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1.  Perinatal iron deficiency predisposes the developing rat hippocampus to greater injury from mild to moderate hypoxia–ischemia 
The hippocampus is injured in both hypoxia–ischemia (HI) and perinatal iron deficiency that are comorbidities in infants of diabetic mothers and intrauterine growth restricted infants. We hypothesized that preexisting perinatal iron deficiency predisposes the hippocampus to greater injury when exposed to a relatively mild HI injury. Iron-sufficient and iron-deficient rats (hematocrit 40% lower and brain iron concentration 55% lower) were subjected to unilateral HI injury of 15, 30, or 45 mins (n = 12 to 13/HI duration) on postnatal day 14. Sixteen metabolite concentrations were measured from an 11 μL volume on the ipsilateral (HI) and contralateral (control) hippocampi 1 week later using in vivo 1H NMR spectroscopy. The concentrations of creatine, glutamate, myo-inositol, and N-acetylaspartate were lower on the control side in the iron-deficient group (P < 0.02, each). Magnetic resonance imaging showed hippocampal injury in the majority of the iron-deficient rats (58% versus 11%, P < 0.0001) with worsening severity with increasing durations of HI (P = 0.0001). Glucose, glutamate, N-acetylaspartate, and taurine concentrations were decreased and glutamine, lactate and myo-inositol concentrations, and glutamate/glutamine ratio were increased on the HI side in the iron-deficient group (P < 0.01, each), mainly in the 30 and 45 mins HI subgroups (P < 0.02, each). These neurochemical changes likely reflect the histochemically detected neuronal injury and reactive astrocytosis in the iron-deficient group and suggest that perinatal iron deficiency predisposes the hippocampus to greater injury from exposure to a relatively mild HI insult.
PMCID: PMC2548275  PMID: 16868555
hippocampus; hypoxia; iron deficiency; ischemia; NMR spectroscopy; rat
2.  Neurochemical Changes after Acute Binge Toluene Inhalation in Adolescent and Adult Rats: A High-Resolution Magnetic Resonance Spectroscopy Study 
Neurotoxicology and teratology  2009;31(6):382-389.
Inhalant abuse in young people is a growing public health concern. We reported previously that acute toluene intoxication in young rats, using a pattern of exposures that approximate abuse patterns of inhalant use in humans, significantly altered neurochemical measures in select brain regions. In this study, adolescent and young adult rats were exposed similarly to an acute (2 × 15 min), high dose (8000 − 12000 ppm) of toluene and high-resolution magic angle spinning proton magnetic resonance spectroscopy (HR-MAS 1H-MRS) was used to assess neurochemical profiles of tissue samples from a number of brain regions collected immediately following solvent exposure. The current investigation focused on N-acetyl-aspartate (NAA), choline-containing compounds, creatine, glutamate, GABA, and glutamine. Contrary to our predictions, no significant alterations were found in levels of NAA, choline, creatine, glutamate, or glutamine in adolescent animals. In contrast to these minimal effects in adolescents, binge toluene exposure altered several neurochemical parameters in young adult rats, including decreased levels of choline and GABA in the frontal cortex and striatum and lowered glutamine and NAA levels in the frontal cortex. One of the more robust findings was a wide-ranging increase in lactate after toluene exposure in adult animals, an effect not observed in adolescents. These age-dependent effects of toluene are distinct from those reported previously in juvenile rats and suggest a developmental difference in vulnerability to the effects of inhalants. Specifically, the results suggest that the neurochemical response to toluene in adolescents is attenuated compared to adults, and imply an association between these neurochemical differences and age-influenced differences in solvent abuse in humans.
PMCID: PMC2771649  PMID: 19628036
Toluene; inhalant abuse; magnetic resonance spectroscopy; N-acetyl aspartate; GABA
3.  Extremely Low Frequency Magnetic Field Modulates the Level of Neurotransmitters 
This study was aimed to observe that extremely low frequency magnetic field (ELF-MF) may be relevant to changes of major neurotransmitters in rat brain. After the exposure to ELF-MF (60 Hz, 2.0 mT) for 2 or 5 days, we measured the levels of biogenic amines and their metabolites, amino acid neurotransmitters and nitric oxide (NO) in the cortex, striatum, thalamus, cerebellum and hippocampus. The exposure of ELF-MF for 2 or 5 days produced significant differences in norepinephrine and vanillyl mandelic acid in the striatum, thalamus, cerebellum and hippocampus. Significant increases in the levels of serotonin and 5-hydroxyindoleacetic acid were also observed in the striatum, thalamus or hippocampus. ELF-MF significantly increased the concentration of dopamine in the thalamus. ELF-MF tended to increase the levels of amino acid neurotransmitters such as glutamine, glycine and γ -aminobutyric acid in the striatum and thalamus, whereas it decreased the levels in the cortex, cerebellum and hippocampus. ELF-MF significantly increased NO concentration in the striatum, thalamus and hippocampus. The present study has demonstrated that exposure to ELF-MFs may evoke the changes in the levels of biogenic amines, amino acid and NO in the brain although the extent and property vary with the brain areas. However, the mechanisms remain further to be characterized.
