CA1039163A - Urea analysis - Google Patents

Abstract

UREA ANALYSIS
ABSTRACT OF THE DISCLOSURE
Disclosed is a method and apparatus for the determination of urea in an aqueous specimen such as blood or urine. The specimen is passed through a bed of immobilized urease to hydrolyze the urea to ammonium ion. The ammonium ion is then converted to ammonia by reaction with a base. The resulting ammonia is then selectively passed as a gas through a hydrophobic, ammonia permeable membrane for potentiometric detect-ion with a pH sensitive electrode.

Description

This invention relates to a method and apparatus for the determination and analysis of urea. More particularly, the present invention relates to the analysis of urea in physiological fluids such as blood and urine, and other aqueous specimens of medical and industrial interest.
There is a great need in the medical field today for a rapid and accurate analytical technique for determining blood urea nitrogen (B~). In the past, such analyses have been performed by cumbersome wet chemical methods, conductivity methods or spectrophotometric and colormetric techniques. While these methods are generally accurate, they are somewhat time consuming and can require careful interpretation.

7'~, More recently there has been research directed toward the development of the so called "enzyme electrode" for determining urea. Enzyme electrodes are made by immobilizing urease on the surface of a cation sensitive electrode. The enzyme electrode is then contacted with the urea specimen and the urea is converted to ammonium ions which are sensed by the electrode. While this system is suitable for some applications, the presence of other monovalent cations such as are present in physiological fluids, interferes with the electrode response.
Accordingly, the present invention overcomes these disadvantages of the prior art by providing an apparatus and method for analysis of urea by hydrolysis thereof with immobilized urease to ammonium ions, conversion of the resulting ammonium ions to ammonia by reaction with a base, permeation of the resulting ammonia through a hydrophobic membrane and into an electrolyte for potentiometric detection with a pH sensitive electrode.
Thus, in accordance with the present teachings, a method is provided for determining urea in an aqueous specimen which contains urea. The method comprises passing the specimen into a hydrolysis zone containing immobilized urease and retaining the specimen in the hydrolysis zone for a time sufficient to hydrolyze the urea to ammonium ions. The resulting hydrolysis mixture is removed from the hydrolysis zone and the pH of the mixture is raised to at least about 11 to convert substantially all of the ammonium ions to an aqueous ammonia solution. The aqueous ammonia solution is contacted with a hydrophobic, ammonia permeable membrane for a time sufficient to allow gaseous ammonia to permeate the membrane. The gaseous ammonia permeating the membrane is determined by dissolving the ammonia in an electrolyte and potentiometrically determining the resulting increase in pH of the electrolyte and converting the ammonia ,~

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10391fà3 determination to the urea equivalent of the specimen.
In accordance with a further embodiment of the present teachings, an apparatus is provided for determining urea in an aqueous specimen which contains urea. The apparatus comprises an hydrolysis chamber which contains immobilized urease with the chamber having a specimen inlet and a hydrolysis product outlet. A mixing chamber is inter-connected with the hydrolysis produ,ct outlet with the chamber including an inlet for base, a mixture positioned for mixing the hydrolysis product with the base and an effluent outlet for the resulting mixture. An hydrophobic, ammonia gas permeable membrane is positioned to have one surface for intimate physical contact with any effluent flowing through the effluent outlet and the opposing surface of the membrane defining a portion of the reservoir of liquid electrolyte in intimate physical contact with the opposing surface of the membrane. The membrane is positioned to separate the effluent from the liquid electrolyte.
A pH sensitive electrode is provided immersed in the electrolyte with the electrode being electrically connected to a pH cell for response to an increase in pH in the electrolyte.
A primary feature of the present invention is that the immobilized urease is physically separated from the --2~-, . . .
,~, .

1039163 G-12~41 potentiometric electrode. This allows the ammonium ions generated by the urease hydrolysis of urea to be removed from the immobilized urease (where the pH must be maintained in the range of 5 to 9 for efficient urease hydrolysis) and mixed with a base to raise the pH to at least about 11 for conversion of the ammonium ion to soluble gaseous ammonia The soluble ammonia gas is then permeated through a hydrophobic membrane for detec-tion with a pH sensitive electrode.
By this technique, the ammonia gas is solely responsible for any change in pH and interference of any cation which may be present in the specimen is prevented. Thus, the present invention is capable of analyzing urea in a wide variety of aqueous specimen with or without the presence of monovalent cations such as sodium, potassium, or lithium. This represents a marked improvement over the "enzyme electrode" types discussed above where the electrode comes in contact with any extraneous cations in the specimen which can interfere with the test results. Furthermore, an enzyme electrode constructed with an ammonia electrode would be inoperative because the pH sufficient to convert ammonium ions to ammonia would deactivate the enzyme immobilized on the electrode.
The present invention will be described with reference to the drawings wherein Fig. 1 is a schematic process flow diagram for practicing the present invention, and Figs. 2 and 3 are cross sectional illustrations of one type of pH electrode cell containing a hydrophobic ammonia permeable membrane for 10;~9163 . G-1244l ¦ practicing the present invention.
¦ Referring now to Fig. 1, an aqueous specimen containing ¦ urea flows into a bed of immobilized urease which functions ¦ as a hydrolysis zone where the specimen is maintained for a ¦ time and at a temperature sufficient to hydrolyze urea to ¦ ammonium ions. Preferably the specimen is maintained in ¦ contact with the immobilized urease for a time sufficient to ¦ hydrolyze substantially all of the urea to ammonium ions.
¦ Typically, this hydrolysis is completed within a few seconds ¦ to 30 minutes or longer at temperatures ranging from 0C to ¦ about 50C and higher. The hydrolysis reaction is believed ¦ to proceed according to the reaction:

