DE2624384A1 - Urea determination in blood - with continuous monitoring for patients receiving artificial kidney treatment - Google Patents

Abstract

Determination of urea in the blood of patients with deficient kidney function, who are being treated with an artificial kidney comprises (a) bringing the dialysate flowing from the artificial kidney into contact with immobilised urease so that ammonium ions are formed by reaction of the urease with the urea in the dialysate, (b) determining the ammonia, which has been formed in the soln. leaving the urease, after adjustment to an alkaline pH, by means of an NH3- gas electrode in a continuous flow measuring cell, (c) calculating the urea concn. in the blood from that measured in the dialysate by means of the electrode and specific factors relating to the dialysis, by means of a computing circuit, and (d) displaying the value of the urea concn. by means of a recorder or print-out system. The method permits the continuous determination of urea concn. of patients with deficient kidney function, under treatment with an artificial kidney, and allows the state of the dialysis to be monitored and regulated at the bedside.

Description

Method for the determination of urea in the blade and device for Carrying out the method The invention relates to a method for determination of urine: f in the blood of patients treated with an artificial kidney with Renal insufficiency and a device for carrying out the procedure.

When using artificial kidneys on patients with renal insufficiency The dialysis or ultrafiltration conditions should be adjusted depending on the degree of the disease and the type of dialyzer used. Presently these are Conditions due to the analysis values for urea, creatinine, electrolytes and The like. Which are obtained in the patient's blood before dialysis is set. However, as more than half a day is required to determine these analytical values It is difficult or impossible to change the dialysis conditions depending on the degree of the disease and the type of dialyzer in the artificial kidney during dialysis in time to discontinue or resign, Currently there are conditions such as Dialysis speed and end point of dialysis based on experiences and perceptions set by the doctor. An exact determination of these conditions is not, however possible, so that the patient experiences numerous complaints such as nausea and Headache.

The object of the invention is to precisely determine the respective state of dialysis to be recognized and monitored. Furthermore, a device is to be made available with which the activity of artificial kier at the patient's bedside is controlled can be.

The invention relates to a method for the determination of urea in the blood of patients with egg failure treated with an artificial kidney, which is characterized in that (1) the emanating from the Christian kidney Brings dialysate into contact with immobilized urease, whereby by reaction of the The urea contained in this dialysate is formed with the urease ammonium ions be, (2) the aminonia-k, which after adjusting to an alkaline pH in the solution separated from the urease is formed with an ammonia gas electrode determined in a flow cuvetto, (3) the urea concentration in the blood from the with the help of the electrode measured urea concentration in the dialysate and certain, dialysis-related factors are calculated with the help of a computer circuit and (4) the value of the urea concentration with the help of a pen and / or printer records.

The device according to the invention for the determination of urea in blood of renal insufficiency patients treated with an artificial kidney characterized by (I) a column or tube packed with immobilized urease, where the dialysate flowing out of the artificial kidney with the immobilized urease is brought into contact, (II) one arranged in a flow cell Ammonia Gaoelectrode for the continuous determination of ammonia, which is produced by setting the solution that has passed through the column is formed to an alkaline pH (III) a ray circuit to determine the urea concentration from the electrode voltage in the blood and certain; calculate dialysis-related factors, and (IV) a recorder and / or printer to record the calculated values.

The invention is based on the fact that the urea concentration in the blood (BUN) of patient with ovarian insufficiency who had an artificial kidney treated, like the product ans [α] X [urea concentration in outflowing dialysate (DUN)], where α is a constant that is given by (1) the type of dialyzer used, (2) the flow rate of the blood during dialysis and (3) the flow rate of the dialysate is determined will. An essential feature of the invention is also a new type of flow cell for the ammonia gas electrode, with which the in the dialysate under the action of the immobilized Urease from the urease ammonia concentration can be determined exactly.

Several references report methods for analyzing constituents in solutions or in blood where a combination of immobilized urease and an electrode is applied. According to one method, for example, is immobilized Urease applied together with a monovalent cation electrode; see G.G. Guilbault and J.G. Montalvo, J. Am. Chem. Soc. 92: 2533-2538 (1970). Furthermore, the use of immobilized urease together with a carbonic acid gas electrode is also possible described; see G.G. Guilbault and P.R. Shu, Anal. Chem., 44: 2161 (1972) until 2166.

However, the values for Na +, K + and HCO3 -1 in the die lysate change of artificial kidneys quantitatively during dialysis very strong, so that the conventional Method does not allow for an accurate determination of urea for the purpose of monitoring from Artificial here can be used.