PMCID: PMC4297757  PMID: 25605992
Amino acid; Biogenic amines; Extremely low frequency magnetic field; Neurotransmitters; Nitric oxide
4.  Cerebral glutamine metabolism under hyperammonemia determined in vivo by localized 1H and 15N NMR spectroscopy 
Brain glutamine synthetase (GS) is an integral part of the glutamate–glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. 15N MRS is an alternative approach to 13C MRS. Incorporation of labeled 15N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate–glutamine cycle. To measure 15N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel 15N pulse sequence to simultaneously detect, for the first time, [5-15N]Gln and [2-15N]Gln+Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized 1H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of 1H and 15N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30±0.050 μmol/g per minute), apparent neurotransmission (0.26±0.030 μmol/g per minute), glutamate dehydrogenase (0.029±0.002 μmol/g per minute), and net glutamine accumulation (0.033±0.001 μmol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification.
PMCID: PMC3318147  PMID: 22167234
15N and 1H MRS; glutamate dehydrogenase; glutamine synthetase; hyperammonemia; net glutamine accumulation
5.  Introduction to power-frequency electric and magnetic fields. 
Environmental Health Perspectives  1993;101(Suppl 4):73-81.
This paper introduces the reader to electric and magnetic fields, particularly those fields produced by electric power systems and other sources using frequencies in the power-frequency range. Electric fields are produced by electric charges; a magnetic field also is produced if these charges are in motion. Electric fields exert forces on other charges; if in motion, these charges will experience magnetic forces. Power-frequency electric and magnetic fields induce electric currents in conducting bodies such as living organisms. The current density vector is used to describe the distribution of current within a body. The surface of the human body is an excellent shield for power-frequency electric fields, but power-frequency magnetic fields penetrate without significant attenuation; the electric fields induced inside the body by either exposure are comparable in magnitude. Electric fields induced inside a human by most environmental electric and magnetic fields appear to be small in magnitude compared to levels naturally occurring in living tissues. Detection of such fields thus would seem to require the existence of unknown biological mechanisms. Complete characterization of a power-frequency field requires measurement of the magnitudes and electrical phases of the fundamental and harmonic amplitudes of its three vector components. Most available instrumentation measures only a small subset, or some weighted average, of these quantities. Hand-held survey meters have been used widely to measure power-frequency electric and magnetic fields. Automated data-acquisition systems have come into use more recently to make electric- and magnetic-field recordings, covering periods of hours to days, in residences and other environments.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1519708  PMID: 8206045
6.  Tissue-specific regulation of avian glutamine synthetase expression during development and in response to glucocorticoid hormones. 
Molecular and Cellular Biology  1987;7(3):1070-1077.
We have isolated a glutamine synthetase cDNA clone derived from chicken retinal RNA. The clone detects a 3.2-kilobase RNA in chicken retina, liver, and brain, based on Northern blotting analysis. The dramatic developmental rise observed for the retinal enzyme, assayed as glutamyl transferase activity, is accompanied by a corresponding rise in this RNA. Injection of hydrocortisone 21-phosphate into the yolk sac of day 10 embryos produces an increase in retinal glutamine synthetase mRNA and glutamyl transferase activity, assayed 4 days after injection. An increase in glutamine synthetase mRNA is also observed within 2 h of incubation of retinal organ cultures with hydrocortisone. Moreover, incubation of these cultures with cycloheximide at a concentration that inhibits protein synthesis by 93% affects neither the basal level nor the hydrocortisone-mediated induction of glutamine synthetase mRNA. Although expression of this RNA is developmentally regulated in the brain, steroid hormone injection does not result in a substantial induction. Hepatic glutamine synthetase mRNA is expressed constitutively between embryonic day 10 and 6 days after hatching and is also not hormone inducible. Southern blotting data with chicken DNA digested with EcoRI, HindIII, and BamHI are best interpreted in terms of the cDNA clone detecting only one gene. If so, several cell-type-specific regulatory mechanisms must function to modulate expression of this gene during development.
PMCID: PMC365178  PMID: 2882415
7.  In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by 15N Nuclear Magnetic Resonance 
Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.
PMCID: PMC91150  PMID: 10049869
8.  Fetal and neonatal iron deficiency causes volume loss and alters the neurochemical profile of the adult rat hippocampus 
Nutritional neuroscience  2011;14(2):59-65.
Perinatal iron deficiency results in persistent hippocampus-based cognitive deficits in adulthood despite iron supplementation. The objective of the present study was to determine the long-term effects of perinatal iron deficiency and its treatment on hippocampal anatomy and neurochemistry in formerly iron-deficient young adult rats.
Perinatal iron deficiency was induced using a low-iron diet during gestation and the first postnatal week in male rats. Hippocampal size was determined using volumetric magnetic resonance imaging at 8 weeks of age. Hippocampal neurochemical profile, consisting of 17 metabolites indexing neuronal and glial integrity, energy reserves, amino acids, and myelination, was quantified using high-field in vivo 1H NMR spectroscopy at 9.4 T (N = 11) and compared with iron-sufficient control group (N = 10).
The brain iron concentration was 56% lower than the control group at 7 days of age in the iron-deficient group, but had recovered completely at 8 weeks. The cross-sectional area of the hippocampus was decreased by 12% in the formerly iron-deficient group (P = 0.0002). The hippocampal neurochemical profile was altered: relative to the control group, creatine, lactate, N-acetylaspartylglutamate, and taurine concentrations were 6–29% lower, and glutamine concentration 18% higher in the formerly iron-deficient hippocampus (P < 0.05).
