Effect of chronic cold and submergence on blood oxygen transport in the turtle, chrysemys picta

Respiration Physiology (1983) 53, 15-29

15

Elsevier

EFFECT OF CHRONIC COLD AND SUBMERGENCE ON BLOOD OXYGEN TRANSPORT IN THE TURTLE, CHRYSEMYS PICTA

L.A. MAGINNISS, S. S. TAPPER and L. S. MILLER Division of Biology and Medicine, Brown University, Providence, RI 02912, U.S.A.

Abstract. Whole blood oxygen equilibrium curves (O2EC's) and related hematologic properties are reported for the turtle Chrysemys picta exposed to two experimental conditions. Summer turtles were maintained at 24°C with free access to air; winter turtles were submerged for 4-12 wk in N2-bubbled water at 3 °C. Half-saturation Po2'S at 3 °C for blood from summer and winter animals were 4.1 and 4.5 Torr, respectively. At 24 °C, summer and winter Ps0's were 20.2 and 22.7 Torr, respectively. The winter turtle Ps0 values were lower than predicted since prolonged submergence effected a severe metabolic acidosis; blood pH's for winter turtles were 0.65 pH unit lower than for summer animals at both temperatures. Cold submergence also had a profound influence on O2EC shape. Winter turtle curves exhibited high 02 affinity below Ps0 while they were distinctly right-shifted above 50% S. Winter animals also exhibited reduced CO2-Bohr coefficients (Alog Po2/ApH) at 3 and 24 °C. Prolonged submergence did not affect the animal's isohemoglobin profile (demonstrated by isoelectric focusing) or [metHb]. The [ATP] and [DPG] in winter turtle red cells, however, decreased significantly; the ratio of organic phosphate ([ATP] + [DPG]) to Hb tetramer fell from 1.4 in summer animals to 0.5 in winter turtles. These findings suggest that the effect of chronic cold and prolonged submergence on turtle O2EC position and shape may result from reduction in RBC organic phosphates. Furthermore, these observed changes in blood oxygen transport may facilitate 02 loading during winter submergence via extrapulmonary gas exchange.

Chrysemys picta CO2-Bohr effect Hill coefficient Metabolic acidosis

Multiple hemoglobins Organic phosphates Oxygen dissociation curve Oxygen equilibrium curve

Freshwater turtles of the family Emydidae exhibit a remarkable capacity for prolonged submergence. At summer temperatures (15-25 °C), these apneic periods may persist for minutes or hours (Lucey and House, 1977; Burggren and Shelton, 1979). During winter, however, when water temperature approaches 0 °C, these reptiles are able to remain continuously submerged for periods of four to six months Accepted for publication 12 April 1983 0034-5687/83/$03.00 © 1983 Elsevier Science Publishers B.V.

16

L.A. MAGINNISS

el al.

(Musacchia, 1959; Ultsch and Jackson, 1982). Survival under these conditions is due in part to the animal's ability to rely on anaerobic metabolism as well as its capacity to deal with the severe metabolic acidosis (Jackson and Silverblatt, 1974; Jackson and Ultsch, 1982). In addition, turtles submerged in aerated water at 3 °C are able to produce a significant portion of their energy requirements from aerobic pathways, deriving oxygen from an extrapulmonary route (Ultsch and Jackson, 1982). This communication reports the effect of prolonged submergence and cold on the oxygen transport properties of whole blood for the turtle Chrysemys picta. These environmental pressures effected a dramatic change in the shape and position of the oxygen equilibrium curve, probably due to the reduction in concentration of red cell organic phosphates. The functional implications of these 02 transport data are evaluated in the context of extrapulmonary gas exchange during prolonged submergence. A preliminary account of these findings has been presented elsewhere (Maginniss et al., 1982). Methods

