Acid-Base Regulation in Response to Hypercapnia in the Lymphatic And Circulatory Systems of the Toad Bufo Marinus

In anuran amphibians, vascular filtrate and water of external origin enter the extensive subcutaneous spaces (Carter, 1979), which act as lymph reservoirs (Kampmeier, 1969). Although the volume of the lymphatic system is unknown, Thorson (1964) estimated 20% of the total body weight of Bufo marinus (L.) to be occupied by interstitial fluid, including lymph. This will, of course, vary with the state of hydration. Lymph is returned from the viscera and subcutaneous spaces to the systemic circulation via contractile lymph hearts, which maintain a proper fluid balance between the extracellular compartments (Middler et al. 1968). The high permeability of amphibian blood capillaries facilitates fluid exchange between the circulatory and lymphatic systems and also allows the movement of all normal plasma proteins into the lymph (Conklin, 1930). Being anionic, the proteins are buffers to H⁺ and should be considered important constituents in acid-base regulation.

The extent of the pH compensation which occurs in Bufo marinus during an acid-base disturbance elicited by high ambient CO₂ levels is greater intracellularly (skeletal muscle 65%, heart muscle 77%; Toews and Heisler, 1982) than extracellularly (blood 17–30%; Boutilier et al. 1979; Toews and Heisler, 1982). Despite the differences in the extent of compensation, both intra- and extracellular compartments exhibit a short-term uncompensated respiratory acidosis resulting in an increase in both arterial and [HCO₃⁻] and a decrease in pH along the in vivo buffer line. A longer-term compensatory phase follows and is marked by rising [HCO₃⁻] and a marginal increase in pH (Boutilier et al. 1979; Toews and Heisler, 1982; Toews and Boutilier, 1986). Studies involving hypercapnia-induced acid-base changes in the extracellular space of amphibians, however, have been confined exclusively to blood and/or plasma with no available literature on responses of the lymphatic system to similar conditions. The present study was conducted to examine whether changes similar to those seen in the circulatory system are apparent in the equally expansive and important lymphatic compartment.

Adult Bufo marinas were anaesthetized by immersion in a 2.0gl⁻¹ 1,3-aminobenzoic acid ethyl ester solution buffered to pH7 with sodium bicarbonate. In each animal, one sciatic artery was cannulated with an indwelling polyethylene cannula (PE 60) filled with heparinized saline (Boutilier et al. 1979). Both posterior lymph hearts were cannulated according to the method of Jones et al. (1992) with a 40cm length of PE50 tubing flared at the distal end. Normal posterior lymph flow did not appear to be compromised by the cannulation as there was no indication of oedema over a 3-to 4-day period following surgery.

Following an 18-to 24-h recovery period, the animals were exposed to 5% CO₂ for 24 h under hydrated conditions (500 ml of chamber water) in a 31 experimental chamber. Just prior to the hypercapnic exposure 300 μl samples of blood and lymph were collected. Additional samples were taken at 1, 12 and 24 h after the onset of hypercapnia and also following a 24 h recovery period from hypercapnia, during which time the experimental chamber was flushed with humidified air. Experiments were conducted at 20±l°C and all samples were analyzed for pH and at 20°C as described by Boutilier et al. (1979). [HCO₃⁻] values were calculated using the Henderson-Hasselbalch equation with a αCO₂ of 0.3203 (mmol l⁻¹ kPa⁻¹; Toews and Stiffler, 1990) and pH-dependent values of pK′ ranging from 6.12 to 6.19 obtained from Heisler (1986). Values for pK′ and αCO₂ were not specifically determined for lymph. Preliminary studies revealed that [Na⁺] and [K⁺] were comparable in lymph and plasma, however, suggesting that the total ionic composition of both fluids was also probably similar. Since this is the primary determining factor of pK′, the determined plasma values were used. Haematocrit readings were taken for the blood samples. For the determination of buffer values, all the lymph and blood were removed from three animals. The samples were centrifuged independently and the supernatant layers of the lymph and plasma were retained for in vitro [HCO₃⁻] analysis using a gas chromatograph (Boutilier et al. 1985).

Statistical analyses included paired t-tests for comparisons between fluids and an analysis of variance (ANOVA) post hoc pairwise comparison for time comparisons within a given fluid. The fiducial limit of significance used in this study was P<0.05.