2H+ + H20 + H2N-C-NH2 Urease)2~H4 + HC03 and/or C02 The urease is believed to be most efficient in hydrolyzing urea at a pH of about 5 to 9. Because urease is most efficient in hydrolyzing urea in the 5 to 9 pH range, the urea specimen, prior to contact with the urease, is usually mixed with an aqueous diluent which is buffered to pH 5 to 9~
The ratio of dilution of the urea specimen in the buffered diluent varies with the concentration of urea in the specimen.
For physicological fluids such as blood or urine having unknown concentration within the expected concentration range, a ratio of 1 part of volume by specimen to 25 to 50 parts of diluent is suitable for an acceptable electrode response. Usually, for efficiency and economy, a small specimen (e.g~ about 10 to 50 microliters)is injected into a stream of buffered diluent flowing at the rate of 0.1 to 10 ml per minute for introduction ¦ G-124~1 j into the bed of immobilized urease. Suitable buffered diluents include O.OlM sodium citrate (pH 6.0); O.OlM sodium maleate (pH 6.2) and O.OlM tris (hydroxymethyl) aminomethane adjusted to pH 7 with HCl.
Additional reagents can be incorporated into the buffered diluent for the purpose of retarding deactivation of the im-mobilized urease and deterioration of the support material.
These include: salts of ethylene diamine tetraacetic acid, to prevent heavy metal ion poisoning of the enzyme; beta-mercapto ethanol, to protect the urease from oxidation; and sodium azide, a bacterial inhibitor.
Any of the known methods for immobilizing urease on an insoluble support can be used in prac~icing the present invention.
For instance, urease can be covalently coupled to a porous glass support with an amino-functional silane coupling agent as disclosed in the article entitled, "Urease Covalently Coupled to Porous Glass," by H. H. Weetall and L. S. Hersh; Biochim.
Biophys. Acta, 185 (1969) 464-465, and U. S. Patent 3,519,538, urease can be coupled to water insoluble diazonium salts as in the article entitled, "Preparation and Properties of Water-insoluble Derivatives of Urease," by E. Riesel and E. Katchalski;
Journal of Biological Chemistry, Vol. 239, No. 5 (1964) 1521;
urease covalently coupled to nylon by the method in the article entitled, "The Immobilization of Enzymes on Nylon Structures and their Use in Automated Analysis," by D. J. Inman and W. E. Hornby;
Biochem. J. (1972) 129, 255-262; urease immobilized on a polyacrylamide gel by the method in the article "A Urea-Specific Enzyme Electrode," by G. G. Guilbault and J. G. Montalvo, Jr.;

G-124~1 ~

Journal of the l~merican Chemical Society, 91, (1969) 2164-5;
urease can be adsorbed on the surface of kaolinite as in the article, "Preparation and Properties of Solid-Supported Urease,"
by P. V. Sundaram and E. M. Crook; Canadian Journal of Biochemistr~
Vol. 49 (1971) 1388-94; and urease can be immobilized with cyanogen bromide according to the method of Patent 3,645,852 entitled, "Method of Binding Water-soluble Proteins and Water-soluble Peptides to Water-insoluble Polymers Using Cyanogen Halide," by R. Axen, J. Porath, and E. Ernbach; and "The Preparation and Characterization of Lyophilized Polyacrylamide Enzyme Gels for Chemical Analysis" by G. P. Hicks and S. J.
Updike appearing in Analytical Chemistry, Vol. 38, No. 6, May 1966 at page 726. Thus, in forming the bed of i~unobilized urease the selection of the support from materials such as porous 15 glass, clay, water insoluble polymers and immobilizing the urease thereon by chemical or physical means is well known in the art and forms no part of the present invention.
After hydrolysis of the urea, the resulting hydrolysis mix-20 ture containing ammonium ions flows from the bed of immobilized urease and is mixed with sufficient base in a suitable mixing chamber to adjust the pH of the mixture to at least about 11.
At this pH and above substantially all of the ammonium ions are converted to an aqueous ammonia solution. The mixing chamber has 25 an inlet for the hydrolyzed urea, an inlet for base, and an outlet for the resulting reaction mixture. Any type of mixer such as an impeller or blade type mixer can be used in the mixing chamber to mix the base with the hydrolyzed urea, although a small magnetically operated mixing bar has been found to be quite 30 satisfactory.