Animals. Adult western painted turtles (Chrysemys picta) of both sexes and weighing 491 to 976 g (av. 647 g) were purchased from Lemberger Associates (Germantown, WI). In the laboratory, animals were maintained in large holding tanks with basking platforms at about 24 °C and with a 12 h : 12 h photoperiod. Turtles were fed several times weekly with vitamin-supplemented dog food and occasionally fresh fish. Implantation of sampling port for blood collection. Turtles were cooled to 3 °C for 24-48 h prior to surgery. A 2 cm hole was trephined through the plastron above the heart and a lucite double-plug was inserted and sealed with dental acrylic. The removable center plug (1.3 cm diameter) provided easy access to the heart for blood sampling. Animals were allowed to recover in air at room temperature for about 8 h and then returned to the holding tanks. Chloramphenicol (50 mg/kg, i.m.) was injected post-operatively and every other day for 2-3 wk thereafter. Experimental blood sampling did not commence for at least 1 wk following surgery. Experimental conditions. Groups of surgically prepared turtles were exposed to two different experimental conditions. Animals designated as summer turtles were maintained in laboratory holding tanks at 24 °C and had free access to air. Winter turtles were submerged for 4-12 wk in nitrogen-bubbled water (Po2 ~< 5 Torr) at 3 °C. The majority of winter turtle experiments, however, were conducted after 8 wk of exposure to cold and anoxia. Blood measurements on summer turtles were performed during summer and fall months; analyses of winter turtle blood were conducted in February and March. Several animals served as both summer and winter turtles.

0 2 TRANSPORTIN

SUBMERGEDTURTLESAT 3 °C

17

Blood collection. Turtles were bled anaerobically (2-6 ml) by ventricular puncture into iced and heparinized syringes through the ventral port. Winter turtles were denied access to air during blood sampling by either covering the head with a water-filled finger cot or by enclosing the anterior of the body in a water-filled latex glove. Blood acid-base status and other hematologic measurements. Blood pH was determined immediately following anaerobic sampling with a thermostatted glass electrode and pH meter (pH 27, Radiometer, Copenhagen) standardized with Radiometer buffers (S 1500 and S 1510). Blood pH was measured at 3 and 24°C for both experimental groups. The corresponding blood Pco2'S were determined by the Astrup method (Siggaard-Andersen and Engel, 1960) using a Radiometer microtonometer (ATM 1). [HCO3-] was calculated from the Henderson-Hasselbalch equation using constants for pK' and CO2 solubility given by Reeves (1976). Hematocrit was determined by centrifugation at 13 000 × g for 6 min in heparinized capillaries. Hemoglobin (Hb) concentration was measured as cyanmethemoglobin at 540 nm (Fisher reagent and standard set 251, Medford, MA) and methemoglobin (metHb) by the method of van Assendelft (1970) at 630 nm. Hb and metHb preparations were centrifuged (8000 × g) to remove the cellular debris (gel formation) characteristic of nucleated red cells. Red cell [ATP] and [DPG] were assayed by the UV methods of Sigma Chemical Co. (St. Louis, MO, Tech. Bull. 366-UV and 35-UV, respectively). For both organic phosphates, the proportions of blood and trichloroacetic acid were modified to compensate for the non-human concentrations of ATP and DPG present in turtle red cells. Hb heterogeneity in summer and winter turtles was evaluated by isoelectric focusing techniques described elsewhere (Maginniss et al., 1980). The focused isohemoglobin bands were cut from the polyacrylamide gels and eluted for 24 h in 0.5 ml degassed distilled water. The relative molar concentrations of the isohemoglobins were measured spectrophotometrically from the elutions at 540 nm in rnicro-cuvettes. Isoelectric points (pI's) were determined by measuring the pH of gel elutions at 24 °C. Oxygen equilibrium curves. Isocapnic oxygen equilibrium c u r v e s (O2EC's) were generated by a micro-method modified from Reeves (1980). Hb saturation (S) for thin blood films was measured by dual wavelength spectrophotometry and blood Po2 by electrode oximetry. (1) Preparation of blood film. A small aliquot (0.5-1.0 #1) of whole blood was gently spread between two 6.4 #m thick Teflon membranes (Dilectrix Div., Fluorocarbon Co., Lockport, NY). The membrane-sample trilayer was then secured by 'O' ring to an opaque acrylic disk with 7 mm center hole and mounted horizontally in the gas-exchanging chamber (fig. 1). High gas permeability of the Teflon membrane promoted rapid oxygen equilibration of the blood film; membrane transparency permitted spectrophotometric determination of Hb saturation.

18

L.A. MAG1NNISS et al.

