| ObjectiveNeurotransmitters play important roles in a variety of functions of the brain as transmitters of nerve messages in mammalian nervous system. They are also involved in the maintaining of brain blood circulation. Neurotransmitters are associated with many diseases such as cerebrovascular disease and craniocerebral trauma. It is meaningful to quantify these neurotransmitters in cerebrospinal fluid for neurophysiology, pathology and clinical diagnosis. This paper is based on some of the existing research, improved the methods,set up a new method which use NBD-F as a derivative to to make the amino acids more detectable and apply high performance liquid chromatography with fluorescence detector to detect the samples. And with this approach to detect the level of amino acid neurotransmitters in cerebrospinal fluid of patients suffering from encephalitis, subarachnoid hemorrhage, cerebroma and acute lymphoblastic leukemia, and determining the monoamine neurotransmitters with method in the literature. These studies provide reference to the diagnosis and differential diagnosis of some disease. Methods1The measurement method of amino acids in cerebrospinal fluid1.1Sample handling:The sample was centrifuged at12,000rpm for10min at4℃.100μL of sample supernatant,175μL of borate buffer solution,200μL of acetonitrile and25μL of NBD-F working solution were mixed in a1.5mL centrifuge tube. The well-mixed solution was allowed to react at60℃in the water bath for7min, excluding light. After cooling to room temperature,10μL of the solution was injected into the equilibrated HPLC system.1.2Chromatographic conditions:Analysis was carried out on a Shimadzu HPLC system equipped with a fluorescence detector. The analyte was separated on an analytical column SHIMADZU shim-pack VP-ODS (250mm length×4.6mm,5μm particle diameter). The mobile phase was composed of a mixture of acetonitrile-phosphate buffer (0.02mol/L, pH6.0;16:84, v/v) filtered through a0.45μm nylon filter and degassed under ultrasound and vacuum for30min. The mobile phase was delivered at a flow rate of1.0mL/min and the column temperature was set to30℃. Determination under the conditions of excitation wavelength (Ex)475nm and emission wavelength(Em) of536nm.2The measurement method of monoamine neurotransmitters in cerebrospinal fluid2.1Sample handling:The sample was centrifuged at12,000rpm for10min at4℃,10μL of sample supernatant was injected into the equilibrated HPLC system.2.2Chromatographic conditionsAnalysis was carried out on a Shimadzu HPLC system equipped with a fluorescence detector. The analyte was separated on an analytical column SHIMADZU shim-pack VP-ODS (250mm length x4.6mm,5μm particle diameter). The mobile phase was composed of a mixture of methanol-sodium acetate solution (0.1mol/L, pH2.5-3;2:98, v/v) filtered through a0.45μm nylon filter and degassed under ultrasound and vacuum for30min. The mobile phase was delivered at a flow rate of1.2mL/min and the column temperature was set to30℃. Determination under the conditions of excitation wavelength (Ex)280nm and emission wavelength(Em) of314nm.Results1.The results of amino acid neurotransmittersdetermination in cerebrospinal fluid1.1Determination of amino acid neurotransmitters in cerebrospinal fluidIn the condition of this experiment, five kinds of amino acid neurotransmitters were separated perfectly in15min. A good linear response was obtained for the five of amino acid neurotransmitters over the range of0.1～100μmol/L with correlation coefficients not less than0.999.Asp:Y=25918X+3517(r=0.9998), the detection limits was2.34×10-3mol/L, the recoveries were98.3%,102.2%and96.6%, the inter-day RSD were2.7%,3.6%and4.2%, the intra-day RSD were4.8%,4.6%and3.9%;Glu:Y=17397X+1952(r=0.9999), the detection limits was1.78×10-3mol/L, the recoveries were98.2%,94.7%and102.7%, the inter-day RSD were3.8%,2.9%and4.4%, the intra-day