the present study performed in human volunteers, and measurements of ventilatory rate and depth were not monitored. Brzezinski et ai. (1967) mea sured Paco2 ...
Journal of Cerebral Blood Flow and Metabolism
9:417-421 © 1989 Raven Press, Ltd., New York
Neuronal pH Regulation: Constant Normal Intracellular pH is Maintained in Brain during Low Extracellular pH 31 Induced by Acetazolamide- p NMR Study
Sissel Vorstrup, Karl Erik Jensen, Carsten Thomsen, Ole Henriksen, Niels A. Lassen, and Olaf B. Paulson Department of Magnetic Resonance, Hvidovre Hospital and Department of Neurology, Rigshospitalet, University of Copenhagen, Denmark.
Summary: The intracellular pH in the brain was studied in six healthy volunteers before and immediately after the administration of 2 g of acetazolamide. Phosphorus-31 nuclear magnetic resonance spectroscopy by a 1.5 tesla whole-body scanner was used. The chemical shift be tween the inorganic phosphate and the phosphocreatine resonance frequencies was used for indirect assessment of the intracellular pH. The mean baseline intracellular pH was 7.05 ± 0.04 (SD). The mean pH changes obtained at 15-min intervals within the first hour of acetazolamide
0.03 ± 0.04 (SD), 0.02 ± 0.03 administration were (SD), and 0.00 ± 0.04 (SD), i.e., no statistically signifi cant pH decrease was observed during the period where extracellular pH is known to drop markedly. Although several factors contribute to the lack of change of the intraneuronal pH, we will discuss that this observation in addition might suggest a direct intracerebral effect of ac etazolamide. Key Words: 31p NMR spectroscopy Intracellular pH-Acetazolamide.
Since the introduction of 31p nuclear magnetic resonance (NMR) spectroscopy, in vivo measure ments of regional pH has become possible. The chemical shift (difference in hertz) between inor ganic phosphate resonance frequency and phos phocreatine resonance frequency permits indirect assessment of pH changes, if other factors such as temperature and ionic strength are stable (Ober haensli et aI., 1986). Hitherto, measurements have derived mainly from experimental studies and mostly from muscular tissues, but the development of whole-body scanners have permitted measure ment of intracerebral pH in humans. Brain tissue is mainly composed of neuronal cells and supportive tissue, the glial cell population. The measured pH
change thus reflects a combination of changes in these two cell types, whereas the extracellular fluid only contains negligible amounts of phosphors. A number of studies have analyzed the extracel lular pH changes induced after administering of ac etazolamide, and all have shown a marked drop (Severinghaus and Cotev, 1968; Heuser et al., 1975). However, due to the technical difficulties in measuring intraneuronal pH, only inferential evi dence has hitherto existed on intracellular pH (pH), and these studies have suggested a concomitant de crease in pH (Kjallquist et aI., 1969; Heuser et aI., 1975, Rothe and Heisler, 1986). The purpose of the present study was therefore to measure directly pHi changes in the human brain following intravenous injection of acetazolamide (Diamox, Lederle).
Received February 2, 1988; accepted December 23, 1988. Address correspondence and reprint requests to Dr. Sissel Vorstrup, Department of Neurology, Rigshospitalet, DK-2JOO Copenhagen, Denmark. Abbreviations used: DMO, dimethyldione; FID, free induction decay; FWHM, full width at half-maximum; NMR, nuclear mag netic resonance; PCr, phosphocreatine; PDE, phosphodiesters; pH., extracellular pH; pHi' intracellular pH; PME, phospho monoesters.
