Jourrlai of Mairr.ociiei,il~tr.y,1976. Vol. 26. pp. 345-352. Pergamon Press. Printed
111
Great Britain.
OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX OF THE RAT IN SEVERE INSULIN-INDUCED HYPOGLYCAEMIA K. NORBERG and B. K. SIESJO From the Brain Research Laboratory, E-blocket, University Hospital, Lund, Sweden (Received 10 April 1975. Accepted 11 July 1975)
Abstract-Measurements were made of organic phosphates, carbohydrate substrates, amino acids and ammonia in the cerebral cortex, as well as of cerebral blood flow and of cerebral metabolic rate for oxygen and glucose in rats that developed an isoelectric EEG pattern (‘coma’) during insulin-induced hypoglycaemia. The results were compared to those obtained in control animals, as well as in hypoglycaemic animals with an EEG pattcrn of slow waves and polyspikes. In animals with slow waves and polyspikes,there was a decrease in all citric acid cycle intermediates except succinate and oxaloacetate. and a decrease in the pool size of intermediates. In animals that had an isoelectric EEG for 5-15 min, there were further decreascs in citrate, isocitrate. u-ketoglutarate, malate and fumarate, but since the concentration of succinate (and oxaloacetate) increased, the pool size remained the same. In isoelectric animals, the results revealed extensive utilization of amino acids by both transamination and deamination reactions. However, since glycogen had disappeared and the amino acid pattern was constant after the first 5min of isoelectric EEG, further oxidation must have occurred at the expense of non-carbohydrate, non-amino acid substrates. There were two- to three-fold increases in cerebral blood flow in animals with slow waves and polyspikes and in animals with isoelectric EEG, and no decrease in the cerebral metabolic rate for oxygen. Since less than half of the oxygen consumption could be accounted for in terms of glucose extraction, the data indicate that severe hypoglycaemia is associated with extensive oxidation of endogenous substrates other than carbohydrates and free acids. Early work revealed that hypoglycaemia is accompanied by a decrease in glutamate and a rise in aspar1950, 1953; CMtate content of the brain (DAWSON, VIOTO et al., 1951). These findings were subsequently confirmed, and later studies have shown that the tissue concentrations of glutamine. GABA and alanine also decrease (DE ROPP& SNEDEKER, 1961; MASSIEU et al., 1970; TEWSet al., 1965; DAVISet al., 1970; LEWISet al., 1974a). The results indicate that amino acid carbon is made available by transamination, but since hypoglycaemia is accompanied by ammonia accumulation in the tissue (KONITZER et al., 1965; TEWS et al., 1965; LEWISet al., 1974~)and by release of ammonia into cerebral venous blood (TEWSet a!., 1965),oxidative deamination of amino acids probably also occurs. Since other results show decreases in the tissue concentration of glycogcn and of glycolytic and citric acid cycle intermediates (GOLDBERG et al., 1966; LEWISet al., 1974u), it is clcar that endogenous carbohydrate and amino acid stores are used as substrates in hypoglycaemia. It is much less clear if proteins, lipids or nudeic acids are useful substrates in hypoglycaemia (DAWSON,1950; TEWSrt al., 1965; DAVIS et ul.. 1970: HINZENet al.. 1970) but two findings warrant special consideration. AEOOD & GEIGER Abbreviations used: AVD,,, arteriovenous difference for glucose; AVDo2,arteriovenous differencefor oxygen CBF, (195% perfusing the vascularly isolated cat brain with cerebral blood flow; CMR,,, cerebral metabolic rate for glucose-free blood, found considerable decreases in the tissue concentrations of proteins and lipids. Furglucose; CMRo., -‘ cerebral metabolic rate for oxygen; T ~ , , & SETCHELL (1973) observed thermore. PAPPENHEIMER total oxygen content.