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1039163 G-l244l Any type of base ~hich does not contain ammonia or ammonium ion can be used to adjust the pH to at least about 11. Suitable bases include the alkali metal hydroxides (e.g.-Ca(OH)2 or ~g (OH)2] although dilute aqueous solutions of alkali metal hydroxides, particularly NaOH, having concentrations in the range of about 0.01 to about lN are preferred for efficiency and economy in pH adjustment.
After adjustment of the pH to at least 11, the resulting aqueous ammonia solution is contacted ~ith a hydrophobic, ammonia permeable membrane for a time sufficient to allo~ gaseous ammonia to permeate throuyh the membrane. Such hydrophobic membranes permit the passage of gaseous ammonia while retaining aqueous solutions and can be in the form of hydrophobic porous and micro-porous plastic films having a thickness of about 0.1 to about 10 mils, a porosity of about 10 to 85% and a pore size diameter of about 0.05 to 10 microns. Preferably such microporous plastic films have a thickness of about 0.5 to 5 mils, a porosity of about 25~o to 80% and anaverage pore size diameter of about 0. 05 to 5 microns. Suitable plastic membranes are commercially available in the form of porous copolymers of acrylonitrile and vinyl chloride on nylon support (AcroporT sold by Gelman Instrument ompany) porous hydrophobic cellulose acetate, porous polytetra-fluoroethylene (Teflon sold by DuPont), microporous polypropy-lene (Celgard sold by Celanese Corporation~, porous polyvinyli-dene fluoride and other membrane materials as disclosed in U.S.Patent 3,649,505. These membranes permit diffusion of gaseous ammonia while monovalent ions such as Na , K , or Li , remain in the aqueous solution G-124~1 ~
1039~63 which does not diffuse through the membrane.
The gaseous ammonia permcating the membrane is then passed to a pH electrode cell which contains an aqueous electrolyte solution. The gaseous ammonia dissolves in this electrolyte solution to increase the pH of the electrolyte solution. This increase in pH is potentiometrically measured with a pH sensitive electrode.
The electrolyte solution is usually a dilute solution of an ammonium salt (e.g. - O.lM ~H4Cl) to provide baseline ammonium ion concentration from which an increase in pH is readily measurable. This increase in pH is a function of the amount of ammonia gas permeating through the membrane and the corresponding potentiometric reading on the pH electrode can be readily converted to the urea equivalent of the original specimen. The urea equivalent of the original specimen is usually reported in mg blood urea nitrogen 'i.e. BUN)/lO0 ml specimen. These units are conventional in clinical applications.
Fig. 2 is a cross sectional illustration of a pH cell for use in the pres~nt invention. Figure 3 is a broken away enlarge-ent of Figure 2 showing the membrane and electrode in detail.In Fig. 2 and 3 pH cell lO comprises an electrode chamber lOa to which membrane housing lOb is engaged by means of screw threads lOc. Chamber lOa contains a pH sensitive electrode ll which can e a conventional glass electrode referenced against a suitable conventional reference standard electrode 12 such as a platinum ire coated with silver/silver chloride. Both of those 1039~63 electrodes are held in position by electrode support 20 equipped with gasket 21. Electrodes 11 and 12 are- electrically connected to a conventional potentiometric pH meter which is not shown.
The sensing tips of electrodes 11 and 12 extend into electrolyte cavity 13 which contains an aqueous 0.lM NH4Cl solution. The bottom of electrolyte cavity 13 is defined by hydrophobic, ammonia permeable membrane 14 and the sensing tip of electrode 11 is positioned adjacent thereto. Membrane 14 is held in contact with electrolyte cavity 13 by membrane housing 10b and membrane holder 22. A'liquid seal is maintained by means of gasket 16. Membrane housing 10b is also provided with a narrow passageway 17 through which the sample containing the ~
ammonia flows in permeation chamber 23. The passageway 17 and permeation chamber 23 are of such dimensions to assure turbulent flow therein for maximum exposure of the sample to membrane 14 to allow effic~ient ammonia permeation. After contact with membrane 14 the specimen residue which is depleted in ammonia leaves through passageway 18. The potentiometric measurement which results from the increase in pH is converted to the urea concentration of the original urea specimen by conventi~nal potentiometric calibration techniques.
In the above technique, a conventional ammonia gas sensing electrode such as a Model 95-10 gas sensing electrode sold by Orion Research Incorporated or an ammonia electrode which electrodes incoporate the hydrophobic ammonia permeable membrane into the pH electrode cell can be employed.