Perinatal iron deficiency was associated with reduced hippocampal size and altered neurochemistry in adulthood, despite correction of brain iron deficiency. The neurochemical changes suggest suppressed energy metabolism, neuronal activity, and plasticity in the formerly iron-deficient hippocampus. These anatomic and neurochemical changes are consistent with previous structural and behavioral studies demonstrating long-term hippocampal dysfunction following perinatal iron deficiency.
PMCID: PMC3170050  PMID: 21605501
Hippocampus; 1H NMR spectroscopy; Iron; Newborn; Perinatal iron deficiency
9.  Simulating Magnetic Nanoparticle Behavior in Low-field MRI under Transverse Rotating Fields and Imposed Fluid Flow 
In the presence of alternating-sinusoidal or rotating magnetic fields, magnetic nanoparticles will act to realign their magnetic moment with the applied magnetic field. The realignment is characterized by the nanoparticle’s time constant, τ. As the magnetic field frequency is increased, the nanoparticle’s magnetic moment lags the applied magnetic field at a constant angle for a given frequency, Ω, in rad/s. Associated with this misalignment is a power dissipation that increases the bulk magnetic fluid’s temperature which has been utilized as a method of magnetic nanoparticle hyperthermia, particularly suited for cancer in low-perfusion tissue (e.g., breast) where temperature increases of between 4°C and 7°C above the ambient in vivo temperature cause tumor hyperthermia. This work examines the rise in the magnetic fluid’s temperature in the MRI environment which is characterized by a large DC field, B0. Theoretical analysis and simulation is used to predict the effect of both alternating-sinusoidal and rotating magnetic fields transverse to B0. Results are presented for the expected temperature increase in small tumors (~1 cm radius) over an appropriate range of magnetic fluid concentrations (0.002 to 0.01 solid volume fraction) and nanoparticle radii (1 to 10 nm). The results indicate that significant heating can take place, even in low-field MRI systems where magnetic fluid saturation is not significant, with careful The goal of this work is to examine, by means of analysis and simulation, the concept of interactive fluid magnetization using the dynamic behavior of superparamagnetic iron oxide nanoparticle suspensions in the MRI environment. In addition to the usual magnetic fields associated with MRI, a rotating magnetic field is applied transverse to the main B0 field of the MRI. Additional or modified magnetic fields have been previously proposed for hyperthermia and targeted drug delivery within MRI. Analytical predictions and numerical simulations of the transverse rotating magnetic field in the presence of B0 are investigated to demonstrate the effect of Ω, the rotating field frequency, and the magnetic field amplitude on the fluid suspension magnetization. The transverse magnetization due to the rotating transverse field shows strong dependence on the characteristic time constant of the fluid suspension, τ. The analysis shows that as the rotating field frequency increases so that Ωτ approaches unity, the transverse fluid magnetization vector is significantly non-aligned with the applied rotating field and the magnetization’s magnitude is a strong function of the field frequency. In this frequency range, the fluid’s transverse magnetization is controlled by the applied field which is determined by the operator. The phenomenon, which is due to the physical rotation of the magnetic nanoparticles in the suspension, is demonstrated analytically when the nanoparticles are present in high concentrations (1 to 3% solid volume fractions) more typical of hyperthermia rather than in clinical imaging applications, and in low MRI field strengths (such as open MRI systems), where the magnetic nanoparticles are not magnetically saturated. The effect of imposed Poiseuille flow in a planar channel geometry and changing nanoparticle concentration is examined. The work represents the first known attempt to analyze the dynamic behavior of magnetic nanoparticles in the MRI environment including the effects of the magnetic nanoparticle spin-velocity. It is shown that the magnitude of the transverse magnetization is a strong function of the rotating transverse field frequency. Interactive fluid magnetization effects are predicted due to non-uniform fluid magnetization in planar Poiseuille flow with high nanoparticle concentrations.
PMCID: PMC2901184  PMID: 20625540
Magnetic nanoparticles; MRI; rotating magnetic field; interactive magnetization; magnetic particle imaging
10.  Neurochemical Effects of Chronic Administration of Calcitriol in Rats 
Nutrients  2014;6(12):6048-6059.
Despite accumulating data showing the various neurological actions of vitamin D (VD), its effects on brain neurochemistry are still far from fully understood. To further investigate the neurochemical influence of VD, we assessed neurotransmitter systems in the brain of rats following 6-week calcitriol (1,25-dihydroxyvitamin D) administration (50 ng/kg/day or 100 ng/kg/day). Both the two doses of calcitriol enhanced VDR protein level without affecting serum calcium and phosphate status. Rats treated with calcitriol, especially with the higher dose, exhibited elevated γ-aminobutyric acid (GABA) status. Correspondingly, the mRNA expression of glutamate decarboxylase (GAD) 67 was increased. 100 ng/kg of calcitriol administration also increased glutamate and glutamine levels in the prefrontal cortex, but did not alter glutamine synthetase (GS) expression. Additionally, calcitriol treatment promoted tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2) expression without changing dopamine and serotonin status. However, the concentrations of the metabolites of dopamine and serotonin were increased and the drug use also resulted in a significant rise of monoamine oxidase A (MAOA) expression, which might be responsible to maintain the homeostasis of dopaminergic and serotonergic neurotransmission. Collectively, the present study firstly showed the effects of calcitriol in the major neurotransmitter systems, providing new evidence for the role of VD in brain function.