OzEC Apparatus OE

OFB

Gas Port ~ Inlets

Licjht Source and Chopper ~I, 42 - 560

nrn

Gos Port Outlets

Spectrophotometer OFB

Fig. 1. Diagrammatic representation of gas-exchanging chamber used for generating continuous dynamic O2EC's and multiple point static O2EC's. The blood film (BF) was secured to the carrier disk by an 'O' ring and mounted in the center (C) compartment of the chamber. Prior to each experiment, the center and outer (O) chamber compartments were flushed with a saturated CO2-N 2 gas mixture. For dynamic measurements, the center compartment gas ports were then closed and an isocapnic O2 gas mixture was circulated through the outer compartments. As oxygen diffused across the rubber silicone membranes (RM), the rise in center compartment P02 was monitored with an 02 electrode (OE). Simultaneously, the change in HbO 2 concentration was determined by dual wavelength spectrophotometry (542, 560 nm); light was relayed to and from the blood film via optical fiber bundles (OFB). For multiple point static curves, the blood film in the center compartment was equilibrated with several isocapnic gas mixtures (mean of 10) containing increasing P02 levels. Blood film P02 and HbO2 saturation were determined at regular intervals between zero and 957~ S.

(2) Gas-exchanging chamber. The chamber described below was employed for generating both dynamic and multiple point static O2EC's. The blood film was mounted in the center chamber compartment which was separated from two outer compartments by gas permeable rubber silicone membranes (fig. 1). Composition and flow of gas to each compartment was controlled independently. During an experiment, all compartments were initially flushed with a water saturated CO2-N2 gas mixture. The desired gas composition was obtained with two gas mixing pumps in series (2M 301/a-F, W6sthoff KG, Bochum, F.R.G.) and water saturation was achieved with a two-stage humidifier. The majority of O2EC's (88~o) were determined by multiple point static measurements of blood film Po, and S. Following the initial deoxygenation stage, the blood sample was equilibrated with several isocapnic gas mixtures containing increasing Po, levels. For each static O2EC, a mean of 10 points were recorded at regular intervals between zero and 95~,, S. For dynamic OREC's, the center compartment gas port was closed following blood film deoxygenation and an isocapnic gas mixture containing 02 was circulated through the outer compartments. Oxygenation of the blood sample occurred in response to diffusive flux of 02 across the rubber silicone membrane barriers; the 02 ramp rate was controlled by regulating the Po~ gradient across the barriers.

0 2 TRANSPORT

IN SUBMERGED TURTLES AT 3 °C

19

Oxygen tension in the gas phase surrounding the blood film was continuously monitored by electrode oximetry (model 731, Transidyne Gen. Corp., Ann Arbor, MI). To maintain the blood film and gases at constant temperature, the chamber, humidifiers, 02 electrode, gas tubing and valves were submerged in a temperature regulated water bath (+_ 0.05 °C). (3) Measurement of Hb saturation. Blood film saturation was determined by dual wavelength spectrophotometry. Light from a tungsten lamp was split and transmitted to two monochromators (model H-10, Instruments SA, Inc., Metuchen, N J) via an optical fiber bundle Y. The emerging wavelengths (542, 560 nm; 4 nm bandpass) were then chopped mechanically (250 Hz), united in a common fiber bundle (1.5 mm diameter), and relayed to the blood film (fig. 1). The selected wavelengths correspond to an absorption peak for oxyHb (542 nm) and a deoxyHb maximum - oxyHb minimum (560 nm). Light passing through the blood film was transmitted to an end-on photomultiplier (9924B, EMI Gencom, Inc., Plainview, NY) by a 6 mm optical fiber bundle. The photomultiplier output was then relayed to an electronic chopper-signal conditioner-log ratio amplifier. During blood film deoxygenation, the output voltages of the two wavelengths were balanced against a reference voltage. With the introduction of oxygen, the fractional change in output voltages for the two wavelengths was directly proportional to the change in HbO 2 concentration. Continuous O2EC's were generated by simultaneously recording the voltage signals from the 02 electrode and spectrophotometer on an X-Y plotter (series 2000, Houston Instrument, Austin, TX); for static O2EC's, the incremental Po2-HbO2 saturation points were also plotted on the X-Y recorder. When oxygen tension in the center compartment effected a 90-9570 S, the blood film was flushed with CO2-O2 (Po2 /> 650 Torr) so that an optical signal for 10070 S could be recorded. (4) Evaluation of 02 equilibrium data. Accuracy of the dynamic O2EC method is critically dependent on the O2 ramp rate. For this continuous curve technique, it is assumed that the oxygen tension recorded by the 02 electrode is equivalent to blood film Po2 ; i.e., there is no phase angle between the 02 electrode and spectrophotometer. As an internal check on the dynamic equilibrium data, the experimental blood samples were evaluated by a time-independent static test as described by Reeves (1980). Mean error in Po2 near half-saturation was less than 270 with 02 ramp rates ranging from 0.3 to 3 Torr/min for the two experimental temperatures. A family of 2 to 5 isocapnic O 2 E C ' s w e r e generated for each animal at Pco2'S which encompassed the in vivo (or in vitro) carbon dioxide tension determined from anaerobic acid-base measurements (3°C, 1-3~o CO2; 24°C, 2-870 CO2). Fresh blood films were prepared for each 24 °C equilibrium curve to minimize the potential effect of erythrocyte metabolism on 02 affinity. The multiple point O2EC's were constructed by best-fit hand-drawn curves. The static and dynamic curves were read for oxygen tension at 5 ~ saturation increments from 5 to 9070 S. The CO2-Bohr coefficients (Alog PoJApH) were then determined by least square regression (5-90~o S) and the standard turtle O 2 E C ' s w e r e calculated for the appropriate blood pH.

