Rapid bioluminescence assay for monitoring rat CES1 activity and its alteration by traditional Chinese medicines

In traditional Chinese medicine herbs(TCM),including Radix Salviae Miltiorrhizae(Danshen),Radix Puerariae Lobatae(Gegen),Radix Angelicae Sinensis(Danggui),and Rhizoma Chuanxiong(Chuanxiong)are widely used for the prevention and treatment of cardiovascular diseases and also often co-administered with Western drugs as a part of integrative medicine practice.Carboxylesterase 1(CES1)plays a pivotal role in the metabolisms of pro-drugs,Since(S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-dihydrothiazole-4-carboxylate(NLMe)has recently been identified by us as a selective CES1 bioluminescent sensor,we developed a rapid method using this substrate for the direct measurement of CES1 activity in rats.This bioluminescence assay was applied to determine CES1 activity in rat tissues after a two-week oral administration of each of the four herbs noted above.The results demonstrated the presence of CES1 enzyme in rat blood and all tested tissues with much higher enzyme activity in the blood,liver,kidney and heart than that in the small intestine,spleen,lung,pancreas,brain and stomach.In addition,the four herbs showed tissue-specific effects on rat CES1 expression.Based on the CES1 biodistribution and its changes after treatment in rats,the possibility that Danshen,Gegen and Danggui might alter CES1 activities in human blood and kidney should be considered.In summary,a selective and sensitive bioluminescence assay was developed to rapidly evaluate CES1 activity and the effects of orally administered TCMs in rats.

Application value of ATP based bioluminescence tumor chemosensitivity assay in the chemotherapy for hydrothorax caused by non-small cell lung cancer

Objective： The aim of the study was to investigate the clinical value and application of ATP based bioluminescence tumor chemosensitivity assay （ATP-TCA） in the chemotherapy for hydrothorax caused by non-small cell lung cancer （NSCLC）. Methods： Hydrothorax specimens from 120 NSCLC patients were analyzed by ATP-TCA and the most sensitive chemotherapeutic drugs were used in NSCLC patients （treatment group）. At the same time, 56 NSCLC patients with hydrethorax were admitted in our Hospital （Department of Oncology, The No. 2 People＇s Hospital of Yibin, China） and given chemotherapy without guidance of the ATP-TCA （control group）. Before the third chemotherapeutic cycle, clinical outcomes were analyzed in the two groups. Results： Effective rate of hydrothorax in treatment group was 67%, while 46% in control group （P 〈 0.05）. In refractory hydrothorax patients, they were 69% and 40% （P 〈 0.05）, respectively.In vitro results correlated well with clinical outcomes （P 〈 0.01）. Conclusion： Effective rate of chemotherapy for hydrothorax in NSCLC is higher in treatment group than that in control group. ATP-TCA is especially helpful for refractory hydrothorax.

Bioluminescent Assay of Microbial ATP in Postmortem Tissues for the Estimation of Postmortem Interval

To study the relationship between changes of microbial ATP in four kinds of murine tis-sues and the postmortem interval （PMI）, healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals. The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentra-tion of microbial ATP in muscle increased with PMI time. The peak appeared at the 7th day after death, and at the 10th day, microbial ATP in muscle tissue increased again. In internal organs, the peaks of microbial ATP were observed at the 8th day after death and the level decreased during 8–10 d. The differences in microbial ATP concentration in liver, spleen and kidney were not statistically significant. During day 0 to day 9 after death, the correlation was best between PMI and microbial ATP in muscle. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.02X3–0.166X2–0.666X＋13.412 （R2=0.989, P〈0.01）. In internal organs, the best correlation was found between PMI and microbial ATP during day 0 to day 10. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.016X3–0.127X2–0.809X＋13.324 （R2=0.986, P〈0.01）. There existed high correlations between PMI and microbial ATP concentration in rat tissues. Since only a small amount of tissue was needed for the detection and the sample was not affected by self-decomposition, the method may extend the time range of PMI estimation.

Changes in ATP Levels in Rabbit Blood and Its Application for Estimation of the Postmortem Interval

Summary： Relationship between ATP changes of rabbit blood and postmortem interval （PMI） was studied. Twenty-four healthy rabbits were sacrificed and randomly divided into 3 groups with 8 rab- bits of each group. The bodies of three groups were placed in calorstat at temperature of 15℃, 25℃ and 35℃, respectively. The blood from the right ventricle was sampled through indwelling needle each 4 h until 72 h after death. ATP levels in the blood samples were measured by using ATP fluo- rescence rapid detection technique at different PMIs. Blood ATP levels slightly increased in the early stage after death and then constantly declined at all temperatures （15℃, 25℃, and 35℃）. Cubic polynomial regression equations with log[ATP] as dependent variable （y） and PMI as independent variable （x） at different temperatures and the optimal time period were established as followed： Under 15℃ and during 16-64 h after death, y=-3.027×10^-5x^3＋0.003x^2-0.096x-10.625 （Ra^2=0.992, P〈0.001）; under 25℃ and during 8-56 h after death, y=-2.921×10^-5x^3＋0.002x^2- 0.059x-11.186 （Ra^2=0.989, P〈0.001）; under 35℃ and during 4-36 h after death, y=-9.769×10^-5x^3＋ 0.005x^2 -0.117x-11.166 （Ra^2=0.991, P〈0.001）. The changes in ATP levels in blood collected from right ven- tricle of rabbit cadavers showed relatively stable and regular degradation within 72 h after death at different temperatures.