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The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine ; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U- 13 C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 mM [U- 13 C]glutamate 85% of the 13 C metabolized was converted to glutamine. The formation of [1,2,3- 13 C 3 ]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2-0.5 mM glutamate, 13 C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [ 13 C]lactate was essentially unchanged within the range of 0.2-0.5 mM glutamate, whereas the amount of [ 13 C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 mM, suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 mM and astrocytes from both rat and mouse brain are consistent with these findings.

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.

Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is converted to pyruvate and CO2 via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37 degrees C in the presence of 0.5 mM malate was 4.15 +/- 0.47 and 11.61 +/- 0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean +/- SEM, n = 18-19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. alpha-Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.

It is well documented that the brain preferentially utilizes alternative substrates for energy during brain development; however, less is known about the use of these substrates by synaptic terminals. The present study compared the rates of 14CO2 production from 1 mM D-[6-14C]glucose, L-[U-14C]glutamine, D-3-hydroxy[3-14C]butyrate, L-[U-14C]lactate and L-[U-14C]malate by synaptic terminals isolated from 17- to 18-day-old and 7- to 8-week-old rat brain. The rates of 14CO2 production from glucose, glutamine, 3-hydroxybutyrate, lactate and malate were 8.55 +/- 0.78, 25.90 +/- 4.58, 42.28 +/- 3.54, 48.42 +/- 2.09, and 9.31 +/- 1.61 nmol/h/mg protein (mean +/- SEM), respectively, in synaptic terminals isolated from 17- to 18-day-old rat brain and 12.95 +/- 1.64, 30.62 +/- 4.19, 16.09 +/- 2.62, 40.33 +/- 6.77, and 8.25 +/- 1.69 nmol/h/mg protein (mean +/- SEM), respectively, in synaptic terminals isolated from 7- to 8-week-old rat brain. In competition studies using unlabelled added substrates, the addition of 3-hydroxybutyrate, lactate or glutamine greatly decreased the rate of 14CO2 production from labelled glucose. Added unlabelled glucose increased the rate of 14CO2 production from 3-hydroxybutyrate in synaptic terminals from 7- to 8-week-old rat brain, but had no effect on 14CO2 production from any other substrates. Lactate also increased 14CO2 production from 3-hydroxybutyrate at 7-8 weeks, whereas the addition of 3-hydroxybutyrate decreased 14CO2 production from lactate only in synaptic terminals from 17- to 18-day-old rat brain. None of the added substrates altered the rate of 14CO2 production from labelled glutamine or malate suggesting that these substrates are metabolized in relatively distinct compartments within synaptic terminals. Overall the data demonstrate that synaptic terminals from both weanling and adult rat brain can utilize a variety of substrates for energy. In addition, the competition studies demonstrate that the interactions of substrates change with age and suggest that there are multiple compartments of energy metabolism (or tricarboxylic acid cycle activity) in isolated synaptic terminals.

Several reports indicate that lactate can serve as an energy substrate for the brain. The rate of oxidation of this substrate by cultured rat brain astrocytes was 3-fold higher than the rate with glucose, suggesting that lactate can serve as an energy source for these cells. Since transport into the astrocytes may play an important role in regulating nutrient use by individuals types of brain cells, we investigated the uptake of L-[U-14C]lactate by primary cultures of rat brain astrocytes. Measurement of the net uptake suggested two carrier-mediated mechanisms and an Eadie-Hofstee type plot of the data supported this conclusion revealing 2 Km values of 0.49 and 11.38 mM and Vmax values of 16.55 and 173.84 nmol/min/mg protein, respectively. The rate of uptake was temperature dependent and was 3-fold higher at pH 6.2 than at 7.4, but was 50% less at pH 8.2. Although the lactate uptake carrier systems in astrocytes appeared to be labile when incubated in phosphate buffered saline for 20 minutes, the uptake process exhibited an accelerative exchange mechanism. In addition, lactate uptake was altered by several metabolic inhibitors and effectors. Potassium cyanide and alpha-cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, alpha-ketoisocaproate, and 3-hydroxybutyrate at 5 and 10 mM greatly attenuated the rate of lactate uptake. These results suggest that the availability of lactate as an energy source is regulated in part by a biphasic transport system in primary astrocytes.