MATERIAL AND METHODS
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Six healthy adults (four men and two women) volun teered for the study. Their age ranged from 22 to 48 years. None of the participants had any history of neurological or arterial disease. A cannula was inserted in a cubital vein. The person was then positioned supine in the mag net, a Siemens Magnetom (Siemens Inc. Erlangen, West
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Germany) 1.5 tesla whole-body scanner, and a three turn solenoid surface coil (8 cm diameter) was positioned 5 cm above the meato-orbital line. The signals from the tem poral and orbicular muscles were minimized with this po sition. Prior to examination, the magnetic field was shimmed with the patient in the magnet using the proton free in duction decay (FID) signal recorded 400 Hz above the water-proton resonance frequency, using the phosphorus tuned surface coil as receiver, thus giving shim in the sensitive volume. The Fourier transform of the proton FID resulted in a water-proton line with a full width at half-maximum (FWHM) below 0.3 ppm in all subjects. After shimming, the transmitter frequency was shifted to the resonance frequency of [31P]phosphocreatine. Our pulse sequence comprised a 900 pulse in the center of the coils sensitive volume and a 500 fLS delay before acquisi tion of the FID. This pulse was repeated 128 times with an interval of 6 s. After the initial baseline measurement, 2 g of acetazol amide (Diamox) corresponding to 25 mg/kg body weight was given over 3-4 min in the vein cannula. Recordings were performed the following hour. Following Fourier transformation the spectra were phase- and baseline-corrected. The chemical shift of in organic phosphate relative to phosphocreatine was mea sured and translated to pH by the use of a standard curve (Petroff et aI., 1985). The time period between sampling points (dwell time) was 500 f.LS and the sampling frequency was 2 kHz. With a vector size (number of sampling points) of 512 points this gave a bandwidth of ±1 kHz. The digital resolution for phosphorus (which is determined by the hardware) was 3.9 Hz or 0.156 ppm, which corresponds to a sensi tivity for absolute pH of 0.108 pH unit. For phosphocreatine, the linewidth at FWHM was 9.5 Hz and the signal amplitude was 70 mV. The correspond ing values for inorganic phosphate were 6.5 Hz and 15 mV. This gives peak definition values of 665 and 95 digital steps for phosphocreatine and for inorganic phosphate, respectively. Thus, it is reasonable to set the sensitivity for relative pH changes at ±0.06 pH unit when the sub jects serve as their own control and area detection of the peaks is used.
RESULTS
'E
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2 0.
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-5.
-10
-15.
-20.
Chemical shift I ppm
FIG. 1. The obtained phosphorus spectra are shown with the frequency (0 ppm equals 25.75 mHz) and signal amplitude. The front spectrum is obtained before injection of acetazol amide and the three signals behind following administering of the drug. Seven frequencies were resolved: Phospho monoesters (7 ppm). inorganic phosphate (5 ppm), phos phodiesters (3.5 ppm), phosphocreatine (0 ppm), -y-ATP (-3 ppm), a-ATP (-7 ppm), and I3-ATP (-15 ppm).
0.04 (SD). Hence, no pHi changes could be detected in any of the 6 subjects. In Fig. 2 the pH of the six subjects is shown as a function of time. Using a Mann-Whitney V-test for paired data no statistical significant decrease in pHi was found. DISCUSSION
In a previous study from our group, we used the 3 same 1p_NMR spectroscopy to study pHi in six young normal adults at various levels of arterial Pco2 (Jensen et aI., 1988). This revealed the ex pected pH variations and allowed to calculate a brain buffer capacity � a log Pco2 brain/a 10g[H +] brain of 2.8, a value agreeing well with the value of 2.3 found by Kjiillquist et aI. (1969a) in animal studies using the carbon dioxide technique. In the present study, our results showed an un=
pH
The acquired spectra from a subject before and following acetazolamide infusion is shown in Fig. 1. Seven frequency peaks were resolved in the spectra (Fig. 1): phosphomonoesters in organic phosphate (PME), (Pi), phosphodiesters (PDE), phosphocre atine (Pcr), ,,(-, a-, and �-ATP. Underlying the spec tra a broad frequency is seen (Fig. 1), this peak represents the phosphorus in the skull and in the brain phospholipids (Oberhaensli et aI., 1986). The mean baseline pHi was 7.05 ± 0.04 (SD). After acetazolamide injection, no chemical shift was noted, and no change in the peak integrals were found in the seven phosphate metabolites. The mean pH changes obtained at IS-min intervals were - 0.03 ± 0.04 (SD), - 0.02 ± 0.03 (SD), and 0.00 ± J Cereb Blood Flow Metab, Vol. 9, No.3,
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B 30
45
mean!1SD
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Time in minutes FIG. 2. The mean pH a function of time.
