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The determination of total blood CO2 and its specific radioactivity in 0.2-milliliter blood samples
ANALYTICAL
BIOCHEMISTRY
The Determination Radioactivity
76, 358-360
(1976)
of Total Blood CO, and its Specific in 0.2-Milliliter Blood Samples
This method was developed to determine CO, and its specific radioactivity and to allow the subsequent analysis of glucose, lactate, and other acid-stable compounds in 0.2-ml samples of rat blood. It compares in accuracy and reproducibility with earlier methods (1,2) but requires less blood. It has been adapted for aceto-acetate determinations by decarboxylation (3) and should be applicable to any estimation of small amounts of CO, and its specific radioactivity. The small scale requires special care to be taken with blood sampling and some operations to be carried out in a nitrogen atmosphere in a glove box. Also, a more convenient seal for the vials was used. Otherwise the method is essentially a scaled-down version of earlier methods (1,2). A standard glass scintillation vial was used for CO, transfer, closed with a Suba-seal stopper (No. 33, Scientific Supplies, London) and containing a glass tube, 35 x 12 mm, holding the trapping agent. This tube was fitted with a polyethylene collar, 1 mm thick, to stop “creep” of the blood extract, An aliquot (0.3 ml) of barium hydroxide (about 0.08 M, containing thymolphthalein indicator and stored under light liquid paraffin) was added to each tube and the vials were immediately sealed. Four were reserved as controls. Blood was added to the rest. Blood was allowed to flow without suction from a cannulated blood vessel directly into a 0.2-ml constriction pipet. This procedure gave more reproducible CO, values than when blood was first collected under light liquid paraffin, from which 0.2-ml samples could only be obtained using suction. The blood was blown gently into the vial using a small teat and the seal was immediately replaced. The vial was cooled in ice to reduce the internal pressure, and perchloric acid (0.8 ml, 1 M) was injected from a syringe through a stopcock (K-75, Pharmaseal Labs., Glendale, Calif.,) and needle. The stopcock was then closed and the needle removed. The blood and acid were mixed by swirling. The vial was shaken for 2-3 hr in a Dubnoff shaker at room temperature and left overnight to complete the transfer of CO, to the trap. Transfer thus took about 18 hr instead of the 48 hr needed in the method of Hinks et al. (1). The trap was then removed from the vial in a glove box filled with cylinder nitrogen. The outside of the trap was washed with deionized water into the vial, the contents of which could then be used for other analyses. Titration was carried out in the glove box with 0.1 M HCl delivered 358 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
COMMUNICATIONS
359
from an Agla micrometer syringe (Scientific Supplies), calibrated by weighing, through a size 17 Luer disposable syringe needle (external diameter 0.5 mm) bent through 90”. The contents of the trap were stirred with a slow stream of nitrogen delivered through a syringe needle (22 gauge, l%A Luer, external diameter 0.7 mm, Sherwood Medical Instruments Ltd., Middx.). The controls were treated identically. The CO2 content was calculated from the differences between the individual titers of the blood samples and the average titer of the four controls (4). After titration the contents of the trap were transferred to a scintillation vial with a Pasteur pipet. Residues were washed in with 0.8 ml of 5% (w/v) EDTA (diaminoethane-tetraacetic acid, disodium salt) in 0.5 M Tris [tri( hydroxymethyl)methylamine] buffer. pH 9, to solubilize the barium carbonate precipitate (1). This was followed by 2 x 6 ml of scintillator [toluene:Triton X-100, 21 by vol, containing 4 g/liter of 2,5-diphenyloxazole (PPO) and 100 mgiliter of 1,4-di-(2(5-phenyloxazolyl))benzene (POPOP)]. Since about 10%’ of the barium carbonate adhered to the bubbler needle. this also was placed in the vial. The contents of the vial were mixed by a Whirlimixer to ensure complete solubilization of the barium carbonate precipitate. The final scintillation mixture contained 1.4 ml of water, which produced a clear, stable solution for counting at 4-5°C. When only count rates were required, omitting the titration. an equivalent amount of water was added. Although transfer of the sample after titration could be simplified and carried out in the glove box using Fox’s method (3). the count rates were less reproducible. In the method of Hinks ef ul. (1) no transfer of the sample was necessary, but adequate mixing of the small volumes of barium hydroxide during titration was difficult and the blood extract was unavailable for further assay. Count rates were determined at 4-5°C using an automatic liquid scintillation counter, Series 4000. and the efficiencies determined using [‘“Cltoluene standards (both from Packard Instrument Ltd., Wembly). Reproducibility was investigated on standard bicarbonate solutions containing NaH’“CO,, (The Radiochemical Centre. Amersham, Bucks.). Recoveries of total CO, were 99.7 2 1.5% (mean 2 SD. 24 samples). The label in the standard was estimated in two ways which gave identical disintegrations per minute. The standard (0.2 ml) was added to 0.3 ml of barium hydroxide to which was added the EDTA-Tris solution and the scintillator described above or to ethanolamine (0.16 ml) to which was added 10 ml of scintillation fluid ( I ,4-dioxane:toluene:Z-ethoxyethanol, 3: 1: 1 by vol, containing 80 g/liter of naphthalene and 10 g/liter of PPO). Recovery of label by the full procedure was 99.0 & 1.8%’ (mean +- SD, 20 samples). Specific radioactivity, when determined, was 99.1 -+ 1.2% (mean ? SD, 16 samples) of that in the standards. Recoveries of ‘-‘C from NaH’%O:, added to blood were 99.7 r 1.0% (mean ?I SD.