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1~9163 G-12441 In the most efficient practice for the clinical laboratory the buffered diluent and the base are pumped continously through the system sho~Jn in Figure l at the rate of about O.l to about lO ml/minutes and usually about l ml/minute.
About 10-25 microliter "shot" of urea specimen is rapidly injected with a syringe directly into the buffered diluent stream at the inlet to the bed of immobilized urease. In ,the bed of immobilized urease, the urea is hydrolyzed to ammonium ions and bicarbonate ions. Upon leaving the bed of immobilized urease the reaction product is mixed with the base to raise the pH to at least about ll to convert the ammonium ions to soluble ammonia gas. The bicarbonate ions are converted to carbonate ions at this increased pH.
.

The buffered diluent stream containing the dissolved ammonia then contacts the hydrophobiG ammonia permeable membrane and ammonia permeation begins. Before equilibrium on both sides of the membrane is reached, however, the concentration of the dissolved ammonia in the buffered diluent has decreased to the point where ammonia diffuses back from the electrode electrolyte solution into the buffered diluent stream.
Because the urea specimen has been injected in a relatively high localized concentration in the buffered diluent stream, this reaction occurs quickly, (e.g. within about 2 or 3 minutes) and produces a rapid increase in the pH which results in a sharp peak in the potentiometric electrode response. The rate of change of pH is a function of the concentration, i.e. the higher the concentration of NH3 in the micro-environrnent of the electrode the greater the slope of the pH curve. The sharpness of the peak is also a function of the rate of change of ~H3 concentration, i.e. how rapidly the pH decreases depends on how rapidly the ~H3 back-diffuses into the diluent buffer stream. Small samples should be removed faster than large samples. The height of this sharp peak is a measure of the concentration of urea. The next urea specimen can then be injected when the potentiometric reading has returned to the base line or a point near enough to the base line such that the next urea determination is not detrimentally affected.

Another method of operation employsthe slow introduction of urea specimen over longer periods of time until equilibrium in ammonia diffusion is reached across the membrane. For instance, a 10-25 microliter urea specimen is slowly and continuously introduced with comp~ete and instantaneous miY~ing over a 10 minute period into a buffered diluent stream flowing at I iO39163 G-12441 1 ml/minute. l'his reaches equilibrium with a constant pH in the electrode electrolyte and a coxrespondingly constant potentiometric response. After the constant reading has been taken, the introduction of urea specimen is stopped and the potentiometric reading returns to the base line. This technique is less preferred because of the longer time period required for each determination.
The invention will be further illustrated in the examples that follow wherein all parts are parts by weight, all percentages are weight percentages, and all temperatures are in C unless stated otherwise.

10~9 1~ 3 G-12441 In Example 1 the buffered diluent is an aqueous O.OlM
solution of tris (hydroxymethyl) aminomethane which has been adjusted to pH 7.0 (with HCl) containing disodium ethylene diamine tetraacetic acid, beta-mercapto ethanol and sodium azide in a concentration of O.OOlM with respect to each of these chemicals.
The base used to adjust the pH is a 0.03N sodium hydroxide solution.
The urease enzyme is obtained from Worthington Biochemical Corporation and has an activity of 139 International Units per milligram.
The support for the immobilization of the urease enzyme is agarose gel (a highly porous polydextran) obtained fro~
Pharmacia Fine Chemicals Inc. under their trademark Sepharose 4B. The electrolyte in cavity 13 is O.lM NH4Cl.
The pH cell is a conventional glass pH electrode employing a silver-silver chloride element in HCl electrolyte referenced against a silver-silver chloride electrode.
The ammonia permeable hydrophobic membrane is a microporous polypropylene film having a thickness of 1 mil, porosity of 35%, an average pore diameter of less than 0.1 microns obtained from Celanese Corporation under the trademark of "Celgard 2400.
Part A
Forty-four ml of a 4~ by weight aqueous dispersion of the agarose gel described above is poured onto a Buchner funnel and washed with distilled water. The agarose on the filter is ~''`' transferred to a beaker and distilled water is added with agitation to yield a dispersion of 40 ml which is then centrifuged in centrifuge tubes at about 1000 rpm for five minutes. After decantation of the supernatant, the agarose in the bottom of the centrifuge tubes is transferred again to a beaker and distilled water is added with stirring to produce a uniform gel having a volume of 40 ml.
Part B
Four hundred mg of the urease described above is dissolved in 20 ml of a 0.05M sodium borate aqueous solution which has been adjusted to pH 9.5 with 6N sodium hydroxide. The resulting solution is stored in an ice bath until ready for use in Part C.
Part C
Four grams of reagent grade cyanogen bromide crystals are added to the agarose gel of Part A and the pH is quickly adjusted to about 11 with 6.0~ sodium hydroxide while stirring continuously. The pH of the mixture is maintained at or near this value by addition of the 6.0N sodium hydroxide as required, during which time the cyanogen bromide crystals slowly dissolve and react. This dissolution reaction requires about 15 minutes during which time crushed ice is added to maintain the temperature below 20C. A total of about 40 ml of 6.ON sodium hydroxide are required over this 15 minute period to maintain the pH at 11.
After the cyanogen bromide has completely dissolved and re-acted, the resulting gel is washed on a sintered glass funnel with 300 ml ofcold 0.05M sodium borate solution adjusted to pH

