HPLC法同时测定复方阿胶补血颗粒中4种氨基酸

目的 建立高效液相色谱(HPLC)法同时测定复方阿胶补血颗粒中4种氨基酸。方法 采用Waters Sunfire C₁₈色谱柱（4.6 mm × 250 mm,5 mm）,以乙腈-0.1 mol·L^-1醋酸钠溶液（用36%乙酸调节pH值至6.5）（7∶93）为流动相A,以乙腈-水（4∶1）为流动相B,梯度洗脱（0~13 min,100% A ® 93% A;13~17.9 min,93% A ® 88% A;17.9~29 min,88% A ® 85% A;29~39 min,85% A ® 66% A;39~45 min,66% A ® 0% A）,柱温43 °C,体积流量1.0 mL·min^-1,检测波长254 nm。结果 L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸进样量分别在0.012 ~ 0.117、0.022 ~ 0.218、0.010 ~ 0.097、0.016 ~ 0.160 mg范围内与色谱峰峰面积呈良好的线性关系;平均回收率（n=6）分别为96.4%、97.3%、97.1%、99.4%;RSD分别为1.2%、1.9%、1.7%、0.9%。结论 该方法操作简单,重复性好,为控制复方阿胶补血颗粒的质量提供了可靠的方法。     

Paracetamol impairs the profile of amino acids in the rat brain

In our experiment we investigated the effect of subcutaneous administration of paracetamol on the levels of amino acids in the brain structures. Male Wistar rats received for eight weeks paracetamol at two doses: 10 mg/kg b.w. (group P10, n = 9) and 50 mg/kg b.w. per day s.c. (group P50, n = 9).The regional brain concentrations of amino acids were determined in the prefrontal cortex, hippocampus, hypothalamus and striatum of control (Con, n = 9) and paracetamol-treated groups using HPLC. Evaluation of the biochemical results indicated considerable decrease of the content of amino acids in the striatum (glutamine, glutamic acid, taurine, alanine, aspartic acid) and hypothalamus (glycine) between groups treated with paracetamol compared to the control. In the prefrontal cortex paracetamol increased the level of γ-aminobutyric acid (GABA). The present study demonstrated significant effect of the long term paracetamol treatment on the level of amino acids in the striatum, prefrontal cortex and hypothalamus of rats.     

氯胺酮麻醉期间大鼠脑内氨基酸递质水平的变化

氨基酸类递质是主要的中枢神经递质之一 ,对于精神意识活动的调整起着关键作用。现认为全麻药 (尤吸入全麻药 )可通过抑制突触前谷氨酸 (Ｇｌｕ)的释放、增强谷氨酸的再摄取及阻滞突触后兴奋性氨基酸受体而发挥全麻作用[1] 。药物对于天冬氨酸 (Ａｓｐ)、γ 氨基丁酸 (ＧＡＢＡ)和甘氨酸(Ｇｌｙ)的突触前作用则较为复杂 ,不如谷氨酸明显和统一[2 ] ,可能不是全麻药的主要作用部位。有些学者认为氯胺酮对各类氨基酸类递质的摄取与释放过程无明显影响[2 ] ,这些都是离体实验的结论 ,活体动物在麻醉状态下 ,中枢神经系统中的氨基酸类递质水平是…     

Lithium-induced changes in the brain levels of free amino acids in stress-exposed rats

The effects of stress and lithium on brain free amino acid levels in rats were investigated. Stress caused a significant decrease in the brain levels of alanine, ammonia, arginine, isoleucine, lysine and phenylalanine. Lithium by itself induced a significant increase in the brain levels of arginine and threonine, and a significant decrease in those of alanine, ammonia, aspartic acid, glutamic acid, glycine, lysine, phenylalanine and tyrosine. Lithium and stress together significantly decreased brain levels of alanine, arginine, aspartic acid, glycine, isoleucine, leucine, lysine, phenylalanine and tyrosine, and significantly increased that of serine.     