This report extends the finding that serum protein(s) (0.5 mg/ml) caused a 50% decrease in the rate of glucose oxidation by dissociated brain cells with only marginal effects on the oxidation of other substrates. Since dissociated cells represent a heterogeneous population, studies were initiated to determine the effect of serum on the rates of substrate oxidation by isolated synaptosomes and cultured rat brain astrocytes. Experiments revealed that the addition of 5% serum v/v to the reaction mixture resulted in a decrease in the rate of 14CO2 production from [6-14C]glucose by isolated synaptosomes by more than 70%. In contrast, the addition of 5% serum had little or no effect on the 14CO2 production from [6-14C]glutamine by the synaptosomes and only marginal effects (20–25 %) on 14CO2 production from [6-14C]lactate and 3-hydroxy[3-14C]butyrate. The effect of serum on the rates of substrate oxidation were similar for synaptosomal preparations obtained from adult animal brains or 18-day-old rats, except that with the latter preparation, 14CO2 production from 3-hydroxy[3-14C]butyrate was more attenuated by the presence of serum than with the former synaptosomal preparation (50 vs. 25%). In contrast to the results with synaptosomes, the presence of 5% serum enhanced the rates of 14C02 production from [6-14C]glucose, 3-hydroxy[3-14C]butyrate and [6-14C]lactate by 61, 35 and 69%, respectively, in cultured rat brain astrocytes. However, this enhancement did not occur when the cells were grown in chemically defined media or when dibutyryl cAMP was added to the media. Collectively the results suggest that the presence of serum protein(s) may contribute to the regulation of substrate oxidation by brain cells in a yet to be defined manner; either by acting directly with enzymes in the metabolic pathway or via an interim (second messenger) factor.

There have been numerous studies on the activity and localization of aspartate aminotransferase (AAT) and glutamate dehydrogenase (GDH) in brain tissue. However, there is still a controversy as to the specific roles and relative importance of these enzymes in glutamate and glutamine metabolism in astrocytes and neurons or synaptic terminals. There are many reports documenting GDH activity in synaptic terminals, yet the misconception that it is a glial enzyme persists. Furthermore, there is evidence that this tightly regulated enzyme may have an increased role in synaptic metabolism in adverse conditions such as low glucose and hyperammonemia that could compromise synaptic function. In the present study, we report high activity of both AAT and GDH in mitochondrial subfractions from cortical synaptic terminals. The relative amount of GDH/AAT activity was higher in SM2 mitochondria, compared to SM1 mitochondria. Such a differential distribution of enzymes can contribute significantly to the compartmentation of metabolism. There is evidence that the metabolic capabilities of the SM1 and SM2 subfractions of synaptic mitochondria are compatible with the compartments A and B of neuronal metabolism proposed by Waagepetersen et al. (1998b. Dev. Neurosci. 20, 310-320).

Since lactate released by glial cells may be a key substrate for energy in neurons, the kinetics for the uptake of L-[U-14C]lactate by cortical synaptic terminals from 7- to 8-week-old rat brain were determined. Lactate uptake was temperature-dependent, and increased by 64.9% at pH 6.2, and decreased by 43.4% at pH 8.2 relative to uptake at pH 7.3. Uptake of monocarboxylic acids was saturable with increasing substrate concentration. Eadie-Hofstee plots of the data gave evidence of two carrier-mediated uptake mechanisms with a high-affinity Km of 0.66 mM and Vmax of 3.66 mM for pyruvate, and a low-affinity system with a Km of 9.9 mM for both lactate and pyruvate and Vmax values of 16.6 and 23.1 nmol/30 s/mg protein for lactate and pyruvate, respectively. Saturable uptake was seen in the presence of 10 mM α-cyano-4-hydroxycinnamate. Lactate transport by synaptic terminals was much more sensitive to inhibition by sulfhydryl reagents than transport in astrocytes. Addition of 0.5 and 2 mM mersalyl decreased the uptake of 1 mM lactate by synaptic terminals by 59.3 and 66.37%, respectively. Pyruvate moderately decreased lactate transport, whereas 3-hydroxybutyrate had little effect. Quercetin, an inhibitor of lactate release, had little effect on the content of 14C lactate in synaptic terminals, supporting the concept that the majority of lactate produced within brain is from glial cells. Oxidation of L-[U-14C]lactate by synaptosomes was saturable, and yielded a Km of 1.23 mM and a Vmax of 116 nmol/h/mg protein. Overall the studies show that synaptic terminals from adult brain have a high capacity for transport and oxidation of lactate, consistent with the proposed role for this compound in metabolic trafficking in brain. Furthermore, the data provide kinetic evidence of two carrier-mediated mechanisms for monocarboxylic acid transport by synaptosomes and demonstrate that uptake of lactate by synaptic terminals is regulated differently than transport by astrocytes. Uptake of lactate by synaptic terminals also has differences from the systems described for neurons.