The mean changes seen in arterial blood and lymph in 11 Bufo marinus during hypercapnic exposure are shown in Fig. 1 and Table 1. The adjustments seen in the blood of Bufo marinus in response to environmental hypercapnia in the present study are quite similar to those seen in previous studies (Boutilier et al. 1979; Toews and Heisler, 1982; Toews and Boutilier, 1986; Boutilier and Heisler, 1988; Toews and Stiffler, 1990). In both compartments there was an initial acidosis followed by a period of compensation. Following recovery from hypercapnic exposure all variables returned to control levels.

Although the control samples of blood and lymph had similar HCO₃⁻ levels, pH was significantly higher and significantly lower in blood than in lymph. This latter observation may be attributed to the diffusion of metabolically produced CO₂ from the tissues into the lymph.

Hypercapnic exposure initially induced significant depressions in the pH of the blood and lymph, from 7.90±0.03 to 7.42±0.02 and from 7.85±0.03 to 7.42±0.02, respectively (Fig. 1; Table 1), as well as significant increases in in both fluids. Although the initial changes in [HCO₃⁻] were not significant, the small decrease in lymph [HCO₃⁻] bears mentioning because it contrasts with the typical increase seen in blood [HCO₃⁻], which is presumed to result primarily from increased CO₂ levels in the erythrocytes (Boutilier et al. 1979). This difference could be attributed to the removal of HCO₃⁻ by the tissues as the lymph passes through the capillary bed and/or to the absence of red blood cells in the lymph.

Previous studies have reported a significant increase in haematocrit during exercise in Bufo marinus (McDonald et al. 1980; Tufts et al. 1987) and during hypercapnia in Cryptobranchus alleganiensis (Boutilier and Toews, 1981). In the present study it is unclear whether the significant increase in haematocrit after 1 h of hypercapnic exposure represents any volume shifts between the blood and lymph or if it is simply a result of mobilization of erythrocytes from the spleen.

After 12h of continual exposure, extracellular pH compensation, marked by rising [HCO₃⁻], appeared to be in progress. Blood samples analyzed prior to 24h (the 1 and 12 h samples, Table 1) of continual hypercapnic exposure, however, represent an unsteady state and equilibrium is not reached in both compartments until about the 24 h mark (Table 1; also Boutilier et al. 1979; Toews and Heisler, 1982; Boutilier and Heisler, 1988). Thus, for clarity, the 12 h samples appearing in Table 1 were omitted from Fig. 1.

After 24 h of hypercapnia, [HCO₃⁻] was elevated above control values in both compartments although the change was only significant in the blood. There were no significant changes in between 1 and 24 h in either lymph or blood, although lymph did increase significantly between 1 and 12 h. The pH compensation arising from these changes 24 h after the onset of hypercapnia was 16% in the blood and 4% in the lymph. The increase in [HCO₃⁻] in blood during this time has been attributed to HCO₃⁻ reabsorption across the skin, bladder and/or kidneys, H⁺ extrusion and/or mobilization of carbonate stores (Tufts and Toews, 1985). The lack of significant differences between blood and lymph [HCO₃⁻] at any given time throughout the experiment probably indicates that there is a similar source of HCO₃⁻ for the two compartments. The lesser increase in lymph [HCO₃⁻] seen over the 24 h of exposure accounts for the lesser degree of pH compensation.

Following the hypercapnic period, the acid-base state of both compartments returned towards normal, with the lymph seemingly responding more quickly than the blood. Boutilier et al. (1979) also found that blood pH recovered quickly while [HCO₃⁻] and recovered slowly. A probably explanation for the observation that lymph [HCO₃⁻] returned to pre-exposure levels more quickly than that of blood is the removal of HCO₃⁻ by the tissues as lymph passes through the capillary bed. The return of blood [HCO₃⁻] to normal would probably involve renal compensation.

The mean buffer value (ΔHCO₃⁻/ΔpH) of lymph was −3.88±1.12 and was not significantly different from that of separated plasma at −5.30±0.77. Preliminary experiments from our laboratory yield a lymph:plasma protein ratio comparable to the value of 0.5 determined for Bufo arenarum (Zwemer and Foglia, 1943), which may account for slight differences in the recorded buffer values.

In summary, while there are some notable differences between the major extracellular fluids lymph and blood, the pattern of acid-base regulation during hypercapnia is quite similar in both. Nonetheless, the large expansive nature of the lymphatic system, and its fairly rapid circulation time, make it necessary to consider this compartment seriously in future amphibian acid-base studies.