±
SO from the six volunteers shown as
NEURONAL pH REGULATION
changed brain pH after acute administering of acet azolamide in all six cases. The digital resolution as determined by the hardware yields a detection limit for absolute pH of 0.108 pH unit. However, as we use detection of the area under the curve, the de tection of a relative change in pH, with the subject serving as his own control, will be much less than 0.108 pH units. The limit for a relative pH change is probably close to 0.06 pH units. Bickler et aI. (1988) described a similar sensitivity for absolute pH as obtained in the present study. Their estimate of rel ative changes in pH was ± 0.02 pH unit, a much smaller value. For the reasons given above, we feel confident that our findings are indeed valid for the conditions studied. Figure 2 presents the mean pH in the four observation periods and the standard deviation. The constant pHi in our study is of particular in terest as acetazolamide administered in the same dose and the same route causes a marked increase in CBF, to about 170-200% of its control level (Ehrenreich et aI., 1961, Vorstrup et aI., 1984), an effect pointing to a sharp drop in extracellular pH as also measured in the animal studies (Severinghaus and Cotev 1968; Heuser et aI., 1975). Severinghaus and Cotev (1968) showed that brain surface pH fell 0.15 pH units, even when the surface CO2 was held constant. Heuser et aI. (1975) found a decrease of 0.203 in cats where Paco2 was kept constant by me chanical ventilation. Bickler et aI. (1988) found de creases of 0.11 and of 0.08 pH units during constant ventilation and during constant brain surface Pco2, respectively. In the study of Bickler et aI. (1988), the effect of acetazolamide on intracellular brain pH was mea sured in rabbits using NMR spectroscopy and ap proximately the same relative dose expressed in milligrams of acetazolamide per kilogram body weight as in the present study. They found a de crease of pHi by 0.07 units (7.08 vs. 7.01, p < 0.05) over the first 1 h following the intraperitoneal injec tion of the drug under conditions where a controlled constant ventilation was used. In contrast, when Pco2 in the extracellular fluid was kept constant before and after acetazolamide by adjusting Paco2, the pHi values were identical [7.02 vs. 7.01 (not significant)]. Brain tissue Pco2 values could not be obtained in the present study performed in human volunteers, and measurements of ventilatory rate and depth were not monitored. Brzezinski et ai. (1967) mea sured Paco2, CSF, brain surface as well as cisternal Pco2 in spontaneously breathing animals before and after acetazolamide using doses of 50-150 mg/kg. They observed a parallel increase in Paco2 (mea-
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sured by in vitro technique) and the brain surface CO2 tension, and concluded that the CO2 tension of the brain could be estimated from P aco2, even after acetazolamide. However, in the human studies (Ehrenreich et aI., 1961, Gotoh and Shinohara 1977, and Vorstrup et aI., 1984) where arterial sampling was performed, Paco2 remained essentially un changed, which is why extrapolation from the ex perimental data to the human studies may not be justified. Actually, it can even be assumed that the precapillary Pco2 value decreases, both due to a moderate hyperventilation and prolongation of the conversion of H2C03 � H20 + CO2, Thus, FeC02 will decrease as will the end-pulmonary capillary Pco2. The circulation time from the alveolar capil laries to the brain averages 5 s (range 3-10 s) as measured in normal volunteers (Vorstrup, 1988). Obrist et ai. (1975) reported a delay time of 7.6 s. The conversion time of H2C03 � CO2 + H20 in the absence of carbonic anhydrase is about 15 s (Maren, 1967). This leads to an estimated Pco2 value of 37 mm Hg in the initial part of the brain capillary, based on the finding of a decrease in FeC02 from 4.4 to 4.0 after acetazolamide (Vorstrup et aI., 1987). Swenson and Maren (1978) estimated a precapillary value of 33 to 38 mm Hg. The measured Paco2 value will furthermore depend on the time elapsed between blood sampling and measurement, as Paco2 due to continual conversion rises steadily with time. Thus, the conclusions drawn from the experimental studies cannot be ap plied in man. There are several reasons to explain why pHi falls less than pHe. According to Siesjo (1973, 1978) pHi changes during hypo- and hypercarbia are mini mized by three mechanisms. First, physicochemical buffering will immediately compensate for pH changes within certain ranges, and as the intracel lular buffer capacity is larger than in the extracel lular fluid, this will give a smaller change in pHi than in pHe. Second, pHi is maintained by adjusting me tabolism to either consumption or production of metabolic acids. Hence, after acetazolamide, a de creased concentration of the glycolytic acids and the citric acid cycle acids is expected. Third, H+ is actively transported out of the cell. Finally, the in creased cerebral oxygenation following acetazol amide administration may influence pHi (''physio logic buffering") and provide a fourth regulatory mechanism as an increase in P02 has been shown to shift the redox state in the mitochondria toward ox ygenation (Hempel et aI., 1977). Such a shift will reduce the concentration of all metabolic interme diate acids in the citric acid cycle, and will thereby tend to alkalinize the cell. The study of Lockwood J Cereb Blood Flow Metab, Vol. 9, No.3,