The present study determined the metabolic fate of [U-13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mM alpha-ketoisocaproate (alpha-KIC). When astrocytes were incubated with 0.2 mM [U-13C]glutamate, 64.1% of the 13C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-(13)C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [13C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [13C]glutamate into aspartate in astrocytes incubated in the presence of alpha-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [13C]glutamate into the 1,2,3-(13)C3-isotopomer of lactate in cells incubated in the presence of alpha-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of alpha-KIC. Altogether more of the [13C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of alpha-KIC than in control cells. Overall, the results demonstrate that the presence of alpha-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.

It is well established that 3-hydroxybutyrate can serve as an energy source for the brain. Since substrate utilization may be regulated in part by transport across the cellular membrane, we investigated the uptake of 3-hydroxybutyrate by primary cultures of rat brain astrocytes. Measurement of the net uptake indicated a saturable system and a Lineweaver-Burke type plot was consistent with a single carrier-mediated mechanism with a Km of 6.03 mM and a Vmax of 32.7 nmol/30 seconds/mg protein. The rate of uptake at pH 6.2 was more than ten times the rate at pH 8.2, with the rate at pH 7.4 being intermediate between these values, suggesting the possibility of cotransport with H+ or exchange with OH- (antiport). Mersalyl had only a slight effect on the transport of 3-hydroxybutyrate, suggesting that sulfhydryl groups are not involved in the transport of this monocarboxylic acid. Phenylpyruvate and alpha-ketoisocaproate also attenuated the transport, but lactate had only a marginal effect. These results suggest that the utilization of 3-hydroxybutyrate as an energy source by astrocytes is regulated in part by carrier-mediated transport and that the uptake system is different from the lactate transport system.

Several recent studies have demonstrated that the metabolism of energy substrates takes place in multiple compartments in both astrocytes and synaptic terminals from brain. There are a number of differences in the metabolism of astrocytes and synaptic terminals primarily due to the localization of key enzymes such as pyruvate carboxylase and glutamine synthetase in astrocytes. The present study determined the rates of 14CO2 production from several energy substrates by primary cultures of astrocytes and cortical synaptic terminals from rat brain. The rates of 14CO2 production from labelled substrates by astrocytes were 0.96 +/- 0.13, 11.13 +/- 0.67, 10.51 +/- 0.35, 24.92 +/- 1.66 and 4.80 +/- 0.50 for D-[6-14C]glucose, L-[U-14C]lactate, D-3-hydroxy[3-14C]butyrate, L-[U-14C]glutamine and L-[U-14C]ma-late, respectively. The rates of 14CO2 production were also measured in the presence of 5 mM aminooxyacetate (AOAA) to determine the effect of inhibiting the malate-aspartate shuttle and other transaminase reactions on the oxidation of energy substrates. In astrocytes the addition of AOAA decreased the rate of glutamine oxidation 5-fold, consistent with other studies showing that glutamine enters the TCA cycle via transamination. AOAA increased the rate of 14CO2 production from labelled glucose 4-fold, suggesting that inhibition of alanine biosynthesis profoundly alters the utilization of glucose by astrocytes. AOAA also increased the oxidation of lactate and 3-hydroxybutyrate 36 and 58%, respectively. The rates of 14CO2 production from labelled substrates by synaptic terminals were 13.12 +/- 1.05, 35.29 +/- 3.58, 17.66 +/- 1.95, 30.18 +/- 1.10 and 9.95 +/- 1.29, respectively, for glucose, lactate, 3-hydroxybutyrate, glutamine and malate, demonstrating that all substrates were oxidized at a higher rate by synaptic terminals than by astrocytes. The addition of AOAA decreased the rate of 14CO2 production from labelled lactate by 57% suggesting that the use of lactate for energy in synaptic terminals is tightly coupled to the activity of the malate-aspartate shuttle. AOAA had no effect on the rate of 14CO2 production from labelled glutamine, demonstrating that exogenous glutamine enters the TCA cycle in synaptic terminals via glutamate dehydrogenase, not via transamination as is the case with astrocytes. AOAA had no significant effect on the rates of oxidation of glucose, 3-hydroxybutyrate and malate by synaptic terminals. These findings demonstrate that inhibiting transamination with AOAA had very different effects on the oxidation of energy substrates in the two preparations, suggesting that the regulation of metabolism is quite different in astrocytes and synaptic terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-[U-14C]malate in primary cultures of rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from 0.01 mM and 0.5 mM malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain. Both the biphasic kinetics and the differential action of many of the effectors on the 14CO2 production from 0.01 mM and 0.5 mM malate provide evidence for the presence of more than one pool of malate metabolism in cultured rat brain astrocytes.