STUDIES of cerebral blood flow (CBF) and of arteriovenous differcnces in hypoglycaemia have shown that there is a proportionally larger reduction in glucose consumption (CMR,,) than in oxygen utilization (CMRoJ, indicating that non-carbohydrate, endogenous substrates are used to support encrgy production (KETYet al., 1948; EISENBERG & SELTZER, 1962; DELLAPORTA et al., 1964; GOTTSTEIN & HELD,1967; PAPPrNHEimR & SETCHELL,1973). Clinical observations and animal experiments show that this production suffices to maintain function and structural integrity for only a limited period of time, and when the hypoglycaemia is sufficiently severe or sufficiently prolonged, there are signs of gross energy failure in the tissue (GEIGER et a/., 1952; TEWSet al., 1965; HINZEN& MULLER,1971; LEWISet a/., 19746), as well as of neuronal damage of the type seen in ischaemia (BRIERLEY et al., 1971). There are two main controversial issues related to the cerebral metabolism in hypoglycaemia. Thus, it has never been clearly established what non-carbohydrate substrates are used, and conflicting data exist regarding the magnitude of oxygen consumption.
345
K. NORBERC and B. K. SIESJO
346
i.u./kg. Anaesthesia was induced with 2-3% haloththat, in hypoglycaemic sheep and rabbits. the molar ane, and the animals were then tracheotomized, immobiratios of glucose utilized to oxygen consumed (6 x lized with tubocurarine chloride and maintained on 70% CMR,,/CMRo,). which was normally 0.93, could N,O and 30% 02.Thus, all results pertain to nitrous oxide remain below 6.4 for periods of more than 2h. They anaesthesia. The electrocorticogram was continuously calculated that, under these conditions, at least recorded in all animals by using gold-plated copper bolts 50pmo1/(100g.min) of oxygen is utilized for oxi- inserted into the skull bone. The animals were grouped dation of non-carbohydrate substrates, and pointed according to the EEG pattern in the following way: (I) out that 0.1 g of lipid/lOOg tissue could cover this normal EEG in control animals, (2) predominantly convulsive, polyspike activity with interspersed slow wave actioxidation for a period of 3 h. Information on CBF and CMRo, in hypoglycaemia vity, lasting about 15 min and (3) isolectric EEG, lasting is controversial. KETY et al. (1948) reported that for S 1 5 m i n . When the EEG showed any of the above hypoglycaemia (mean blood glucose concentration patterns the brain tissue was frozen for metabolite studies 19 mg%) and hypoglycdemic coma (mean glucose (series A) or the CBF and CMR,,, were determined (series concentration 8mg%) did not change CBF in the B).Series A. In these animals, only one femoral artery was schizophrenic patients studied, but that both CMR,, catheterized, exposure of the sinus was omitted and the and CMR, , decreased progressively with the severity tissue was prepared for in situ freezing with liquid nitrogen of hypoglycaemia. In five comatose patients there was (PONTENrt al., 1973). At the time of freezing, arterial blood a decrease in C M k , to a mean value of 55% of nor- was taken for measurements of Po,, P,,, and glucose. The mal. Subsequent studies in man confirmed that even brain was then chiselled out during irrigation with liquid moderate hypoglycaemia is accompanied by a fall in nitrogen and stored at - 80°C. The upper part of the fronCMR-,. However, these studies showed that CMRo, tal-parietal cortex of both hemispheres was cut out and was either unchanged (GOTTSTEIN & HELD,1967) or analysed for pyruvate, citrate, isocitrate, cc-ketoglutarate, even increased (EISENBERG & SELTZER, 1962). Further- succinate, fumarate, malate. aspartate, glutamate, glutamore, in a study of insulin coma, DELLAPORTA et mine, alanine, GABA, NH:, phosphocreatine, creatine, ATP, ADP and AMP. a/. (1964) found an increase in CBF in 10 out of 14 Series B. The main objective of the present experiments comatose patients (see also EISENBERG & SELTZER,was to study cerebral circulatory and metabolic events in 1962) and there was no change in CMR,,. Animal isoelectric ('comatose') animals that did not develop experiements tend to support these observations. hypoxia, hypercapnia or hypotension. Since the objectives Thus, neither