PMCID: PMC4277014  PMID: 25533012
vitamin D; calcitriol; neurotransmitter systems; brain function
11.  Medial-frontal cortex hypometabolism in chronic phencyclidine exposed rats assessed by high resolution magic angle spin 11.7T proton magnetic resonance spectroscopy 
Neurochemistry International  2012;61(1):128-131.
Proton magnetic resonance spectroscopy (1H-MRS) clinical studies of patients with schizophrenia document prefrontal N-acetylaspartate (NAA) reductions, suggesting an effect of the disease or of antipsychotic medications. We studied in the rat the effect of prolonged exposure to a low-dose of the NMDA glutamate receptor antagonist phencyclidine (PCP) on levels of NAA, glutamate and glutamine in several brain regions where metabolite reductions have been reported in chronically medicated patients with schizophrenia.
Two groups of ten rats each were treated with PCP (2.58 mg/kg/day) or vehicle and were sacrificed after 1 month treatment. Concentrations of neurochemicals were determined with high resolution magic angle (HR-MAS) 1H-MRS at 11.7Tesla in ex-vivo punch biopsies from the medial frontal and cingulate cortex, striatum, nucleus accumbens, amygdala and ventral hippocampus.
PCP treatment reduced NAA, glutamate, glycine, aspartate, creatine, lactate and GABA in medial frontal cortex. In the nucleus accumbens, PCP reduced levels of NAA, aspartate and glycine; similarly aspartate and glycine were reduced in the striatum. Finally the amygdala and hippocampus had elevations in glutamine and choline, respectively.
Low-dose PCP in rats models prefrontal NAA and glutamate reductions documented in chronically-ill schizophrenia patients. Chronic glutamate NMDA receptor blockade in rats replicates an endophenotype in schizophrenia and may contribute to the prefrontal hypometabolic state in schizophrenia.
PMCID: PMC3387321  PMID: 22522288
phencyclidine; N-acetyl aspartate; glutamate; schizophrenia; 1H-MRS
12.  Behavioral teratology of ethylene glycol monomethyl and monoethyl ethers. 
A recent addition to the field of teratology has been the inclusion of functional assessment techniques of offspring after prenatal exposure to exogenous agents. The present paper reviews the behavioral teratogenic effects of ethylene glycol monomethyl ether (EGME, 2-methoxyethanol) and ethylene glycol monoethyl ether (EGEE, 2-ethoxyethanol). Groups of 15 pregnant Sprague-Dawley rats were exposed via inhalation to 25 ppm EGME or to 100 ppm EGEE on gestation days 7 to 13 or 14 to 20. An equal number of sham-exposed controls were included for both periods of gestation. The only effect noted in the maternal animals was a slightly prolonged gestation in the group exposed to 100 ppm EGEE on days 14 to 20. Litters were culled to four female and four male pups on the day of birth. Pups of each sex from all litters were tested on a variety of behavioral tasks (including tests of neuromuscular ability, activity, and learning ability) extending from postnatal days 10 to 90. In addition, brains from newborn and from 21-day-old offspring were removed and analyzed for concentrations of the neurotransmitters acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine (serotonin). Both the behavioral testing and the neurochemical evaluations revealed functional alterations in the litter groups experiencing prenatal exposure to EGME and EGEE at concentrations which produced no observable effects in the maternal animals.
PMCID: PMC1568271  PMID: 6499817
13.  Assessment of Genotoxic and Cytotoxic Hazards in Brain and Bone Marrow Cells of Newborn Rats Exposed to Extremely Low-Frequency Magnetic Field 
The present study aimed to evaluate the association between whole body exposure to extremely low frequency magnetic field (ELF-MF) and genotoxic , cytotoxic hazards in brain and bone marrow cells of newborn rats. Newborn rats (10 days after delivery) were exposed continuously to 50 Hz, 0.5 mT for 30 days. The control group was treated as the exposed one with the sole difference that the rats were not exposed to magnetic field. Comet assay was used to quantify the level of DNA damage in isolated brain cells. Also bone marrow cells were flushed out to assess micronucleus induction and mitotic index. Spectrophotometric methods were used to measure the level of malondialdehyde (MDA) and the activity of glutathione (GSH) and superoxide dismutase (SOD). The results showed a significant increase in the mean tail moment indicating DNA damage in exposed group (P < 0.01, 0.001, 0.0001). Moreover ELF-MF exposure induced a significant (P < 0.01, 0.001) four folds increase in the induction of micronucleus and about three folds increase in mitotic index (P < 0.0001). Additionally newborn rats exposed to ELF-MF showed significant higher levels of MDA and SOD (P < 0.05). Meanwhile ELF-MF failed to alter the activity of GSH. In conclusion, the present study suggests an association between DNA damage and ELF-MF exposure in newborn rats.
PMCID: PMC3471024  PMID: 23091355
14.  Manganese Neurotoxicity: Lessons Learned from Longitudinal Studies in Nonhuman Primates 
Environmental Health Perspectives  2008;117(3):325-332.
Exposure to excess levels of the essential trace element manganese produces cognitive, psychiatric, and motor abnormalities. The understanding of Mn neurotoxicology is heavily governed by pathologic and neurochemical observations derived from rodent studies that often employ acute Mn exposures. The comparatively sparse studies incorporating in vivo neuroimaging in nonhuman primates provide invaluable insights on the effects of Mn on brain chemistry.
The purpose of this review is to discuss important aspects of Mn neurotoxicology and to synthesize recent findings from one of the largest cohorts of nonhuman primates used to study the neurologic effects of chronic Mn exposure.
