Ionization Chamber Method for Continuous Detection of Pyruvate-1-¹⁴C and Acetate-1-¹⁴C Oxidation in Normal and Thiamine-Deficient Rats
Ngo Manh Tran

Donner Laboratory of the Lawrence Radiation Laboratories, University of California, Berkeley

          Despite the availability of considerable biochemical information concerning the functions of thiamine, the effective early diagnosis of beri-beri awaits the development of simple, rapid techniques for screening populations for the metabolic defects attendant to this vitamin-deficiency state. The present work is directed toward development of such techniques.

         An in-vivo ionization chamber method was used for continuous measurement of ¹⁴CO₂ production in the breath of normal rats and thiamine-deficient rats following intravenous

administration of #1-¹⁴C-labeled pyruvate and acetate.

   The ionization chamber device consists of the following described components. Compressed air is passed through the animal cage containing the rat at a constant flow rate determined by a precision flow regulator. Air mixed with expired gases from the rat exits from the animal cage and passed through a water absorber. Subsequent to removal of water, the gases are serially passed through a 0.377-liter ionization chamber containing a vibrating-reed electrometer, an infrared carbon dioxide analyzer, and a paramagnetic oxygen analyzer. Continuous graphical plotting of the ¹⁴CO₂, CO₂, and O₂ data was achieved by using a multichannel chart recorder.

   Twenty-seven male Buffalo rats (Simonsen Laboratory, Gilroy, California) were used in ¹⁴CO₂ experiments. The diet of the control rats had the following composition expressed as percentages: vitamin-free casein, 20.0; sucrose, 67.5; cotton-seed oil, 5.0; UCB-Irb salts, 5.5; choline bitartrate, 1.22; vitamins A, D, E premix, 1.0; and vitamins B premix, 2.0. The deficient diet contained the same formula as above except that it had no thiamine. The experiments were conducted 14 to 20 days after initiation of these diets, when the thiamine-deficients rats showed weight loss and generalized anesthesia.  

   Results in ¹⁴CO₂ studies show that approximately 65% of ¹⁴C administered as pyruvate-1-¹⁴C appears in the breath within 60 minutes (comparable to the amount obtained following administration of H¹⁴CO₃  about 61%), providing confirmation that in vivo pyruvate metabolism occurs almost exclusively via decarboxylation to acetyl CoA (a metabolic step requiring the presence of thiamine pyrophosphate). On the contrary, only approximately 53% of ¹⁴C administered as acetate-1-¹⁴C appears in the breath as ¹⁴CO₂ within 60 minutes, suggesting that a small but measurable amount of acetate is fixed in more slowly catabolized compounds (e.g., fatty acids) as opposed to direct oxidation to CO₂ in the TCA cycle.

   For comparison of ¹⁴CO₂ production curves, two parameters were used: the first parameter is the slope of the ¹⁴CO₂ production curve, expressed as T_1/2 in min, and the second parameter is the integral amount of ¹⁴C administered in the first 60 minutes, expressed as percent. Numbers of animals in each group were noted in parentheses.

   There is a significant delay in oxidation of #1-¹⁴C-pyruvate in  thiamine-deficient rats (7) with T_1/2 ± S.E. = 19.678 ± 0.241 (p < 0.01) and ¹⁴C cumulative ± S.E. = 42.9687 ± 4.1139 (p < 0.01) as compared to normal rats (6) with T_1/2 ± S.E. = 10.728 ± 0.222 and ¹⁴C cumulative ± S.E. = 64.8095 ± 2.2642. Similarly, there is a significant delay in oxidation of #1-¹⁴C-acetate in thiamine-deficient rats (3) with T_1/2 ± S.E. = 27.530 ± 1.497 (p < 0.01) and ¹⁴C cumulative ± S.E. = 46.720 ± 0.683 (p < 0.01) as compared to normal rats (3) with T_1/2 ± S.E. = 18.474 ± 0.593 and ¹⁴C cumulative ±  S.E.= 52.233 ± 4.193.

   Within 2 days after intramuscular injection of thiamine hydrochloride into previously thiamine-deficient rats (7), the ¹⁴CO₂ appearance curves following injection of #1-¹⁴C-pyruvate return to normal limits with T_1/2 ± S.E. = 12.317 ± 0.3910 (p > 0.05) and ¹⁴C cumulative in 60 min ± S.E.= 57.1520 ± 3.4390 (p > 0.05). Similarly, 45 minutes after intravenous injection of thiamine hydrochloride into previously thiamine-deficient rats (3), the ¹⁴CO₂ appearance curves following injection of acetate-1-¹⁴C return to normal limits with T_1/2 ± S.E. = 19.184 ± 0.477 (p > 0.05) and ¹⁴C cumulative ± S.E. = 55.645 ± 3.213 (p > 0.05).

   There is no significant change in ¹⁴CO₂ production following administration of H¹⁴CO₃ in thiamine-deficient rats (4) with ¹⁴C cumulative ± S.E. = 68.8246 ± 2.2626 as compared to normal rats (4) with ¹⁴C cumulative ± S.E. = 61.1227 ± 3.0906 (p > 0.05). This result shows that the differences in ¹⁴CO₂ appearance in thiamine-deficient animals given pyruvate or acetate-1-¹⁴C are not due to the alterations of the bicarbonate pool produced by the thiamine deficiency.

   The overall results of these studies suggest the possibility of diagnosis of thiamine deficiency in man by determination of ¹⁴CO₂ appearance in the breath after administration of #1-¹⁴C-pyruvate or acetate prior to, and subsequent to, the administration of thiamine. Furthermore, the ionization chamber method provides an useful tool for future studies of metabolic disorders in vivo.

References:

1) Ngo Manh Tran, Vietnamese American Medical Research Foundation, Exhibition, 2002

2) Ngo Manh Tran, H.S. Winchell, M.A. Williams, and N.N. Finley: Decased 14CO2 Production in Thiamine-Deficient Rats Given 1-14C-Pyruvate and Acetate: A Possible Means for Early Diagmosis of Beri-Beri?, J. Nuclear Med., 10: 676-682, 1969

Ngo Manh Tran, M.D., Ph.D.

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