J. P. Jasper¹, T. M. Schelhorn², and J. L. Treadway^2
1Molecular Isotope Technologies, LLC, 8 Old Oak Lane, Niantic CT 06357-1815. ²Pfizer Inc., Central Research Division, Groton, CT 06340.

Indirect calorimetry has been performed by a variety of methods, including accumulation of CO² etc. (Tanis et al., 1998) from a variety of organisms. It is believed to be a useful base-line technique for evaluating the efficacy of pharmaceuticals on test organisms. Recently, the stable isotopic composition of exhaled CO² in human breath was used to measure the time-varying consumption of subjects’ glycogen (Tanis et al., 1998). The time-variation was taken to be an index of the subject’s gross metabolism. We suggest that stable isotopic indirect calorimetry of a mouse may be undertaken in an in vivo sample production system.

The design and function of a stable isotopic indirect calorimeter (SIIC) for mice is described here. The SIIC is a nearly atmospherically-benign (pCO² = 200-450 ppm) system for the extraction of mouse-produced carbon dioxide (CO²) for the indirect calorimetry of metabolism. Atmospheric air is pumped (at ~4.8 l/min) through a CO² scrubber to remove CO². The CO²-free air continues into a mouse chamber where the rate of incoming-air dilution by the mouse’ metabolic CO² efflux determines the effluent pCO². The effluent gas, containing mouse-sourced CO², is advected to an open split where the gas flow is reduced from ~4800 ml/min to ~28 ml/min. A capillary flow restrictor combined with a downstream, low-vacuum pump decrease the pressure downstream of the open split from 1.0 atmosphere to ~0.85 atmosphere, permitting separation of atmospheric gases (N², O², Ar etc.) during cryogenic distillation of the biologically-derived CO². The distilled CO² is then ampoulated for stable isotopic analysis.

The SIIC is composed of two major parts: a Carbon Dioxide Production Unit (CPU) and a Carbon Dioxide Extraction Unit (CEU). Mouse-produced CO² is released into a CO² -free air stream in the CPU. Approximately 0.6% of the gas stream emanating from the CPU is transferred to the CEU via the open split. With a flow rate of ~28 ml/min, 2 to 3 µmol of CO² is transferred to sample ampoules in ~7 to 9 minutes. Calibration results are recorded for (i) the air flow rate though the mouse chamber, (ii) the flow rate as determined by the flow restricting capillary, and (iii) the volume of the volumetric calibration tee. The flow rate of the capillary leak is highly predictable, indicating the possibility to either exchange or to vary the capillary column in the SIIC as flow rates in the CEU require. Another useful variable in the system is the CO² mass flux emanating from the mouse chamber (Rm). Experimentation shows that with an ob/ob mouse and a CPU flow of ~4.8 l/min that the mouse’ Rm decreased from ~250 to 68 mmol CO² in ~8 minutes and thereafter remained approximately constant. Initial assessment indictes that sample collection at may begin at ~8 minutes. Since the pCO²is inversely proportional to the CO²-dilution rate by CO²-free air, adjustment of the dilution rate (relative to the mouse’s production rate) permits determination of the pCO² of the effluent air, a critical variable in determining the mouse’s atmospheric pCO² environment and the CO² sampling time.

The Stable Isotope Indirect Calorimeter was first used to obtain baseline data for metabolic parameters in the ob/ob mouse. A dietary regimen for hepatic glycogen loading was established, consisting of 48 hr of fasting followed by 16 hr of refeeding with a naturally ¹³C-enriched corn syrup diet. Liver glycogen repletion was confirmed by biochemical analysis as described by Hassid and Abraham (1959). Mice were placed into the SIIC, where their biologically-derived CO² was continuously monitored (pCO²) and intermittently collected (~30 min intervals). Results of stable carbon isotopic (¹³C/¹²C) analyses showed that mice metabolized stored hepatic glycogen-carbon in a time-dependent manner, spanning from typical C⁴ carbon-isotopic values ( ¹³C-CO² = -12‰ vs VPDB) to C³ values (-25‰) in ~4 + 1 hr (or ~3.3‰/hour). These data formed the baseline against which the effect of pharmaceutical agents on mice will be compared in future experiments. In summary, we have developed a Stable Isotope Indirect Calorimeter that can be used to quantitatively determine the effects of either dietary manipulation or pharmaceutical agents by a non-invasive, in vivo mouse-metabolic method.