Arterial acid-base changes in unanaesthetized rats in acute hypoxia

ARTERIAL ACID-BASE CHANGES IN UNANAESTHETIZED RATS IN ACUTE iIYPOXIA

Abstract. Arterial PO*, P,,, and pH, as well as lactate and pyruvate concentrations, were measured in unanaesthetized rats exposed to air or to gas mixtures containing from 15.30 to 5.07% 0, for 5 to 30 min. In air-breathing controls the Pa+, was 91 mm Hg, Pqo, 41 mm Hg and pH 7.43. With reduction in inspired O2 the Pao, decreased to minimal values of 21 mm Hg (SO/,OJ, the Pko, fell to minimal values of 13 ta 14 mm Hg (5% 0,) and the pH increased to maximal values of 7.661 (7% 0,). The decrease in Pa,-+ and increase in pH occurred already at 15.3% 0,. With 5% 0, there was a marked but variable nonrespiratory acidosis that either limited the alkaline shift in pH, or caused a net acid shift in pH. Accumulation of lactate occurred with l3”/ O,, and with 1I”/ 0, there was an increase in lactate/pyruvate ratio. However. a significant pyruvate accumulation occurred only when the inspired 0, was decreased to less than 7% Acid-base balance Hypoxia

Lactate Metabolic acidosis

For many studies of the physiological responses to hypoxia it is a distinct advantage to use small animals like rats. When anaesthetized animals are used sampling of arterial blood is easily performed, and measurements of arterial and venous blood gases and acid-base parameters can be combined with studies of pulmonary ventilation (Turek, Frans and Kreuzer, 1970). However, since anaesthesia may influence the response to hypoxia there have been many attempts to sample blood from unanaesthetized animals in a way that causes minimal interference with the acid-base state. Although it has been claimed that values for Pco2, pII and [HCO;] measured after heart puncture are correct (Simmons, Kahn and Guze, 1966) the results obtained using indwelling arterial catheters (Popovic and Popovic, 1960) indicate that heart puncture leads to erroneous values for e.g. the Pa,-o, (Altland et al,, 1967; Ponttn and Siesjo, 1967, Burlington, Maher and Sidel, 1969).

312

PLASMA ACID-BASE CHANGES IN HYPOXIA

313

Ahland et al. (1967) measured the arterial oxygen and carbon dioxide contents and the arterial pH of rats acutely exposed to 10.4, 8.3, 6.6 and 4.9% 0,. The Pko2, calculated from the Singer and Hastings (1948) nomogram for human blood, decreased from 38 to 30 mm Hg at 10.4% and to 17 mm Hg at 4.9% 0,. However, since hypoxic gas mixtures containing more than 10.4% 0, were not used, it was not established at what degree of hypoxia a decrease in Pk,, first appears. These authors also measured lactate and pyruvate in arterial blood. At 6.6% 0,, they found increased lactate and pyruvate concentrations and at 4.9% there was an increase in the excess lactate concentration calculated according to Huckabee (1958). The authors concluded that the degree of hypoxia at which excess lactate appears is greater in the rat than in man (Huckabee, 1958) or the dog (Cain and Dunn, 1964; Cain, 1965). In the experiments of Altland et al. (1967) arterial pH was not analysed in those animals in which lactate and pyruvate were measured, and the Pao,, was not recorded. We therefore undertook a study of arterial acid-base changes in unanaesthetized rats exposed to oxygen concentrations ranging from 20.9% (room air) to .5.10/,,measuring PaoZ, P+o,, pH, lactate and pyruvate during the first 30 min period. The objectives of the study was to define the relationship between the inspired 0, concentration and the arterial P02, Pco, and pH and to evaluate changes in the blood lactate and pyruvate concentrations. Methods

Male SPF rats of the Wistar strain (Mellegaard, Copenhagen), weighing 280425 g, were used. The animals had unlimited access to water and commercial rat pellets (San-Bolagen, Malmii). A tail artery catheter was introduced under halothane anaesthesia after induction with divinyl ether as described previously (Ponten and Siesjii, 1967). A short syringe needle was connected to the catheter and closed with a rubber stopper after flushing the needle and catheter with heparinized Krebs solution. Approximately one third of the exterior part of the catheter and the tail were covered with circular turns of adhesive tape. The animals were then left to recover from the anaesthesia until the following day. Each animal was kept in a separate cage. Blood samples from animals breathing room air were taken while they were still in their cages. Great care was taken not to disturb the animals by the sampling. In this manner the samples were obtained without the rat moving and, if asleep, often without awakening. After an initial intraarterial injection of heparin (300 I.U./kg body weight) two samples were taken at least 15 min apart. If the Pco, values did not agree within 5% additional samples were taken until this criterion was met. A rectal temperature probe (Type TE 3, Elektrolaboratoriet, Copenhagen) was then inserted and the animal was transferred to a 20 x 14 x 7 cm plastic box that had an inlet and outlet for gas. The end of the catheter and the lead to the temperature probe were passed through the outlet. The animal was then allowed to accommodate to his new surroundings for about 10 min before being exposed to

