CSF HCO3− regulation in isosmotic conditions: The role of brain PCO2 and plasma HCO3−

Respiration Physiology (1978) 33, 177-198 © Elsevier/North-Holland Biomedical Press

CSF HCO3- REGULATION IN ISOSMOTIC CONDITIONS: THE ROLE OF BRAIN Pco, AND PLASMA H C O ; i

E.E. NATTIE and LEWIS ROMER Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755, U.S.A.

Abstract. We have studied in anesthetized cats the response of CSF HCO 3- to ( I ) a n increase in CSF Pco2, plasma HCO3- controlled at the normal value ; (2) an increase in plasma HCOT, CSF Pco2 controlled at the normal value ; and (3) an increase in both CSF Pco2 and plasma HCO F . Plasma H C O f was controlled via HCO3-/CI- exchange without altering plasma Na concentration or osmolarity using the technique of peritoneal dialysis. The results suggest that CSF HCO F regulation in these conditions is dependent on (1) a Pco,-dependent process in which HCO3- ions are formed in brain cells or choroid plexus and (2) an ionic movement'of H C O f from plasma to CSF dependent on an increase in plasma H C O f . The first process is largely completed by 3 hours; the second takes 6 hours or more. An analysis of plasma and CSF HCO~, C I - and unmeasured anions (UA), (Na + + K + - C1- - HCO3-), suggests that CSF HCO3- regulation may be complex involving ionic exchanges of three or more anions between brain extracellular fluid, plasma and brain cells. Brain metabolism Cerebrospinal fluid

Extracellular fluid Ionic movements

Cerebrospinal fluid (CSF) pH is closely regulated in both metabolic and' respiratory acid-base disturbances, though less well in the latter (see Leusen, 1972; Siesj6, 1972, for review). This regulation depends on mechanisms that can affect (a) CSF Pco2, e.g. ventilation and cerebral blood flow, and (b)CSF HCOf. This paper focuses on CSF HCO3- regulation. Recent observations support a general hypothesis for this regulation. Nattie and Tenney (1976) from an analysis of their study on the effects of potassium depletion on CSF HCO F regulation suggested that both CSF Pco_~and plasma AcceptedJor publication 2 December 1977 l This investigation was supported in part by an allocation from Grant 5S07-RR-05392 from the Biomedical Research Support Branch, Division of Research Resources, NIH, by a Young Pulmonary Investigator Award, HL 18351, to Dr. Nattie from the Heart and Lung Institute, NIH, and by a Public Health Service Research Grant, HL 02888. 177

178

E.E. NATTIE AND L. ROMER

H C O 3 when above some 'threshold' value are independent determinants of CSF HC03 in conditions of increased plasma HCOF and CSF Pco~. They reviewed the literature for experiments in which Pco~ and plasma HCO~ had been for other reasons varied independently and found that in all cases examined the data supported the hypothesis. In studies of the specific role of plasma HCO 3 in CSF HCO 3 regulation during acute respiratory acid-base disturbances, acid or base has been infused intravenously to control or change plasma HC03. In acute respiratory alkalosis, infusion of NaHCO 3 prevented the usual decrease of plasma HCO 3 and the observed decrease in CSF HCO~ was less than when plasma HC03 was allowed to decrease (Chorea and Kazemi, 1976; Pelligrino and Dempsey, 1976). In acute respiratory acidosis, infusion of HC1 prevented the usual increase in plasma HC03 and the observed increase in CSF HCO 3 was less than when plasma HCO 3 was allowed to increase (Hasan and Kazemi, 1976). It was concluded that in these conditions CSF HC03 was in part determined by the plasma H C O 3. After accounting for this effect of plasma HCO~, the remaining change in CSF HCO 3 seemed to correlate with the change in Pco,. In respiratory alkalosis the remaining decrease in CSF HCO 3 when plasma HCO~ was controlled correlated with the increase in CSF lactate, a metabolic change determined largely by tissue Pco.~(Wichser and Kazemi, 1975). In respiratory acidosis it was found that intracerebral acetazolamide diminished that part of the increase in CSF HCO£ that could not be accounted for by changes in plasma HC03- (Wichser and Kazemi, 1975; Hasan and Kazemi, 1976). From these results it was proposed that CSF HCO F is determined by (1) processes occurring in brain cells dependent on Pco2 and inhibited by acetazolamide and (2) processes at the blood-brain barrier which reflect the plasma H C O 3 c o n c e n t r a t i o n . A similar hypothesis had been proposed earlier in a less specific form (Adaro et al., 1969). It is important to note that in the experiments which evaluate the role of plasma HCO~, the intravenous solutions used were not isosmotic with body fluids. DiMattio et al. (1975) have shown that bulk flow of fluid into or out of the CSF can be altered significantly by very small changes in serum osmolarity. That this effect is not dependent on changes in brain tissue water content suggests that it is located at the blood-brain barrier. Intravenous infusions of non-isosmotic solutions could alter bulk flow of fluid into or out of the CSF and therby affect the CSF HCO 3 concentration independent of passive or active ionic movement. Similarly, the ionic movement of HC03 could be affected by changes in the balance of other ions not controlled in the experiment (Nattie and Tenney, 1976). This paper reports experiments testing the role of brain Pco2 and plasma HCO~ in CSF HCOf regulation at normal and elevated Pco2 and plasma HCOF values. Control of plasma HCOf, Na +, K +, CI- and osmotic pressure was achieved using the techniques of peritoneal dialysis.