20

l~ A. M A G I N N I S S et a/.

Results Blood acid-base status

Table 1A reveals the severe acidosis effected by prolonged cold and submergence; in vivo blood pH for winter turtles was 0.65 pH units lower than for summer animals when measured at 3°C. The substantial reductions in [HCO3] and buffering capacity (A[HCOf]/ApH) coupled with only a slight rise in Pco~ indicate the acidosis was due primarily to strong ion changes in body fluids. Comparable acid base disturbances have been reported for chronically catheterized C. picta exposed to prolonged anoxic submergence at 3 °C (Ultsch and Jackson, 1982). These investigators demonstrated that the metabolic acidosis resulted primarily from anaerobic lactate production. Similar differences between winter and summer turtle acid-base conditions were also observed at 24 °C (table 1B). The effect of temperature change on blood pH (ApH/AT) was 0.018 0.019 for both animal groups and similar to values reported for other ectothermic vertebrates (Reeves, 1977). Hematological properties ~

Hematocrit values for winter and summer turtles were not statistically different (table 2), but were low by comparison with other turtle species (Pough, 1980). The reason for reduced hematocrit is unknown; surgery resulted in little or no blood loss and the majority of experimental animals were sampled only once or twice. Mean corpuscular hemoglobin concentration (MCHC) was significantly lower (P < 0.001) among the winter turtles suggesting a change in red cell volume regulation with prolonged cold submergence. MetHb represented approximately 1~, of the total [Hb] in both animal groups. TABLE 1 Blood acid-base status of winter and s u m m e r turtles measured at 3 and 24 °C Winter a

Summer b

A. 3 ° C

pH Pcoz(T°rr) [ H C O f ] (mmol/l) A[HCOf]/ApH

7.44 11.3 12.1 -7.3

± 0.07 ± 1.2 ± 2.4 ±1.3

(6) c (6) (6) (6)

8.09 7.5 34.8 -12.7

± 0.02 +_ 0.4 + 2.0 ±2.0

(9) (8) (8) (6)

7.05 35.1 12.9 -10.0

_+ 0.06 ± 4.6 ± 2.9 ±2.3

(6) (5) (5) (5)

7.71 26.5 38.2 -11.6

_+ 0.02 _+ 2.3 _+ 1.6 +_1.2

(15) (14) (14) (15)

B. 2 4 ° C

pH Pco.~ (Torr) [HCO~] (mmol/1) A[HCO/]/ApH

a Winter turtles submerged for 4-12 wk in N2-bubbled water at 3 °C. b Summer turtles maintained at 24 °C with free access to air. c Mean _+ 1 SEM (N).

21

02 TRANSPORT IN SUBMERGED TURTLES AT 3 °C TABLE 2 Hematologic properties of winter and summer turtles Winter Hematocrit (~o) [Hb] (g/dl blood) MCHC (g Hb/dl RBC) MetHb (~o total Hb) [ATP] (#mol/ml RBC) [DPG] (/~mol/ml RBC) [ATP] + [DPG] a (mol/mol) [Hb]

14.4 + 3.29 ± 22.2 ± 1.0 ± 1.33 ± 0.25 ± 0.46

Summer 1.6 (10) 0.46 (9) 1.1 (9) 0.5 (6) 0.21 (6) 0.06 (10)

17.6 ± 1.0 5.11 + 0.37 28.8 + 0.9 1.2 ± 0.3 5.92 +_0.17 0.43 + 0.05

(15) (15) (15)** (14) (15)** (14)*

1.42

* P < 0.05; t-test (Snedecor and Cochran, 1967). ** P < 0.001. a MW 64 500 assumed for Hb tetramer.