According to the invention, the following advantages are achieved: (1) The urea concentration in the blood of patients with renal insufficiency who are treated with an artificial herd can be determined continuously without direct blood sampling is required.

(2) The status of urea dialysis in patients with renal insufficiency can be monitored by the bed.

Conventional artificial kidneys can be used in the method according to the invention become, for example, spiral type, hollow fiber type and modified ones Standard hollow fiber type.

The immobilized urease is fed into the column by conventional methods or hose packed and inserted.

A small amount of the dialysate leaking from the artificial kidney can be fed directly to the monitoring device through a hose. Another There is a possibility of collecting samples from flowing out in reference glasses Dialysates from several artificial kidneys one after the other on the monitoring device to feed.

The flow cell used in the process according to the invention is used together with the ammonia gas electrode to determine the amount caused by the contact of the Dialysate with the immobilized urease ammonia formed. To increase the The flow cuvette has a stirring rod and above the determination accuracy Liquid surface on a pressure compensation hole. By stirring appropriately and by tilting the electrode, the introduction of bubble cavities into the flow cell be avoided.

As mentioned earlier, the value of the constant 'depends on the type of Dialyzer (F1), the flow rate of the blood (F2) and the flow rate of the dialysate (F3).

As will be explained in the experimental section below, α can be easily determined experimentally. In practice the three variables F1, F2 and F3 as electrical arithmetic signals into the arithmetic circuit using conventional methods entered. The voltage signal obtained from the electrode, that of the ammonia concentration in the dialysate that has passed through the column is transmitted to the circuit whereby the value for the urea concentration in the patient's sheet is continuous can be obtained.

Fig. 1 shows schematically an apparatus of the invention.

Figures 2 and 3 show the influence of changes in flow rates of blood and dialysate to the urea concentration in the dialysate at constant Concentration of urea in the blood.

Fig. 4 explains the relationship of those determined by conventional methods Urea concentration in the blood to the corresponding with the help of the device of Fig. 1 determined concentration.

Fig. 5 shows a typical continuous with the device according to 1 carried out urea determination.

FIGS. 6 and 7 show the results of clinical trials shown.

Fig. 8 shows a flow cell.

Figures 9 and 10 are flow charts of two embodiments the device according to the invention.

The functioning of the device according to the invention is based on Fig. 1 explains. The device shown there is used for k continuous Determination of the heart rate from which the dialysis status can be read leaves.

The reference numerals in Fig. 1 have the following meaning: 1 is a flow-through air trap, which removes the air from the dialysate or ultrafiltrate of the artificial here. 2 represents a pipe or a hose for supplying a standard urea or ammonia solution for calibration purposes. The reference number 3 denotes an electromagnetic three-way valve, which allows either the inflow of the liquid from 1 or the standard solution from 2. 4 Is a pump that delivers the solution at a constant rate. 5 with a column packed with immobilized urease is designated. 6 is a Flow cell and 7 a pump for sucking off the solution at a constant gas speed. The parts designated by the reference numerals 1 to 7 are in the order given arranged in the flow direction in the current path 8. This current path is between the pump t and the column 5 with a tube or hose 9 for the supply of phosphate buffer and between the column 5 and the flow cell 6 with a tube or a hose 10 connected to the supply of NaOH solution. These solutions are generated by the pump 11 or by the pump 12 at a constant speed supplied. Into the flow cell 6, an ammonia electrode 13 is immersed. There is also a flow-through cuvette in this a stirring rod 15, which is driven by the magnetic stirrer located outside the flow cell 6 16 is driven. A pen 14 is connected to the electrode 13 over the circuit 17, which in turn is connected to the cable 18, via that of the artificial Kidney information, such as blood and dialysate flow rates, be transmitted. The column 5, the electrode 13 and the magnetic stirrer 19 are located in a thermostatically controlled container.

The mode of operation of the monitoring device explained above is as follows: The dialysate coming from the artificial kidney (not shown) or ultrafiltrate passes through the air trap 1, where all existing Air bubbles are removed. Subsequently, the liquid is with a constant Aspirated speed of 0.2-0.5 ml / minute. The liquid comes over the valve 5 in the pump 4 and then in the column 5. Between the pump 4 and the dialysate or ultrafiltrate with phosphate buffer of pH 7 is transferred to column 5 added in an amount of 0.2 to 1.0 ml / minute. The one in the dialysate or ultrafiltrate The urea contained in the column 5 is immobilized by the urcase broken down into ammonia and carbon dioxide.