CSF HCO;: BRAIN Pco2 AND PLASMA HCO3-

179

Methods

Thirty-eight cats of both sexes weighing from 2.5 to 6.0 kg were anesthetized with Dial urethane 0.75 ml/kg injected i.p. This provided deep surgical anesthesia for the 2-3 hours of preparation due to both the phenobarbital and the urethane. Over the longer course of the experiment as the phenobarbital effects wore off the level of anesthesia lightened. The cats were intubated and the femoral artery and vein catheterized. A teflon catheter of 4 mm diameter was positioned within the peritoneum through a midline ventral incision and sewn into place. The animals were fixed in a stereotaxic head holder and the neck muscles were dissected to expose a small part of the atlanto-occipital membrane. An 18 gauge needle was placed in the cisterna magna through the atlanto-occipital membrane using a micro-manipulator. An adapter on the proximal end of the needle held an 80-100 pl Radiometer glass capillary tube connected with tubing to a 1 ml syringe. Body temperature was maintained at 37-38 °C with a heating pad and a Yellow Springs Instrument proportional temperature controller. Brain temperature in the region of the cisterna magna was recorded at the end of 6 representative experiments using a small bead thermistor inserted rostrally 1 cm into the brain through the atlanto-occipital membrane puncture site. PAco2 was monitored with a Beckman infrared C O 2 analyzer, blood pressure with a Statham pressure transducer and the outputs recorded on a Grass Instrument Co. oscillograph. Immediately after intubation and catheter placement the animals were paralyzed with gallamine triethiodide 2-3 ml i.v. and ventilated with a 30~ O2, 70~ N 2 gas mixture at a volume and frequency to maintain PAco., in the 25-30 mm Hg range, i.e. the normal range for the awake cat. This situation was maintained for 1-2 hours before the onset of the experiment. There were four experimental groups: Group 1, normal Pco2, normal bicarbonate; Group 2, normal Pco_- high bicarbonate; Group 3, high Pco_,, normal bicarbonate; Group 4, high Pco:, high bicarbonate. The terms 'normal' and 'high' Pco~, referred to CSF values. Normal CSF Pco., was maintained by keeping the PAco, at 2530 mm Hg throughout the experiment. High CSF Pco: was achieved by switching to a 3~o CO2, 30~o O2, 67~ N2 mixture for the experimental period and adjusting the ventilation as needed. The terms 'normal' and 'high' bicarbonate referred to blood plasma. Normal HCO3- animals were dialyzed with a normal HCO3- dialysis solution, high HCO3- animals with a high HCO;, low C1- solution. The ionic constituents of the normal dialysis solution were: NaHCO3 0.02 M, NaC1 0.15 M, KCI 0.005 M, CaC1 0.001 M, MgC1 0.001 M and glucose 100 mg~o; for the high HCO 3 solution: NaHCO3 0.160 M, NaC1 0.02 M, KC1 0.01 M, CaC1 0.00! M, MgC1 0.001 M and glucose 100 mg~. Control measurements Were made at time 0, then the Pco,, was changed if appropriate and the dialysis solution previously warmed to body temperature was injected i.p. 100 ml/kg body weight. The solution remained in the peritoneum for the duration of the experiment. Measurements were made at 2, 4 and 6 hours after the onset of the experiment. Arterial blood was analyzed for pH, Pco: and Po_, with Radiometer electrodes and meters calibrated before and after

180

E.E. NATTIE AND L. ROMER

each measurement with Radiometer precision buffers and with Pco.~ and Po_, gas mixtures calibrated by Scholander micro-gas analysis. The Pco_, and Poe electrodes were also checked periodically with tonometered blood samples. The measurements were made at 37.7 '~C and were corrected to body temperature if necessary. Blood plasma and CSF were analyzed for total CO2 (T CO2) with a Natelson microgasometer, for Na and K with a flame photometer, for C1 with a Cotlove chloridometer and for osmolarity with a vapor pressure osmometer. For evaluation of CSF pH and Pco2 the dead space of the needle, capillary tube, syringe apparatus was discarded and the next portion was analyzed within 5 sec for pH in the Radiometer electrode, calibrated before and after each measurement. Duplicate CSF samples agreed to within 0.01 pH unit. As measurements of brain temperature in the region of the cisterna magna in 6 representative animals showed it to be 1 'C lower than body temperature, CSF pH values were corrected in all animals to a temperature 1 :C lower than the measured body temperature. CSF Pco_~ was calculated from the measured CSF pH and CSF T CO2 (Nattie and Tenney, 1976).

Results

There were 19 successful experiments as defined by the following criteria: (a) mean arterial pressure was maintained at greater than 80 mm Hg for the entire experiment; (b) PaO2 was maintained at greater than 110 mm Hg for the entire experiment; (c) the CSF samples were clear, i.e. unhemolyzed; (d) appropriate changes in CSF Pco,, and plasma H C O 3 were produced by the protocols ; and (e) isosmotic conditions were maintained throughout the experiment. Table 1 shows the mean values for serum sodium, potassium and osmolarity of the four groups for the four measurement times. The Na and osmolarity values at 0 hours were similar and the changes in these values over the course of the experiment were very small in all four groups. The mean serum osmolarity increased slightly over 6 hours from 4 to 10 mOsm/1 in three of the four groups. These changes in serum Na and osmolarity were considered trivial in comparison to what must occur in experiments in which NaHCO3 solutions up to 1 M in concentration are infused intravenously. Mean serum K decreased in the high H C O f dialysate groups by about lmM/1 in spite of the increased KC1 levels in this dialysate solution. This hypokalemia was associated with high plasma H C O f but not necessarily with metabolic alkalosis as Group 4 arterial pH was essentially unchanged but serum K decreased to the same degree as in Group 2. The mean CSF Na values are shown as a function of time in fig. 1. The initial mean CSF Na values of Groups 1 and 3 tended to be slightly higher than observed in Groups 2 and 4. With dialysis, little change in mean CSF Na occurred in any groups with the exception of the slight decrease in the 6 hour value of Group 4. Mean CSF osmolarity initially was very much the same in all groups and did not change over the course of the experiment. CSF K data were not shown as it was essentially the same in all groups at all measurement times. The mild hypokalemia associated with the high

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H C O 3 dialysate was not severe enough to be reflected in the CSF K values. The total range of the mean CSF K values observed was from 2.7-3.4 mM/1. The response of blood and CSF CI to the protocols is shown in fig. 2. The initial mean blood (serum) C1 levels were similar in all four groups. Groups 1 and 3 with normal H C O ; ,

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normal C1 dialysis showed a slight increase in mean blood C1 over the 6 hour duration of the experiment. Mean serum C1 of Groups 2 and 4 decreased to a similar degree with a similar time course reflecting the effects of the high HCO3-, low C1 dialysis. The initial mean CSF C1 was similar in all four groups. Over the time course of the experiment Group I showed no essential change in mean CSF C1, the Group 3 mean CSF C1 decreased even though plasma CI was unchanged or slightly increased, and the Groups 2 and 4 mean CSF C1 decreased with a roughly similar time course and degree reflecting the decrease in plasma C1. Note that the quantitative change in mean CSF C1 in Groups 2 and 4 was less than the change in mean plasma C1 (see Discussion). The results in terms of the independent variables of the experiment, CSF Pco.~ and blood HCO 3, and the dependent variable, CSF HCO3, are shown in fig. 3. The initial mean values of all three variables were similar for all four groups. For Group 1 normal HCO3 normal Pco2 control animals, mean arterial HCOF was essentially unchanged for the duration of the experiment. Mean arterial Pco2 (not shown) ranged from 25.5 to 28.5 mm Hg and CSF Pco: remained essentially unchanged. Mean CSF HCO£ decreased slightly (1.1 mM/kg H20 at 6 hours). The origin of this small change was unclear (see later Discussion). In Group 2 normal Pco, high HCOy animals, mean blood HCO 3 increased, a change that was essentially, complete at 2 hours and stable for the duration of the experiment. Mean arterial Pco., (not shown) ranged from 27.1 to 27.9 mm Hg while CSF Pco: increased