RSD were5.6%,4.2%and3.7%;Gly:Y=28913X+2074(r=0.9997), the detection limits was8.33x10-3mol/L, the recoveries were99.4%,105.2%and102.7%, the inter-day RSD were4.2%,3.4%and4.6%, the intra-day RSD were5.2%,4.8%and5.1%;Tau:Y=36726X+6736(r=0.9995), the detection limits was5.75x10-3mol/L, the recoveries were96.3%,98.6%and104.5%, the inter-day RSD were3.4%,2.9%and4.1%, the intra-day RSD were4.7%、5.6%and5.2%;GABA:Y=19475X+3638(r=0.9998), the detection limits was 7.26x10-3mol/L, the recoveries were106.1%,97.3%and99.6%, the inter-day RSD were3.1%,3.6%and4.7%, the intra-day RSD were4.2%,6.0%and4.3%.1.2The relationship between amino acid neurotransmitters in cerebrospinal fluid and diseaseThe significant difference in the contents of Asp Glu, Gly, Tau were found between patients with encephalitis and control (p<0.05).The contents of Asp, Glu, Gly, Tau, GAB A in subarachnoid hemorrhage patients group were significantly higher than the control group (P<0.05).The contents of Asp, Gly in cerebroma patients group were significantly lower than the control group (P<0.05).The contents of Glu, Gly in acute lymphocytic leukemia patients group were significantly higher than the control group (P<0.05).2. The results of monoamine neurotransmitter determination in cerebrospinal fluid2.1Determination of monoamine neurotransmitters in cerebrospinal fluidIn the condition of this experiment, four kinds of monoamine neurotransmitters were separated perfectly in20min. A good linear response was obtained for the four kinds of monoamine neurotransmitters over the range of0.02～10μmol/L with correlation coefficients not less than0.999.NE:Y=77573X+5638(r=0.9997), the detection limits was3.67×10-4mol/L, the recoveries were97.8%,98.2%and103.6%, the inter-day RSD were3.2%,2.6%and4.3%, the intra-day RSD were4.5%,5.2%and3.7%;E:Y=173671X+7663(r=0.9999), the detection limits was2.16×10-4mol/L, the recoveries were104.2%,99.8%and98.6%, the inter-day RSD were4.1%,3.9%and3.4%, the intra-day RSD were4.5%,5.2%and3.7%;DA:Y=55632X+2241(r=0.9997), the detection limits was7.87x10-4mol/L mol/L, the recoveries were103.4%,97.2%and102.5%, the inter-day RSD were4.5%,3.1%and4.2%, the intra-day RSD were5.0%,4.6%and4.8%;5-HT:Y=98126X+5633(r=0.9996), the detection limits was 4.29x10-4mol/L mol/L, the recoveries were99.1%,105.6%and97.4%, the inter-day RSD were3.8%,4.9%and4.7%, the intra-day RSD were5.6%,4.5%,5.3%.2.2The relationship between monoamine neurotransmitters in cerebrospinal fluid and diseaseThe significant difference in the contents of5-HT were found between patients with encephalitis and control (p<0.05).The contents of NE in subarachnoid hemorrhage patients group were significantly higher than the control group (P<0.05), the contents of5-HT were significantly lower than the control group (P<0.05).The contents of NE in cerebroma patients group were significantly lower than the control group (P<0.05), the contents of5-HT were significantly higher than the control group (P<0.05).Conclusions1. An optimized HPLC-FLD method for determination of Asp, Glu, Gly, Tau and GAB A in cerebrospinal fluid was developed. The attractive advantages of this method are inexpensive simple, rapid and accurate. All of these characteristics indicate that it is suitable to analyse amino acids in biological samples in clinical and biochemical research..2. The determination of neurotransmitters is meaningful to the diagnosis and antidiastole of viral encephalitis and suppurative encephalitis.3. The determination of neurotransmitters can provide some basis for the early diagnosis and treatment of subarachnoid hemorrhage.4. Studying the changing level of neurotransmitters in CSF is helpful to the treatment of cerebroma.5. The determination of amino acids neurotransmitters can provide some basis for the treatment of acute lymphocytic leukemia. |
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