The resulting solution then reaches the flow cell 6, wherein. a 0.1 to 0.2 N NaOH solution was previously added in an amount of 0.2 to 1.0 ml / minute has been.

By rotating the stirring rod 15 in the flow-through cuvotte 6 the solution is made homogeneous. The electrode 13 generates one of the ammonia concentration dependent electrical signal, this signal goes through the Rechon circuit 1 7, in which signals also corresponded to the flow rates of blood and dialysate, be fed in. As a result, the urea concentration recorded by the pen 14 can be obtained read directly in the blood, the solution is dispensed with the aid of the pump 7 together with air pumped from the flow cell 6 at a rate of 1.5 to 4.0 ml / minute. The temperature is, for example, thermostatted to 30 + 0.500 by circulating warm air.

The air trap 1 prevents the penetration of air bubbles, the measurement errors could cause into the current path 8. Furthermore, the air trap 1 allows sampling of dialysate or ultrafiltrate alone.

The electromagnetic three-way valve 3 is used to supply standard urea or ammonia solutions in the current path 8 to the immobilized at suitable intervals To calibrate and test urease and the electrode 13. The pumps 4, 7, 11 and 12th for the supply of solution at constant speed are Behlauch core-priming pumps, which preferably have more than 4 channels and are very stable. The flow rate is determined by the diameter of the pipe or hose used.

The SL: ule 5 contains immebilized urease, with which the urea specifically, rapidly and effectively decomposed to ammonia under mild conditions. Since rapid response and sustained stability (constant reaction speed) are required, the slow diffusion of urea and ammonia, represent a stable flow rate and the maintenance of enzyme activity are important Factors.

As a result, an immobilized urease that does not die will die fermable carrier, for example glass beads, is bound. In particular, being immobilized Urease is used by coating Glaspelen with a, by uni setting of urease and a polyamic acid with acidic amino acid structural units, whose Oarboxyl group (s) converted into acid azide groups has been obtained. A A method for producing such an immobilized enzyme is disclosed in Japanese Patent Publication 109579/1974 described. This immobilized urease exhibits 95 percent after a period of use of 6 months and 95 percent after a period of use 91 percent of its original enzyme activity after 1 year.

In the Durohflusskilvette 6, which is the electrode for determining the ammonia concentration contains, a pressure compensation hole is provided above the liquid level, to maintain a constant fluid pressure. There is also the cuvette placed in a thermostatic vessel to keep the temperature in the cuvette constant to keep. As already mentioned, the stirring rod 15 serves to make the solution homogeneous to hold, thereby increasing the responsiveness and accuracy of the electrode will.

In Figure 8, a preferred embodiment is one according to the invention used flow cell shown. The individual reference symbols have the following Conditions: 21 electrode tube, 22 pH electrode, 23 membrane, 24 flow cell, 24a chamber for the liquid sample, 24b outlet for the liquid, 24e inlet for the liquid, 25 comparison electrode, 26 Ppmpe, 27 waste water pipe, 28 pump, 29 inlet pipe, 30 stirring rod, 31 Magnot, 32 rotor, 33 timer, 34 switches and 35 Pressure Equalization Hole The experimental results shown below will be obtained with the aid of the circuit 17 and the recorder 14.

With a complete decomposition of the urea, the obtained is Ammonia concentration twice the urea concentration. The urea concentration in the blood is o (times the corresponding concentration in the dialysate or ultrafiltrate. The meaning of the constant α is explained at the beginning.

Corresponding test results are shown in FIGS. 2 and 3.

If the variables from the artificial kidney in the form of electrical Signals are entered into the arithmetic or working circuit, the of the Electrode 13 transmitted the value of the ammonia concentration to the corresponding urea concentration in the Dlut converted and can be read directly on the recorder depending on the time been. As is apparent from Fig. 4, there is a close relationship between the after conventional methods determined urea concentration in blood and the corresponding word determined by the device according to the invention. As an ammonia electrode is used, the following applies: In [NH3] = 2.303 [pH] + C; Considered If one assumes that the ammonium is produced by the decomposition of urea, then this equation can be written also write as follows: In [urea] = 2.303 [pH] + C '; On the other hand changed the urea concentration is theoretically based on the following equation: [urea] t = [Urea] @ e-kt; or in 7urea, 7 = -kt + a; t Obtained through substitution one finally has the following equation: 2.303 AP2t = -kt - a '; in the C, O ', a and a' Constants are, t means time and In means the natural logarithm.

If the pH value (the electrode potential) is plotted against time, thus the linear relationship shown in Fig. 5 is obtained.