184

E. E, NATTIE A N D L. ROMER

slightly at 2 hours, was stable over 4 hours, and then increased slightly at 6 hours such that the Group 2 mean CSF Pco2 at 6 hours was greater than that of Group 1 (P < 0.01) but less than that of Groups 3 and 4 (P < 0.01). The mean CSF HCO~response was a steady, almost linear increase over 6 hours such that the mean CSF HCO~ at 6 hours was 30.1 m M / k g H_~O, a value very close to the mean arterial HCO3 at that time, 31.1 mM/kg H:O. In Group 3 high Pco2 normal HCO 3 animals, arterial HCO3 remained unchanged and at a level similar to Group 1 animals over the course of the experiment. Mean arterial Pco: (not shown) was 56.4, 52.7 and 53.6 mm Hg at 2, 4 and 6 hours respectively. The resultant mean CSF Pco2 was increased to a value of 63.3 mm Hg at 2 hours and remained roughly at this level over the duration of the experiment. In response, mean CSF H C O 7 increased from 22.7 to 25.0 mM/kg H:O by 2 hours with only a small further increase to 25.9 mM/kg H:O by 6 hours. In Group 4 high Pco_, high HCO~ animals, arterial HCO~ was increased by 2 hours to a mean value of 33.8 mM/kg H20 and it further increased slightly to 36.3 mM/kg H,O at 6 hours. Group 4 mean arterial HCO3 was slightly higher than observed in Group 2 but in both cases the change was complete by 2 hours and the values were maintained at a relatively stable level over the duration of the experiment. Mean arterial Pco_,was increased to 47.2, 48.8 and 50.3 mm Hg at 2, 4 and 6 hours respectively, a change in arterial Pco~ that was less than that in Group 3. The reason for this was that the Group 4 animals had a wider (CSF-arterial) Pco~ difference and as the goal of the experiment was to maintain similar CSF Pco: values in Groups 3 and 4, a lower arterial Pco~, was needed. The resultant mean CSF Pco~, of Group 4 animals was increased by 2 hours to 57.3 mm Hg and further increased to 65.5 and 67.6 mm Hg by 4 and 6 hours respectively. While the time course of the change in CSF Pco~ was slightly different in Group 4 compared to Group 3, the general characteristics of the response were very similar. While Group 4 was not perfectly comparable to Groups 2 and 3 in respect to the degree of change in plasma HCO 3 or the time course of the change in CSF Pco~, the agreement was sufficient to allow a qualitative comparison of the response. The Group 4 CSF HCO 3 response qualitatively reflected the responses observed in Groups 2 and 3, There was an initial increase in mean CSF H C O 3 by 2 hours that was greater than observed in either Groups 2 or 3 and the remaining time course of the CSF HCO3 change was steady, linear, and almost parallel to that observed in Group 2. At all times the mean CSF HCO7 of Group 4 was greater than that observed in either Groups 2 or 3. The mean arterial and CSF pH values for the four groups at the four time periods are shown in fig. 4. The mean control arterial and CSF values were similar in all four groups, CSF being more acid than blood. Group 1 mean CSF pH decreased slightly reflecting the small decrease in CSF HCO3. In Group 2, mean arterial pH increased significantly and was reflected in the CSF at 4 and 6 hours. In Group 3, mean arterial and CSF pH decreased with about the same time course and the change in CSF pH was less than that observed in blood. In Group 4, mean arterial pH was about the same as in Group 1 while in CSF the mean pH decreased

CSF H C O ~ : BRAIN Pco_~ A N D PLASMA HCO F

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and ranged between the values observed in Groups 2 and 3 though closer to the Group 3 data. An evaluation of the changes in all anions was made. For this evaluation, plasma lactate was measured and plasma and CSF unmeasured anions (UA) were calculated defining UA as (Na + + K + - C 1 - - H C O 3 ) . In plasma there was essentially no change in the UA in Groups 1 and 3 (table 2) nor was there any significant trend in the measured lactate values. In Groups 2 and 4 the plasma UA increased significantly over the course of the experiment and the sum of the increased plasma HCO 3 and UA exactly balanced the decrease in CI- in these groups. The measured TABLE 2 Plasma unmeasured anions and lactate (in parentheses). Unmeasured anions are defined as (Na + + K + C I - - HCO~). The data are expressed as mM/kg H20 assuming plasma is 0.93 H20. The values are means _+ SEM 0

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186

E . E . N A T T I E A N D L. R O M E R

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Fig. 5. The changes in CSF CI -, HCO F and unmeasured anion (UA) for each group for the three time periods 0-2, 0 4 and 0-6 hours are shown in the figure. Values above 0 for the open and cross-hatched bars on the left of each set representing HCO~ and UA, indicate an increase in these variables, below 0 indicate a decrease. For the stippled bars on the right of each set representing CI-, values above 0 indicate a decrease in C1- and vice versa. So, for example, in Group 2 over the 0 2 hour time period, CSF HCO S increased almost 2 mM and CSF UA increased just over 4 mM for a total increase of ~ 6 7 mM which was almost exactly balanced by the decrease in CSF CI-. lactate increased significantly only in G r o u p 2 a n d here it c o n t r i b u t e d b u t a small p a r t o f the increase in U A . In the C S F , to m i n i m i z e the effect o f the large v a r i a t i o n in the c a l c u l a t e d values for the U A , the c h a n g e in U A f r o m 0 to 2, 0 to 4, a n d 0 to 6 h o u r s was c a l c u l a t e d for each a n i m a l . T h e m e a n results for each g r o u p are shown in fig. 5 a n d r e l a t e d to the o b s e r v e d changes in H C O S a n d C1- for the same time periods. In G r o u p 1 there was an increase in U A - p r o b a b l y the result o f the small increase in C S F N a in this g r o u p (fig. 1), while the changes in C S F C1- a n d H C O 3 were r o u g h l y equivalent. In G r o u p s 2, 3 a n d 4 the s u m o f the increase in H C O £ a n d U A was equivalent to the decrease in C1-. H o w e v e r , in G r o u p 2 the increase in U A was s u b s t a n t i a l while in G r o u p s 3 a n d 4 there was little increase in U A a n d the C I - a n d H C O 7 changes a l o n e were a p p r o x i m a t e l y equivalent.

