing rapidly many samples of specimen. Thus, the auto mated urea nitrogen technique utilizing only a salicylate and a hypochlorite donating reagent has been unexpectedly found to utilize reagents that are neither hazardous or dangerous to equipment. Additionally, the discard resulting from the operation of the system illustrated schematically above is nonacidic in nature. Hence, waste materials may be easily disposed of.
Several distinct advantages over available automated methods for urea nitrogen analysis are provided by the new technique. The diacetyl monoxime procedure, for example, entails the use of a hot mixture of concentrated sulfuric and phosphoric acids. This solution is not only hazardous to personnel, but it is also highly corrosive and presents a serious disposal problem. Furthermore, the results obtained with this technique are not linear and hence, Beers law is not satisfied.
Also, there have been advocated for automated urea nitrogen assay the use of phenate-hypochlorite reagent. This mixture adversely afiects the polyvinyl chloride (Tygon) transmission tubing conventionally used in such system as well as the methyl methacrylate (Lucite) dialyzer plates also conventionally utilized in the system. Therefore, glass tubing must be used from the point of entry of phenatehypochlorite reagent into the recipient stream to the position where the mixture leaves the colorimeter.
To summarize briefly, the present invention relates to a diagnostic test for the detection of urea nitrogen in biological solutions which is highly accurate and entails the use of reagents that are neither hazardous nor damaging to instrumentation.
1. A method for the automatic analysis of urea nitrogen in a specimen of a biological fluid which consisting essentially of adding the specimen to an amount of urease which reacts with urea nitrogen in said biological fluid to liberate ammonia, dialyzing the so-formed solution against a salt of a salicylate and developing color by adding a hypochlorite donating agent to the recipient stream of the dialysate.
2. A method for the automatic analysis of the urea nitrogen in a specimen of a biological fluid as defined in claim 1 wherein the salt of a salicylate utilized is an alkali metal salt thereof and the hypochlorite donating agent utilized is an alkali metal salt of dichloroisocyanurate in an alkaline medium.
3. A method for the automatic analysis of urea nitrogen in a specimen of a biological fluid as defined in claim 1 wherein the salt of a salicylate utilized is sodium salicylate and the hypochlorite donating agent utilized is sodium dichloroisocyanurate in an alkaline medium.
References Cited UNITED STATES PATENTS 2,797,149 6/1957 Skeggs ..195--103.5 X 2,879,141 3/1959 Skeggs 23-253 3,432,395 3/1969 Reardon 103.5
ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 195-127