Detection of catecholamine metabolites in urine based on ultra-high-performance liquid chromatography-tandem mass spectrometry.

The excretion of neurotransmitter metabolites in normal individuals is of great significance for health monitoring. A rapid quantitative method was developed with ultra-performance liquid chromatography-tandem mass spectrometry. The method was further applied to determine catecholamine metabolites vanilymandelic acid (VMA), methoxy hydroxyphenyl glycol (MHPG), dihydroxy-phenyl acetic acid (DOPAC), and homovanillic acid (HVA) in the urine. The urine was collected from six healthy volunteers (20-22 years old) for 10 consecutive days. It was precolumn derivatized with dansyl chloride. Subsequently, the sample was analyzed using triple quadrupole mass spectrometry with an electrospray ion in positive and multireaction monitoring modes. The method was sensitive and repeatable with the recoveries 92.7-104.30%, limits of detection (LODs) 0.01-0.05 µg/mL, and coefficients no less than 0.9938. The excretion content of four target compounds in random urine samples was 0.20 ± 0.086 µg/mL (MHPG), 1.27 ± 1.24 µg/mL (VMA), 3.29 ± 1.36 µg/mL (HVA), and 1.13 ± 1.07 µg/mL (DOPAC). In the urine, the content of VMA, the metabolite of norepinephrine and adrenaline, was more than MHPG, and the content of HVA, the metabolite of dopamine, was more than DOPAC. This paper detected the levels of catecholamine metabolites and summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.

Saikosaponin a increases interleukin-10 expression and inhibits scar formation after sciatic nerve injury.

Nerve scarring after peripheral nerve injury can severely hamper nerve regeneration and functional recovery. Further, the anti-inflammatory cytokine, interleukin-10, can inhibit nerve scar formation. Saikosaponin a (SSa) is a monomer molecule extracted from the Chinese medicine, Bupleurum. SSa can exert anti-inflammatory effects in spinal cord injury and traumatic brain injury. However, it has not been shown whether SSa can play a role in peripheral nerve injury. In this study, rats were randomly assigned to three groups. In the sham group, the left sciatic nerve was directly sutured after exposure. In the sciatic nerve injury (SNI) + SSa and SNI groups, the left sciatic nerve was sutured and continuously injected daily with SSa (10 mg/kg) or an equivalent volume of saline for 7 days. Enzyme linked immunosorbent assay results demonstrated that at 7 days after injury, interleukin-10 level was considerably higher in the SNI + SSa group than in the SNI group. Masson staining and western blot assay demonstrated that at 8 weeks after injury, type I and III collagen content was lower and nerve scar formation was visibly less in the SNI + SSa group compared with the SNI group. Simultaneously, sciatic functional index and nerve conduction velocity were improved in the SNI + SSa group compared with the SNI group. These results confirm that SSa can increase the expression of the anti-inflammatory factor, interleukin-10, and reduce nerve scar formation to promote functional recovery of injured sciatic nerve.

[Preparation and characterization of monoclonal antibody against HCCR protein].

AIM: Preparation of monoclonal antibody (mAb) to HCCR, which is a candidate biomarker for human hepatocellular carcinoma (HCC). METHODS: The recombinant protein HCCR-1(167-360); was expressed and was used as immunogen to immunize mouse for generation of mAb against HCCR. The protein Ep-HCCR, which displayed a epitope of HCCR, was also expressed and purified to use to detect serum antibody titer and to screen the positive clones of hybridmas. The properties of HCCR antibody were analyzed by ELISA, Western blot, immunofluorescence and immunohistochemistry. RESULTS: A hybridmas clone, which secreted anti-HCCR mAb, was obtained. The affinity constant (Kaff) of the mAb is 5.4×10(6); L/mol analyzed by ELISA; Western blot showed that the mAb could specifically recognize HCCR-1 and HCCR-2 expressed in HepG2 cells; The mAb was also used to detect the expression of HCCR proteins in hepatoma cells and HCC tissues. The results of immunofluorescence indicated that HCCR proteins mainly localized on the plasma membrane and cytoplasm of HepG2 cells. In addition, HCCR was found high-expressed in HCC tissues but not in normal liver tissue detected by Immunohistochemistry. CONCLUSION: A specific mAb against HCCR was successfully generated, which laid the foundation for establishing HCC detection method based on HCCR.

[Generation of monoclonal antibody to GP73 based on antigen epitope].

AIM: Preparation of monoclonal antibody (mAb) against GP73 protein. METHODS: The N-terminal peptide (AAAERGAVELK) of GP73 protein was displayed on T7 phage, the recombinant phage was amplified and used as the immunogen to immunize mouse to produce antibody. The titer of the antiserum and the positive hybridoma clones which secreted the mAb against GP73 protein were detected by ELISA. The mAb specificity was assayed by ELISA and Western blot. RESULTS: The high specificity mAb against GP73 protein was selected from the mouse immunized with the recombinant T7 phages displaying the epitope of GP73 by cell fusion and screening. CONCLUSION: The appropriate protein epitope displayed on T7 phage could be used as alternative antigen to immunize animals to make specific antibody against the corresponding native protein.

[Production and identification of a monoclonal antibody against human HPPCn].

AIM: To make monoclonal antibody(mAb) against human HPPCn for the use in research on HPPCn's function and its relationship with liver diseases. METHODS: The female BALB/c mice were immunized with the recombinant HPPCn proteins. Splenocytes and Sp2/0 cells were fused with PEG-1500. The positive clone was identified through indirect ELISA and then subcloned by limited dilution. Indirect ELISA, Western blot and Ig sub-class identification kit were used to identify the mAb's properties. By immunofluorescence experiments, we studied the cellular localization of HPPCn. The mAb epitope was also analyzed using peptide phage display technology. RESULTS: An anti-HPPCn mAb, named W2-D5, was obtained. It belongs to IgG1 subclass. It could specially bind to human HPPCn. Furthermore, by immunofluorescence results, wo confirmed HPPCn located in the nucleus and our mAb could combined with the natural protein. With the mAb, the minimal detectable concentration was 0.1 µg/L for HPPCn; The peptide sequence of HPPCn7₋13;(IHLELRN)was identified as the epitope of the mAb. CONCLUSION: An anti-HPPCn mAb with high specificity and high affinity was successfully obtained.