Discussion In terms o f e x p e r i m e n t a l design, the desired changes in the p r o p o s e d i n d e p e n d e n t variables, p l a s m a H C O 3 a n d C S F PcQ, were p r o d u c e d u n d e r w e l l - c o n t r o l l e d i s o s m o t i c c o n d i t i o n s with m a i n t e n a n c e o f n o r m a l s e r u m s o d i u m c o n c e n t r a t i o n . Technically, in the 19 e x p e r i m e n t s t h a t f o r m the basis o f this r e p o r t the a n i m a l s

CSF HCOF: BRAIN Pco_, AND PLASMA HCOf

187

were well oxygenated and perfused and, as shown in the control group, the 6 hour duration of the experiment per se had little effect on the two independent variables or the dependent variable, CSF HCO3. Two technical points deserve further comment. First, brain temperature, which has been shown to differ from peripheral blood temperature in the awake (Baker and Hayward, 1967) and anesthetized cat (McCook et al., 1962; McElligott and Melzack, 1967), was measured in brain rostral to the cisterna magna sampling site in six animals. It was found to be 1 °C below rectal temperature and all CSF values were corrected to a temperature 1 ~C below the measured rectal temperature. Second, the experiments were performed under anesthesia, in this case Dial urethane which consists of diallylbarbituric acid, urethane, and monoethyl urea. The effects of these substances on blood-brain barrier function is unknown. Barbiturates do have demonstrable effects on brain tissue metabolism (Nilsson and Siesj6, 1974) while the effect of the other agents on brain metabolism is unknown. Some caution must be used in applying these results to experiments using other anesthetic agents or no anesthesia at all. C S F Pco2 The significance of the Pco2 of CSF is controversial. Some workers feel that CSF Pco2 closely reflects brain venous (and thereby brain tissue) Pco2 (Pappenheimer et al., 1965). Others have postulated that CSF Pco2 more closely reflects a value of blood Pco~ midway between the arterial and venous end of the capillary (Pont6n and Siesj6, 1966). Experimental evaluation of the problem has been hindered by the technical difficulty in accurately measuring pH or Pco_,in CSF (Fencl et al., 1966; Davies, 1976) and by the many factors that appear to affect the Pco~ difference between arterial blood and CSF, the APco 2 (CSF-a). These factors include cerebral blood flow (CBF) (Siesj6 et al., 1967), anesthesia, ventriculo-cisternal perfusion and positive pressure ventilation (Berkenbosch, 1971), the degree of blood acidity via the 'charged membrane hypothesis' (Davies et al., 1973; Davies and Gurtner, 1973; Razavi et al., 1977) and hypotension and ischemia (Siesj6 and Zwetnow, 1970). In our results the mean control values of APco~ (CSF-a) for the four groups ranged from 10.0 to 14.3 mm Hg, and agree well with those reported for anesthetized animals with positive pressure ventilation (Berkenbosch, 1971 ; Pavlin and Hornbein, 1975a~t ; Davies and Gurtner, 1973). However, all of these values for APco~(CSF-a) are greater than those reported in awake goats (Pappenheimer et al., 1965; Fencl et al., 1966), in rats (Caronna et al., 1974; Nattie and Tenney, 1976) and in man (Plum and Price, 1973). In our experiments we utilize anesthesia and positive pressure ventilation, factors which are known to widen the z~Pco, (CSF-a) (Berkenbosch, 1971), and we evaluate changes in acid-base balance in blood and brain, factors which can have different effects on APco~ (CSF,a) depending on conditions. To evaluate this latter possibility, our mean control values of APco: (CSF-a) and the subsequent mean values under the isosmotic conditions of these experiments have been plotted as a function of arterial [ H ÷] in fig. 6 and connected by the solid line. Note that the APco: (CSF-a) is inversely related to arterial [H +].

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Fig. 6. The Pea2 difference between arterial blood and CSF, APco: (CSF-a), is shown as a function of the arterial hydrogen ion concentration. This value, labeled H+a, is calculated from pH measurements assuming an activity coefficient of 1.0. The mean values at each measurement time for each of the four groups of the main experiment are shown using the same symbols as in the previous figures. These values are connected by the solid line drawn by eye and labeled "isosmotic dialysis'. The mean values at the same time intervals for four animals dialyzed to increase plasma H C O ~ to the same degree as in G r o u p 2, with normal Paco: but with a decrease in plasma osmolarity, are connected by the lower dashed line labeled "hyposmotic dialysis'. The actual values at the same time intervals for two animals dialyzed to increase plasma H C O 7 to the same degree as in Group 2, with normal Paco, and with an increase in plasma osmolarity, are connected by the upper dashed line labeled 'hyperosmotic dialysis'. S~e text for further details.

These results do not support the 'charged membrane hypothesis' which predicts that APco: (CSF-a) is directly related to arterial [H +]. Expressing the data as a function o f C S F [ H + ] also results in an inverse relationship of APco: (CSF-a) and [ H + ] . Insofar as arterial and CSF [ H +] in these non-steady-state experiments reflect brain interstitial fluid [ H + ], the results support the idea that with low [ H + ] in the vicinity of cerebral vessels vasoconstriction occurs and decreased cerebral blood flow widens the APco: (CSF-a) (Caronna et al., 1974). The results of two additional experiments are also shown in fig. 6. Four animals were dialyzed such as to result in a 5')~; decrease in plasma osmolarity and serum Na with plasma HCO3 increased roughly to the same degree as in Group 2 and maintenance of a normal Paco:. The mean APco ~ (CSF-a) of these animals is below that of the isosmotic dialysis group in the low arterial [ H + ] range. Two further animals were dialyzed with hyperosmotic solutions again at constant Paco: and increased plasma [ H C O 3 ] . The plasma osmolarity in these animals was increased by 6-7%o, serum Na was increased by 6'!J~, in one case, in the other it remained unchanged. In these hyperosmotic animals the APco: (CSF-a) was quite high in the low arterial [ H +] range. The results suggest that changes in osmolarity can be added to the list of factors that affect the APco: (CSF-a). The mechanism of the osmotic pressure effect is unclear.