PAPPENHEIMER & SETCHELL (1973), in necessitated measurements of CBF and CMRo2at varying spite of drastic reductions in blood glucose con- times after an isoelectric record was obtained, it was less centrations, nor GEIGER et al. (1952), when perfusing practical to use tracer methods that require long periods the brain with glucose-free blood, found a clear de- of saturation and desaturation. For this reason, CBF was et measured with a rapid tissue uptake method (LANDAU crease in CMRo,. The present experiments were undertaken to evalu- a[., 1955; REIVICHet al., 1969) as applied to [14C]ethanol ( E K L ~ et F af., 1974) and CMR,, was calculated from the ate utilization of carbohydrate and amino acid subCBF derived for frontal and parietal cortex and arteriostrates in severe hypoglycaemia, and to measure venous differences for oxygen and glucose, using venous CMR,, and C M k , in situations that lead to deple- blood from the superior sagittal sinus. However, since the tion of the substrate stores. The specific questions results were somewhat unexpected, control experiments raised were the following. To what extent can the were performed with the KETY & SCHMIDT (1948) techcitric acid cycle and the amino acid pools be utilized nique using '33Xeand measurements of tracer activities as substrates'! Is there a fall in CMRo, when the in arterial and cerebral venous blood during desaturation & SIESJO,1974). stores of these substrates have been exhausted, or is (NORBERG When the ['4C]ethanol method was used, the prothere evidence of oxidation of non-carbohydrate, nonamino acid substrates? In order to provide answers cedures were as follows. Both femoral arteries and femoral to those questions animals were made hypoglycaemic veins were cannulated. One of the femoral arteries was used for continuous recording of blood pressure and the by means of insulin, and CMR,,, CMRo,. organic other for removal of consecutive blood samples. The phosphates, carbohydrate intermediates, amino acids femoral veins were used for continuous infusion of and ammonia were measured after the EEG had been ['4C]ethanol and for injection of saturated potassium isoelectric for 5 1 5 min ('coma'). The results were chloride at the end of the experiments. Blood samples for compared to those obtained in control animals, as determination of the total oxygen (To,) and glucose conwell as in animals showing an EEG pattern with slow tents were taken from the fcmoral artery and the superior sagittal sinus 2-3 min prior to and immediately before the waves and polyspikes. METHODS Animals
Male Wistar rats (325-36Og) were fasted for 18-26 h before the experiments but had free access to water. Two hours before operation they were given an intraperitoneal injection of crystalline zinc insulin (Vitrum) in a dose of
start of the infusion of ['4C]ethanoI. Arterial samples were also drawn to measure haemoglobin content, pH, Pco2and Po?. ['4C]Ethanol was infused intravenously for 30 S. Arterial blood was sampled every 3 s during the infusion. Immediately after the last sample, saturated KCI was injected intravenously, the rat was decapitated and the head was frozen in liquid nitrogen. The brains were dissected at - 12°C and the parietal and frontal cortex were selected and counted to allow estimation of cortical CBF and meta-
Brain metabolism in severe hypoglycaemia
347
bolic rates. However, cerebellar and brain stem samples
in animals showing a slow wave-polyspike pattern. Animals with an isoelectric EEG pattern will be prostructures. visionally denoted as ‘comatose’ (see LEWISet ul., The ‘33Xe method was used in three animals with a 1974a). Since normal animals and animals with slow slow wave-polyspike activity in the EEG, and in four others that showed an isoelectric EEG. The period of satu- waves and polyspikes have been extensively studied ration was 15-20 min. Arterial and cercbral venous blood before (LEWISet al., 1974u,b), and since the results for determination of total oxygen content (To*) was obtained in the isoelectric animals were very consisobtained just before and 2-4min after the beginning of tent, the numbers of animals in the group were 4, desaturation. During the sampling period, a slow infusion 4 and 