Neurochemical and behavioral profiling of the selective GlyT1 inhibitors ALX5407 and LY2365109 indicate a preferential action in caudal vs. cortical brain areas

Selective inhibitors of the glycine transporter 1 (GlyT1) have been implicated in central nervous system disorders related to hypoglutamatergic function such as schizophrenia. The selective GlyT1 inhibitors ALX5407 (NFPS) and LY2365109 {[2-(4-benzo[1,3]dioxol-5-yl-2-tert-butylphenoxy)ethyl]-methylamino}-acetic acid increased cerebrospinal fluid levels of glycine and potentiated NMDA-induced increases in dialysate levels of neurotransmitters in the prefrontal cortex (PFC) and the striatum. However, higher doses produced both stimulatory and inhibitory effects on motor performance and impaired respiration, suggesting significant involvement of cerebellar and brain stem areas. A dual probe microdialysis study showed that ALX5407 transiently elevated extracellular levels of glycine in the PFC with more sustained increases in the cerebellum. In support of these findings, immuno-staining with pan-GlyT1 and GlyT1a antibodies showed a higher abundance of immunoreactivity in the brain stem/cerebellum as compared to the frontal cortical/hippocampal brain areas in four different species studied, including the mouse, rat, monkey and human. In addition, the inhibitory effects of ALX5407 on cerebellar levels of cGMP in the mouse could be reversed by the glycine A receptor antagonist strychnine but not the glycine B receptor antagonist L-701324. We propose that the adverse events seen with higher doses of ALX5407 and LY2365109 are the result of high GlyT1 inhibitory activity in caudal areas of the brain with sustained elevations of extracellular glycine. High levels of glycine in these brain areas may result in activation of strychnine-sensitive glycine A receptors that are inhibitory on both motor activity and critical brain stem functions such as respiration.     

A microdialysis study of glycinamide, glycine and other amino acid neurotransmitters in rat frontal cortex and hippocampus after the administration of milacemide, a glycine pro-drug

Milacemide is a glycinamide derivative which readily enters the brain and is metabolised to glycine. As its mechanism of action as an anticonvulsant drug is unknown we used the technique of microdialysis to study the temporal inter-relationship of glycinamide, glycine and other amino acid neurotransmitters in the extracellular fluid of rat hippocampus and frontal cortex. After milacemide administration (400 or 800 mg/kg i.p.), glycinamide concentrations rose linearly and dose-dependently in both hippocampus and frontal cortex. In contrast, whilst glycine concentrations rose in the hippocampus, glycine was unaffected in the frontal cortex. Concomitant increases in taurine hippocampal concentrations were observed. An increase in serine and a decrease in alanine concentrations was only observed at the highest milacemide dose (800 mg/kg). Other amino acids were unaffected. Thus, while glycinamide appears to be universally distributed throughout the brain, its metabolism to glycine and its effects on brain amino acids appear to be region specific.     

The effect of CRF and α-helical CRF(9-41) on rat fear responses and amino acids release in the central nucleus of the amygdala

The effects of intracerebroventricular injections of CRF and a non-selective CRF receptor antagonist, α-helical CRF_(9-41), on the release of glutamate, aspartate, and GABA in the central nucleus of the amygdala (CeA), were examined in the course of testing rat anxiety-like behaviour in the conditioned fear test (a freezing response), using the microdialysis technique. It was found that CRF (1 μg/rat), given to animals exposed to the stress of novelty only, insignificantly increased the glutamate concentration in the CeA, up to 200% of the control level. In the fear-conditioned animals, the influence of CRF on the local concentration of aspartate, glutamate, and Glu/GABA ratio was much more pronounced (up to a 400% increase above the baseline level of aspartate concentration), preceded an increased expression of anxiety-like responses, and appeared as early as 15 min after the drug administration. The intracerebroventricular administration of α-helical CRF_(9-41) (10 μg/rat) significantly decreased the rat freezing responses and increased the local concentration of GABA during the first 30 min of observation. In sum, these are new findings, which show an important role of CRF in the CeA in the regulation of fear-controlled amino acids release and suggest an involvement of amino acids in the central nucleus of the amygdala in the effects of this neurohormone on the expression of conditioned fear.     

Milacemide effects on the temporal inter-relationship of amino acids and monoamine metabolites in rat cerebrospinal fluid

The temporal inter-relationship of various amino acids and monoamine metabolites in rat cerebrospinal fluid was examined after acute administration of milacemide (100, 200 or 400 mg/kg i.p.), a glycine prodrug. Glycine concentrations rose linearly and dose dependently (20–190%) but were only significantly elevated at the higher milacemide dose (200 and 400 mg/kg). In animals given 400 mg/kg, glycine values were still significantly elevated 8 h later. A concomitant increase (20–25%) in serine and taurine and a decrease in alanine cerebrospinal fluid values were observed at the highest milacemide dose. Other amino acids were unaffected. While cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations were unaffected, the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, exhibited a linear dose-dependent reduction. However, only homovanillic acid values were significantly decreased after 400 mg/kg milacemide. Cerebrospinal fluid analysis may be useful as a first screen in ascertaining putative neurochemical changes associated with drug administration.     