CSF HCOT: BRAIN Pco~, AND PLASMA HCO F

189

Given the uncertainty with respect to the determinants of the APco., (CSF-a) it is difficult to conclude precisely that CSF Pco2 at all times reflects mean brain tissue Pco~,- But in the isosmotic conditions of these experiments we feel that the CSF Pco: is a reasonable approximation of the brain tissue value.

Effect 0[" increased CSF Pco: on CSF HCO~ with maintenance Qf constant, normal plasma HCO3 These G r o u p 3 experiments were designed to evaluate the time course and magnitude of these response o f C S F HCO3 to the isolated stimulus of an increase in tissue Pco,~Comparing the 2, 4 and 6 hour values of CSF HCO 3 with the 0 hour value, the increase in CSF H C O ; was 2.3, 3.1 and 3.2 mM/kg H20. The response was 72% completed by 2 hours, and 97~o completed by 4 hours. Comparing the 6 hour CSF H C O ; value with the 0 hour value and using an average value for the increased CSF Pco~, the ACSF H C O 3 / A C S F Pco, was 0.152 m M • mm Hg-~. This value agrees well with others in the literature obtained under experimental conditions in which CSF Pco_,was increased in the same range as in this report with plasma HCO3- kept constant or decreased by infusion of acid. F r o m the results of Kazemi et al. (1976) in rats with 2 hours exposure to CO2, the calculated value was 0.166 m M • mm Hg -~ ; from Hasan and Kazemi (1976) in dogs with 4 hours exposure to CO 2, 0.169 mM • mm H g - l ; and from Siesj6 and Pont~n (1966) in rats with 6 hours exposure to CO,, 0.170 m M . mm Hg -1. Thus in anesthetized animals a 10 mm Hg increase in brain tissue Pco_,will result in a 1.5-1.7 mM increase in CSF HCO3. This response is insufficient to regulate CSF pH perfectly. In fig. 4 it can be seen that CSF pH is most acid in Group 3. We can calculate that with a 10 mm Hg increase in CSF Pco2 and no change in CSF HCOc, CSF [ H + ] would increase from the normal value of 43.4 nM to 54.2 nM. With an increase of CSF H C O 3 of 1.52 mM (0.152 m M . mm Hg -1 x 10 mm Hg), CSF [ H + ] would be 50.7 mM. The increase in CSF HCO3 resulting from the change in tissue Pco,~ alone would decrease the change in CSF [ H +] by 32%. However, for complete CSF pH regulation another source for H C O c must exist. Effect of increased plasma HCO3 on CSF HCO3 with maintenance of constant, normal CSF Pco.~ In these Group 2 experiments, with a 12-13 mM increase in plasma HCO3 completed within 2 hours, CSF H C O ; increased steadily over the 6 hours of the protocol. The change in CSF H C O ; at 2, 4 and 6 hours compared to the 0 hour control value was 1.9, 4.6 and 8.4 mM respectively. While Paco~ was well controlled at the normal value, CSF Pco.~ increased by 4-5 mm Hg at 2 and 4 hours and by 11.5 mm Hg at 6 hours. This increasing APco~ (CSF-a) most probably reflected decreased cerebral blood flow accompanying the slightly alkaline brain ecf pH (see above). The small increase in CSF Pco,~is unlikely to account for the very different magnitude and time course of the CSF HCO3 response in these Group 2 experiments in comparison to G r o u p 3. Using the value for AHCO~/ACSF Pco_~of 0.152 mM • mm Hg-1, an 11.5 mm

190

E.E. NATTIE A N D L. ROMER

increase in CSF Pco~ would account for only a 1.8 mM increase in CSF HCO;. Our interpretation of the results is that the increased plasma HCO; is being reflected in the CSF due to a process of ionic movement. To quantitate the dtCSF HCO3/A plasma HCO3 the observed Group 2 CSF HCO3 values at 2, 4 and 6 hours have been corrected for the HCO~ contributed by the small increase in CSF Pco, using the value of 0.152 raM. mm Hg -~ derived from the Group 3 experiments. The dICSF HCO;/A plasma HCO~- values so calculated are 0.09, 0.30 and 0.54 at 2, 4 and 6 hours respectively. Other estimates of this relationship, A CSF HCO3/A plasma HCO3, in conditions of metabolic alkalosis with some attempt to control Pco- are present in the literature. In most cases a small increase in CSF Pco,~ accompanied the metabolic alkalosis and the increase in CSF HCOz due to this Pco_~component has been corrected as above. Pavlin and Hornbein (1975b) infused 0.9 N NaHCO 3over 6 hours to increase plasma HCO; in dogs. CSF Pco: increased 5.4 mm Hg and the corrected ACSF HCO~/A plasma HCO3 is 0.43. Chazan et al. (1969) produced chronic metabolic alkalosis in dogs via gastric HC1 depletion. CSF Pco: increased 13 mm Hg and the corrected value for ACSF HCO~/A plasma HCOf is 0.45. Hasan and Kazemi (1976) infused 1 M NaHCO3 over 4 hours to increase plasma HCO~ in dogs while inhibiting the mechanism of brain Pco:-dependent CSF HCO3 formation by the use of intracerebral acetazolamide. The value for ACSF HCO~/di plasma HCO~ is 0.40. The corrected value in our Group 2 experiments at 6 hours, 0.54, is higher than those calculated from the literature. The explanation for this variation is not apparent though both the species used and the technical details of the experiments are different (see below). While roughly half of an isolated increase in plasma H C O ; is reflected in the CSF in 6 hours, in respect to CSF pH regulation the importance of the hindrance to ionic HCOf movement at the blood-brain barrier can be seen in fig. 4. In the Group 2 results, plasma pH increases to a value of ~ 7.630, while CSF pH increases only to 7.377. Were the bloo&brain barrier freely permeable to HCO~, CSF pH in these circumstances would become quite alkaline. With normal blood-brain barrier function the small rise in CSF Pco~, even with Paco~ held constant, further minimizes the increase in CSF pH. If hypoventilation in response to the metabolic alkalosis were allowed to occur, PacQ would increase and therefore even with the same APco: (CSF-a) CSF Pco,,would increase still further with a concomitant decrease in CSF pH towards the normal value. EfJbct o f increased C S F PcQ and plasma HCO3 on C S F HCO~ The response ofCSF HCO 3 to the combined stimuli of an increase in CSF Pco~ and plasma HCO 3 is greater than the response to either stimulus alone. The general nature of the time course of the response appears to represent the sum of the responses to the stimuli presented individually. There is a rapid increase in CSF HCO 3 by 2 hours, followed by a steady increase over 4 and 6 hours. The magnitude of the stimuli and the responses were such that while plasma pH became slightly alkaline,