314

L. D. LEWIS, U. PONTEN AND 3. K. SIESJ6

the gas mixtures. Control measurements showed that air-breathing rats within the box had identical acid-base values as those studied in the cages. Thegas mixturesusedcontained 20.92(room air), 15.30,13.24, 11.11,9.23,7.03, 5.12 and 5.07% 0, in nitrogen, as determined with Scholander analyses. The gases were flushed through the box at a rate of 2 L/min. Arterial blood was sampled for analyses either 5, 15 and 30 min, or 10, 20 and 30 min after the hypoxic gas mixture was introduced. When sampling the blood, 2-3 drops were first allowed to emerge from the tip of the catheter before filling 0.2 ml glass capillaries. The total volume of blood drawn in an experiment was 1.0 to 1.5 ml, which is less than 6% of the rat’s total blood volume. At each sampling, the rectal temperature and blood pressure were recorded. Immediately after sampling, the blood was analysed for pH with a Radiometer capillary glass electrode, and for PO, and PcoZ, using micro electrodes (Eschweiler and Co., Kiel). Ali electrodes were maintained at 37.0 “C and appropriate corrections were made for temperature (Kelman and Nunn, 1966). The pH measurements were referred to the phosphate buffers of the National Bureau of Standards (6.841 and 7.383 at 37 “C). No correction was made for the suspension effects of the erythrocytes. The Po, and Pea, electrodes were calibrated with gases before each measurement, and frequently controlled by means of equilibration of blood with known 0, and CO, tensions, using Laue tonometers (La&, 1951). The plasma HCO; concentration was calculated from pH and PcoZ, using a pK; for carbonic acid corrected for temperature and pH (Severinghaus, 1965). The actual blood base excess was calculated by means of the equation given by Siggaardml blood as observed Andersen ( 1966) using a mean value of 14.6 g hemoglobin/l~ in these rats. The base excess for oxygenated blood (B&,) was derived from the nomogram of Thews (1967) using an oxygen dissociation curve with a P,, of 38.8 mm Hg. For measurements of lactate and pyruvate arterial blood was sampled directly in liquid nitrogen and stored at - 85 “C until analysis. The samples were weighed and extracted with HCl-methanol at - 22 “C. Lactate and pyruvate were then analysed with enzymatic fluorometric techniques (see FolbergrovB, MacMillan and Siesjo, 1972). The one-tailed probabilities given in the text were calculated using Student’s t-test. Results ACID-BASE

PARAMETERS AND PO, IN ANIMALS BREATHING

AIR

Table 1 compares the present control data (room air) with those previously obtained in unanaesthetized rats. The present Pao,, is in excellent agreement with that reported by Burlington et al. (1969). The Pko, and pH values are very similar to those previously reported from this laboratory (Ponten and SiesjB, 1967), and to those of Burlington et al. (1969), but the Pace, is much higher than that obtained

315

PLASMA ACID-BASE CHANGES fN HYPOXIA TABLE 1 Arterial blood gas and acid-base parameters in the rat breathing air Source

No. of observations

Pro,

Pa% mm Hg

PH

mm Hg

WO,I meq/l

Simmons er al. (1966) * Altland et al. ( 1967) Ponten and Siesjo (1967) Burlington er al. (1969) Present data

52 9

-

2I

27.1 (20.5-34.5) 39.8 & 1.04

Actual

19.7**

Base excess meq/l

7.48 (7.34-7.56) 7.3910.005

- 1.1

23.1 iO.6

-0.7**

3s.5*0.5

7.47f0.006

27.3 kO.5

-t 4.2**

(11.5to -3.3)

6

91.7& 1.7

40.7i 1.8

7.42+0.011

25.2*0.08

+ 2.0**

128

90.6& 1.5

40.9kO.4

7.43 * 0.004

27.8 k 0.2

+4.2+0.2

Values are arithmetic meanfS.E.M. or (range). * Values corrected to 37°C. ** Determined from the mean value of Pa,-o, and pH, using the Siggaard-Andersen alignment nomogram (1963).

by heart puncture (Simmons et aE., 1966). The results demonstrate that arterial pH, [HCO;] and Base Excess are slightly higher in the rat than in man, but that the Pao, and Pace, are similar. ARTERIAL ACID-BASE CHANGES IN HYPOXIA

Figure 1 shows the changes in Pao,, Paeoz and pH with time in the animals exposed to 11.11 and 7.03% 0,. There were no changes in any of these parameters after 10 min of exposure. Since similar results were obtained with the other gases, the data obtained at 15, 20 and 30 min were pooled. Table 2 shows the Pro,, Paoz and the acid-base parameters in the various groups (means&-SD.). The Pao, decreased continuously from 91 mm Hg with room air to 21 mm Hg in the animals breathing 5.12% 0,. There was a significant decrease in Pat,, (p< 0.001) and increase in pH (p
316

I_. D. LEWIS, U. PONTEN AND B. K. SIBSJO

Fig. I. Arterial

PO,.