The relationship between the contents of 13 amino acids in brain tissues and the progression of NAFLD via C57BL/6 model mice

Non-alcoholic fatty liver disease (NAFLD) is the steatosis of liver parenchyma unrelated to alcoholism, autoimmunity, and viral infection. It is also a metabolism-related syndrome, which has an unseparated relationship with adipose tissue dysfunction and obesity. Hepatic encephalopathy (HE) is one of the severe complications of chronic liver disease and one of the end-stage syndromes of liver disease. Some researchers have suggested that NAFLD, like other forms of liver injury, may be related to the metabolic disorder of branched-chain amino acids (BCAAs), which have been approved to be associated with HE influencing ammonia and energy metabolism. However, several studies have revealed the relationship among amino acids in serum, HE, and chronic liver disease; there are few studies on the contents of amino acids in brain tissues of an animal model with NAFLD. In the present research, we established a NAFLD mouse model with C57BL/6 mice and determined the contents of 13 amino acids in brain tissues of model mice by HPLC-FLD derivatization method using ortho-phthalaldehyde (OPA) to explore the relationship between the contents of amino acids in brain tissues and the progression of NAFLD. Moreover, the study showed that the changes of amino acid contents in the brain of the C57BL/6 mice were associated with the advancement of NAFLD, and this change might be related to the mechanism of HE.     

Simultaneous determination of amino acids in discrete brain areas in Suncus murinus by high performance liquid chromatography with electrochemical detection

An improved and simple reversed-phase high performance liquid chromatography method with electrochemical detection for the simultaneous determination of amino acids in brain tissue of Suncus murinus was developed. Homogenates from 5 different brain areas were derivatized with o-phthalaldehyde in the presence of sodium sulphite. Subsequent separation was achieved using linear gradient elution over 30 min. The derivatives were stable for up to 20 h at 4 °C. The method was accurate, reproducible, and showed good linearity. The recoveries were >88% for aspartate, glutamine, glutamate, glycine and γ-aminobutyric acid, with the limit of quantification varying from 5 to 30 pmol. The method was successfully applied for the measurement of amino acids under fed and fasted conditions.     

Specific antagonism of excitant amino acids in the isolated spinal cord of the neonatal rat.

The specificity of the neurodepressant actions of D-alpha-aminoadipate, alpha,epsilon-diaminopimelic acid, HA-966 (HAP) and Mg2+ has been investigated. On the isolated spinal cord of the neonatal rat, ventral root depolarizations produced by kainate, substance P, carbachol and noradrenaline were relatively unaffected by the same concentrations (0.25--1 mM) of the agents as those which reduced synaptic activity and ventral root depolarizations produced by N-methyl-D-aspartate (especially), L-aspartate and L-glutamate. The same or higher concentrations of the agents did not affect excitatory transmission in the isolated rat superior cervical ganglion. It is proposed that the agents specifically block synaptic transmission mediated by an excitatory amino acid.     

The antagonizing effect of aspartic acid on the brain levels of monoamines and free amino acids during the development of tolerance to and physical dependence on morphine

Since it has been shown in a previous study that aspartic acid prevents the development of physical dependence on and tolerance to morphine and antagonizes the abstinence syndrom signs, the biochemical bases of that prevention were investigated in the present study. The brain contents of serotonin, DA, NA, and free amino acids of the rats given aspartic acid and morphine separately and in combination were determined. It has been observed that most of the morphine-induced changes in the brain were normalized in the group given aspartic acid and morphine together. The relative ineffectiveness of aspartic acid in normalizing some amino acid levels decreased by morphine was discussed and some logical explanations were found.     

Biochemical correlates in mouse-killing behavior of the rat: prolonged isolation and brain cholinergic function

After 30 days of isolation, 45% of the rats exhibited mouse-killing behavior. The killing response was suppressed by atropine (5 mg/kg and 8 mg/kg, IP) and scopolamine (8 mg/kg, IP), whereas methylatropine was ineffective. Acetylcholine (ACh) content and acetylcholinesterase (AChE) activity were measured in 5 discrete areas of rat brain. As compared with the aggregated rats only the killer rats exhibited higher ACh levels in the diencephalon. The activity of AChE in all brain areas was unchanged by isolation; no significant difference was found between the killer and nonkiller rats. These results suggest that central cholinergic mechanisms participate in the mediation of mouse-killing behavior in the rat.     