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6 samples) without titration and 98.2 +- 1.4% (mean + SD, 7 samples) with titration. The method was compared with that of Hinks rt nl. (1). which required 1 ml of blood, by analyzing three specimens of blood for total CO,, in triplicate or quadruplicate. by both methods. The mean values obtained by theirs were 96.7, 99.2, and 97.0% of those by mine. The coefficient of deviation of replicates was 1.1% in their method and 1.8% in mine. REFERENCES 1. 2. 3. 4.
Hinks, N. T.. Mills. S. Fox, R. M. (1971) Anal. Passingham, B. J.. and Conway, E. J. (1947) pp. 189-200. Crosby
C.. and Setchell. B. P. (1966) Alla/. Biochem. 17, 551-553. Biochem. 41, 578-589. Barton, R. N. (1975)Anul. Biochrm. 65, 418-421. Microdiffusion Analysis and Volumetric Error, Revised Lockwood, London.
J. G. Experimental Pathology qf Trauma Section MRC Toxicology Unit Medical Research Council Laboratories Woodmansterne Road Carlshnlton. Surrey SM5 4EF. England Received January 27. 1976: accepted June 30. 1976
ed..
ROSE
BIOCHEMISTRY
The Determination Radioactivity
76, 358-360
(1976)
of Total Blood CO, and its Specific in 0.2-Milliliter Blood Samples
This method was developed to determine CO, and its specific radioactivity and to allow the subsequent analysis of glucose, lactate, and other acid-stable compounds in 0.2-ml samples of rat blood. It compares in accuracy and reproducibility with earlier methods (1,2) but requires less blood. It has been adapted for aceto-acetate determinations by decarboxylation (3) and should be applicable to any estimation of small amounts of CO, and its specific radioactivity. The small scale requires special care to be taken with blood sampling and some operations to be carried out in a nitrogen atmosphere in a glove box. Also, a more convenient seal for the vials was used. Otherwise the method is essentially a scaled-down version of earlier methods (1,2). A standard glass scintillation vial was used for CO, transfer, closed with a Suba-seal stopper (No. 33, Scientific Supplies, London) and containing a glass tube, 35 x 12 mm, holding the trapping agent. This tube was fitted with a polyethylene collar, 1 mm thick, to stop “creep” of the blood extract, An aliquot (0.3 ml) of barium hydroxide (about 0.08 M, containing thymolphthalein indicator and stored under light liquid paraffin) was added to each tube and the vials were immediately sealed. Four were reserved as controls. Blood was added to the rest. Blood was allowed to flow without suction from a cannulated blood vessel directly into a 0.2-ml constriction pipet. This procedure gave more reproducible CO, values than when blood was first collected under light liquid paraffin, from which 0.2-ml samples could only be obtained using suction. The blood was blown gently into the vial using a small teat and the seal was immediately replaced. The vial was cooled in ice to reduce the internal pressure, and perchloric acid (0.8 ml, 1 M) was injected from a syringe through a stopcock (K-75, Pharmaseal Labs., Glendale, Calif.,) and needle. The stopcock was then closed and the needle removed. The blood and acid were mixed by swirling. The vial was shaken for 2-3 hr in a Dubnoff shaker at room temperature and left overnight to complete the transfer of CO, to the trap. Transfer thus took about 18 hr instead of the 48 hr needed in the method of Hinks et al. (1). The trap was then removed from the vial in a glove box filled with cylinder nitrogen. The outside of the trap was washed with deionized water into the vial, the contents of which could then be used for other analyses. Titration was carried out in the glove box with 0.1 M HCl delivered 358 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
COMMUNICATIONS
359