.

~039163 9.5 with NaOH.
The resulting agarose gel is transferred to a 100 ml beaker and the cold urease solution prepared in Part B is added immediately while stirring. The urease/agarose gel is then S frozen quickly and kept frozen for 5 minutes. It is then maintained at 0C for 20 hours ~.~hile stirring gently with a magnetic stirrer.
The resulting immobilized urease/agarose composite gel is filtered on a sintered glass funnel with suction and ~ashed first with a 0.5M sodium chloride solution and then with distilled, deionized water until the filtrate washings are free of urease as shown by the absence of any absorption at the characteristic wavelength 272 nm. The immobilized urease/
agarose composite gel is stored in 0.05M tris (hydroxymethyl) aminomethane buffer solution (pH 7.5) at 0-5C.
Part D
The activity of the immobilized urease/agarose composite gel of Part C is determined by mixing a kno~n quantity of the gel-in a 0.15M solution of urea which is 0.005 molar in tris (hydroxymethyl) aminomethane and 1.0 x 10 3 molar in disodium ethylenediaminetetraacetic acid and measuring the change in pH
with time. The rate of change of pH is converted to enzyme activity by the method of L. Jaco~sen, K. Lindstrom~Lang, M. Ottesen and D. Glick Ed., in "'~'ethods o~ Biochemical Analysis," Vol. IV, Interscience Publishers, N~ Y., 1957, p. 171 , . -14-.,,"; . "

1 ~ 9 16 3 G-12441 This analytical technique gives an activity for urease of lO00 I~u~/ml of gel which decreases to about 400 I.U./ml of gel after storage for 2 months at 0-4C in 0.05M tris (hydroxymethyl) aminomethane.
Part E
A glass column is prepared from a 75 mm borosilicate glass capillary tube with an inside diameter of 2.8 mm and an outside diameter of 6 mm. A 400 mesh nylon disc is attached to one end of the column. The immobilized urease/agarose composite gel of Part C is charged thereto to fill the tube. The urease/
agarose gel packs into the column by gravity and the other end of the column is also fitted with a 400 mesh nylon disc after the column is filled with the urease/agarose gel.
The column ends are then fitted with a plastic tubing fittings one of which is in the form of a "tee" for sample injection. The injection tee is provided with a rubber membrane for sample injection with a hypodermic needle.
Part F
The buffered diluent and base solutions described above are pumped through the apparatus described in Fig. l at a rate of l.0 ml/min. for each stream. Duplicate lO microliter samples of each of the a~ueous urea specimen concentration describedkelCw are quickly injected with a hypodermic needle through the injection "tee" into the immobilized enzyme column as shown in Figure l.
The analysis takes place as described above in conjunction with the drawing. Correspondins millivolt readings obtained are:
. -15-lOJ916;~ G-1244 1 ¦Aq~_ous Urea Concentration EMF Change ~in Millivolts) On pH Meter ¦ 0.10M 194.5 - - 194.0 l 0.01M 138.5 1 137.5 0.001M 78.0 78.0 A calibration graph is prepared by plotting the millivolt change against the logarithm of the urea concentration. This graph is essentially a straight line which indicates Nernstian behavior.
A blood serum specimen of unknown urea concentration is analyzed by injecting 10 microliter specimens in the column using the procedure described above. A maximum millivolt reading is obtained about one minute after specimen injection. Successive samples are injected into the column at approximately oAe minute intervals. A total of 50 specimens of the serum provides an EMF
change of 130~1.0 millivolts This change in EMF correspond to a concentration of 7.50 x 10 molar urea from the above calibration graph. The concentration corresponds to a blood urea nitrogen (BUN) value of 21.0- 0.8 mg nitrogen/100 ml of serum.
Similar results are obtained in the above procedure for BUN
analysis when the immobilized enzyme bed is prepared from a -cross-linked polydextran obtained from Pharmacia Fine Chemicals Inc. under the trademark of Sephadex G-200. Thus, one gram of the cross-linked polydextran, 4 grams of cyanogen bromide, 6.5 ml of 6N sodium hydroxide and 200 mg of urease are reacted by the above procedures to yield an immobilized urease/polydextran composite gel having an activity of about 400 I.U./ml of gel.

