Method for isolating urea while removing objectionable CO2

Abstract

A method for isolating urea and removing CO.sub.2 from plasma samples, comprising the following steps: a) providing a plasma sample; b) adding an acid so as to partially remove CO.sub.2; c) lyophilizing the sample so as to further remove CO.sub.2 and obtain a dried sample; and d) redissolving the dried sample and neutralizing to a pH value of 4 to 7 using a buffer solution, wherein optionally a filtration step is carried out before adding the acid.

Claims

1. A method for determining the .sup.13C isotope ratio of urea in a plasma sample, comprising the following steps in the following order: a) collecting a plasma sample from a person to whom a .sup.13C-labeled urea precursor has been administered; b) precipitating material comprising proteins and lipids from the plasma sample, then separating the precipitated material from the plasma sample by filtration, and then adding an acid to the plasma sample, whereby CO.sub.2 is partially removed from the plasma sample; c) lyophilizing the sample treated in step b), whereby residual CO.sub.2 is removed from the sample and a dried sample is obtained; d) redissolving the dried sample, thereby obtaining a sample solution, neutralizing the sample solution with a buffer solution to a pH value of 5 to 6; e) degassing the sample solution at reduced pressure to remove CO.sub.2 introduced into the sample solution by the buffer solution so that as a result of steps b) through e), the sample solution is CO.sub.2-free as determined by isotope ratio mass spectrometry (IRMS); f) rinsing the sample solution with inert gas; g) adding urease to the rinsed sample solution so as to produce CO.sub.2; h) incubating the sample solution after urease has been added thereto; i) adding an acid to the incubated sample solution so as to release CO.sub.2 that has been produced in the incubated sample solution; and j) measuring the .sup.13C isotope ratio of the released CO.sub.2.

2. The method according to claim 1, wherein, in steps b) and i), the acid is phosphoric acid.

3. A method according to claim 1, wherein, in step d), the buffer solution is phosphate buffer solution having a pH of 9.0.

4. The method according to claim 1, wherein the plasma sample collected in step a) is of a volume of 0.3 ml, and in step g), 20 to 100 units of the urease are added to the rinsed sample solution.

5. A method for diagnosing urea metabolism in a person, comprising: determining, for each of a plurality of plasma samples collected from the person, the .sup.13C isotope ratio in the plasma sample by the method of claim 1, wherein each of the blood samples collected after a first of the blood samples is collected, is collected at a predetermined time interval after the previous blood sample has been collected.

6. The method according to claim 5, wherein the plasma samples are collected over a time period of 15 to 240 minutes.

7. The method according to claim 5, wherein the predetermined time interval is 15 minutes.

8. The method according to claim 1, wherein the plasma sample collected in step a) is of a volume 0.2 to 0.3 ml.

9. The method according to claim 1, wherein the degassing of the sample solution in step e) is effected by applying to the sample solution for 2 to 3 hours a vacuum in a range of 1 to 10 mbar.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the reproducibility of the method according Example 3.

(2) FIG. 2 shows the calibration curve of the spiking experiment according to Example 4.

(3) FIG. 3 shows the reproducibility of the method according to Example 5.

(4) FIG. 4 shows the calibration curve of the spiking experiment according to Example 6.

(5) FIGS. 5 and 6 show the results of the measurement of the urea production according to Example 7.

DETAILED DESCRIPTION OF THE INVENTION

(6) The method will be described in more de ail by way of the following examples.

Example 1: Urea Isolation Using Filtration

(7) Plasma was obtained from a blood sample. 300 l plasma was diluted with 200 l deionized water. 100 l acetonitrile containing 1% formic acid was added to the sample. A precipitation formed in the vessel. The samples were filtered using a HybridSPE column.

(8) The filtrate was mixed with 50 l M phosphoric acid, frozen and lyophilized. The sample was adjusted to a pH value of 5.5 using a degassed 0.5 M phosphate buffer solution, pH 9. The sample container (Vacutainer) was rinsed with helium gas. Thereafter, 70 l of a solution containing 15 mg/ml Jack Bean Urease Type III from Sigma in phosphate buffer was added. The sample was incubated for one hour at 36 C. Thereafter, 60 l 20% phosphoric acid was injected through the septum so as to stop the urease reaction and release CO.sub.2. The released CO.sub.2 was used to determine the isotope ratio by means of IRMS.

Example 2: Urea Isolation Using No Filtration

(9) The method as in Example 1 was carried out, however the addition of acetonitrile and formic acid, and the filtration step were dispensed with, which is to say the plasma sample was directly lyophilized after acid was added. The remainder of the method was carried out in identical fashion.

Example 3: Reproducibility of the Method

(10) A plasma sample was divided into five samples, which were each subjected separately from each other to the method according to Example 1. Each CO.sub.2 sample that was obtained was measured five times. The differences are minimal; refer to FIG. 1.

Example 4: Spiking Experiment

(11) 99% labeled .sup.13C urea was added to the plasma samples from Example 1 in quantities of 0.01 mg, 0.25 mg, 0.5 mg, 0.1 mg, 0.2 mg, and 0.3 mg, and the sample was treated in accordance with method 1. FIG. 2 shows the corresponding measurement values. The calibration curve is located on a straight line with a correlation coefficient of 0.99923.

Example 5: Reproducibility

(12) The measurement regarding reproducibility according to Example 3 was repeated for the method using no filter according to Example 2. FIG. 3 shows the results. Again, excellent reproducibility exists.

Example 6: Spiking Experiment

(13) The spiking experiment according to Example 4 was repeated, wherein the method according to Example 2 was employed. The corresponding straight calibration line is apparent from FIG. 4. The coefficient of correlation thereof was R=0.99959.

Example 7: Measurement of the Urea Production

(14) Blood was collected from two subjects, and from 300 l of the plasma that was obtained from each of the blood samples, urea was isolated using the method according to the invention, and the .sup.13C/.sup.12C isotope ratio of the urea was determined.

(15) Each sample was measured 5 times and the basal value was determined by way of the average value. The subjects were then administered 27 mg/kg 99% .sup.13C-labeled Na-acetate.

(16) The procedure of collecting blood and determining the .sup.13C/.sup.12C isotope ratio of the isolated urea was repeated at 15-, 30-, 45-, 60-, 75-, 90-, 120-, 180-, and 240-minute intervals. A smaller portion of the .sup.13C-labeled acetate is converted into urea in the body and can be detected because of the sensitivity of the measurement. The kinetics of the newly produced urea was determined by measuring the delta values (increasing the .sup.13C/.sup.12C isotope ratio). The kinetics of the urea production is shown in FIGS. 5 and 6.