Pro2 and pW chirnges with time in unanaesthetized rat$ breathing 7.03 (0) and If.lf (0) % 0,. Each point represents the mean_+S.E.M. of 6 values.

TABLE 2 Arterial blood gas and acid-base parameters in the rat in hypoxia.

_--.

.__I __~~_.

I

128 12 12 17 ‘?ff

0.20% 0.1530 0.1324 Q.1111 0~0923

t9 8 8 _a--

O.O7i)3 0.0512 0.0507

___. ...---.

146.OIt:3,Y 90.6&-I I.3 40.9&3.3 108.7*2.9 58.9 +4.3 36.8 k2.1 91.3*2.1 47.5 * 4.2 31.4k2.4 78.2i1,2 40*2* 2.5 30.0 + 2.1 HO& 1.8 33.7i3.f 24.4 2 3.1 i7.8+ 1.3 25.9 i 3.4 4u+ f”3 36.3 SO. t 21.2+2.f5 13.7+ I.4 37.Qk 1.2 21.1 k2.9 20.4 + 2.6 ___““,“_ e.*w”-

Values are arithmetic means_tS.D. Two ob~er~a~joRs~a~~mai(only one obsrvarion

--“-.tl

L.._~__..

.-

7.43 * 0.03 27.8F 1.9 7.47 ,+.$.o?; 27.1 f 2.0 7.49rl:O.Q4 23.5 i: 0.8 7.5z~a.03 25.5i2.2 7.57 f 0.1)3 22.7 - 3,6 18.Xi2.2 Zdi f0.M 7,56&rl.O6 i 2.4 k 2.4 7,25 * 0.06 8.4i_ 1.4 11,” _-___-L__ ______.I.

on one of the animals in the iI and 7% groups).

PLASMA ACID-BASE

CHANGES

317

IN HYPOXIA

I

I

I

I

I

I

I

0

5

10

15

20

25

30 Mmutes

Fig. 2. Arterial 5.12 (0) 2, 0,

P,,, Pm, indicating

and

pH changes

with time in unanaesthetized

the variability in response at this degree the mean + S.E.M. of 4 values.

rats breathing

of hypoxia.

Each

5.07 (0) point

and

represents

those reported by Altland et ul. (1967) but since our pyruvate values are higher the L/P ratios are lower (cf. Huckabee, 1958). There was a significant increase in lactate already at 13:4 0, (p < 0.001) and significant increases in L/P ratio (p < 0.001) with 0, concentrations of ll”/,, or lower. Since the pyruvate concentration did not increase significantly until in the 5% 0, group, the increases in L/P ratio were due to increases in lactate concentration. Table 3 also gives the excess lactate concentrations, calculated according to Huckabee (1958). Like the lactate, the excess lactate concentration increased significantly at 0, concentrations of 13%, (p< 0.02) or lower. Figure 3 relates the lactate concentrations in blood to the B&,, calculated for the comparable blood samples. The B&,Xat 13% 0, deviated markedly from those obtained at 15, 11 and 9%. Regression analysis on the entire material shows a high correlation coefficient between increase in lactate and decrease in base excess (r=0.96) and a slope of - 1.9. In other words, the change in base excess was almost twice that in lactate concentration (see Discussion).

318

L. D.

LEWIS, U. PONY&N AND B. K. SIESJ6

TABLE 3 ~1--

Arterial lactate and pyruvate concentrations in the rat after 30 min of hypoxia. “-l.._~_ - ..^_-.- I-.-~_____.___I. “I_.-_-- _... ..__~

No. of observations

Fto,

Lactate

Pyruvate

meq/L

meq/L

~“-~26 5 6 4 5 4 4

Lactate, pyruvate ratio

“. 0.2092 0.1530 0.1324 O.iI11 0.0923 0.0703 0.0507

1.09*0.31 1.34kO.72 1.60&0.35* 1.95*0.55* 3.50+ I.34* 527 f 1.t4* 13.89* E.02’

Excess lactate meq/L -- _-ll-l~..l--__.