We reviewed our recent in vivo and ex vivo studies that have significantly advanced the understanding of Mn-induced neurotoxicity. In those studies, we administered weekly doses of 3.3–5.0 (n = 4), 5.0–6.7 (n = 5), or 8.3–10.0 mg Mn/kg (n = 3) for 7–59 weeks to cynomolgus macaque monkeys. Animals expressed subtle deficits in cognition and motor function and decreases in the N-acetylaspartate-to-creatine ratio in the parietal cortex measured by magnetic resonance spectroscopy reflective of neuronal dysfunction. Impaired striatal dopamine release measured by positron emission tomography was observed in the absence of changes in markers of dopamine neuron degeneration. Neuropathology indicated decreased glutamine synthetase expression in the globus pallidus with otherwise normal markers of glutamatergic and GABAergic neurotransmission. Increased amyloid beta (A4) precursor-like protein 1 gene expression with multiple markers of neurodegeneration and glial cell activation was observed in the frontal cortex.
These findings provide new information on mechanisms by which Mn affects behavior, neurotransmitter function, and neuropathology in nonhuman primates.
PMCID: PMC2661898  PMID: 19337503
cognitive function; dopamine; manganese; neurodegeneration; neurotoxicity; non human primates; Parkinson disease; positron emission tomography
15.  Glutamine supplementation in a child with inherited GS deficiency improves the clinical status and partially corrects the peripheral and central amino acid imbalance 
Glutamine synthetase (GS) is ubiquitously expressed in mammalian organisms and is a key enzyme in nitrogen metabolism. It is the only known enzyme capable of synthesising glutamine, an amino acid with many critical roles in the human organism. A defect in GLUL, encoding for GS, leads to congenital systemic glutamine deficiency and has been described in three patients with epileptic encephalopathy. There is no established treatment for this condition.
Here, we describe a therapeutic trial consisting of enteral and parenteral glutamine supplementation in a four year old patient with GS deficiency. The patient received increasing doses of glutamine up to 1020 mg/kg/day. The effect of this glutamine supplementation was monitored clinically, biochemically, and by studies of the electroencephalogram (EEG) as well as by brain magnetic resonance imaging and spectroscopy.
Treatment was well tolerated and clinical monitoring showed improved alertness. Concentrations of plasma glutamine normalized while levels in cerebrospinal fluid increased but remained below the lower reference range. The EEG showed clear improvement and spectroscopy revealed increasing concentrations of glutamine and glutamate in brain tissue. Concomitantly, there was no worsening of pre-existing chronic hyperammonemia.
In conclusion, supplementation of glutamine is a safe therapeutic option for inherited GS deficiency since it corrects the peripheral biochemical phenotype and partially also improves the central biochemical phenotype. There was some clinical improvement but the patient had a long standing severe encephalopathy. Earlier supplementation with glutamine might have prevented some of the neuronal damage.
PMCID: PMC3495849  PMID: 22830360
Glutamine supplementation; Glutamine synthetase; Chronic encephalopathy; Neonatal onset seizures; Hyperammonemia; Qatar consanguinity; Therapeutic trial; GABA; Neurotransmitter replenishment; SLC38
The effect of zinc and glutamine on brain development was investigated during the lactation period in Swiss mice.
Malnutrition was induced by clustering the litter size from 6–7 pups/dam (nourished control) to 12–14 pups/dam (undernourished control) following birth. Undernourished groups received daily supplementation with glutamine by subcutaneous injections starting at day 2 and continuing until day 14. Glutamine (100 mM, 40–80μl) was used for morphological and behavioral studies. Zinc acetate was added in the drinking water (500 mg/L) to the lactating dams. Synaptophysin (SYN) and myelin basic protein (MBP) brain expressions were evaluated by immunoblot. Zinc serum and brain levels and hippocampal neurotransmitters were also evaluated.
Zinc with or without glutamine improved weight gain as compared to untreated, undernourished controls. In addition, zinc supplementation improved cliff avoidance and head position during swim behaviors especially on days 9 and 10. Using design-based stereological methods, we found a significant increase in the volume of CA1 neuronal cells in undernourished control mice, which was not seen in mice receiving zinc or glutamine alone or in combination. Undernourished mice given glutamine showed increased CA1 layer volume as compared with the other groups, consistent with the trend toward increased number of neurons. Brain zinc levels were increased in the nourished and undernourished-glutamine treated mice as compared to the undernourished controls on day 7. Undernourished glutamine-treated mice showed increased hippocampal GABA and SYN levels on day 14.
We conclude that glutamine or zinc protects against malnutrition-induced brain developmental impairments.
PMCID: PMC2909626  PMID: 20371167
malnutrition; hippocampus; stereology; ontogeny behavior; suckling mice
17.  Magnetic-field-induced DNA strand breaks in brain cells of the rat. 
Environmental Health Perspectives  2004;112(6):687-694.
