Analysis of Pesticide Raid^(█) in Feed of Wistar Rat by High-Pressure Liquid Chromatography (HPLC)

The distribution of pesticide by-product in tissues of wistar rats were analyzed using high pressure liquid chromatography. The limit of detection of the HPLC was 0.1 μg. Results show bioaccumulation factor of pesticide “Raid?” in lipid, up to three times that of the feed at the first concentration and gradually decreased as the concentration increased in the muscle > (0.7), brain > (0.5) and liver > (0.3) as indicated in the text. At higher concentration of 961 μg/g, bioaccumulation factor decreased in the lipid to 1.2 and 0.6 in the muscle, 0.03 in the brain and 0.08 in the liver respectively. High Pressure Liquid Chromatography (HPLC) analysis of raid extract suggests the presence of micprothrin and palethrin. The implications are numerous, but simply put that accidental ingestion of chlorinated hydrocarbon as in “Raid?” may involve convulsions, collapse and coma after only brief excitation and ataxia at the onset.

Plasma amino acid profiling of cancer patients with abnormal Savda based on high-performance liquid chromatography

OBJECTIVE: To investigate metabolic signatures in plasma of cancer patients with abnormal Savda using plasma-free amino acid profiles, and to evaluate the diagnostic potential of these profiles for the detection and explanation of the mechanisms of different symptoms in traditional Uyghur medicine.METHODS: Plasma samples from cancer patients with abnormal Savda(n=85) or non-abnormal Savda(n=105) and a healthy control group(n=65)were analyzed using high-performance liquid chromatography(HPLC). Orthogonal projection to latent structures with discriminant analysis was used for the classification and prediction of abnormal Savda, and spectral profiles were subjected to Student's t-tests to assess statistical significance.RESULTS: Compared with the healthy group, the levels of aspartic acid, glutamate, glycine, histidine,arginine, threonine, alanine, proline, methionine,isoleucine, leucine and phenylalanine decreased significantly in plasma of cancer patients with abnormal Savda(all P<0.05). Serine, cystine, tyrosine,valine and lysine levels showed no significant differences(all P>0.05). Compared with non-abnormal Savda syndrome patients, abnormal Savda syndrome patients showed high concentrations of glutamate, serine, valine, isoleucine, leucine and phenylalanine(all P<0.05). The remaining plasma amino acids showed no significant differences(all P>0.05).CONCLUSION: Plasma-free amino acid profiling has the potential to assist in understanding and determining abnormal Savda. A HPLC-based metabonomic platform could be a powerful tool for the classification of symptoms in traditional medicine.

Clinical and genetic characteristics of Chinese hereditary nonpolyposis colorectal cancer families

AIM： To analyze the clinical characteristics of Chinese hereditary nonpolyposis colorectal cancer （HNPCC） families and to screen the germline mutations of human mismatch repair genes hMLH1 and hMSH2 in the probands. METHODS： Thirty-one independent Chinese HNPCC families were collected in Zhejiang Province. All of them met Chinese HNPCC criteria. Clinical data about patient gender, site of colorectal cancer, age of onset, history of multiple colorectal cancer, associated extracolonic cancer were recorded. PCR and denaturing high performance liquid chromatography （DHPLC） were employed to screen the mutations. Sequencing analysis was used to find out the exact mutation site and characteristics of the samples showing abnormal DHPLC profiles. RESULTS： One hundred and thirty-six malignant neoplasms were found in 107 patients including 14 multiple cancers. One hundred and six of the 136 neoplasms （77.9%） were diagnosed as colorectal cancer, with an average age of onset at 48.57 ±29.00 years. Gastric cancer was the most common extracolonic cancer （10.3%） in these families. Twenty-three different sequence variations in hMLHI and hMSH2 genes were detected in these 17 families. Fifteen sequence variations were located in the exons, including 5 SNPs, 3 silent mutations, 3 missense mutations, 2 nonsense mutations and 2 frameshift mutations. The latter seven mutations seemed to be pathogenic. CONCLUSION： Germline mutations of hMLH1 and hMSH2 genes are identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria. Chinese HNPCC families have some particular clinical characteristics, such as a left-sided predominance, less synchronous or metachronous colorectal cancer, and frequent occurrence of gastric cancer.

Analysis of Hupehenine in the Total Alkaloids from Fritillaria hupehensis by HPLC-ELSD

A reverse-phase high pressure liquid chromatography （HPLC） method with evaporative light scattering detection （ELSD） has been developed for the quantitative analysis of hupehenine in the total alkaloids from Fritillaria hupehensis. Samples were analyzed on a reverse-phase column （Hypersil C-18） with a mobile phase of methanol：water：chloroform： triethylamine （85：15：1：0.6）. The ELSD was set at the drift tube temperature of 68.3℃ and gas flow rate of 1.8 L/min. Hupehenine＇s retention time was 13.7 min with an asymmetry factor of 1.2. The validity of the method has been verified with linearity, limit of detection, accuracy and precision. The logarithmic linear curve was obtained from 8.936 to 134.04 μg/mL （r=0.9993）. The detection limit （S/N〉3） of hupehenine was 1.79 μg/mL on the column. Intra-day RSD was 1.42% and inter-day RSD was 2.26% （3 days within a week）. The average recovery of hupehenine was 101.50%, and RSD was 1.62%.