Prolonged submergence at 3 °C caused a significant reduction in the concentration of red cell organic phosphates (table 2). [ATP] was reduced to less than 25~o of the summer turtle level, while [DPG] was reduced by about one-half. It should be noted that the Sigma ATP assay measures all nucleotide triphosphates. However, ATP represents about 90~o of the total nucleotide triphosphates in the pancake tortoise Malacochersus tornieri (Wood et al., 1978), while Bartlett (1980) reported the [ATP] in red cells of four turtle species to be approximately 20 times greater than the concentration of guanosine triphosphate. Inositol pentaphosphate, an organic phosphate reported in low concentrations for several other turtle species (Isaacks et al., 1978; Bartlett, 1980), was not measured for C. picta. Hemoglobin heterogeneity Isoelectric focusing revealed two major hemoglobin bands referred to as Hb 1 and Hb 2 (fast and slow components, respectively) for both winter and summer turtles (table 3). These results are consistent with observations by Manwell and Schlesinger (1966) and Sullivan and Riggs (1967a) for C. picta blood. Among both experimental groups, a minor component (more anodal) was occasionally noted in association with each major isohemoglobin. Maginniss et al. (1980) reported similar findings for Pseudemys scripta and suggested the additional bands might represent tetramertetramer polymers frequently observed with reptilian blood (Sullivan and Riggs, 1964). The pI's and relative molar concentrations for the two major isohemoglobins were not statistically different among winter and summer turtles (table 3). Hence, prolonged submergence at 3 °C had no apparent effect on the animal's isohemoglobin profile.

22

L.A. M A G I N N I S S et al.

TABLE 3 Isohemoglobin profiles for winter and s u m m e r C h r y s e m ) . s p i c t a , lsohemoglobin separation performed on 10 winter and 10 s u m m e r turtles by IEF techniques Winter

lsoelectric points

Hb 1 Hb 2

Relative molar concentrations (~o)

Hb 1 Hb 2

Summer

6.97 _+ 0.02 7.56 _+ 0.02

7.00 _+ 0.01 7.61 _+ 0.03

25.6 _+ 1.5 74.4 _+ 1.5

23.0 _+ 1.2 77.0 ± 1.2

Oxygen equilibrium data Mean whole blood O2EC's for winter and summer turtles at both 3 and 24 °C are

presented in fig. 2. These equilibrium data correspond to the measured blood pH values for each experimental condition. At 3 °C, the Po~ at half-saturation (Ps0) for

Human//)_,/~

~.~._---'~*~

Winter

0.~ 5°C OzEC's P02 Torr

,~ S

2~

Hu /

30 . . . . . . . - ..... Winter

.....

0.5

1

,

r/

LZ,'~/ / ' o

P02 Tort '-

3'0. . . .

6'0

'

9~-

Fig. 2. Whole blood O2EC's for winter and summer turtles (dashed and solid curves with data points) measured at 3 °C (upper panel) and 24 °C (lower panel). Equilibrium curves were generated by both multiple point static methods and continuous dynamic techniques at the acid-base conditions shown in table 1. The data plotted from 5 to 907,~ S at 5% S increments are mean Po.~'s from 6 to 10 individual experiments; the horizontal bars are _+ 1 SEM. Half-saturation P o / s at 3 °C were 4.5 and 4.1 Torr for winter and s u m m e r turtles, respectively; at 24 °C, P50's were 22.7 and 20.2 Torr for winter and summer animals, respectively. To demonstrate the non-standard shapes of winter and s u m m e r curves, O2EC's for h u m a n blood (Severinghaus, 1979), scaled to the mean turtle P_s0 at each temperature, are also illustrated (thin curves).