0.140~0.034 0.15510.072 O,f68+0,051 0. I55 _t 0.044 0.148 40.076 0.148~0‘038 0.362 f 0.072%

8.1i2.5 8.6i 1.3 Q.Qk 1.6 12.7i 2.3* 27.2+8.t* 36.0 & 2.6* 40.2% 12.8X

_

0.00 li:0.26 0.09 _t 0.25 0.25F0.18” 0.70~0.30’ 2.41+0.92* 4.08 & 0.86* 10.97I: t,59*

Values are arithmetic means+S.D. One observation/animal except in the air breathing animals in which 2 observations

were made.

Excess lactate= Lt,ynox,a - (Phypuxiax g) (Huckabee, 1958). 81r * Significantly different from the corresponding values in the air breathing animals (p c 0.001, except the excess lactate in the 0.1324 group in which p c: 0.02).

i

I

I

I

0

5

10

15

[Lactate]

mEq/ iifer

Fig, 3, Changes in arterial lactate concentration and base excess of oxygenated blood in unanaest~et~zed rats after 30 mm of breathing a gas containing either 5.07, 7.03, 9.23, It.ll, 13.24, 15.30 or 20.92% 0, as shown in the parenthesis beside the mean values (n = 4,4, 5,4,6, 5 and 26, respectively), The regression equation as determined by the meMIod of least squares using the individual values for each animal was [B.E.] =4.2- 1.85 [Lactate].

Discussion The present results have shown that when unanaesthetized rats are acutely exposed to 0, concentrations ranging from that of room air to 5%, the Pka decreases from 91 to 21 mm Hg. ~~m~ltaneo~sly~ the Pz+~, falls from 41 mm Hg to minimal values of 13-14 mm Hg. The decrease in Pk-,, is significant already with 15.300/,

PLASMA

ACID-BASE

CHANGES

IN HYPOXIA

319

O,, corroborating previous results in man (Lutz and Schneider, 1919-20; Dripps and Comroe, 1947) and in the dog (Watt, Dumke and Comroe, 194243; Chambers et al., 1947) in showing that the ventilation is increased even at very moderate reductions in Pao,,. Associated with the decrease in Pace, there is an increase in pH until an inspired O2 concentration of about 7% is reached. At still lower 0, concentrations (Pao,, of below 25 mm Hg) a progressive nonrespiratory acidosis reduces the shift of pH in the alkaline direction, and in many animals a marked acid shift in pH occurs. With 5% O2 the response of the animals is varied and unpredictable, although the PaoZ is rather uniformly reduced to 21-23 mm Hg. Thus, in some animals the Pco, is reduced to lo-15 mm Hg and the pH increased to values of about 7.6, while others show a lesser degree of Pa,-o, reduction and a marked nonrespiratory acidosis. In this group the arterial oxygen saturation may therefore vary within wide limits (8-27x). The development of a marked acidosis at these degrees of hypoxia is accompanied by a falling blood pressure and it seems probable that it reflects a variable response of cardiac function to the reduction in Po, (Cross et al., 1963; Bullard and Kollias, 1966; Downing, Talner and Gardner, 1966). The relationship between the lactate and the base excess concentrations at all degrees of hypoxia indicates that the increase in the lactate concentration can account for only about 50% of the decrease in base excess. These results could mean that other acids than lactic acid significantly contribute to the nonrespiratory acidosis at low 0, tensions, or that lactate is removed from the blood stream without a corresponding amount of H+. However, the findings could at least partly be explained by the fact that calculation of acid-base changes due to hemoglobin desaturation based on an in vitro buffer curve does not take into account the redistribution of HCO; between blood and extracellular fluid, occurring in vivo (see Michel, 1968). The present results demonstrate that lactate accumulates when the 0, concentration is reduced to 13x, and at 11% there is a significant increase in the lactate/ pyruvate ratio. Since progressive reductions in inspired 0, concentration are associated with decreases in both the arterial Po, and Pco, it becomes difficult to evaluate the cause of the increased lactate concentrations. Hypocapnia is known to induce increases in blood lactate and pyruvate concentrations, and it has been reported that moderate hypoxia is unassociated with lactacedemia unless accompanied by hypocapnia (Eichenholz et al., 1962). Furthermore, alkalosis increases the rate of glycolysis in tissues in vitro (Domonkos and Huszak, 1959; Delcher and Shipp, 1966; Scheuer and Berry, 1967). Thus, it is conceivable that the increased lactate concentrations at 13% 0, are secondary to tissue alkalosis elicited by the hyperventilation. However, if we assume that an increased lactate/pyruvate ratio (or an excess lactate concentration, see Huckabee, 1958) reflects anaerobic production of lactic acid due to tissue hypoxia, exposure of rats to about 11% 0, is obviously accompanied by tissue hypoxia. At any rate, the data indicate that the rat is at least as sensitive to hypoxia as is man (Huckabee, 1958) or the dog (Cain and Dunn, 1964; Cain, 1965).

320

L. D. LEWIS,

U. PONTiN

AND B. K. SlESJij

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14

denerva-