CSF HCO~ : BRAIN Pco., AND PLASMAHCOF

191

CSF pH decreased such that it was between the values observed in response to each individual stimulus (fig. 4). Quantitatively, we can use the values for ACSF H C O f / A CSF Pco,, and ACSF HCO3-/A plasma HCO 3 to predict the increase in CSF HCO 3 in response to both stimuli presented simultaneously. At 2 hours the predicted increase in CSF HCO S is 3.6 raM, the observed 4.3 mM; at 4 hours, the predicted value is 8.3 raM, the observed 6.9 raM; and at 6 hours the predicted value is 13.4 raM, the observed 9.7 raM. The agreement is good at 2 hours but the predicted values become progressively greater than the observed values at 4 and 6 hours. The primary determinant of these later predictions is the ACSF HCO3/A plasma HCO£ values from the Group 2 experiments, which suggests that the mechanisms determining the CSF HCO~ response to an increase in plasma HCO3 may be different quantitatively in the Group 2 and 4 experiments. This difference may be due to an effect of the difference in Pco_,in the two groups (Pelligrino and Dempsey, 1977), the difference in pH on the brain side of the barrier (Hasan and Kazemi, 1976) or the difference in pH on the blood side of the barrier (Woodbury, 1971). Mechanisms

In this section we discuss the possible mechanisms involved in (1) the Pco,-related increase in CSF HCO£ and (2) the plasma HCOF-related increase in CSF HCO£. There are at least three mechanisms which could contribute to the increase in CSF HCOf observed following an increase in CSF Pco2, plasma HCOf held stable: (1) Pco, could affect either the active or passive characteristics of the barrier separating brain ecf and blood such as to alter the usual steady-state distribution of H C O f ; (2)there could be increased secretion of HCOf by choroid plexus (? or glial) cells; (3)neurons or glia could increase cellular HCO 7 via buffering or metabolic events and contribute HCO£ ions via transmembrane flux to brain ecf. Fencl et al. (1966) in their studies using ventriculo-cisternal perfusion in awake goats found that the steady-state concentration difference for HCOf between mean capillary plasma and the CSF perfusate at zero net HCO3 flux changed in value in different metabolic acid-base states. The determinants of this steady-state HCOf concentration difference between CSF and blood are unknown. The presence of a small DC potential difference (PD) of a few millivolts between CSF and blood, CSF being positive, and the fact that this PD varies inversely with blood pH (Held et al., 1964) has led to many studies designed to evaluate whether passive or active forces are involved in this steady-state HCOf distribution. Usually, the PD and the ionic concentrations are used to calculate electrochemical potentials. The application of equations for electrochemical equilibrium to situations like this in which factors other than the membrane itself are involved in determining the ionic distributions yields results difficult to interpret (Rapoport, 1976) but still of interest. Pavlin and Hornbein (1975a d) measured simultaneously in dogs over a 6 hour time period the CSF-plasma PD and the concentrations of H + and HCOf in CSF and plasma during acid-base disturbances and reviewed the pertinent literature. In

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almost all cases the calculated electrochemical potential for H + and HCO3- returned to a value close to that in the control state after an initial pertubation, suggesting that passive forces were involved. However, as emphasized by the investigators (Pavlin and Hornbein, 1975d), the interpretation of the findings depends on many assumptions. It is possible that in our experiments the change in Pco2 affected the active or passive characteristics of the brain ecf plasma barrier such as to result in a new steady-state HCO£ distribution. However, this may be difficult to evaluate for in addition to the theoretical problems mentioned above, the CSF-blood PD in the cat is (a)CSF negative (Pannier et al., 1971; McLeskey et al., 1977) and (b) unstable over time (McLeskey et al., 1977). The fact that the cat differs from other species in respect to the CSF-blood PD but not in respect to CSF HCO3 regulation suggests that the PD may not be of major importance in CSF HCO3 regulation. The role of choroid plexus (? and glial) cells in Pco,-determined CSF HCO3 formation (Husted and Reed, 1977) catalyzed by carbonic anhydrase has been emphasized by Maren (1972) and Wichser and Kazemi (1975). Intracerebral use of carbonic anhydrase inhibitors has inhibited part of the CSF HCO3 response to an increase in Pco, (Maren, 1972; Vogh and Maren, 1975; Wichser and Kazemi, 1975; Hasan and Kazemi, 1976; Kazemi et al., 1976). Whether the effect of the enzyme inhibitor is an actual CO 2 hydration within the cells or on some subsequent process, e.g. transcellular ionic movement, is unclear as brain tissue HCO£ increased to the same extent following CO 2 stimulation in both the normal and inhibited state while CSF HCO3 was increased to a greater degree in the normal state (Wichser and Kazemi, 1975; Kazemi et al., 1976). Net HCO3 secretion by these cells into brain ecf would also involve H + movement into blood to prevent intracellular acidosis. The concept that cell metabolites might play a role in CSF pH regulation was suggested by Siesj6 and Kj~llquist (1969). They hypothesized that a primary choroid plexus secretion rich in HCO 3 would be altered as it passed through the brain by metabolic acids produced by cell metabolism. Cerebral metabolism does respond to changes in Pco,,. Following an increase in Pco2, there is within minutes a decrease in glucose utilization (Miller et al., 1975; Borgstr6m et al., 1976) while oxygen consumption remains unchanged (Miller et al., 1975). The tissue levels of citric acid cycle intermediates, lactate, pyruvate, glutamate and aspartate all fall (Messeter and Siesj6, 1971c; Folbergrovfi et al., 1972b, 1974, 1975) but the cerebral energy state as reflected by high energy phosphates and the NADH/NAD + ratio is unaffected (Folbergrov/t et al., 1972a). The interpretation of these results is that the brain utilizes endogenous substrates to maintain energy production in hypercapnia (Miller et al,, 1975; Folbergrovfi et al., 1975). lnsofar as some of the endogenous substrates are organic acids the response may also represent a metabolic means of cell acid-base regulation (Folbergrovfi et al., 1972b). If the increase in cell HCO3 so generated can be reflected in brain ecf via transmembrane flux, then these brain cells can be a source of CSF HCO3. An additional cell biochemical mechanism