In previous research, we found that rats acutely (2 hr) exposed to a 60-Hz sinusoidal magnetic field at intensities of 0.1-0.5 millitesla (mT) showed increases in DNA single- and double-strand breaks in their brain cells. Further research showed that these effects could be blocked by pretreating the rats with the free radical scavengers melatonin and N-tert-butyl-alpha-phenylnitrone, suggesting the involvement of free radicals. In the present study, effects of magnetic field exposure on brain cell DNA in the rat were further investigated. Exposure to a 60-Hz magnetic field at 0.01 mT for 24 hr caused a significant increase in DNA single- and double-strand breaks. Prolonging the exposure to 48 hr caused a larger increase. This indicates that the effect is cumulative. In addition, treatment with Trolox (a vitamin E analog) or 7-nitroindazole (a nitric oxide synthase inhibitor) blocked magnetic-field-induced DNA strand breaks. These data further support a role of free radicals on the effects of magnetic fields. Treatment with the iron chelator deferiprone also blocked the effects of magnetic fields on brain cell DNA, suggesting the involvement of iron. Acute magnetic field exposure increased apoptosis and necrosis of brain cells in the rat. We hypothesize that exposure to a 60-Hz magnetic field initiates an iron-mediated process (e.g., the Fenton reaction) that increases free radical formation in brain cells, leading to DNA strand breaks and cell death. This hypothesis could have an important implication for the possible health effects associated with exposure to extremely low-frequency magnetic fields in the public and occupational environments.
PMCID: PMC1241963  PMID: 15121512
18.  Rapid Enhancement of Glutamatergic Neurotransmission in Bipolar Depression Following Treatment with Riluzole 
Neuropsychopharmacology  2009;35(3):834-846.
Glutamatergic abnormalities may underlie bipolar disorder (BD). The glutamate-modulating drug riluzole may be efficacious in bipolar depression, but few in vivo studies have examined its effect on glutamatergic neurotransmission. We conducted an exploratory study of the effect of riluzole on brain glutamine/glutamate (Gln/Glu) ratios and levels of N-acetylaspartate (NAA). We administered open-label riluzole 100–200 mg daily for 6 weeks to 14 patients with bipolar depression and obtained imaging data from 8-cm3 voxels in the anterior cingulate cortex (ACC) and parieto-occipital cortex (POC) at baseline, day 2, and week 6 of treatment, using two-dimensional J-resolved proton magnetic resonance spectroscopy at 4 T. Imaging data were analyzed using the spectral-fitting package, LCModel; statistical analysis used random effects mixed models. Riluzole significantly reduced Hamilton Depression Rating Scale (HAM-D) scores (d=3.4; p<0.001). Gln/Glu ratios increased significantly by day 2 of riluzole treatment (Cohen's d=1.2; p=0.023). NAA levels increased significantly from baseline to week 6 (d=1.2; p=0.035). Reduction in HAM-D scores was positively associated with increases in NAA from baseline to week 6 in the ACC (d=1.4; p=0.053), but was negatively associated in the POC (d=9.6; p<0.001). Riluzole seems to rapidly increase Gln/Glu ratios—suggesting increased glutamate–glutamine cycling, which may subsequently enhance neuronal plasticity and reduce depressive symptoms. Further investigation of the Gln/Glu ratio as a possible early biomarker of response to glutamate-modulating therapies is warranted.
PMCID: PMC3055603  PMID: 19956089
bipolar disorder; depression; glutamate; N-acetylaspartate; riluzole; MRS; Depression; Unipolar/Bipolar; Imaging; Clinical or Preclinical; Glutamate; Psychopharmacology; MRS; N-acetylaspartate; plasticity; riluzole
19.  Assessing human exposure to power-frequency electric and magnetic fields. 
Environmental Health Perspectives  1993;101(Suppl 4):121-133.
This paper reviews published literature and current problems relating to the assessment of occupational and residential human exposures to power-frequency electric and magnetic fields. Available occupational exposure data suggest that the class of job titles known as electrical workers may be an effective surrogate for time-weighted-average (TWA) magnetic-field (but not electric-field) exposure. Current research in occupational-exposure assessment is directed to the construction of job-exposure matrices based on electric- and magnetic-field measurements and estimates of worker exposures to chemicals and other factors of interest. Recent work has identified five principal sources of residential magnetic fields: electric power transmission lines, electric power distribution lines, ground currents, home wiring, and home appliances. Existing residential-exposure assessments have used one or more of the following techniques: questionnaires, wiring configuration coding, theoretical field calculations, spot electric- and magnetic-field measurements, fixed-site magnetic-field recordings, personal- exposure measurements, and geomagnetic-field measurements. Available normal-power magnetic-field data for residences differ substantially between studies. It is not known if these differences are due to geographical differences, differences in measurement protocols, or instrumentation differences. Wiring codes and measured magnetic fields (but not electric fields) are associated weakly. Available data suggest, but are far from proving, that spot measurements may be more effective than wire codes as predictors of long-term historical magnetic-field exposure. Two studies find that away-from-home TWA magnetic-field exposures are less variable than at-home exposures. The importance of home appliances as contributors to total residential magnetic-field exposure is not known at this time. It also is not known what characteristics (if any) of residential electric and magnetic fields are determinants of human health effects.
PMCID: PMC1519693  PMID: 8206021
20.  Ethanol Exposure during the Early First Trimester Equivalent Impairs Reflexive Motor Activity and Heightens Fearfulness in an Avian Model 
Alcohol (Fayetteville, N.Y.)  2010;45(1):57-63.