The slope of this straight line corresponds to the dialysis speed. The points at which the dialysis conditions [pH] t = -kt are changed are marked with Marked with arrows. Thus it is not only possible to change the dialysis conditions but one can determine by continuously measuring and recording the Urea concentration in the dialysate or ultrafiltrate as well the dialysis endpoint foresee.

Furthermore, it is also possible to use appropriate devices to provide warning signals to give up if there are sudden changes in dialysis conditions or if dialysis comes to a standstill. Furthermore, the dialysis speed can be entered by entering of the electrical signal generated by this device into the artificial kidney regulated so that the possibility of imbalance is reduced.

According to one embodiment of the device according to the invention the vaccination trap 1 is replaced by a sampler that takes samples of dialysate or ultrafiltrate, that comes from several artificial kidneys takes. The three-way valve is with the Sampler connected, the information corresponding to the respective samples will be in this case nods via the label 18, but via other appropriate devices transferred to the artificial kidneys. Thus, it is possible continuously and to carry out urea analyzes on a number of patients at the same time Device can also be used to monitor and measure the amounts of creatinine, ilarnic acid, Glucose, amino acids and the like that get into the dialysate or ultrafiltrate, be used. In these cases, immobilized creatininase (E.C. 3.5. 4.21), uricase, glucose oxidase, amino acid oxidase, oxygen or ammonia electrodes used in the appropriate combinations.

The monitoring device according to the invention can be used in any desired manner artificial kidneys based on dialysis, ultrafiltration or dialysate-ultrafiltrate recirculation be applied. In addition to the use on dialyzers or ultrafilters of artificial The device according to the invention can also be used for kidney monitoring of dialysates or ultrafiltrates from small analytical dialyzers or ultrafilters that connected to the bloodstream.

As already explained, the device according to the invention enables Continuous eating and recording of the concentrations of urea, creatinine, Uric acid, glucose and amino acids in the blood by looking at the concentrations of these Substances in the dialysate or ultrafiltrate after reaction with immobillsierton euzymes determined in appropriate columns, the concentrations of the substances mentioned in the Dialysate or ultrafiltrate are potsntiometrically arranged in flow cells Blektrodan for sure. The values obtained in this way are determined by means of a working or Calculation circuit converted into the corresponding blood values and then recorded.

Accurate and quick measurements are thus possible. In addition to the Information on the concentrations of Urineff and other metabolic products in the blood and the rates of dialysis and ultrafiltration of these substances (Variables that are necessary to determine the course of the dialysis) it is also possible to experience sudden changes in dialysis or ultrafiltration conditions and to decide when the dialysis is due at the bottom of the patient. Through this This results in less stress for doctors and patients.

In the method according to the invention, it is not necessary to test blood to be taken, so that a disquiet of the patient and corresponding pain be avoided. This is due to the fact that, according to the invention, a measurement in the dialysate or ultrafiltrate, which was previously discarded, is possible without the function of the artificial kidney is impaired.

Finally, the device according to the invention, its dimensions are very low, even in the patient's home or in small hospitals be used. Large hospitals can use a variety of monitoring devices can be checked at the same time when the scribes are set up in a room will.

In the case of the errors shown in FIGS. 2 and 3, the artificial kidney CD-1 from Nikkisch Co., Inc. and the dialysis instrument HFAK-M-4 (Hollow fiber type artificial kidney) made by Cordis Dow Co. was used.

In the case of FIg. 2 is the value QD (flow velocity of the Dialysate 250 ml and in Fig. 3 the value QB (flow rate of the blood) 200 ml. If the experiments are repeated using the artificial kidney CD = 1 from Nikkisoh Co., Inc., and the dialyzer EX-23 (artificial kidney from Spiral type) from Extracorporeal Co. with the values QD = 500 ml and QB = 200 ml, similar results are obtained.

The following experiments illustrate the invention.

V e r s u c h 1 There are clinical trials with an inventive Device for monitoring artificial kidneys carried out.

For this purpose, urease immobilized on poly-methyl-D-glutamate is used.

The examinations are carried out according to the flow chart of FIG. In the period from September 1974 to February 1975 he was in the dialysis center of Toranomom General Hospital monitored 23 patients during dialysis treatment.

The column is with 2.5 ml of immobilized urease with urease activity packed by 20 U. The urease is immobilized using the azide method and attached to poly γ-methyl-D-glutamate, which is applied to glass beads with a mesh size of 50 to 80 is fixed.