6, respectively. A larger series of animals was of blood from donor rats was given intravenously (see used to measure CBF, CMR,, and CMR,, since the & SIESJO, 1974). In order to avoid changes in initial results were somewhat unexpected. Table 1 NORBERG blood glucose concentration the donor rats were rendered gives the blood glucose concentration, the mean arterhypoglycaemic by means of insulin. ial blood pressure as well as the arterial Pol, Pml and pH in the two series. The body temperature was Analytical tuckniques close to 37”C, and the haemoglobin concentration 16The arterial Po2. Pa2 and pH were measured with microelectrodesat 37°C (Eschweiler & Co., Kiel, and Radi- 18 g/100 ml (not shown). All animals, including those ometer, Copenhagen). The activity of [‘4C]ethanol in with an isoelectric EEG, had adequate blood pressure brain tissue and blood was determined in a Nuclear Chi- and arterial Po,, and the mean arterial Pco, was close cago scintillation counter. The frozen brain tissue was to 35mm Hg. Series A. The ATP, ADP and AMP concentrations, extracted at - 22°C with HC1-methanol, and the neutralized extracts were used to measure the metabolites with the sum of adenine nucleotides, the ATP/ADP ratios, & PASSON- and the phosphocreatine (PCr) concentrations are the enzymic fluorometric techniques of LOWRY NEAU (1972). The analytical conditions have been given presented in Table 2. The results confirm our previous et al., 1974). The findings that there is an unchanged energy state in before (for references, see FOLBERGROVA oxaloacetate was not measured but calculated from the animals with a polyspike-slow wave pattern, and that aspartate aminotransferase reaction, using a K,, of 6.7 a major derangement of energy mctabolism occurs & KORNBEKG, 1957).TheTo2of arterial and venous (KREBS in isoelectric animals (LEWISet ul., 1974h). In that blood was measured in 25pl samples (BORGSTROM et al., study, only three animals had an isoelectric pattern 1974) by the polarographic method of FABEL& LUBBERS in the EEG lasting longer than 3min, and of these, (1964). two were hypotensive. In the present isoelectric Statistical methods group, the results were remarkably consistent (see When the results appeared to follow a normal distribu- ranges in Table 2). Thus, the derangement of the ceretion curve the two-sample r-test or Aspin Welch’s test were bral energy state scemed to develop during the first used for statistical evaluation. Otherwise, the non-para- 5 min of isoelectricity. and the pattern observed was metric Mann--Whitney U-test was applied. The following then maintained even if the ‘comatose’ condition was symbols are used: P < 0.05 = *, P < 0.01 = ** and prolonged to 15min. The changes observed in gluP < 0.001 = ***. cose, glycogen and glycolytic intermediates were very similar to those reported previously (LEWISet al., 1974~).All animals with an isoelectric EEG had very RESULTS low tissue glycogen concentrations (0.02-0@l pmol/g). I n the metabolite series (A), the main emphasis was Thus, since the tissue was virtually depleted of glucose et ul., 1974~)there laid on the pattern obtained in animals having an even before ‘coma’ ensued (LEWIS isoelectric EEG for 5 to 15min, but the results were were no carbohydrate stores to support oxidation compared to those obtained in control animals, and during the period of isoelectricity. were also counted to permit estimation of CBF in these
TABLEI , PHYSIOLOGICAL PARAMETERS No. of
animals Series A (metabolites)
4
4 6
Series B (CBF)
17
EEG
normal slow wavespolyspikes isoelectric
normal
Blood glucose (Pmol!d
14
slow wavespolyspikes isoelectric
MABP
(mm Hg)
Pa0, (mm Hg)
Pam, (mm Hg)
8.78
148
138
33.9
*074 2.0 1 f0.17 1.02 +0,10 12.0
k7
T 18 148 k6 137 f7 128
f 1.2
0.8 8
AND BLOOD GLUCOSE CONCENTRATIONS
1.53 f 0.1 1 0.98 0.06
139 f 3
146 t l l 133
+3 139 +8
146 +4
The values are means + S.E.M. MABP. mean arterial blood pressure.
f 6
I20 f8 146 +5
PH
36.9
7.390 * 0.001 7.387
- 1.9
+0018
32.9
*35.2
7,386
1.0
T 0.023
+0.7
36.1
7.370 * 0.010 7.357
& 1.1
fool2
+
32.4
7.438
1.0
rfIO.018
+_
K. NORBERG and B. K. SIESJO
348
TABLE2. CONCENTRATIONS OF PHOSPHOCREATINE (per) ATP, ADP, AMP AND THE SUM OF ADENINE NUCLEOTIDES (
111
Great Britain.
OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX OF THE RAT IN SEVERE INSULIN-INDUCED HYPOGLYCAEMIA K. NORBERG and B. K. SIESJO From the Brain Research Laboratory, E-blocket, University Hospital, Lund, Sweden (Received 10 April 1975. Accepted 11 July 1975)
Abstract-Measurements were made of organic phosphates, carbohydrate substrates, amino acids and ammonia in the cerebral cortex, as well as of cerebral blood flow and of cerebral metabolic rate for oxygen and glucose in rats that developed an isoelectric EEG pattern (‘coma’) during insulin-induced hypoglycaemia. The results were compared to those obtained in control animals, as well as in hypoglycaemic animals with an EEG pattcrn of slow waves and polyspikes. In animals with slow waves and polyspikes,there was a decrease in all citric acid cycle intermediates except succinate and oxaloacetate. and a decrease in the pool size of intermediates. In animals that had an isoelectric EEG for 5-15 min, there were further decreascs in citrate, isocitrate. u-ketoglutarate, malate and fumarate, but since the concentration of succinate (and oxaloacetate) increased, the pool size remained the same. In isoelectric animals, the results revealed extensive utilization of amino acids by both transamination and deamination reactions. However, since glycogen had disappeared and the amino acid pattern was constant after the first 5min of isoelectric EEG, further oxidation must have occurred at the expense of non-carbohydrate, non-amino acid substrates. There were two- to three-fold increases in cerebral blood flow in animals with slow waves and polyspikes and in animals with isoelectric EEG, and no decrease in the cerebral metabolic rate for oxygen. Since less than half of the oxygen consumption could be accounted for in terms of glucose extraction, the data indicate that severe hypoglycaemia is associated with extensive oxidation of endogenous substrates other than carbohydrates and free acids. Early work revealed that hypoglycaemia is accompanied by a decrease in glutamate and a rise in aspar1950, 1953; CMtate content of the brain (DAWSON, VIOTO et al., 1951). These findings were subsequently confirmed, and later studies have shown that the tissue concentrations of glutamine. GABA and alanine also decrease (DE ROPP& SNEDEKER, 1961; MASSIEU et al., 1970; TEWSet al., 1965; DAVISet al., 1970; LEWISet al., 1974a). The results indicate that amino acid carbon is made available by transamination, but since hypoglycaemia is accompanied by ammonia accumulation in the tissue (KONITZER et al., 1965; TEWS et al., 1965; LEWISet al., 1974~)and by release of ammonia into cerebral venous blood (TEWSet a!., 1965),oxidative deamination of amino acids probably also occurs. Since other results show decreases in the tissue concentration of glycogcn and of glycolytic and citric acid cycle intermediates (GOLDBERG et al., 1966; LEWISet al., 1974u), it is clcar that endogenous carbohydrate and amino acid stores are used as substrates in hypoglycaemia. It is much less clear if proteins, lipids or nudeic acids are useful substrates in hypoglycaemia (DAWSON,1950; TEWSrt al., 1965; DAVIS et ul.. 1970: HINZENet al.. 1970) but two findings warrant special consideration. AEOOD & GEIGER Abbreviations used: AVD,,, arteriovenous difference for glucose; AVDo2,arteriovenous differencefor oxygen CBF, (195% perfusing the vascularly isolated cat brain with cerebral blood flow; CMR,,, cerebral metabolic rate for glucose-free blood, found considerable decreases in the tissue concentrations of proteins and lipids. Furglucose; CMRo., -‘ cerebral metabolic rate for oxygen; T ~ , , & SETCHELL (1973) observed thermore. PAPPENHEIMER total oxygen content.