Prenatal alcohol exposure is a leading cause of childhood neurodevelopmental disability. The adverse behavioral effects of alcohol exposure during the second and third trimester are well documented; less clear is whether early first trimester-equivalent exposures also alter behavior. We investigated this question using an established chick model of alcohol exposure. In ovo embryos experienced a single, acute ethanol exposure that spanned gastrulation through neuroectoderm induction and early brain patterning (19–22hr incubation). At 7 days post-hatch the chicks were evaluated for reflexive motor function (wingflap extension, righting reflex), fearfulness (tonic immobility), and fear/social reinstatement (open field behavior). Chicks exposed to a peak ethanol level of 0.23–0.28% were compared against untreated and saline-treated controls. Birds receiving early ethanol exposure had a normal righting reflex and a significantly reduced wingflap extension in response to a sudden descent. The ethanol-treated chicks also displayed heightened fearfulness, reflected in increased frequency of tonic immobility, and they required significantly fewer trials for its induction. In an open field test, ethanol treatment did not affect latency to move, steps taken, vocalizations, defecations, or escape attempts. The current findings demonstrate that early ethanol exposure can increase fearfulness and impair aspects of motor function. Importantly, the observed dysfunctions resulted from an acute ethanol exposure during the period when the major brain components are induced and patterned. The equivalent period in human development is 3–4 weeks post-conception. The current findings emphasize that ethanol exposure during the early first trimester equivalent can produce neurodevelopmental disability in the offspring.
PMCID: PMC3011049  PMID: 20705421
fetal alcohol spectrum disorders; chick embryo; tonic immobility; motor coordination; fearfulness; neurobehavior
21.  Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate 
Gut  2003;52(2):224-230.
Background and aims: Factors that induce luminal bacteria to cross the intestinal epithelium following injury remain poorly defined. The aim of this study was to investigate the interaction between glutamine metabolism, energy supply, and inflammatory mediators in determining the translocation of non-pathogenic bacteria across cultured enterocytes.
Methods: The effect of tumour necrosis factor α (TNF-α) on translocation of Escherichia coli C25 across Caco-2 epithelial monolayers was studied in the presence of products and inhibitors of glutamine metabolism. Simultaneous measurements of transepithelial electrical resistance (TEER) and flux of lucifer yellow were used to assess effects on the paracellular pathway. Lactate dehydrogenase release was used to monitor enterocyte integrity. Imaging of monolayers in these experimental conditions was undertaken with transmission electron microscopy.
Results: Exposure to basolateral TNF-α (20 ng/ml) for six hours induced translocation of E coli across Caco-2 but only if accompanied by simultaneous glutamine depletion (p<0.01). Translocation was inhibited by addition of glutamine for two hours (p<0.01) but not by an isonitrogenous mixture of non-glutamine containing amino acids. Inhibition of glutamine conversion to α-ketoglutarate, but not blockade of glutathione or polyamine synthesis, also induced translocation in the presence of TNF-α. Manipulations that induced bacterial translocation were associated with a marked reduction in enterocyte ATP levels. No effect of these treatments on paracellular permeability or lactate dehydrogenase release was observed. Conditions in which translocation occurred were associated with the presence of bacteria within enterocyte vacuoles but not the paracellular space.
Conclusions: In inflammatory conditions, the availability of glutamine as an enterocyte fuel substrate is essential for the preservation of a functional barrier to microorganisms. In conditions of acute glutamine depletion, cytokine mediated bacterial translocation appears to be primarily a transcellular process.
PMCID: PMC1774948  PMID: 12524404
intestinal barrier; sepsis, cytokines; Caco-2 cells; glutamine; tumour necrosis factor
22.  Prolonged continuous intravenous infusion of the dipeptide L-alanine- L-glutamine significantly increases plasma glutamine and alanine without elevating brain glutamate in patients with severe traumatic brain injury 
Critical Care  2014;18(4):R139.
Low plasma glutamine levels are associated with worse clinical outcome. Intravenous glutamine infusion dose- dependently increases plasma glutamine levels, thereby correcting hypoglutaminemia. Glutamine may be transformed to glutamate which might limit its application at a higher dose in patients with severe traumatic brain injury (TBI). To date, the optimal glutamine dose required to normalize plasma glutamine levels without increasing plasma and cerebral glutamate has not yet been defined.
Changes in plasma and cerebral glutamine, alanine, and glutamate as well as indirect signs of metabolic impairment reflected by increased intracranial pressure (ICP), lactate, lactate-to-pyruvate ratio, electroencephalogram (EEG) activity were determined before, during, and after continuous intravenous infusion of 0.75 g L-alanine-L-glutamine which was given either for 24 hours (group 1, n = 6) or 5 days (group 2, n = 6) in addition to regular enteral nutrition. Lab values including nitrogen balance, urea and ammonia were determined daily.
Continuous L-alanine-L-glutamine infusion significantly increased plasma and cerebral glutamine as well as alanine levels, being mostly sustained during the 5 day infusion phase (plasma glutamine: from 295 ± 62 to 500 ± 145 μmol/ l; brain glutamine: from 183 ± 188 to 549 ± 120 μmol/ l; plasma alanine: from 327 ± 91 to 622 ± 182 μmol/ l; brain alanine: from 48 ± 55 to 89 ± 129 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test).
Plasma glutamate remained unchanged and cerebral glutamate was decreased without any signs of cerebral impairment. Urea and ammonia were significantly increased within normal limits without signs of organ dysfunction (urea: from 2.7 ± 1.6 to 5.5 ± 1.5 mmol/ l; ammonia: from 12 ± 6.3 to 26 ± 8.3 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test).