ll G-12441 10391~i3 i Similar results are obtained when the hydrophobic ammonia permeable membrane is a copolymer of acrylonitrile and vinyl chloride sold by Gelman Instr~lent Company under the trademark of Acropor ANH-3000 instead of the microporous polypropylene membrane.-Thirteen 20 microliter specimens of a serum sample having been chemically analyzed to contain 15.9 mg BUN/l00 ml by conventional clinical spectrophotometric techniques are analyzed by the procedures of Example l. The results indicate a BUN value ;l0 of 16.l mg/l00 ml with a standard deviation of 0.2 mg/l00 ml.
Similarly precise results are obtained when the analysis is repeated with chemically analyzed specimens containing 49 mg BUN/
l00 ml.
EXAMPLE 3 .
lS '` Part A
~wenty ml of distilled water is mixed with l0 ml (about l gram) of agarose used in Example l to form a suspension of agarose. Ten ml of a 10% by weight solution of cyanogen bromide is added to the above agarose suspension and the pH adjusted to ll.0 by addition of 3.0N NaOH while the temperature of the agarose suspension is maintained at about 22 to 27C by the addition of ice as necessary.
After pH adjustment, the agarose is quickly washed by vacuum filtration with approximately one liter of cold aqueous 0.2M sodium borate buffer at pH 8.5. One half of the resulting cyanogen bromide "activated" agarose is added to a solution of urease prepared by dissolving 20 mg of urease (92 International Units/mg) in 5 ml of the above 0.2M sodium borate buffer ~.~, .

1~9163 G-12441 which has been cooled to 0C. The resulting urease/agarose suspension is stirred overnight at 0-3C to complete the im-mobilization reactions.
The activity of the resulting immobilized urease is determined by a laboratory pH meter (Model pHR sold by Sargent-Welch)and a calibrated monovalent cation electrode (Beckman Model 39137) referenced to a standard calomel electrode. The activity of the immobilized urease is determined to be approximately 500 International Units per gram of urease/agarose composite gel.
1.5 ml of the immobilized urease/agarose composite gel is placed into a 4 mm inside diameter glass tube and the glass tube is fitted at each end with a disc of 400 mesh nylon to form a small column filled with immobilized urease. Distilled water is passed through the column at the rate of 1 ml/minute to hydraulically pack the immobilized urease as a bed.
The tube containing immobilized urease/agarose composite gel is connected as the immobilized urease bed in Example l. A
calibrated ammonia electrode containing a hydrophobic ammonia permeable membrane and pH electrode cell (available from Orion Corporation as electrode Model 95-10) is employed.
Six blood serum specimens from six different human patients are chemically analyzed in a hospital laboratory. Three of these samples are analyzed to have a BUN value of 14.0 mg/100 ml and three specimens are analyzed to have a BUN value of 17.0 mg/100 ml. These serum specimens are diluted in 0.01 M tris (hydroxymethyl) aminomethane buffer in the 10~9163 G-1~441 ratio 1 to 25 and the diluted serum specimens are introduced into the column of immobilized urease of this example at the ~low rate of about 1 ml/minute. About 1 ml/minute of 0.2M
sodium hydroxide is used as the base to adjust the pH to 13.
Under these conditions, chemical equilibrium is attained and change in EMF (in millivolts) is measured. The corresponding BUN value is determined from the calibration graph. The results are set forth below.

Serum BUN Value by Chemical Analysis BU~ Value by Invention Sample (mq BUN/100 ml serum sample) (mq BU~/100 ml serum) 1 14 13.8