STUDIES of cerebral blood flow (CBF) and of arteriovenous differcnces in hypoglycaemia have shown that there is a proportionally larger reduction in glucose consumption (CMR,,) than in oxygen utilization (CMRoJ, indicating that non-carbohydrate, endogenous substrates are used to support encrgy production (KETYet al., 1948; EISENBERG & SELTZER, 1962; DELLAPORTA et al., 1964; GOTTSTEIN & HELD,1967; PAPPrNHEimR & SETCHELL,1973). Clinical observations and animal experiments show that this production suffices to maintain function and structural integrity for only a limited period of time, and when the hypoglycaemia is sufficiently severe or sufficiently prolonged, there are signs of gross energy failure in the tissue (GEIGER et a/., 1952; TEWSet al., 1965; HINZEN& MULLER,1971; LEWISet a/., 19746), as well as of neuronal damage of the type seen in ischaemia (BRIERLEY et al., 1971). There are two main controversial issues related to the cerebral metabolism in hypoglycaemia. Thus, it has never been clearly established what non-carbohydrate substrates are used, and conflicting data exist regarding the magnitude of oxygen consumption.
345
K. NORBERC and B. K. SIESJO
346
i.u./kg. Anaesthesia was induced with 2-3% haloththat, in hypoglycaemic sheep and rabbits. the molar ane, and the animals were then tracheotomized, immobiratios of glucose utilized to oxygen consumed (6 x lized with tubocurarine chloride and maintained on 70% CMR,,/CMRo,). which was normally 0.93, could N,O and 30% 02.Thus, all results pertain to nitrous oxide remain below 6.4 for periods of more than 2h. They anaesthesia. The electrocorticogram was continuously calculated that, under these conditions, at least recorded in all animals by using gold-plated copper bolts 50pmo1/(100g.min) of oxygen is utilized for oxi- inserted into the skull bone. The animals were grouped dation of non-carbohydrate substrates, and pointed according to the EEG pattern in the following way: (I) out that 0.1 g of lipid/lOOg tissue could cover this normal EEG in control animals, (2) predominantly convulsive, polyspike activity with interspersed slow wave actioxidation for a period of 3 h. Information on CBF and CMRo, in hypoglycaemia vity, lasting about 15 min and (3) isolectric EEG, lasting is controversial. KETY et al. (1948) reported that for S 1 5 m i n . When the EEG showed any of the above hypoglycaemia (mean blood glucose concentration patterns the brain tissue was frozen for metabolite studies 19 mg%) and hypoglycdemic coma (mean glucose (series A) or the CBF and CMR,,, were determined (series concentration 8mg%) did not change CBF in the B).Series A. In these animals, only one femoral artery was schizophrenic patients studied, but that both CMR,, catheterized, exposure of the sinus was omitted and the and CMR, , decreased progressively with the severity tissue was prepared for in situ freezing with liquid nitrogen of hypoglycaemia. In five comatose patients there was (PONTENrt al., 1973). At the time of freezing, arterial blood a decrease in C M k , to a mean value of 55% of nor- was taken for measurements of Po,, P,,, and glucose. The mal. Subsequent studies in man confirmed that even brain was then chiselled out during irrigation with liquid moderate hypoglycaemia is accompanied by a fall in nitrogen and stored at - 80°C. The upper part of the fronCMR-,. However, these studies showed that CMRo, tal-parietal cortex of both hemispheres was cut out and was either unchanged (GOTTSTEIN & HELD,1967) or analysed for pyruvate, citrate, isocitrate, cc-ketoglutarate, even increased (EISENBERG & SELTZER, 1962). Further- succinate, fumarate, malate. aspartate, glutamate, glutamore, in a study of insulin coma, DELLAPORTA et mine, alanine, GABA, NH:, phosphocreatine, creatine, ATP, ADP and AMP. a/. (1964) found an increase in CBF in 10 out of 14 Series B. The main objective of the present experiments comatose patients (see also EISENBERG & SELTZER,was to study cerebral circulatory and metabolic events in 1962) and there was no change in CMR,,. Animal isoelectric ('comatose') animals that did not develop experiements tend to support these observations. hypoxia, hypercapnia or hypotension. Since the objectives Thus, neither