Methanol extract of Nigella sativa seed induces changes in the levels of neurotransmitter amino acids in male rat brain regions

Context: Nigella sativa L. (Ranunculaceae) (NS) has been used for medicinal and culinary purposes. Different parts of the plant are used to treat many disorders.

Objective: This study investigates the effects of NS methanol extract on brain neurotransmitter amino acid levels.

Materials and methods: We measured the changes in aspartate, glutamate, glycine and γ-aminobutyric acid in five brain regions of male Wistar rats after methanol extract treatment. Animals were injected intraperitoneally with saline solution (controls) or NS methanol extract (equivalent of 2.5?g/kg body weight) and sacrificed 1?h later or after administering 1 daily dose for 8?days. The neurotransmitters were measured in the hypothalamus, cortex, striatum, hippocampus and thalamus by HPLC.

Results and discussion: Results showed significant changes in amino acids compared to basal values. Glutamate increased significantly (16–36%) in the regions analyzed except the striatum. Aspartate in the hypothalamus (50 and 76%) and glycine in hippocampus (32 and 25%), thalamus (66 and 29%) and striatum (75 and 48%) also increased with the two treatment intervals. γ-Aminobutyric acid significantly increased in the hippocampus (38 and 32%) and thalamus (22 and 40%) but decreased in the cortex and hypothalamus although in striatum only after eight days of treatment (24%).

Conclusion: Our results suggest that injected methanol extract modifies amino acid levels in the rat brain regions. These results could be of interest since some neurodegenerative diseases are related to amino acid level imbalances in the central nervous system, suggesting the prospect for therapeutic use of NS against these disorders.     

The effect of 3-mercaptopropionate,an inhibitor of glutamate decarboxylase,on the levels of GABA and other amino acids,and on presynaptic inhibition in the rat cuneate nucleus

Rats were anaesthetized with urethane and treated with the reversible competitive glutamate decarboxylase (GAD) inhibitor 3-mercaptopropionate (3-MP: 150mg/kg) in order to lower the endogenous levels of GABA. Within 15min GAD activity and GABA levels had decreased by about 50 and 30% respectively in the region of the caudal medulla containing the dorsal column nuclei (den). The decreased GABA levels were subsequently maintained for at least 45 min. In unanaesthetized animals, running fits and tonic clonic convulsions occur within 15 min coincident with the rapid fall in GABA levels. Urethane appeared to protect against such convulsant activity without itself altering GABA levels or the decrease in levels produced by 3-MP. Taurine and glycine levels were unaffected by 3-MP, glutamine levels rose progressively, glutamate levels rose transiently, aspartate and serine levels fell progressively and alanine levels fell sharply within 5 min, subsequently rising to the pre-treatment level. The level of presynaptic inhibition in the dcn was also decreased by 3-MP, as assessed from the size of the surface recorded P-wave produced by supramaximal, electrical stimulation of the ipsilateral forepaw. The observed similarity in time course between the alteration of GABA levels and the decrease in presynaptic inhibition supports previous studies suggesting that GABA is the transmitter mediating synaptic inhibition in the den.     

Environmental modulation of the response to amphetamine: dissociation between changes in behavior and changes in dopamine and glutamate overflow in the rat striatal complex

Rationale: We have previously shown that environmental novelty enhances the behavioral activating effects of amphetamine and amphetamine-induced expression of the immediate early gene c-fos in the striatal complex, particularly in the most caudal portion of the caudate. In contrast, we found no effect of novelty on the ability of amphetamine to induce dopamine (DA) overflow in the rostral caudate or in the core of the nucleus accumbens. Objectives: The twofold aim of the present study was to determine the effect of environmental novelty on (1) amphetamine-induced DA overflow in the shell of the nucleus accumbens and in the caudal portions of the caudate, and (2) glutamate and aspartate overflow in the caudal portions of the caudate. Methods: Two groups of rats with a unilateral 6-hydroxydopamine lesion of the mesostriatal dopaminergic system received amphetamine (0.5 mg/kg, i.v.) in physically identical cages. For one group, the cages were also the home environment, whereas, for the other group, they were a completely novel environment. In vivo microdialysis was used to estimate DA, glutamate, and aspartate concentrations. Results: Environmental novelty enhanced amphetamine-induced rotational behavior (experiments 1–3) but did not alter amphetamine-induced DA overflow in either the shell of the nucleus accumbens (experiment 1) or the caudate (experiment 2). In addition, the ability of environmental novelty to enhance amphetamine-induced behavioral activation was not associated with changes in glutamate or aspartate efflux in the caudate (experiment 3). Conclusions: The present data indicate that the psychomotor activating effects of amphetamine can be modulated by environmental context independent of its primary neuropharmacological actions in the striatal complex. Received: 27 July 1999 / Accepted: 30 November 1999     