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et al. (1984) confirmed that the cytochrome oxidase redox state was shifted toward oxygenation after acetazolamide administration as well. Despite the factors described above, the lack of change in pHi seen after acetazolamide is still some what surprising. These findings contrast the results observed after hypercarbia. Messeter and Siesjo (1971a,b) using 11% CO2 inhalation observed a marked pHi drop after 15 min (pHi decreased from 7.09 ± 0.01 to 6.94 ± 0.01) in the rat. pHi was derived from the HC03 concentration and the mean CO2 tension. After 45 min, slight pH adjust ments had taken place (pHi increased to 6.98) but after 3 h pHi had adapted and had increased to a stable although somewhat reduced level (7.04). These findings were corroborated by Arieff et al. (1976) who applied the dimethadione (DMO) tech nique to a quite similar experimental design in the dog: A significant decrease in pHi was observed after 1 h returning to the baseline value after 3 h. The studies performed by Kjallquist et al. (1969a,b) in spontaneously breathing rats suggested that pHi decreases after acetazolamide. They ob served an increase in total CO2, a finding that nor mally would indicate brain alkalosis, but as con comitant measurements showed that the lactate and pyruvate concentrations were reduced, a decrease in pHi on the basis of carbonic acidosis was as sumed. Severinghaus et al. (1968, 1969) also sug gested from their studies of acetazolamide that pHi would decrease along with the drop in pHe' maybe even more so. In the present study, the pHi changes obtained at 15-min intervals were - 0.03, - 0.02 and 0.00 pH units. Although none of the values dif fered significantly from the baseline value of 7.05, the time course might be compatible with an initial modest pH decrease that is adapted for over time. For comparison, the pHi values obtained with the same technique in six volunteers changed - 0.05 ± 0.04 and 0.06 ± 0.05 during 7% CO2 inhalation (18 min) and hyperventilation (18 min), respectively, with both observations significant at the 0.05 level (Jensen et al., 1988). What can explain the discrepancy between the pHi decrease during hypercarbia and the unchanged pH after acetazolamide, for a given decrease in ex tracellular pH? The intracellular pH buffering may be expected to be the same during both conditions. Likewise, the physiologic buffering owing to the in creased tissue oxygenation is probably of similar magnitude, as comparable flow increases during constant CMR02 prevails. During hypercarbia, however, CO2 is constantly administered, and de spite the increase in ventilatory rate a new steady state at a higher Pco2 has to be reached. Conse�
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quently, the brain is flooded with freely diffusible CO2, which is hydrated to H2C03 yielding H+ in all compartments. As described above, after acetazol amide, the increased ventilation will tend to lower Paco2, alkalinizing intracellular pH. Still, if the met abolic end-products in the Krebs' cycle were CO2 and H20 (and not H2C03), then the brains hydrogen ion concentration would be expected to increase. If, on the other hand, H2C03 is the primary end product as already indicated by Severinghaus et al. (1969), this could explain why intracellular pH re mains stable along with an increase in HC03 In this case, acetazolamide will to a certain degree im pede the reaction H2C03 � H+ + HC03 H2C03 will freely diffuse over the cell membranes, and in the extracellular fluid with much less buffer capac ity, the dissociation of H2C03 will cause pH to de crease. In conclusion, we have demonstrated by the use 3 of 1 P- NMR technique that intracellular brain pH in spontaneously breathing human volunteers re mained essentially unchanged after administration of acetazolamide, a drug that is known to cause a marked pHe decrease (Severinghaus and Cotev, 1968; Heuser et al., 1975). Although methodological factors limits our detectability of the intracellular pH changes to about 0.06 pH units, chemical shift ing was not observed in any of the six cases studied. Intracellular pH buffering, changes in cerebral me tabolism with increased intracellular metabolic acid consumption, and enhanced transmembrane trans portation of H+ in addition to a moderate hyper ventilation explains the intracellular pH adjust ment, but not why a nearly stable pHi is maintained after acetazolamide, contrasting with the pHi de crease seen during hypercarbia. The discrepancy between the unchanged pHi in face of the significant extracellular hydrogen ion increase after acetazol amide is a remarkable observation that could sug gest that H2C0 3 is the primary metabolic end product in the citric acid cycle, and that acetazol amide has a direct intracerebral effect. �.