PAPPENHEIMER & SETCHELL (1973), in necessitated measurements of CBF and CMRo2at varying spite of drastic reductions in blood glucose con- times after an isoelectric record was obtained, it was less centrations, nor GEIGER et al. (1952), when perfusing practical to use tracer methods that require long periods the brain with glucose-free blood, found a clear de- of saturation and desaturation. For this reason, CBF was et measured with a rapid tissue uptake method (LANDAU crease in CMRo,. The present experiments were undertaken to evalu- a[., 1955; REIVICHet al., 1969) as applied to [14C]ethanol ( E K L ~ et F af., 1974) and CMR,, was calculated from the ate utilization of carbohydrate and amino acid subCBF derived for frontal and parietal cortex and arteriostrates in severe hypoglycaemia, and to measure venous differences for oxygen and glucose, using venous CMR,, and C M k , in situations that lead to deple- blood from the superior sagittal sinus. However, since the tion of the substrate stores. The specific questions results were somewhat unexpected, control experiments raised were the following. To what extent can the were performed with the KETY & SCHMIDT (1948) techcitric acid cycle and the amino acid pools be utilized nique using '33Xeand measurements of tracer activities as substrates'! Is there a fall in CMRo, when the in arterial and cerebral venous blood during desaturation & SIESJO,1974). stores of these substrates have been exhausted, or is (NORBERG When the ['4C]ethanol method was used, the prothere evidence of oxidation of non-carbohydrate, nonamino acid substrates? In order to provide answers cedures were as follows. Both femoral arteries and femoral to those questions animals were made hypoglycaemic veins were cannulated. One of the femoral arteries was used for continuous recording of blood pressure and the by means of insulin, and CMR,,, CMRo,. organic other for removal of consecutive blood samples. The phosphates, carbohydrate intermediates, amino acids femoral veins were used for continuous infusion of and ammonia were measured after the EEG had been ['4C]ethanol and for injection of saturated potassium isoelectric for 5 1 5 min ('coma'). The results were chloride at the end of the experiments. Blood samples for compared to those obtained in control animals, as determination of the total oxygen (To,) and glucose conwell as in animals showing an EEG pattern with slow tents were taken from the fcmoral artery and the superior sagittal sinus 2-3 min prior to and immediately before the waves and polyspikes. METHODS Animals
Male Wistar rats (325-36Og) were fasted for 18-26 h before the experiments but had free access to water. Two hours before operation they were given an intraperitoneal injection of crystalline zinc insulin (Vitrum) in a dose of
start of the infusion of ['4C]ethanoI. Arterial samples were also drawn to measure haemoglobin content, pH, Pco2and Po?. ['4C]Ethanol was infused intravenously for 30 S. Arterial blood was sampled every 3 s during the infusion. Immediately after the last sample, saturated KCI was injected intravenously, the rat was decapitated and the head was frozen in liquid nitrogen. The brains were dissected at - 12°C and the parietal and frontal cortex were selected and counted to allow estimation of cortical CBF and meta-
Brain metabolism in severe hypoglycaemia
347
bolic rates. However, cerebellar and brain stem samples
in animals showing a slow wave-polyspike pattern. Animals with an isoelectric EEG pattern will be prostructures. visionally denoted as ‘comatose’ (see LEWISet ul., The ‘33Xe method was used in three animals with a 1974a). Since normal animals and animals with slow slow wave-polyspike activity in the EEG, and in four others that showed an isoelectric EEG. The period of satu- waves and polyspikes have been extensively studied ration was 15-20 min. Arterial and cercbral venous blood before (LEWISet al., 1974u,b), and since the results for determination of total oxygen content (To*) was obtained in the isoelectric animals were very consisobtained just before and 2-4min after the beginning of tent, the numbers of animals in the group were 4, desaturation. During the sampling period, a slow infusion 