This report is an extension of recent studies indicating the presence of a factor in serum that preferentially inhibits 14CO2 production from labeled glucose. Experiments with dissociated cells revealed that the inhibitory effects of serum were only slightly changed over more than a 50-fold range in initial glucose concentration. Serum had no effect on the rate of glucose transport (uptake of 1,3[3H]2-deoxyglucose). The inhibitory effect of serum was greater on 14CO2 production from [6-14C]glucose than [1-14C]glucose. Other studies revealed that 14CO2 production from [1-14C]pyruvate was more than 5 times the rate obtained using [3-14C]pyruvate; however, the inhibitory effect of serum was much greater on the latter (20% vs 60% inhibition respectively) at 2 mM pyruvate and in the presence of 1% fetal bovine serum. Attempts to characterize the factor using Amicon filtration showed the highest inhibitory activity in a 10,000 mol. wt. fraction, although some inhibitory activity was found in commercial preparations of bovine serum albumin. Delipidation of serum had no effect. Based on these results, we postulate that the observed decrease in labeled CO2 production reflects the regulation of substrate utilization at the pyruvate carboxylase step by one or more factors in serum (with a mol. wt. of approximately 10,000).

— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase (l-glutamine amido-hydrolase EC 3.5.1.2) and glutamate dehydrogenase (l-glutamate: NAD oxidoreductase EC 1.4.1.2). The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase (l-glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood. In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α-ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.

Glycerol, which decreased circulating levels of ketone bodies in adults, was tested for its antiketonemic action in developing rats. Following intraperitoneal injection of glycerol in rats from 2 to 90 days of age, blood levels of 3-hydroxybutyrate (beta HOB) decreased and reached minimum values in 35-40 minutes. The pattern of recovery in suckling animals (2, 7 and 14 days of age) differed from that of 24-, 35- and 90-day-old rats. Glycerol did not change blood acetoacetate (Ac2) levels in suckling rats but caused marked reductions in fasted older animals. The effect of glycerol on the ration of beta HOB:Ac2 was strikingly different for 90-day-old rats compared to all other groups. Glycerol did not raise blood glucose levels in neonatal or suckling rats. These developmental patterns of response to glycerol differed remarkedly from those to alanine, suggesting differential antiketonemic mechanisms for these two compounds on in vitro rats of beta HOB synthesis in liver homogenates from rats injected with either glycerol or alanine.

Extract: Studies of substrate oxidation by cultured skin fibroblasts from an infant (CC) with severe recurrent episodes of ketoacidosis showed normal rates of oxidation of pro-pionate (CC 55. ± 8 versus control 65 ± 13 μμmoles/mg protein/hr), and succinate (73. ± 16 versus 45 ± 8 μμmoles/mg protein/hr). In contrast, both oxidation and uptake of glucose by these cells were decreased in comparison with controls. Essentially neither glucose-6-14C nor glycerol-U-14C was converted to 14CO2 by fibroblasts of the patient, and glucose-1-14C was oxidized at 45–65% of control rates. In the absence of glucose, oxidation of pyruvate-2-14C did not differ from that of normal controls (710 versus 940 μμmoles/mg protein/hr). In the presence of 2.5 mM glucose, however, fibroblasts of the patient did not show the expected decrease in pyruvate-2-14C oxidation whereas control cells showed a fourfold decrease. Studies of glucose uptake demonstrated that with glucose as the only substrate extended incubation periods of 12–18 hr were necessary before the cells of the patient began to utilize measurable quantities of glucose. The results of the metabolic studies in skin fibroblasts, when viewed in conjunction with the clinical manifestations and laboratory studies, have excluded the known causes of infantile ketoacidosis. The data reported indicate a correlation of this form of ketoacidosis with a block in the terminal pathway of glycolysis. Speculation: A defect in glycolysis may produce intracellular hypoglycemia resulting in the activation of unknown mechanisms in peripheral tissue that initiate lipolysis and ketosis. It could also be that a yet undefined block in ketoacid metabolism might result in the observed irregular glucose oxidation. The basic abnormality may be a result of an in-balance in redox cofactors, an absence of a critical intermediate, or a reduction of an end product. These possibilities are currently being investigated.

