Effect of Qingzao Jiufei Tang on Rate-limiting Enzymes HK2, PFK2 and PKM2 in Glycolytic Pathway in Lewis Lung Cancer-bearing Mice Cells / 中国实验方剂学杂志

Objective::To study the effect of Qingzao Jiufei Tang on the expression of key limiting enzymes hexokinase 2(HK2), phosphofructokinase 2(PFK2) and pyruvate kinase M2 (PKM2), and the glucose content in Lewis mice colon cancer cells. Method::A total of 50 male C57BL/6J mice were randomly divided into model group, chemotherapy group, and high, middle and low-dose Qingzao Jiufei Tang groups, with 10 mice in each group. The lung cancer cell model was established by injecting Lewis lung cancer cells into the right axilla. The high, middle and low dose groups were administered at the doses of 11, 5.5, 2.75 g·kg-1·d-1 for 2 weeks before modeling. The drug was administered through intraperitoneal injection at a dose of 50 mg·kg-1·(2 d)-1 in the chemotherapy group. The model group was intragastrically administered with an equal volume of normal saline. After the inoculation, the drug was administered for two weeks. Two weeks later, all of the mice were put to death, and tumor tissues were collected. The mRNA expression of HK2 was detected by Real-time PCR. the protein expression of PFK2 was detected by Western blot, the PKM2 activity was detected by enzyme-linked immunosorbent assay (ELISA). Result::Compared with the model group, mRNA expressions and activity of PKM2 in lung cancer cells of treatment groups were significantly declined, and glucose content increased significantly, with significant differences from those of model group (P<0.01). The PFK2 protein expressions in lung cancer cells of treatment groups (high, medium and low-dose groups) were significantly decreased (P<0.05, P<0.01). Conclusion::Qingzao Jiufei Tang could inhibit Lewis proliferation, and decrease the glucose intake in lung cancer cells. The effect targets may be the key rate-limiting enzymes HK2, PFK2, PKM2.

Simultaneous Determination of Glutamate, Glycine, and Alanine in Human Plasma Using Precolumn Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate and High-Performance Liquid Chromatography

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method has been validated for determining concentrations of glutamate, glycine, and alanine in human plasma. Proteins in plasma were precipitated with perchloric acid, followed by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Simultaneous analysis of glutamate, glycine, and alanine is achieved using reversed-phase HPLC conditions and ultraviolet detection. Excellent linearity was observed for these three amino acids over their concentration ranges with correlation coefficients (r)>0.999. The intra- and inter-day precision were below 10%. This method utilizes quality control samples and demonstrates excellent plasma recovery and accuracy. The developed method has been successfully applied to measure plasma glutamate, glycine, and alanine in twenty volunteers.