The coating is carried out by making a solution of 6 parts Poly-γ-methyl-D-glutamate, 3 parts polyurethane diisocyanate and 1 part epichlorohydrin used in chloroform as a solvent.

One is supplied by Ajinomote Co., Inc. in August 1974 artificial kidney used. The clinical examinations will be carried out on 10 patients during the whole of September 1 974 and a; 'i the other 13 patients during 40 Days from the end of January to the end of February 1975. The monitoring device remains unused at room temperature from October 1974 to January 1975 to be able to determine the aging of the urease.

9 dialysers of the spiral type, 10 of the hollow fiber type and 4 of the modified standard high-fiber type used.

The phosphate buffer and sodium hydroxide solutions are used every 2 weeks freshly made. The hoses in the hose pump are changed every month. The electrolyte solution and the electrodes' membranes are also changed every month.

The immobilized urease is not during the entire duration of the experiment exchanged.

The urea concentration of the outflowing dialysate can be changed with Using the monitoring device according to the invention quickly and accurately continuously determine. It can also be confirmed that the urea concentration in the dialysate agrees well with the concentration of urea in the blood. The device can thus use it to display important factors in dialysis treatment. the Dialysis conditions, such as the flow rate, can be based on the determination according to the urea concentration in the dialysate. Accordingly the end of dialysis can also be easily determined.

The monitoring device works for the entire duration of the experiment with success. During the shutdown period, bacterial growth will occur in the with the immobilized enzyme packed column detected. However, this reduces the enzyme activity hardly affected. By changing the flow rate of the samples through the Column no difficulties arose.

Heretofore, artificial kidneys were generally used for a predetermined one Operated for a period of time. The condition of the patient is done through blood tests Supervised. Since the examinations of the blood analysis are generally only a half Day later, the dialysis conditions were generally based on experience determined by the attending physician. When using the monitoring device according to the invention Not only does it eliminate blood loss through frequent blood tests, but it does can also weaken the physical condition of the patient due to excessive Dialysis should be avoided.

In the method according to the invention, the immobilized by polyglutamate plays Urease plays an important role. A specific reaction with the urea, a constant one Reactivity of the enzyme and a rapid response are essential for the success of the invention Procedural important. The urease used in the experiments described here is stable and highly active and thus fulfills the aforementioned requirements.

Experiment 2 Artificial kidney: CD-1 from Nikkisoh Co., Inc.

Dialyzer: EX-23 (artificial kidney of the spiral type) from Extracorporeal Co. and HFAK-M-4 (hollow fiber type artificial kidney) made by Cordis Dow Co.

4 patients with renal insufficiency are each using a device treated by type EX-23. 4. other patients who also have renal insufficiency are treated with an HFAK-M-4 device.

The values for the urea concentration in the blood of the patients are determined according to the invention. In doing so, the outflowing points in time are taken into account Individual dialysate samples with a volume of 1 to 2 ml are taken. The dialysis conditions (F1, F2 and F3) are stored according to the individual samples during dialysis in the Calculation cycle set. The values are printed out with the printer.

Furthermore, the patient is treated simultaneously with the during dialysis Samples of the outflowing dialysate, blood samples taken and their urea concentration examined. The results are compiled in FIGS. 6 and 7. In Fig.

10 shows the flow diagram of this experiment.

Claims (2)

P a t e n t a n s p r ü c h e 1. Method for the determination of urea in the blood by using an artificial The patients treated here with renal insufficiency, d u 1 e n g e n n n notices that (1) the dialysate flowing out of the artificial kidney brings into contact with immobilized urease, whereby by reaction of the in this Pialysate contained urea with the urease ammonium ions are formed, (2) the ammonia, which after setting an alkaline pH in that of. the Urease is formed with an ammonia gas electrode in a separated solution Flow cuvette determines (3) to) the Ilarrlstoff concentration in the blood from the with the help of the electrode measured urea concentration in the dialysate and certain, dialysis-related factors are calculated with the help of a computer circuit and (4) the value of the urea concentration with the help of a pen and / or printer records. 2. Apparatus for performing the method according to claim 1, g e k e n n n e c h n e t by (I) a column packed with immobilized urease, where the dialysate flowing out of the artificial kidney with the immobilized urease is brought into contact, (II) one arranged in a flow cell Ammonia gas electrode for the continuous determination of ammonia by setting the solution passed through the column is formed to an alkaline pH value, (III) a computing circuit to determine the urea concentration from the electrode release voltage in the blood and certain factors related to dialysis and (IV) one Schreiber ufld / odev printer for recording the calculated values