Attenuation of <Emphasis Type="Italic">d</Emphasis>-amphetamine self-administration by baclofen in the rat: behavioral and neurochemical correlates

Rationale Recent reports have demonstrated that gamma-aminobutyric acid (GABA)-ergic compounds attenuate the reinforcing effects of cocaine in rats. Baclofen, a GABA_B receptor agonist, appears to be particularly effective in this respect, suggesting that GABA_B receptor activation is critically involved in mediating anti-cocaine effects. Amphetamine, like cocaine, is a psychomotor stimulant with high abuse potential in humans.Objectives The purpose of the present investigation was to determine whether baclofen may attenuate the reinforcing effects of d-amphetamine (dAMPH) in rats. Dose–response curves were generated to examine the effect of three doses of baclofen (1.8, 3.2 or 5.6 mg/kg, IP) on dAMPH intravenous self-administration (IVSA). Separate groups were trained to self-administer two doses of dAMPH (0.1 mg/kg or 0.2 mg/kg per injection) under either a fixed-ratio (FR) or progressive ratio (PR) schedule of reinforcement. Microdialysis was performed in an additional group of rats to examine the effect of baclofen on dAMPH-induced increases in dopamine (DA) efflux in the nucleus accumbens (NAc).Results Pretreatment with baclofen produced dose-dependent reductions in responding for dAMPH under both the FR and PR schedules, and attenuated dAMPH-induced increases in DA levels in the NAc.Conclusion These results add to previous findings showing that baclofen attenuates the reinforcing effects of psychostimulant drugs, and suggest that further investigation into the effects of GABA_B receptor agonists on drug self-administration is warranted.     

Intracerebroventricular injection of propionic acid, an enteric bacterial metabolic end-product, impairs social behavior in the rat: implications for an animal model of autism

Environmental, dietary, and gastrointestinal factors may contribute to autism spectrum disorders (ASD). Propionic acid (PPA) is a short chain fatty acid, a metabolic end-product of enteric bacteria in the gut, and a common food preservative. Recent evidence indicates that PPA can cause behavioral abnormalities and a neuroinflammatory response in rats. Social behavior was examined in similarly-treated pairs of adult male Long-Evans rats placed in an open field following intracerebroventricular (ICV) injection of PPA (4 microl of 0.26 M solution) or control compounds. Behavior was analyzed using both the EthoVision behavior tracking system and by blind scoring of videotapes of social behaviors. Compared to controls, rats treated with PPA displayed social behavior impairments as indicated by significantly greater mean distance apart, reduced time spent in close proximity, reduced playful interaction, and altered responses to playful initiations. Treatment with another short chain fatty acid, sodium acetate, produced similar impairments, but treatment with the alcohol analog of PPA, 1-propanol, did not produce impairments. Immunohistochemical analysis of brain tissue taken from rats treated with PPA revealed reactive astrogliosis, indicating a neuroinflammatory response. These findings suggest that PPA can change both brain and behavior in the laboratory rat in a manner that is consistent with symptoms of human ASD.     

Histamine-induced excitation of spontaneously active medullary neurones in the rat brain is mediated by H2-receptors : A microiontophoretic study using H1 and H2-agonists and antagonists

The effects of histamine, applied by microiontophoresis onto spontaneously-active medullary neurones were investigated in the rat. Histamine caused current-dependent excitation of these neurones, an action that is at variance with previous studies in the cat. The nature of the receptor mediating these effects was examined using a number of agonists with differing potencies at peripheral H₁- and H₂-receptors. The precursor of histamine, l-histidine and the metabolite, N-telemethylhistamine did not mimic the effects of histamine while the H₂-agonist, 4-methylhistamine caused similar but weaker excitation. The extent of excitations produced by the H₁-agonists, 2-pyridylethylamine, 2-methylhistamine and 2-thiazolylethylamine could be related to their activity at H₂-receptors. Metiamide was ineffective in antagonising responses to histamine and related agonists as was mepyramine. The H₂-antagonist ranitidine, however, proved a good antagonist of responses to histamine and the H₁- and H₂-agonists, despite an unrelated excitatory action which may be linked to inhibition of cholinesterase. It is concluded that the excitatory effects of microiontophoretically-applied histamine and the agonists on medullary neurones in the rat is probably a result of activation of H₂-receptors.