from an Agla micrometer syringe (Scientific Supplies), calibrated by weighing, through a size 17 Luer disposable syringe needle (external diameter 0.5 mm) bent through 90”. The contents of the trap were stirred with a slow stream of nitrogen delivered through a syringe needle (22 gauge, l%A Luer, external diameter 0.7 mm, Sherwood Medical Instruments Ltd., Middx.). The controls were treated identically. The CO2 content was calculated from the differences between the individual titers of the blood samples and the average titer of the four controls (4). After titration the contents of the trap were transferred to a scintillation vial with a Pasteur pipet. Residues were washed in with 0.8 ml of 5% (w/v) EDTA (diaminoethane-tetraacetic acid, disodium salt) in 0.5 M Tris [tri( hydroxymethyl)methylamine] buffer. pH 9, to solubilize the barium carbonate precipitate (1). This was followed by 2 x 6 ml of scintillator [toluene:Triton X-100, 21 by vol, containing 4 g/liter of 2,5-diphenyloxazole (PPO) and 100 mgiliter of 1,4-di-(2(5-phenyloxazolyl))benzene (POPOP)]. Since about 10%’ of the barium carbonate adhered to the bubbler needle. this also was placed in the vial. The contents of the vial were mixed by a Whirlimixer to ensure complete solubilization of the barium carbonate precipitate. The final scintillation mixture contained 1.4 ml of water, which produced a clear, stable solution for counting at 4-5°C. When only count rates were required, omitting the titration. an equivalent amount of water was added. Although transfer of the sample after titration could be simplified and carried out in the glove box using Fox’s method (3). the count rates were less reproducible. In the method of Hinks ef ul. (1) no transfer of the sample was necessary, but adequate mixing of the small volumes of barium hydroxide during titration was difficult and the blood extract was unavailable for further assay. Count rates were determined at 4-5°C using an automatic liquid scintillation counter, Series 4000. and the efficiencies determined using [‘“Cltoluene standards (both from Packard Instrument Ltd., Wembly). Reproducibility was investigated on standard bicarbonate solutions containing NaH’“CO,, (The Radiochemical Centre. Amersham, Bucks.). Recoveries of total CO, were 99.7 2 1.5% (mean 2 SD. 24 samples). The label in the standard was estimated in two ways which gave identical disintegrations per minute. The standard (0.2 ml) was added to 0.3 ml of barium hydroxide to which was added the EDTA-Tris solution and the scintillator described above or to ethanolamine (0.16 ml) to which was added 10 ml of scintillation fluid ( I ,4-dioxane:toluene:Z-ethoxyethanol, 3: 1: 1 by vol, containing 80 g/liter of naphthalene and 10 g/liter of PPO). Recovery of label by the full procedure was 99.0 & 1.8%’ (mean +- SD, 20 samples). Specific radioactivity, when determined, was 99.1 -+ 1.2% (mean ? SD, 16 samples) of that in the standards. Recoveries of ‘-‘C from NaH’%O:, added to blood were 99.7 r 1.0% (mean ?I SD.
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COMMUNICATIONS
6 samples) without titration and 98.2 +- 1.4% (mean + SD, 7 samples) with titration. The method was compared with that of Hinks rt nl. (1). which required 1 ml of blood, by analyzing three specimens of blood for total CO,, in triplicate or quadruplicate. by both methods. The mean values obtained by theirs were 96.7, 99.2, and 97.0% of those by mine. The coefficient of deviation of replicates was 1.1% in their method and 1.8% in mine. REFERENCES 1. 2. 3. 4.
Hinks, N. T.. Mills. S. Fox, R. M. (1971) Anal. Passingham, B. J.. and Conway, E. J. (1947) pp. 189-200. Crosby
C.. and Setchell. B. P. (1966) Alla/. Biochem. 17, 551-553. Biochem. 41, 578-589. Barton, R. N. (1975)Anul. Biochrm. 65, 418-421. Microdiffusion Analysis and Volumetric Error, Revised Lockwood, London.
J. G. Experimental Pathology qf Trauma Section MRC Toxicology Unit Medical Research Council Laboratories Woodmansterne Road Carlshnlton. Surrey SM5 4EF. England Received January 27. 1976: accepted June 30. 1976
ed..
ROSE