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REFERENCES Arieff lA, Kerian A, Massry SG, DeLima J (1976) Intracellular pH of brain: alterations in acute respiratory acidosis and alkalosis. Am J Physiol 230:804-8 12 Bickler PE, Litt L, Bonville DL, and Severinghaus JW (1988) Effects of acetazolamide on cerebral acid-base balance. J Appl Physiol (in press) Brzezinski J, Kjallquist A, Siesj6 BK (1967) Mean carbon diox ide tension in the brain after carbonic anhydrase inhibition. J Physiol 188: 13-23 Ehrenreich DL, Burns RA, Alman RW, Fazekas JF ( 196 1) In fluence of acetazolamide on cerebral blood flow. Arch Neu raI5:227-232 Hempel FG, J6bsis FF, LaManna JL, Rosenthal MR, Saltzman HA (1977) Oxidation of cerebral cytochrome aa3 by oxygen
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NEURONAL pH REGULATION plus carbon dioxide at hyperbaric pressures. J Appl Physiol 43:873--879 Heuser D, Astrup J, Lassen NA, Betz E ( 1975) Brain carbonic acid acidosis after acetazolamide. Acta Physiol Scand 93: 385-390 Jensen KE, Thomsen C, Henriksen 0 (1988) In vivo measure ment of intracellular pH in human brain during different ten sions of carbon dioxide in arterial blood. Acta Physiol Scand 134:295-298 Kjiillquist A, Nardini M, Siesjo BK (l969a) The regulation of extra- and intracellular acid-base parameters in the rat dur ing hyper- and hypocapnia. Acta Physiol Scand 76:485-494 Kjiillquist A, Nardini M, Siesjo BK (l969b) The effect of acet azolamide upon tissue concentrations of bicarbonate, lac tate, and pyruvate in the cat brain. Acta Physiol Scand 77:241-251 Lockwood A, LaManna JC, Snyder S, Rosenthal M (1984) Ef fects of acetazolamide and electrical stimulation on cerebral oxidative metabolism as indicated by the cytochrome oxi dase redox state. Brain Res 308:9-14 Maren TH (1967) Carbonic anhydrase: chemistry, physiology and inhibition. Physiol Rev 47:595-781 Messeter K, Siesjo BK (I 97 1a) Regulation of the CSF pH in acute and sustained respiratory acidosis. Acta Physiol Scand 83:21-30 Messeter K, Siesjo BK ( 1971b) The intracellular pH in the brain in acute and sustained hypercapnia. Acta Physiol Scand 83:210-219 Obrist WD, Thompson HK, Wang HS, Wilkenson WE (1975) Regional cerebral blood flow estimated by xenon-l33 inha lation. Stroke 6:245-256 Rothe KF, Heisler N (1986) Corrections of metabolic alkalosis by HCl and acetazolamide: effects of extracellular and in-
tracellular acidlbase status in rats in vivo. Acta Anaesthesiol Scand 30:566-570 Oberhaensli RD, Hilton-Jones D, Bore PJ, Radda GH (1986) P-3 1 magnetic resonance studies of human brain at 2 T. Magn Reson Imaging 4:417-419 Petroff OAC, Prichard JW, Behar KL, Alger JR, den Hollander JA, RG Shulman (1985) Cerebral intracellular pH by 31p nuclear magnetic resonance spectroscopy. Neurology 35: 781-788 Severinghaus JW, Cotev S (1968) Carbonic acidosis and cerebral vasodilation after diamox. Scand J Clin Lab Invest 1 (Suppl 102) Severinghaus JW, Hamilton FN, Cotev S ( 1969) Carbonic acid production and the role of carbonic anhydrase in decarbox ylation in brain. Biochem J 114:703-705 Siesjo BK (1973) Metabolic control of intracellular pH. Scand J Clin Lab Invest 32:97-104 Siesjo BK (1978) Brain Energy Metabolism. Chichester, John Wiley, pp. 304-23 Swenson ER, Maren TH ( 1978) A quantitative analysis of CO2 transport at rest and during maximal exercise. Respir Phys iol 35: 129-159 Vorstrup S (1988) Tomographic cerebral blood flow measure ments in patients with ischemic cerebrovascular disease and evaluation of the vasodilatory capacity by the acetazolamide test. Acta Neurol Scand (suppl 1 14) 77: 1-48 Vorstrup S, Henriksen L, Paulson OB (1984) Effect of acetazol amide on cerebral blood flow and cerebral metabolic rate for oxygen. J Clin Invest 74: 1634-1639 Vorstrup S, Boysen G, Brun B, Engell HC (1987) Evaluation of the regional cerebral vasodilatory capacity before carotid endarterectomy by the acetazolamide test. Neurol Res 9: 10- 18
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