02 TRANSPORT IN SUBMERGED TURTLES AT 3 °C

23

winter and summer animals were 4.5 + 0.4 (X + 1 SEM) and 4.1 + 0.2 Torr, respectively. These Ps0 values were not statistically different (0.4 > P > 0.2), even though blood pH at 3 °C for the two experimental groups differed by 0.65 pH units (table 1). At 24 °C, blood O2-affinity was reduced for both groups, but again, the halfsaturation values were not different (0.2 > P > 0.1). The effect of temperature on Hb-O2 affinity was similar for winter and summer turtles. The apparent heat of oxygenation (AH) was calculated from the van't Hoff equation : AH = 2.303 R(Alog Ps0)/A(1/T) where R is the gas constant and T the absolute temperature (K). The enthalpy of Hb-O2 affinity was -12.5 kcal/mol O2 for winter turtles and -12.4 kcal/mol O2 for summer animals. Figure 2 also reveals the non-standard shape of C. picta equilibrium curves. By comparison with the Severinghaus curve (1979) for human blood, the summer turtle O2EC's exhibited greater oxygen affinity below Ps0 (less sigmoid) and were distinctly flattened (right-shifted) above 50~o saturation at both temperatures. Prolonged submergence at 3 °C had a profound effect on curve shape. Below half-saturation, winter turtle blood exhibited high oxygen affinity similar to summer turtles. Above Ps0, however, the curves diverged significantly at both temperatures; blood oxygen affinity for the winter animal was much reduced at these higher saturations. The effect of chronic cold and anoxic submergence on O2EC shape is also reflected by the Hill plots (log S/(1 - S) vs log Po2) presented in fig. 3A. The Hill relationships for summer turtle blood were reasonably linear at both temperatures; the Hill coefficients (n) were similar to n values determined for air-breathing Pseudemys scripta (Burggren et al., 1977; Maginniss et al., 1980). Hill plots for winter turtle blood, on the other hand, exhibited steep limbs at the low and high saturations joined by segments of reduced slope in the mid-curve region. Differences between winter and s u m m e r OEEC shapes are also revealed by plots of Hill's n vs S (fig. 3B). The winter turtle equilibrium data exhibited greater excursions in the Hill coefficient as a function of saturation. Figure 4 illustrates the CO2-Bohr coefficients (Alog PoJApH) plotted as a function of Hb-O2 saturation for both experimental groups at 3 and 24 °C. The effect of CO2 on oxygen affinity of winter turtle blood was less pronounced than for the summer animal at both temperatures. At 3 °C, the Bohr coefficients for winter and summer turtles at Ps0 were -0.14 and -0.39, respectively. The corresponding Alog Ps0/ApH values at 24 °C were -0.30 and -0.41, respectively. For both experimental groups at 3 °C, the CO2-Bohr effect appeared reasonably saturation-independent below 60~ S; at higher saturations, the Bohr shift increased dramatically.

L A. MAGINN1SS et al.

24

5£ lOt-

Hill Plots

Summer Turtles ? 5 / fog FS /'

05

/

24°C,

.7/

5°C

24 °C "

t

ooi

/

i

i //

I

4,

!

/

-05[

,l

/'

I-Ss

? ,/

5°0/

I

Wmter Turtles

/

o'

/

/

,o~,/

o.,

,,'

log P02

/

/

/

¥

j' .,,,'

o

5B

i

log Poz

~

Hill's n vs Hb Saturation

3 n

n

Summer Turtles 24oc

.,J°

, - -/.'" ~ ' \

Winter Turtles

24°C

."

.X

./:,<,. ,....

./ ..... . .

°.,

•

S 0.1

0.3

0.5

0.7

0.9 ol

os

o5

o7

09

Fig. 3. A. Hill relationships lbr summer and winter turtles at 3 and 24 °C plotted between 10 and 90~£ S at 5% S increments. Plotted points correspond to oxygen equilibrium data illustrated in fig. 2. Best-fit Hill coefficients for summer animals between 20 and 80% S were 1.70 (3 °C) and 2.03 (24 °C). B. Hill coefficients (n) for summer and winter turtles plotted as a function of Hb 02 saturation.

CO2 Bohr Effect &log PO2/ApH

,,"t

-0.6

-02

3oc...t.-""

L~.r ~

Wnter ..L'"'"'I

t ....t.......t.......f d ........t...... t Saturation

0,0

Oi'l

0;~

0.15

0,17

0.~9

Fig. 4. CO2-Bohr coefficients for summer and winter turtles as a function of Hb 02 saturation at 3 and 24 °C. Plotted points are mean Bohr slopes determined from 6- I0 animals: the vertical bars are + 1 SEM.