Study on Purifying Technology of Andrographolide by Supercritical CO_(2) Secondary Gradient Crystallization

The effects of the secondary gradient crystallizing pressure, temperature and time on the purity and crystallization ratio of andrographolide were investigated via single factor experiments. The shape of crystal was observed with scanning electron microscopy (SEM). The purity of andrographolide was determined by high pressure liquid chromatography (HPLC). It was found that the optimized parameters were pressure 14 MPa, temperature 55℃, time 60 min, and CO2 flow rate 15 L min-1, under these conditions the particle of andrographolide was much smaller, the crystal of andrographolide was distributed in floccule on crystal board,with the purity of andrographolide 92.5%, the crystallization ratio 48.9%.

Genetic Mutation of Vitamin K-dependent Gamma-glutamyl Carboxylase Domain in Patients with Calcium Oxalate Urolithiasis

To investigate the exon mutation of vitamin K-dependent gamma-glutamyl carboxylase （GGCX or VKDC） in patients with calcium oxalate urolithasis, renal cortex and peripheral blood samples were obtained from severe hydronephrosis patients （with or without calculi）, and renal tumor patients undergoing nephrectomy. GGCX mutations in all 15 exons were examined in 44 patients with calcium oxalate urolithiasis （COU） by polymerase chain reaction （PCR） and denatured high pressure liquid chromatography （DHPLC）, and confirmed by sequencing. Mutation was not found in all COU samples compared to the controls. These data demonstrated that functional GGCX mutations in all 15 exons do not occur in most COU patients. It was suggested that there may be no significant association between the low activity and mutation of GGCX in COU.

Determination of berberine in Phellodendron amurense from different sites of Changbai Mountain

Phellodendron amurense has been used for many years as a medical plant in traditional Chinese medicine and has shown great prospect in recent clinical trials for future applications. Berberine is an essential active compound contained in P. amurense. Our objective in this study was to quantify the content of berberine in P.amurense from sites at different elevations on Changbai Mountain. We collected samples of P. amurense from five different elevations on Changbai Mountain. Berberine in samples was extracted by ultrahigh pressure extraction（UPE）. And the quantity was measured by high performance liquid chromatography（HPLC）. First, the optimal HPLC conditions for berberine were identified with satisfactory precision（relative standard deviation, R SD／5.6 %）, good accuracy（relative error, R E／ 3.6 %） and good linear relation（R2= 0.9998） in the range of 6.576–328.8 mg L-1. Second, the combination of UPE and HPLC methods in quantitative analysis of berberine showed high repeatability（R SD= 3.28 %）, reproducibility（R SD= 4.72 %）,stability（R SD／ 1.27 %） and good recovery（99.54 %） for real plant materials. Samples from Heilongjiang Province at the lowest elevation contained the highest amount of berberine.Similarly, the lowest amount of berberine was recorded in samples from Changbai Forest Bureau of Jilin Province collected at the highest elevation in this paper. The proposed UPE–HPLC method is simple, reliable and low-cost for quantitative analysis of berberine. Content of berberine in P.amurense varied significantly by site on Changbai Mountain.

Effects of Different Compounding of Formulae on Content of Gardenoside in Yin Chen Hao Decoction

In order to observe the effects of the ground and intact Zhi Zi (Fructus Gardeniae) and different combinations of the ingredients and refined single Chinese drug granules in Yin Chen Hao Decoction compound prescription on the contents of gardenoside (an effective component of the prescription), the contents of gardenoside were determined with reversed phase high performance liquid chromatography (HPLC), with acetonitrile-water (15:85) as mobile phase, at wave length 238nm. The results indicated that the gardenoside-decocted-out rates in the decoctions prepared by different combinations of the ingredients with the ground Zhi Zi (Fructus Gardeniae) all were higher significantly than those in the decoction with intact Zhi Zi (Fructus Gardeniae), and generally, different combinations of the ingredients in the decoction had only little effect on the gardenoside-decocted-out rate.