High dose L-alanine-L-glutamine infusion (0.75 g/ kg/ d up to 5 days) increased plasma and brain glutamine and alanine levels. This was not associated with elevated glutamate or signs of potential glutamate-mediated cerebral injury. The increased nitrogen load should be considered in patients with renal and hepatic dysfunction.
Trial registration NCT02130674. Registered 5 April 2014
PMCID: PMC4227121  PMID: 24992948
23.  Epidemiological appraisal of studies of residential exposure to power frequency magnetic fields and adult cancers. 
OBJECTIVES: To appraise epidemiological evidence of the purported association between residential exposure to power frequency magnetic fields and adult cancers. METHODS: Literature review and epidemiological evaluation. RESULTS: Seven epidemiological studies have been conducted on the risk of cancer among adults in relation to residential exposure to power frequency magnetic fields. Leukaemia was positively associated with magnetic fields in three case-control studies. The other two case-control studies and two cohort studies did not show such a link. Brain tumours and breast cancer have rarely been examined by these studies. Based on the epidemiological results, the analysis of the role of chance and bias, and the criteria for causal inferences, it seems that the evidence is not strong enough to support the putative causal relation between residential exposure to magnetic fields and adult leukaemia, brain tumours, or breast cancer. Inadequate statistical power is far more a concern than selection bias, information bias, and confounding in interpreting the results from these studies, and in explaining inconsistencies between studies. CONCLUSIONS: Our reviews suggested that the only way to answer whether residential exposure to magnetic fields is capable of increasing the risks of adult cancers is to conduct more studies carefully avoiding methodological flaws, in particular small sample size. We also suggested that the risk of female breast cancer should be the object of additional investigations, and that future studies should attempt to include information on exposure to magnetic fields from workplaces as well as residential exposure to estimate the effects of overall exposure to magnetic fields.
PMCID: PMC1128532  PMID: 8983460
24.  Ethanol- and/or Taurine-Induced Oxidative Stress in Chick Embryos 
Journal of Amino Acids  2013;2013:240537.
Because taurine alleviates ethanol- (EtOH-) induced lipid peroxidation and liver damage in rats, we asked whether exogenous taurine could alleviate EtOH-induced oxidative stress in chick embryos. Exogenous EtOH (1.5 mmol/Kg egg or 3 mmol/Kg egg), taurine (4 μmol/Kg egg), or EtOH and taurine (1.5 mmol EtOH and 4 μmol taurine/Kg egg or 3 mmol EtOH and 4 μmol taurine/Kg egg) were injected into fertile chicken eggs during the first three days of embryonic development (E0–2). At 11 days of development (midembryogenesis), serum taurine levels and brain caspase-3 activities, homocysteine (HoCys) levels, reduced glutathione (GSH) levels, membrane fatty acid composition, and lipid hydroperoxide (LPO) levels were measured. Early embryonic EtOH exposure caused increased brain apoptosis rates (caspase-3 activities); increased brain HoCys levels; increased oxidative-stress, as measured by decreased brain GSH levels; decreased brain long-chain polyunsaturated levels; and increased brain LPO levels. Although taurine is reported to be an antioxidant, exogenous taurine was embryopathic and caused increased apoptosis rates (caspase-3 activities); increased brain HoCys levels; increased oxidative-stress (decreased brain GSH levels); decreased brain long-chain polyunsaturated levels; and increased brain LPO levels. Combined EtOH and taurine treatments also caused increased apoptosis rates and oxidative stress.
PMCID: PMC3628655  PMID: 23606945
25.  Chronic Binge Ethanol Mediated Acidemia Reduces Availability of Glutamine and Related Amino Acids in Maternal Plasma of Pregnant Sheep 
Alcohol (Fayetteville, N.Y.)  2008;42(8):657-666.
Heavy drinking during pregnancy can result in Fetal Alcohol Syndrome (FAS), of which, fetal and postnatal growth retardation and central nervous system deficits are cardinal features. While a number of mechanisms have been proposed, none fully account for these deficiencies. We have previously reported that maternal ethanol exposure (1.75 g/kg) results in transient acidemia in the mother and fetus. Alterations in pH are known to regulate glutamine homeostasis. Therefore, we hypothesized that chronic binge ethanol mediated acidosis reduces glutamine concentrations in maternal plasma that result in decreases in the circulating levels of amino acids related to glutamine metabolism. Pregnant ewes were divided into three groups: ethanol (1.75 g/kg), saline control, and acidemia (inspired fractional CO2 was manipulated to mimic the maternal arterial pH pattern created by ethanol). The experiment was conducted on three consecutive days followed by four days without treatment beginning on day 109 of gestation, continuing to day 132. Plasma samples were analyzed for nutrients and metabolites using HPLC and spectrophotometric methods. Maternal plasma concentrations of glutamate increased (58%), while those of glutamine and related amino acids decreased (between 14 and 53%) in response to an acute challenge following the chronic exposure in ethanol treated ewes. No differences in these amino acid concentrations were noted between the ethanol and acidemic group subjects. Maternal plasma lactate increased by ~100% in response to ethanol while glucose and urea did not change in any group. We conclude that maternal chronic binge ethanol consumption results in acidosis mediated reductions in circulating levels of glutamine and related amino acids that could be responsible for neuronal deficits, altered fetal growth, development, and programming. We also speculate that the consequent increase in fetal glutamate during critical periods of brain development may contribute to the pathogenesis of FAS.
PMCID: PMC2662367  PMID: 19038697
FAS; FASD; alcohol; acidosis; amino acids; glutamate

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