02 TRANSPORTIN SUBMERGEDTURTLESAT 3 °C

25

Discussion

Effect of chronic cold and anoxic submergence on oxygen affinity and 02EC shape The blood 02 transport properties for summer Chrysemys picta at 24 °C were similar to those of the aquatic turtle Pseudemys scripta (25 °C) with respect to oxygen affinity and O2EC shape (Maginniss et al., 1980). Both turtles exhibited non-standard equilibrium curve shapes when compared with the Severinghaus curve (1979) for normal human blood. Furthermore, the pI's and relative molar concentrations of isohemoglobins for these closely related ectotherms were similar. Maginniss et al. (1980) proposed that non-standard OzEC shapes obtained with P. scripta blood resulted from the presence of functionally distinct multiple hemoglobins. These investigators reported that the simultaneous occurrence of two or more isohemoglobins with different 02 affinities, Hill coefficients, temperature coefficients, and/or affinity differences for other ligands (e.g., H +, CO2, organic phosphates) could potentially affect equilibrium curve shape. The non-standard curves observed for summer C. picta might also be attributed to hemoglobin heterogeneity. With prolonged submergence at 3 °C, O2EC's for Chrysemys blood became more distorted at both experimental temperatures; the winter equilibrium curves exhibited high Hb-O 2 affinity below Ps0 and they were distinctly flattened (right-shifted) at the higher saturations (fig. 2). In addition, O2 affinity at half-saturation for winter and summer animals were similar at each temperature, even though blood pH differed by 0.65 pH units. There are several possible explanations for these changes in blood 02 transport with cold submergence: (1) changes in the number and/or relative molar concentration of red cell isohemoglobins could account for these observations; (2) oxidation of hemoglobin to the metHb form has been shown to affect 02 affinity and equilibrium curve shape in turtles (Sullivan and Riggs, 1967b); and (3) changes in concentration of red cell organic phosphates (allosteric modifiers of Hb function) could account for these results. Based on the findings of this study, proposals (1) and (2) can be eliminated. The isohemoglobin profiles for winter and summer turtles were similar and metHb represented only 1~o of total [Hb] in each experimental group. The significant reduction in organic phosphate concentration associated with long-term submergence provides the most plausible explanation for the observed shape and position of winter turtle O2EC's. For summer animals, the ratio of organic phosphate (ATP + DPG) to Hb tetramer was 1.4; for winter turtles, the ratio fell to less than 0.5 (table 2). Previous investigations of mammalian and avian hemoglobin solutions have demonstrated anomalous O2EC shapes when the ratio of organic phosphate (inositol hexaphosphate (IHP), DPG, or ATP) to Hb tetramer was reduced below unity (Benesch et al., 1968; Vandecasserie et al., 1971; Ochiai et al., 1972; Petschow et al., 1977; Maginniss and Reeves, 1980). In addition, these equilibrium data revealed complex Hill relationships, similar to those obtained with winter turtle blood (fig. 3). These non-standard O2EC's appear to represent the net effect of two different hemoglobin populations; a phosphate-complexed hemoglobin

26

L. A,. M A G I N N I S S et al.

with low 02 affinity and an unliganded hemoglobin with high 02 affinity. Support for this working hypothesis would require evidence that (1) the turtle red cell organic phosphates (ATP and DPG) act as allosteric modifiers of Hb-O, affinity and (2) the liganded and unliganded hemoglobin populations of winter turtle blood exhibit the same O2EC shape as 3 °C summer turtle blood. A preliminary study has examined the 02 binding properties of purified C. picta hemoglobin in the presence and absence of ATP (Maginniss and Tapper, unpublished). Winter turtle hemoglobin was stripped of small ions and organic phosphates, the Hb solution was brought to a 1 mM tetramer concentration in 120 mM KCI Ringer buffered with 16 mM HCO3, and O2EC's were generated at 3 °C. At pH 7.24, addition of 5 mM ATP increased the half-saturation Po.~ by 3.4 times, from a Ps0 of 2.1 Torr in the absence of organic phosphate to 7.1 Torr. The equilibrium curve shapes for the two Hb solutions were similar, but not identical, to the 3 °C summer turtle O2EC. The Hill coefficient was higher in the absence of ATP and lower in the presence of ATP by comparison with summer turtle data, such that the Hb solution O2EC's straddled the whole blood curve when all data were scaled to a common Ps0These findings suggest that the reduction in organic phosphate concentration may contribute significantly to the observed changes in 02 transport by turtle blood. An investigation of the oxygen binding properties for the individual turtle isochemoglobins would undoubtedly provide more insight. Furthermore, the intracellular pH of winter turtle red cells is probably more alkaline than would be predicted from the change in plasma pH because of the reduction of non-diffusible anions (ATP and DPG). This too must be considered when evaluating the winter turtle data.