CSF HCOF : BRAIN Pco, A N D PLASMA HCO~

193

suggested to play a role in brain cell acid-base regulation involves the glutamine - glutamate - ammonia system (Kazemi et al., 1973, 1976; Weyne et al., 1973, 1976). Following an increase in Pco2, brain tissue glutamine, ammonia, and gammaaminobutyric acid are increased while glutamate is decreased (Messeter and Siesj6, 1971 c; Folbergrovfi et al., 1972b; Kazemi et al., 1973, 1976 ; Weyne et al., 1973, 1976). The increased glutamine - whatever its source - has been interpreted as a source for ammonia via deamination, the ammonia then acting as a buffer for the excess acid (Kazemi et al., 1973; Weyne et al., 1976). Alternatively, the use of amino acids as endogenous substrates for energy metabolism requires deamination of some amino acids. The ammonia produced would be detoxified via the glutamate glutamine pathway (Weyne et al., 1976). The cause and function of the observed changes in this metabolic system are as yet unclear. However, in the range of Pco., levels used in our experiments the reported changes in brain ammonia, glutamine and glutamate are small and variable (Kazemi et al., 1976; Folbergrovfi et al., 1975) such that it is difficult to assign a significant quantitative role to these mechanisms in our experiments. Mechanistic interpretation of our Group 3 results then is difficult given these different possibilities. We find it interesting that the time course of the CSF HCO 3 response to an increase in Pco~ agrees well with the time course of the calculated intracellular HCO3- increase as well as with the time course of the changes in cell metabolites possibly related to this increase (Messeter and Siesj6, 1971a~c; Folbergrovfi et al., 1972b). Our analysis of CSF and plasma anions lends support to the possibility that HCO3 formed within cells can move to the ecf. In Group 3, the increase in CSF HCO3- was roughly equivalent to the decrease in CSF C1- (fig. 5). Plasma C1- had increased slightly. These data are consistent with a brain tissue ecf HCO3-/C1- exchange. In respect to the mechanisms that determine the increase in CSF HCO~ in response to an increase in plasma HCO3, Pco., held constant, the measured values of HCO3 obtained from cisternal fluid reflect the result of a number of processes. These include secretory and possibly non-secretory movement of HCO3 at the choroid plexus, movement of HCO3 at the blood capillary-brain ecf sites, possible net exchange of HCO3- ions between extra- and intracellular fluid, the movement of the HCO£ ions in the ecf space from the pericapillary region to the ependymal and pial glial barriers and the movement of HCO3 across these barriers. The choroid plexus appears to play little part in the CSF HCO£ increase following an increase in plasma HCO3-. The HCO3- concentration of cat choroid plexus fluid evaluated in an isolated chamber in situ does not change following changes in plasma HCO£ (Husted and Reed, 1977). The movement of HCO 3 at the capillary-brain ecf site has been proposed to be both an active (Severinghaus et al., 1963; Fencl et al., 1966) and a passive process (Siesj6 and Kj~llquist, 1969; Pavlin and Hornbein, 1975a-d), though the evidence for either case is inferential using, as does this study, CSF and plasma HCO 3 values. Studies using large labeled molecules have demonstrated movement from ventricular fluid to tissue that is consistent with

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diffusion across the ependymal and pial glial barriers and through the ecf space (Fenstermacher and Patlak, 1975). Results of studies using ventriculo-cisternal perfusion of HCO3 have also been interpreted as being consistent with relatively free movement of HCO; ions via diffusion across the ependymal and pial glial barriers and through the ecf space (Pappenheimer et al., 1965; Fencl et al., 1966). Interpreting our Group 2 results in the light of this evidence we feel that the increase in CSF HCO3- over 6 hours following an increase in plasma HCO£ probably reflects an active or passive exchange process at the capillary-ecf barrier with subsequent diffusion of HCO3 through the ecf space and ependyma to CSF. The role of net HCO3- exchange with intracellular contents in these conditions is unknown and changes in the HCO3- concentration of the primary choroid plexus secretion appear not to be important. The relative contributions of the capillary-ecf exchange process, the diffusion process and the possible net HCO~ exchange between ecf and cell contents to the total time course of the increase in CSF HCO3 are at present not calculable. However, it is useful to compare values obtained in this study for the time course of CSF HCO; equilibration following a step change in plasma HCO3 maintained for the duration of the experiment with similar values obtained in studies of other molecular species. CSF glucose reaches equilibrium in 80-100 min following a step increase in plasma concentration (Pappenheimer and Setchell, 1973). 24Na and 3sC1 reach CSF equilibrium roughly at 5-6 hours following a step increase in plasma levels (Davson and Welch, 1971). In both studies there are interactions between ecf and the cell. Glucose is taken up by cells for metabolic utilization, and the distribution spaces for Na and C1 are greater than the brain ecf space determined by larger molecules. If we assume that the process of HCO 7 equilibration in brain following a step increase in plasma includes no component of interaction with the intracellular space or its contents, then the time required for HCO£ to cross the blood ecf space barrier and diffuse to CSF is longer than presumed for these other molecules. This analysis suggests that (1) either the bloodecf barrier permeability for H C O ; is less than or these other species or that there are some other forces opposing HCO~ movement from blood to CSF, e.g. the negative CSF-plasma PD in the cat ; (2) there is a hindrance to HCO 3 diffusion in brain ecf; or (3) that the assumption concerning the lack of interaction between ecf HCO£ and the intracellular space or its contents is incorrect. We cannot at present distinguish among these possibilities. We do gain some further information from the analysis of changes in plasma and CSF anions. The nature and source of the UA shown in fig. 5 are unknown. In respect to Group 2, that the origin of the UA may be in brain tissue or in plasma has different implications for understanding the time course of CSF HCO3 equilibration as well as the mechanisms determining CSF HCO~-. For example, if the UA in this group reflect organic acid anions that titrate ecf HCO3, then our measured time course of CSF HCO~ equilibration cannot simply reflect the blood-ecf movement and subsequent diffusion through the ecf space. If there are in fact no cell-ecf interactions the net exchange of HCO3 at the blood-ecf barrier appears to be complex, involving both CI- and the UA.