4 and 6, respectively. A larger series of animals was of blood from donor rats was given intravenously (see used to measure CBF, CMR,, and CMR,, since the & SIESJO, 1974). In order to avoid changes in initial results were somewhat unexpected. Table 1 NORBERG blood glucose concentration the donor rats were rendered gives the blood glucose concentration, the mean arterhypoglycaemic by means of insulin. ial blood pressure as well as the arterial Pol, Pml and pH in the two series. The body temperature was Analytical tuckniques close to 37”C, and the haemoglobin concentration 16The arterial Po2. Pa2 and pH were measured with microelectrodesat 37°C (Eschweiler & Co., Kiel, and Radi- 18 g/100 ml (not shown). All animals, including those ometer, Copenhagen). The activity of [‘4C]ethanol in with an isoelectric EEG, had adequate blood pressure brain tissue and blood was determined in a Nuclear Chi- and arterial Po,, and the mean arterial Pco, was close cago scintillation counter. The frozen brain tissue was to 35mm Hg. Series A. The ATP, ADP and AMP concentrations, extracted at - 22°C with HC1-methanol, and the neutralized extracts were used to measure the metabolites with the sum of adenine nucleotides, the ATP/ADP ratios, & PASSON- and the phosphocreatine (PCr) concentrations are the enzymic fluorometric techniques of LOWRY NEAU (1972). The analytical conditions have been given presented in Table 2. The results confirm our previous et al., 1974). The findings that there is an unchanged energy state in before (for references, see FOLBERGROVA oxaloacetate was not measured but calculated from the animals with a polyspike-slow wave pattern, and that aspartate aminotransferase reaction, using a K,, of 6.7 a major derangement of energy mctabolism occurs & KORNBEKG, 1957).TheTo2of arterial and venous (KREBS in isoelectric animals (LEWISet ul., 1974h). In that blood was measured in 25pl samples (BORGSTROM et al., study, only three animals had an isoelectric pattern 1974) by the polarographic method of FABEL& LUBBERS in the EEG lasting longer than 3min, and of these, (1964). two were hypotensive. In the present isoelectric Statistical methods group, the results were remarkably consistent (see When the results appeared to follow a normal distribu- ranges in Table 2). Thus, the derangement of the ceretion curve the two-sample r-test or Aspin Welch’s test were bral energy state scemed to develop during the first used for statistical evaluation. Otherwise, the non-para- 5 min of isoelectricity. and the pattern observed was metric Mann--Whitney U-test was applied. The following then maintained even if the ‘comatose’ condition was symbols are used: P < 0.05 = *, P < 0.01 = ** and prolonged to 15min. The changes observed in gluP < 0.001 = ***. cose, glycogen and glycolytic intermediates were very similar to those reported previously (LEWISet al., 1974~).All animals with an isoelectric EEG had very RESULTS low tissue glycogen concentrations (0.02-0@l pmol/g). I n the metabolite series (A), the main emphasis was Thus, since the tissue was virtually depleted of glucose et ul., 1974~)there laid on the pattern obtained in animals having an even before ‘coma’ ensued (LEWIS isoelectric EEG for 5 to 15min, but the results were were no carbohydrate stores to support oxidation compared to those obtained in control animals, and during the period of isoelectricity. were also counted to permit estimation of CBF in these
TABLEI , PHYSIOLOGICAL PARAMETERS No. of
animals Series A (metabolites)
4
4 6
Series B (CBF)
17
EEG
normal slow wavespolyspikes isoelectric
normal
Blood glucose (Pmol!d
14
slow wavespolyspikes isoelectric
MABP
(mm Hg)
Pa0, (mm Hg)
Pam, (mm Hg)
8.78
148
138
33.9
*074 2.0 1 f0.17 1.02 +0,10 12.0
k7
T 18 148 k6 137 f7 128
f 1.2
0.8 8
AND BLOOD GLUCOSE CONCENTRATIONS
1.53 f 0.1 1 0.98 0.06
139 f 3
146 t l l 133
+3 139 +8
146 +4
The values are means + S.E.M. MABP. mean arterial blood pressure.
f 6
I20 f8 146 +5
PH
36.9
7.390 * 0.001 7.387
- 1.9
+0018
32.9
*35.2
7,386
1.0
T 0.023
+0.7
36.1
7.370 * 0.010 7.357
& 1.1
fool2
+
32.4
7.438
1.0
rfIO.018
+_
K. NORBERG and B. K. SIESJO
348
TABLE2. CONCENTRATIONS OF PHOSPHOCREATINE (per) ATP, ADP, AMP AND THE SUM OF ADENINE NUCLEOTIDES (