Functional significance of winter turtle 02EC During winter, aquatic turtles may remain submerged for periods of weeks or months, especially in the ice-covered ponds of northern regions (Carr, 1952). In the laboratory, Jackson and colleagues demonstrated the capacity of Chrysemys to endure submergence in both normoxic and anoxic waters at 3 °C for periods up to 6 months (Ultsch and Jackson, 1982; Jackson and Ultsch, 1982). Their results revealed greater survival with normoxic submergence (water bubbled with air), although their conclusions were somewhat limited by the prevalence of a skin fungal condition restricted to animals in air-saturated water. The acidosis was less severe among the turtles in aerated water. After 8 wk of submergence, arterial pH had decreased by 0.3 pH units as compared with 0.6 pH units for anoxic turtles. In addition, the fall in [HCO3] was less dramatic, and plasma lactate increased to about one-half the level in anoxic turtles. Although arterial Po2 was only about 1 Torr for animals in normoxic water, the acid-base data indicate that a significant fraction of the animals' energy requirements were provided by aerobic metabolism via extrapulmonary gas exchange. At 3 °C, winter turtle blood would be 7-8~o saturated with oxygen at an arterial Po_~of 1 Torr. Although this saturation is low, it represents a blood 02 concentration more than 40 times higher than the dissolved [02]. For the summer turtle data at

02 TRANSPORTIN SUBMERGEDTURTLESAT 3 °C

27

3 °C, a blood O2 saturation of only about 4 ~ would be predicted at the in vivo pH for winter animals (pH 7.44). Hence, the unexpectedly high oxygen affinity of winter turtle blood at saturations below Ps0 may greatly facilitate Oz loading during winter submergence. It should also be appreciated that the high temperature sensitivity of turtle Hb (-12.5 kcal/mol 02) provides an apparent adaptive advantage for cutaneous Oz exchange by the submerged winter animal. In addition, the low CO2-Bohr effect at saturations below Ps0 would limit the reduction in 02 affinity with further pH changes during winter submergence. The reduced affinity and increased CO2-Bohr effect observed at the higher saturations for the winter animal would be of little functional importance, since the submerged turtle would never operate on that segment of the curve. The 7 - 8 ~ S predicted for diving turtles in normoxic water was based on equilibrium data determined for anoxic animals. It would seem highly unlikely, however, that the observed changes in 02 binding properties for winter turtles would not have occurred had the animal's arterial Po2 been 1 Torr rather than zero. Wood and Johansen (1972) demonstrated that hypoxia caused an increased blood O: affinity associated with a decrease in red cell [ATP] for the eel Anguilla anguilla. In addition, normoxic submergence of adult snapping turtle (Chelydra serpentina) for 20 days at 10 °C effected a three-fold decrease in the red cell concentration of ATP + DPG (Maginniss and Ultsch, unpublished). Furthermore, the submerged snapping turtle revealed the same relative changes in oxygen affinity, O2EC shape and CO2-Bohr effect as observed with winter Chrysemys blood. It appears, therefore, that the hypoxia induced by submergence (anoxic or normoxic) was the primary stimulant for the observed changes in the oxygen transport properties of winter turtle blood, and that "these changes are not unique to the western painted turtle.

Acknowledgements This investigation was supported by the National Science Foundation (PCM 8202702) and the National Institutes of Health (BRSG RR05664-14). The authors gratefully acknowledge Georg Panol for his generous and expert machinist support in the construction and modification of experimental equipment. The authors are also indebted to Dr. Robert Blake Reeves for helpful discussions, Dr. Donald C. Jackson for providing the gas-mixing pumps for this study and critically reviewing the manuscript, and Christine V. Herbert for technical assistance.

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Ultsch, G. R. and D. C. Jackson (1982). Long-term submergence at 3 °C of the turtle, Chrysemys picta bellii, in normoxic and severely hypoxic water. I. Survival, gas exchange and acid-base status. J. Exp. Biol. 96:11-28. Van Assendelft, O. W. (1970). Spectrophotometry of Haemoglobin Derivatives. Assen, The Netherlands, Van Gorcum, 152 p. Vandecasserie, C., A.G. Schnek a.nd J. Leonis (1971). Oxygen-affinity studies of avian hemoglobins. Chicken and pigeon. Eur. J. Biochem. 24: 284-287. Wood, S. C. and K. Johansen (1972). Adaptation to hypoxia by increased HbO 2 affinity and decreased red cell ATP concentration. Nat. New Biol. 237: 278-279. Wood, S.C., G. Lykkeboe, K. Johansen, R.E. Weber and G.M.O. Maloiy (1978). Temperature acclimation in the pancake tortoise, Malacochersus tornieri: metabolic rate, blood pH, oxygen affinity and red cell organic phosphates. Comp. Biochem. Physiol. 59A: 155-160.