2 14 14.0

3 14 13.0

4 17 17.0 17 16.1 6 17 16.8 Urease is immobilized on a particulate porous alumina support by mixing 100 mg urease and 1.0 g of particulate alumina in 200 ml of O.OlM tris (hydroxymethyl) aminomethane (adjusted to pH 8.2 with HCl) at 40C and stirring for one hour. The partic-ulate alumina has a particle size in range of from -50 to +100 mesh (U.S. sieve screen) and an average pore size diameter of about 0.1 to 0.2 microns, The immobilized urease/alumina reaction product is allowed to stand overnight at 0.
The immobilized urease reaction product is then vacuum filtered on a scintered glass funnel and washed first with 500 ml of 0.5 M NaCl, followed by washing with 1 to 2 liters of distilled water. The washed immobilized urease reaction product is stored in 10-20 ml of 0.01 M tris (hydroxymethyl) aminomethane buffer until ready for use. The activity of the immobilized urease/alumina product is analyzed to be 1500 I.U./
cm3.
This activity decreases sharply upon use in urea hydrolysis . due to leaching of the urease from the alumina support, and has a relatively short service life when used for urea analysis according to Example 1. .
~ EXAMPLE 5 A solution of 1,2-dibromoethane is prepared by diluting 0.25 ml of 1,2-dibromoethane in 20.0 ml of methanol. This dibromoethane solution is added to 200 ml of a tris (hydroxymethyl) aminomethane at pH 8.2 buffered solution. -~
The pH of the resulting solution is adjusted to and maintained at 8.2 by the dropwise addition of 1.0 M HCl.
100 mg of urease and 1.0 g porous alumina powder (the same alumina powder used in Example 4) are slowly added to the buffered dibromoethane solution with stirring while keeping the temperature at 40C. This reaction mixture is stirred for one hour at 40C and allowed to stand overnight at 0. After filtering and washing the immobilized urease/

G-124~1 1 10391~;3 alumina composite is assayed and determined to have an ¦ activity of 618 I.U./cm . The activity of this composite ¦ decreases very slowly in use and has prolonged service life ¦ when used for urea analysis according to Example 1. The ¦ dibromoethane apparently functions as a cross-linking agent ¦ in immobilizing the urease on the porous alumina.

l _ .
¦ Part A
Urease is immobilized on porous alumina as in Example

5 except that 0.25 ml of 1,3-dibromopropane is used as the cross-linking agent instead of the 1,2-dibromoethane The immobilized urease/alumina composite has an activity of approximateiy 1,000 I.U./cm .
This immobilized urease/alumina composite is pacXed in a column and used to analyze urea samples following the procedure of Example 1. A calibration graph i5 prepared by plotting the change in EMF (in millivolts) against urea concentration of three serum samples having 14, 28, and 70 mg BUN/100 ml serum. These serum samples produce average millivolt change of 70, 90, and 111 respectively.
Part B
Two blood serum specimens having been analyzed in a hospital laboratory to contain 12.2 and 30.7 mg BUN/100 ml of serum are analyzed using the method and procedures of Part A
of this Example. Based on the calibration graph, the potentiometric response indicates that the serum samples have 12.2l 0 4 and 30.8~ 0.6 mg BUN/100 ml of serum, respectively.

¦ Similar results are obtained in the above proccdure for ¦ BUN analysis when the immobiliæed enzyme bed is a urease/
¦ acrylamide composite gel prepared by the method of the Hicks ¦ and Updike article discussed above. Such a composite gel ¦ can be prepared by reacting 1.0 ml of a 0.1 M phosphate buffer solution (pH 7.4) containing 400 mg of acrylamide, 4.0 ml of a solution of the same buffer containing 23 mg of N,N
methylenebis (acrylamide); 1.0 ml of the same buffer solution containing 10 mg of urease; together with 0.03 mg of riboflavin and 0.03 mg of potassium persulfate to catalyze a photo-polymerization reaction. The reaction mixture isstirred in an ice bath while photolyzing with a flood lamp. Gellation occurs in about 10 minutes. The gel is mechanically dispersed and washed with 0.1 M phosphate buffer before use in the procedure of Example 1.
Similar results are obtained in the above procedure for BUN analysis when the immobilized enzyme bed is prepared from a ureas~ kaolinite composite prepared by the method of the articl~
of Sundaram and Crook described above.Such a composite can be prepared by reacting 200 g powdered kaolinite (average particle size less than 0.1 micron) suspended in 8 ml of tris (hydroxymethyl) aminomethane buffer with 12 mg of urease in a stirred reactor for 20 minutes at 30C. The resulting immobilized urease/kaolinite composite is washed to remove residual soluble urease before use in the procedures of Example 1.