The rates of [6-14C]-glucose oxidation by reconstituted systems of cytosol and mitochondria or cytosol and synaptosomes were essentially the same as the rate of oxidation of [3-14C]-3-hydroxybutyrate. However, the rate of [U-14C]-glutamine oxidation by mitochondria was 2.5 times that by synaptosomes. The addition of glutamine (5 mM) caused a reduction in the rates of oxidation of [6-14C]-glucose of 20% and 40% by mitochondria and synaptosomes, respectively. Conversely, the addition of glucose (5 mM) had little or no effect on the rate of [U-14C]-glutamine oxidation by either organelle. Amino-oxyacetate decreased [U-14C]-glutamine oxidation by mitochondria more than 35% but had little or no effect on the rate of glutamine oxidation by synaptosomes. When glucose (5 mM) was added to [3-14C]-3-hydroxybutyrate, the rates of oxidation by the mitochondria and synaptosomes were increased by 65% and 77%, respectively. However, in the reverse situation the addition of 3-hydroxybutyrate decreased [6-14C]-glucose oxidation by synaptosomes (35%) but did not decrease the rate by mitochondria. These results suggest that differences in the rates of substrate utilization by mitochondria and synaptosomes and differences in substrate interactions in these two subcellular organelles may contribute to metabolic compartmentation in the brain.

— Newborn rats from dams fed on a high fat diet developed increased ketonemia and significant hypertriglyceridemia i.e. “hyperketonemic pups”. This perinatal metabolic stress led to an alteration in the developmental pattern of glycolytic intermediates in their brains. In control rats, the concentration of glucose 6-phosphate (G6P) in the brain was high at birth, and gradually decreased to adult values by the third week of life. In contrast, the fructose-1,6-diphosphate (FDP) concentration was low at birth and increased thereafter. The lactate concentration was also high at birth but decreased to the adult level by the first day of life. In the brains of control pups, lactate and pyruvate concentrations remained relatively constant during the first 3 weeks of life. In the brains of hyperketonemic pups, the concentration of G6P was the same as in the control animals at birth but decreased significantly during the first days of life. During early development the concentrations of FDP and pyruvate were significantly lower and the concentration of lactate, higher in the hyperketonemic pups as compared to the control group. The alteration in the concentration of these glycolytic intermediates in the brains of hyperketonemic pups indicated a change in the developmental pattern of glycolysis. The ratio of [lactate]/[pyruvate] also suggested an increased cytoplasmic redox potential in the brains of hyperketonemic pups during the first week of life.

A readily available apparatus is described for the simultaneous development of three chromatographic columns. Also described is a single-stage, nonlinear gradient elution system capable of the uninterrupted separation of the adenine nucleotides of human erythrocytes. The practicality of these methods is exemplified in their application to the determination of the ATP content of red cells of normal adults and full-term newborn infants. The results show a significantly higher level of the triphosphate in the cells of the infant as compared to those of the adult.

Oral administration of pCPA produces in the adult rat a series of metabolic imbalances which do not entirely mimic human phenylketonuria. Using low dosages of this enzyme inhibitor and additional amino acids in the diet, it was possible to lower rat liver phenylalanine hydroxylase to levels comparable to hyperphenylalaninemic or phenyl-ketonuric patients who may have vestigial (14) or no detectable (15) enzymatic capability. Under these conditions, only a minor disturbance is produced in serotonin metabolism as now appears to be the case in those genetic defects, as studied in man (16). The most apparent divergency, besides the introduction of a nonnatural, substituted amino acid, is that serum tyrosine changes are generally not parallel to the human inborn error. However, tryptophanemia has an inverse correlation with serum phenylalanine and this pattern is common to phenylketonuria.

THE molecular architecture of the red cell membrane formed mainly by lipids and proteins is not known; but it is possible to obtain information about its surface structure with the aid of agents which act on components located at the outer surface of the erythrocyte. This approach has been used to show the role of sialic acids in the electrokinetic behaviour of erythrocytes in an electrolyte medium1.

Plasma tryptophan can be extracted from spots on filter paper with dilute ethanol and estimated directly by spectrofluorometry in alkaline medium. The use of an inert support simplifies the required deproteinization step permitting good recovery of the free amino acid. This procedure enables the assay of a large number of samples in a short period. Results obtained for normal fasting adults are in agreement with previously published values.