Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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Animals, materials and blood collection

Rana sylvatica were collected from ponds around Ottawa during May and June of 1997. The frogs were maintained in containers containing damp sphagnum moss at 4°C until use. Animals were kept for a maximum of 1 month prior to use. Animals were sacrificed by pithing followed by exsanguination. Unless otherwise indicated, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). For glucose utilization experiments, blood from 4-6 frogs was collected into tubes containing a 1:2 mixture of 100 mM EDTA and Buffer A (25 mM Hepes, 120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂-H₂O, 1.0 mM NaH₂PO₄-H₂O, pH 7.4 at 25°C). For ³¹P-NMR spectroscopy, Buffer A was replaced with a phosphate-free Buffer B (25 mM Hepes, 120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂-H₂O, pH 7.4 at 25°C). Cells were washed three times and diluted to approximately 70% haematocrit. Washed cells were used immediately.

Spectroscopy

NMR spectroscopy was performed on a Brüker AM-400 spectrometer equipped with an ASPECT 3000 computer with a 16 bit analog-digital converter. Samples were held in a 5 mm probe operating at 100.6 MHz (¹³C) or 162 MHz (³¹P). Free induction decays were collected using continuous composite-pulse broad-band decoupling. For ¹³C-NMR, the pulse width was 0.9 micro s (9.2° rotation angle) and scans were in collected in 1 h blocks of 8700 scans with no relaxation delay (0.41 s/scan). For ³¹P-NMR, the pulse width was 5 micro s (37.5° rotation angle) and scans were collected in 1 h blocks of 3500 scans with a 0.2 s relaxation delay (0.82 s/scan). Data was analyzed using the software provided by the instrument.

To measure ¹³C-glucose utilization, a 250 ul aliquot of the 70% haematocrit blood sample was placed into a 5 mm tube. Into this sample was placed a 2.5 mm (O.D.) capillary tube containing p-phenylenediamine (Aldrich Chemical Co., Milwaukee, WI) dissolved in D₂O and the instrument was shimmed and tuned. A 13.2 ul aliquot of a 200 mM [2-¹³C]D-glucose solution (Omicron Biochemicals Inc., South Bend, IN) in Buffer A was then added with mixing. [2-¹³C]D-glucose peaks were measured relative to the standard contained in the capillary tube. Glucose concentrations were then calculated by comparison with peak ratios using known concentrations of glucose measured under conditions identical to those used to follow glucose metabolism.

¹⁴C-glucose experiments

Aliquots of erythrocytes (130 ul) were placed in Eppendorf tubes and allowed to equilibrate at one of four different temperatures (4°C, 12°C, 17°C and 23°C). A 10 ul aliquot of 136 mM glucose plus 0.6 mCi [U-¹⁴C]D-glucose (Amersham, Mississauga, ON) was added to each tube and 10 ul samples were removed at 0, 0.5, 1, 2, 3, 5 h. Samples were placed into 100 ul of Buffer A and centrifuged at 3000g for 3 min. The supernatant was removed, the pellet re-suspended in an additional 100 ul Buffer A and re-centrifuged. The supernatants were pooled and the pellet was treated with 100 ul of NCS-II tissue solubilizer (Amersham) followed by a 1 hour incubation at 50°C. All samples were counted in a scintillation counter (Wallac 1410, Pharmacia, Uppsala, Sweeden) after addition of 10 ml of Redi-Gel (Beckman, Mississauga, ON).

Other measurements

The hemoglobin content of the cell suspensions from the ¹³C-NMR experiments taken after termination of the metabolic portion of the experiment as well as from freshly prepared cell suspensions were measured with Drabkin's reagent (Sigma Chemical Co, Kit #525-A). The change in optical density was followed at 550 nm (filter) with a Dynatech MR5000 microplate spectrophotometer (Chantilly, VA). Erythrocytes in the same freshly prepared cell suspensions were counted by microscopy after appropriate dilution. Haematological analysis of R. sylvatica erythrocyte preparations gave a value of 6.9 ± 1.1 x 10⁷ cells per mg hemoglobin. A comparison of haematocrit values and cell counts showed that 0.50 ± 0.06 ml of packed cells corresponded to 9.9 ± 1.3 x 10⁸ cells or that each cell occupied a volume of 0.5 ± 0.1 nl (N = 2, mean ± SD).

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