G-124~1 10391bi3 Similar results are obtained in the above procedure for BUN analysis when the immobilized enzyme bed is prepared by the method of U. S. Patent 3,519,538 discussed above. Such a composite can be prepared by chemically coupling urease to 96~ silica glass powder (about 100 mesh) having an average pore size of approximately 0.1 micron. Thus, 1.0 g of porous glass is combined with 50 ml of a 10~ solution of ~-aminopropyltrietho-silane in toluene. This mixture is stirred overnight with continuous refluxing, filtered and washed with acetone. After further reaction with p-nitrobenzoic acid, reduction of the incorporated pendant nitro group and its subsequent diazotization the thus activated porous glass is reacted with 10 mg of urease in 10 ml of 0.05 M tris (hydroxymethyl) aminomethane l buffer solution (pH 7.5). The mixture is stirred overnight ¦ at 5C, filtered and washed with buffer before use in the l procedures of E~ample 1.
¦ Similar results are obtained in the above procedure for BUN analysis when the immobilized enzyme bed is a urease nylon l composite prepared by the method of the Inman and Hornby ¦ article discussed above. The nylon is a low molecular weight type 6 polymer in powder form (120-150 mesh). Pre-treatment of the nylon with glutaraldehyde is carried out by suspending 250 mg of the powdered nylon in 10.5 ml of 12.5% (weight per volume) glutaraldehyde~ The latter reagent is dissolved in 0.10 M sodium borate buffer adjusted to a pH of 8.5. The nylon-glutaraldehyde mixture is stir~ed rapidly at 0 for 20 minutes an~ then filtered on a scintered glass funnel and washed with ¦ 0.2 M sodium.boratc buffer. The washed activated nylon powder ¦ is suspended in 5 ml of a urease solution containing 10 mg ¦ urease, 25 micromoles of ethylene diamine tetraacetic acid and ¦ 5 micromole of mercaptoethanol in 0.05 M KH2P04 buffer adjusted tc ¦ pH 7.0 with dilute sodium hydroxide. The urease nylon mixture ¦ is stirred for 16 hours at about 1C. The suspension of ¦ immobilized urease nylon composite is washed free of unreacted ¦ urease with a 0.2 M sodium chloride solution before use in the ¦ procedures of Exampl.e 1.

Claims (13)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for determining urea in an aqueous specimen containing urea, comprising the sequential steps of:
passing said specimen into a hydrolysis zone containing immobilized urease, retaining said specimen in said hydrolysis zone for a time sufficient to hydrolyze said urea to ammonium ions, removing the resulting hydrolysis mixture from said hydrolysis zone, raising the pH of said hydrolysis mixture to at least about 11 to convert substantially all of said ammonium ions to an aqueous ammonia solution, contacting said aqueous ammonia solution with a hydrophobic, ammonia permeable membrane for a time sufficient to allow gaseous ammonia to permeate said membrane, determining the gaseous ammonia permeating said membrane by dissolving said ammonia in an electrolyte and potentiometrically determining the resulting increase in pH of said electrolyte, and converting said ammonia determination to the urea equivalent of said specimen.

2. The method of claim 1 when substantially all of said urea is hydrolyzed to ammonium ions.

3. The method of claim 1 wherein said hydrolysis zone comprises a bed of urease immobilized on a solid support.

4. The method of claim 1 wherein said aqueous specimen containing urea is an aqueous solution buffered to a pH in the range of about 5 to 9.

5. The method of claim 1 wherein said membrane comprises a thin sheet of porous plastic having a thickness of about 0.1 to about 10 mils, a porosity of about 10% to about 85%
with an average pore size diameter of about 0.05 to about 5 micron.

6. The method of claim 5 wherein said membrane comprises a thin sheet of porous plastic having a thickness of about 0.5 to about 5 mils, a porosity of about 25% to about 80%
with an average pore size diameter of about 0.05 to about 5 micron.

7. The method of claim 1 wherein said liquid electrolyte comprises a dilute aqueous solution of ammonium chloride.

8. Apparatus for determining urea in an aqueous specimen containing urea, comprising in combination, an hydrolysis chamber containing immobilized urease, said hydrolysis chamber having a specimen inlet and a hydrolysis product outlet, a mixing chamber interconnected with said hydrolysis product outlet, said mixing chamber including an inlet for base, a mixer positioned for mixing said hydrolysis product with base, an effluent outlet for the resulting mixture, an hydrophobic, ammonia gas permeable membrane having one surface positioned for intimate physical contact with any effluent flowing through said effluent outlet, said membrane having its opposing surface defining a portion of a reservoir of liquid electrolyte in intimate physical contact with said opposing surface of said membrane, said membrane being positioned to separate said effluent from the liquid electrolyte, and a pH sensitive electrode immersed in said electrolyte said electrode being electrically connected to a pH cell for response to an increase in pH in said electrolyte.

9. The apparatus of claim 8 wherein said hydrolysis chamber contains a bed of urease immobilized on a solid support.

10. The apparatus of claim 8 wherein said mixer is magnetically activated.

11. The apparatus of claim 8 wherein said membrane comprises a thin sheet of porous plastic having a thickness of about 0.1 to about 10 mils, a porosity of about 10% to about 85% with an average pore size diameter of about 0.05 to about 10 micron.

12. The apparatus of claim 11 wherein said membrane comprises a thin sheet of porous plastic having a thickness of about 0.5 to about 5 mils, a porosity of about 25% to about 80% with an average pore size diameter of about 0.05 to about 5 micron.

13. The apparatus of claim 8 wherein said liquid electrolyte comprises a dilute aqueous solution of ammonium chloride.