CSF HCOF: BRAIN Pco, AND PLASMAHCO3A 50

20

CSF [ H C O ; ] mM/Kg H20 (33.8)

[ ,

B

30

195

z

(19.6) ,

,

,

csF [HCO~]

:

mM/Kg H20 "

20

I

~

/

,

, CSF,PCO2 ,

....~4~

T (31,1 /

(363)

(21.6)

(19,1)

35

40

,

,

45

50

i 55

CSF PCO a 60

65

J 70

Fig. 7. CSF H C O f is shown as a function of CSF Pco2 with plasma H C O f values in parentheses. Panel A represents the mean values (+ SEM) of the four experimental groups at the 2 hour time period, panel B at the 6 hour time period. The symbols are those used in fig. 1. Roughly isoplasma H C O f values are connected by the lines.

Summary Figure 7 summarizes the main findings of the study, the relationship of the dependent variable, CSF HCO3-, to the two independent variables, CSF Pco2 and plasma HCOf. The numbers in parentheses represent the mean plasma HCO3- values. At 2 and 6 hours and at roughly the same plasma HCO3- values both in the normal and high range, CSF HCO3 increases in response to an increase in CSF Pco2. At a given CSF Pco2, an increase in the plasma H C O 3 is associated with an increase in the CSF HCO3- and this effect is greater at 6 hours than at 2 hours. As shown in fig. 3, the time courses of these two relationships differ. The CSF HCOf response to an isolated increase in Pco~ occurs more quickly and probably involves choroid plexus cells as well as brain tissue (neurons and glia). The CSF HCOF response to an isolated increase in plasma HCOf occurs more slowly over 6 hours and probably reflects ionic movement of HCO3 from capillary to brain ecf and diffusion to CSF. The mechanisms involved in both processes are unclear and analysis of the changes in HCO3-, C1- and UA in these isosmotic experiments suggests they may be complex.

Acknowledgements The authors thank Susan Knuth and Heryun Kim for their expert echnical assistance.

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References Adaro, F. V. M., E.E. Roehr, A. R. Viola and C. W. deObrutzky (1969). Acid-base equilibrium between blood and cerebrospinal fluid in acute hypercapnia. J. Appl. Physiol. 27: 271-275. Baker, M.A. and J.N. Hayward (1967). Carotid rete and brain temperature of cat. Nature (London) 216: 139-141. Berkenbosch, A. (1971). CSF-arterial Pco,, differences with and without ventriculo-cisternal perfusion in the anaesthetized cat. p[liigers Arch. 330:99 110. Borgstr6m, L., K. Norberg and B.K. Siesj6 (1976). Glucose consumption in rat cerebral cortex in normoxia, hypoxia and hypercapnia. Acta Physiol. Scand. 96: 569-574. Caronna, J.J., F. Plum and B. K, Siesj6 (1974). Pco2 gradients between blood and CSF in rat during alterations of acid-base balance. Am. J. Physiol. 227:1173 1177. Chazan, J.A., F. M. Appleton, A.M. London and W.B. Schwartz (1969). Effects of chronic metabolic acid-base disturbances on the composition of cerebrospinal fluid in the dog. Clin. Sci. 36:345 358. Chorea, L. and H. Kazemi (1976). Importance of changes in plasma H C O ; on regulation of CSF H C O f in respiratory alkalosis. Respir. Physiol. 26:265 278. Davies, D.G. and G. H. Gurtner (1973). CSF acid base balance and the Wien effect. J. Appl. Physiol. 34 : 249-254. Davies, D.G., R.S. Fitzgerald and G . H . Gurtner (1973). Acid base relationships between CSF and blood during acute metabolic acidosis. J. Appl. Physiol. 34:243 248. Davies, D. G. (1976). Cerebrospinal fluid sampling technique and Astrup pH and Pco2 values. J. Appl. Physiol. 40:123 125. Davson, H. and K. Welch (1971). The permeation of several materials into the fluids of the rabbits" brain. J. Physiol. (London) 218:337 351. DiMattio, J., G. M. Hochwald, C. Malhan and A. Wald (1975). Effects of changes in serum osmolarity on bulk flow of fluid into cerebral ventricles and on brain water content. Pflfigers Arch. 359: 253 264. Fencl, V., T. B. Miller and J. R. Pappenheimer (1966). Studies on the respiratory response to disturbances of acid base balance, with deductions concerning the ionic composition of cerebral interstitial fluids. Am. J. Physiol. 210: 459472. Fenstermacher, J. D. and C. S. Patlak (1975). The exchange of material between cerebrospinal fluid and brain. In : Fluid Environment of the Brain, edited by H. F. Cserr, J. D. Fenstermacher and V. Fencl. New York, Academic Press, pp. 201 214. Folbergrovfi, J., V. MacMillan and B.K. Siesj6 (1972a). The effect of moderate and marked hypercapnia upon the energy state and upon the cytoplasmic N A D H / N A D + ratio of the rat brain. J. Neurochem. 19:2497 2505. Folbergrovfi, J., V. MacMillan and B.K. Siesj6 (1972b). The effect of hypercapnic acidosis upon some glycolytic and Krebs cycle associated intermediates in the rat brain. J. Neurochem. 19: 2507-2517. Folbergrov~i, J., U. Pont6n and B. K. Siesj6 (1974). Patterns of changes in brain carbohydrate metabolites, amino acids and organic phosphates at increased carbon dioxide tensions. J. Neurochem. 22: 1115 1125. Folbergrovfi, J., K. Norberg, B. Quistorff and B.K. Siesj6 (1975). Carbohydrate and amino acid metabolism in rat cerebral cortex in moderate and extreme hypercapnia. J. Neurochem. 25 : 457~62. Hasan, F. M. and H. Kazemi (1976). Dual contribution theory of regulation o f C S F HCO3- in respiratory acidosis. J. Appl. Physiol. 40: 559-567. Held, D., V. Fencl and J.R. Pappenheimer (1964). Electrical potential of cerebrospinal fluid. J. Neurophysiol. 27: 942-959. Husted, R.F. and D.J. Reed (1977). Regulation of cerebrospinal fluid bicarbonate by the cat choroid plexus. J. Physiol. (London) 267:411~t28. Kazemi, H., N.S. Shore, V.E. Shih and D.C. Shannon (1973). Brain organic buffers in respiratory acidosis and alkalosis. J. Appl. Physiol. 34: 478~,82.

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