BIOCHEMICAL

MEDICINE

12, 376-379 (1975)

A Simple

Apparatus

14C02 NEELAKANTAN

for

Activities

VAIDYANATH

Determining

in Blood AND RAPIER H. MCMENAMY~

Departments of Biochemistry and Surgery, State University of New York at Buffalo. Buffalo, New York 14214 Received December 2, 1974

The measurement of 14C0, produced from oxidation of 14C labeled substrates is an index of their utilization for energy purpose. A simple, rapid, accurate, and inexpensive apparatus for the determination of 14C0, from blood is described. EXPERIMENTAL The apparatus and its assembly is shown in Fig. 1. Into the glass scintillation vial (Packard Instrument Company) (b) containing 1 ml of methylcellosolve-ethanolamine solution (2 : 1, v/v) was placed the upright inner vial (d) (15 x 30 mm) cut from 1 dram vial containing 0.6 ml of 1 N H,SO, and the magnetic stirring flea (e) (7 x 2 mm) (BelArt Products). The scintillation vial was then tightly closed with the serum stopper (Size 20, Sleeve Type, VWlX Scientific No. 16170-167) and polyethylene insert (c) (13 mm length, 6.25 mm i.d., 9.5 mm o.d.). Prior to use the stopper unit was soaked 15 min in 0.1 N H,SO, and dried. This latter prevents CO, absorption. The polyethylene insert provides a firm fit of the stopper with the vial. The vial and its contents were then tared, placed on a magnetic stirrer, and with rapid stirring, 0.2-0.25 gm of freshly drawn blood from a syringe injected into the inner vial via a 27 gauge needle (approximately 15 drops). The vial was left on the stirrer for one-half hour, weighed to obtain the amount of blood added, the stopper removed, the inner vial lifted, the residue from the sides of the inner vial carefully rinsed into the scintillation vial with 1 ml of methylcellosolve-ethanolamine solution, and finally 10 ml of scintillation medium of two parts of toluene and one part of methylcellosolve containing 5.5 gm/liter of 2,5 diphenyloxazole (l), added to the scintillation vial. The scintillation vial was capped and counted. The weight of blood in the vial is divided by 1.03 to obtain its volume. The pmoles COz per 1 Supported by the National Institutes of General Medical Sciences, Grant GM1.5768. 376 Copyright All rights

@ 1975 by Academic Press, Inc. of reproduction in any form reserved.

DETERMINATION

OF 14C02 IN BLOOD

377

FIG. 1. Apparatus for determining ‘TO, activity in blood. (a) Assembled apparatus. (b) Glass scintillation vial. (c) Serum stopper containing a polyethylene insert. (d) Inner vial. (e) Magnetic stirring flea.

liter of blood are used for calculations of the specific activity Substrates specific activities are also calculated in micromoles.

of CO,.

RESULTS AND DISCUSSION Studies with 14C02 in 0.01 N NaOH added directly to either the methylcellosolve-ethanolamine solution (control) or to blood in the inner vial were conducted to determine the stirring time necessary for recovery, and the precision of recovery (Fig. 2). Recovery was 100% at 30 minutes. With the nine determinations of stirring times of 30 minutes or greater (Fig. 2) the range of the determinations varied only 2%. Table 1 is a recovery study where the isotope was mixed with the blood prior to addition to the vial. Again, based on controls where the isotope was added directly to the methylcelIosolve-ethanolamine solution, recovery was 99.6% with a range of 3.5%. Increasing the amount of blood to 0.5 gm or the addition of 100 ~1 of 0.5 M NaHCO, to the blood did not affect the recovery of 14C0,. An important consideration was the rinsing of the sides of the inner vial. This must be done with the methylcellosolve-ethanolamine solution, not the scintillation fluid. The short soak of the stopper assembly in 0.1 N H,SO, is also important. It increased recovery as much as 10% in some instances and led to much more stable values. The mouth size of

378

VAIDYANATH

AND

MCMENAMY

100 -

?’

s L !$ 95 3 L

j++

4 ae SO0 STIRRING

I

I

30

SO

TIME (min)

FIG. 2. Recovery of 14C02 at different stirring times. All activities were counted for 5000 counts.

some of the scintillation vials vary and it was necessary to screen them for a snug fit with the serum stopper polyethylene insert unit. Transporting the vials to the hospital, holding the vials with the hands for injection of blood, stirring, etc., was found not to introduce appreciable errors in the tared values, but obviously one must exercise care to not alter the tared weights appreciably. It is important to allow the vials TABLE

1

RECOVERY OF '*CO, FROMBLOODAFTER30 MINUTESSTIRRING TIME Weight of isotope solution added (mgm)

cm (less background)

cpm per gm isotope solution

Percent recovery

Controls* 1 2 3 Mean

254 272 272

543 584 587

2140 2147 2159

99.6 100 100.5 100

Experiments* 1 2 3 4 5 6 7 8 Mean

253 258 261 264 264 264 267 272

544 548 561 572 575 563 559 572

2150 2127 2149 2167 2182 2137 2095 2104

100 99 100 100 101 99.5 97.5 98 99.6

* In the controls, the isotope solutions were added to the methylcellosolve-ethanolamine trap directly. In the experiments it was added to blood which was in the inner vial.

DETERMINATION

OF

14C02

IN

BLOOD

379

to reequilibrate with the environment in which they were first tared before reweighing with the blood inside. With blank vials (no blood added) the tared weights after mock manipulation never changed more than 2 mgm. Brisk stirring should be commenced in the inner vial before blood is added. Otherwise, the magnetic flea may become lodged in a clot, which sometimes forms, and proper mixing of the contents cannot be obtained. SUMMARY An apparatus and conditions are described for determining blood 14C0, activity. The equipment is easily obtained and inexpensive. Such measurements in conjunction with the total blood CO, are very valuable in steady state isotope infusion experiments where 14C labeled substrates are catabolized. REFERENCES 1. Jeffary,

H. and Alvaret,

J., Anal.

Chem.

33, 612-615

(1961).