J. Tyson Tildon

Gastrointestinal abnormalities in children with autistic disorder.

OBJECTIVES Our aim was to evaluate the structure and function of the upper gastrointestinal tract in a group of patients with autism who had gastrointestinal symptoms. STUDY DESIGN Thirty-six children (age: 5.7 +/- 2 years, mean +/- SD) with autistic disorder underwent upper gastrointestinal endoscopy with biopsies, intestinal and pancreatic enzyme analyses, and bacterial and fungal cultures. The most frequent gastrointestinal complaints were chronic diarrhea, gaseousness, and abdominal discomfort and distension. RESULTS Histologic examination in these 36 children revealed grade I or II reflux esophagitis in 25 (69.4%), chronic gastritis in 15, and chronic duodenitis in 24. The number of Paneths cells in the duodenal crypts was significantly elevated in autistic children compared with non-autistic control subjects. Low intestinal carbohydrate digestive enzyme activity was reported in 21 children (58.3%), although there was no abnormality found in pancreatic function. Seventy-five percent of the autistic children (27/36) had an increased pancreatico-biliary fluid output after intravenous secretin administration. Nineteen of the 21 patients with diarrhea had significantly higher fluid output than those without diarrhea. CONCLUSIONS Unrecognized gastrointestinal disorders, especially reflux esophagitis and disaccharide malabsorption, may contribute to the behavioral problems of the non-verbal autistic patients. The observed increase in pancreatico-biliary secretion after secretin infusion suggests an upregulation of secretin receptors in the pancreas and liver. Further studies are required to determine the possible association between the brain and gastrointestinal dysfunctions in children with autistic disorder.

Transport ofl-lactate by cultured rat brain astrocytes

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 ofl-[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 α-cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, α-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.

Succinyl-CoA: 3-Ketoacid CoA-Transferase Deficiency. A CAUSE FOR KETOACIDOSIS IN INFANCY

To explain the cause of a unique form of severe and intermittent ketoacidosis in an infant who expired after 6 months of life, tissue culture fibroblasts and post mortem tissue were examined for enzyme activities that catalyze glucose and ketoacid oxidation. No measurable succinyl-CoA: 3-ketoacid CoA-transferase (CoA-transferase) activity could be detected in homogenates of the post mortem brain, muscle and kidney tissue, or in the cultured skin fibroblasts. Since seven other enzyme activities involving both glycolysis and ketone body oxidation were present in these same tissues, it was reasonable to conclude that the observed absence of CoA-transferase activity was not an artifact of homogenate preparation. It was concluded that the absence of CoA-transferase activity resulted in a loss of intracellular homeostasis leading to ketoacidosis. In addition, the absence of this enzyme appears to be a reasonable explanation for the alteration in glucose metabolism that was previously reported in fibroblasts from this patient.

Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells.

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.

Energy metabolism in cortical synaptic terminals from weanling and mature rat brain: evidence for multiple compartments of tricarboxylic acid cycle activity.

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.

Elevation of Amino Acids in the Interstitial Space of the Rat Brain following Infusion of Large Neutral Amino and Keto Acids by Microdialysis: Leucine Infusion

A microenvironment similar to that found in maple syrup urine disease was created in the brain of free-moving, awake rats by the infusion of leucine into the brain using microdialysis. To determine the effects on amino acid homeostasis the eluate of the probe was analyzed. Perfusion with leucine elevated the interstitial levels of large neutral amino acids suggesting hetero exchange of large neutral amino acids from neuronal cells into the interstitial space. The data also demonstrated the inter relationship of leucine and glutamine, both of which may be nitrogen sinks in the brain. Elevation of large neutral amino acids in the interstitial space suggests a decreased concentration in neurons which might have an effect on the synthesis of serotonin and catecholamines and suggests a mechanism by which elevated leucine may affect neuronal function in maple syrup urine disease.

Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats.

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C‐lactate and14C‐glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 μmol/L fluorocitrate. However, 20 and 100 μmol/L fluorocitrate reduced lactate oxidation by 55 ± 20% and 68 ± 12%, respectively (p < 0.05 for both). Twenty and 100 μmol/L fluorocitrate reduced 14C‐glucose oxidation by 50 ± 14% (p < 0.05) and 24 ± 19% (ns), respectively. Addition of non‐radioactive lactate to 14C‐glucose plus fluorocitrate decreased 14C‐glucose oxidation by an additional 29% and 38%, respectively. These results indicate that astrocytes oxidize about 50% of the interstitial lactate and about 35% of the glucose. By subtraction, neurons metabolize a maximum of 50% of the interstitial lactate and 65% of the interstitial glucose.

CoA transferase in the brain and other mammalian tissues

Abstract Succinyl-CoA: 3-ketoacid CoA-transferase (EC 2.8.3.5) activity was demonstrated in the brain of man, rat, mouse, guinea pig, and pigeon, but not of the dog, calf, and pig. In contrast to the enzyme from heart tissue, the brain enzyme was a particulate protein which sedimented with the mitochondrial fraction. The protein could be solubilized in 0.3% deoxycholate, or by freezing and thawing, and fractional salt and acid precipitation, and DEAE-cellulose chromatography yielded a 300-fold purification of the rat brain enzyme. Double reciprocal plots of one substrate vs velocity at several concentrations of the second substrate resulted in parallel lines which suggested a “ping-pong” mechanism as has been described for the pig heart enzyme. The Km values for succinate, acetoacetate, and succinyl CoA were 3.9 × 10−2, 2.9 × 10−4, and 6.7 × 10−4 m , respectively. In the rat, the thermal lability, acrylamide gel electrophoretic mobility, and substrate specificity were essentially the same for the enzymes obtained from either heart, brain, or kidney. These findings support the suggested physiological role for the enzyme in ketone oxidation in central nervous tissue.

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free‐moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min‐fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C‐amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U‐14C]glutamate and [U‐14C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.

Glucose Transport in Astrocytes: Regulation by Thyroid Hormone

Abstract: Primary cultures of astrocytes from newborn rat brain showed evidence of a substrate‐saturable process for glucose transport. The system shows a relatively high affinity for the substrate, with an apparent Km of approximately 1 mM. Maintenance of the cells in medium containing thyroid‐hormone‐free serum for 3, 6, or 9 days resulted in significantly reduced rates of hexose transport. Addition of exogenous triiodothyronine to the transport incubation medium of these “hypothyroid′’ cells markedly increased the net rate of 2‐deoxyglucose uptake within 60 s to values equal to or above those of control cultures (cells maintained in normal serum). These findings support a key role for thyroid hormone in the transport of glucose across plasma membranes of brain cells and demonstrate the presence of this regulatory system in astrocytes.