Relationship between cardiotonic activity of Fuzi(Radix Aconiti Lateralis Preparata)and its fingerprint determined by liquid chromatography-mass spectrometry

OBJECTIVE:To investigate the relationship between the cardiotonic activity of Fuzi(Radix Aconiti Lateralis Preparata,RALP)and its fingerprint determined by liquid chromatography-mass spectrometry(LC-MS).METHODS:First,the fingerprints of six processed products of RALP were established by high performance liquid chromatography quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS)followed by analysis of the principal component of the relative peak area of its common peaks.Next,the scores of the first five principal components were used as input for an artificial neural network(ANN).Additionally,the therapeutic effect of RALP was assessed by measuring the hemodynamic indexes of heart failure model rats.Subsequently,fluorescence semi-quantitative polymerase chain reaction and an enzyme-linked immunosorbent assay kit were used to determine the effects of RALP-processed products on the serum levels of noradrenaline(NA),angiotensin-Ⅰ(Ang-Ⅰ),and the expression ofβ-norepinephrine receptor m RNA(β-NRm)in the rat cardiac tissues.P<0.05 was used as the output of the ANN.Finally,a network was constructed to display the relationship between the LC-MS fingerprints and the cardiotonic activity of the RALP-processed products.RESULTS:Several types of RALPs can improve diastolic function,systolic function and heart rate.On the basis of the findings from the principal component analysis(PCA)of 16 common peaks of fingerprints of six RALP-processed products,it was revealed that the first five principal components may include 100%of the information of the original data.As observed from the multilayer perceptron neural network analysis,principal component 4 presented with the strongest effects on serum levels of NA and Ang-Ⅰin rats,while principal component1 exerted the greatest effect onβ-NRm expression in cardiac tissue.CONCLUSION:The key findings obtained from this study indicated that the network constructed by the PCA-ANN may predict pharmacodynamic effects of the main ingredients of Traditional Chinese Medicine(TCM).This method may serve as a new approach to identify the relationship between LC-MS fingerprints and the pharmacodynamic effects of TCM ingredients.

Relationship between a novel polymorphism of lipoprotein lipase gene and coronary heart disease

OBJECTIVE: To investigate polymorphisms in the gene for lipoprotein lipase (LPL) in Chinese populations with coronary heart disease (CHD) and to inquire into the relationship between these polymorphisms in LPL gene and CHD. METHODS: Genomic DNA was extracted from patients with CHD and normal control subjects using a salting out method. The entire coding region and flanking sequences of all coding exons of the LPL gene were amplified by PCR technique and PCR products were detected by denaturing high-performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method. RESULTS: A novel polymorphic site, G830A, that is within the fifth exon of the LPL gene was found. The 192 codon CGA was changed into CAA and resulted in the substitution of glutamine for arginine. Between the control and CHD groups, chi-square test showed no significant difference in the frequencies of the A/A genotype and A allele (P > 0.05). However, the frequencies of A/A genotype and A allele (0.653 and 0.786) in CHD patients with high plasma triglyceride/lowed plasma high density lipoprotein cholesterol were higher than those (0.415 and 0.642) in CHD patients without hyperlipidemia (P Gln substitution polymorphism and CHD, but there is a significant positive correlation between the A/A genotype of the LPL gene and CHD associated with high triglyceride/lowed high density lipoprotein cholesterol. This study may provide new data for exploring the molecular mechanism of CHD.

Dynamic Study of the Effect of Electroacupuncture on Dopamine in the Striatum for Rats with Cerebral Ischemia and Reperfusion

Object： To observe the dynamic changes of dopamine （DA） in the striatum with the process of duplicating cerebral ischemia and reperfusion （CIR） model on rats. And observe the effect of electroacupuncture （EA） on such changes. Methods： Using microdialysis sampling, high performance liquid chromatography （HPLC） detecting technology, dynamic sampling from experimental animals at multiple moments to observe the changes of dopamine in striatum with ischemia. And observe the effect of EA at Fengchi （GB 20） on such changes. Result： About the extracellular level of DA in normal group, sham-operation group and sham-operation ＋ EA group, no significant changes were observed during the measuring period. The extracellular level of DA peaked at 15-45 min after MCAO and 0-30 min （P〈0.05） after reperfusion. A rising trend was also observed following 120 min after reperfusion. After EA, the extracellular level of DA was significant lower than ischemia group following 90 min after reperfusion （P 〈0.05）. And there was no peak again. Conclusion： EA at Fenchi （GB 20） can regulate the disorder of DA. EA can improve the integrity neurological function. This may be one of the mechanisms of acupuncture treating the lesion of cerebral ischemia and reperfusion.

Efficacy of phospholipid complex of flavonoids from persimmon leaves on atherosclerosis, and possible mechanism

OBJECTIVE:To investigate the efficacy of phospholipid complex of flavonoids from persimmon leaves(PLF-PC)on atherosclerosis,and to study its mechanism behind the action.METHODS:To clarify the constituents of the flavonoids from persimmon leaves(PLF),an ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry method was established.To enhance the anti-atherosclerotic effect of PLF,a newly emerging approach based on the combination of phospholipid complexation technique was employed.PLF-PC was prepared by the solvent-evaporation method then characterized using Fourier transform infrared spectroscopy,Powder X-Ray Diffractometry and Scanning electron microscopy.A model of oxidized lowdensity lipoprotein-induced injury on human umbilical vein endothelial cells was established to investigate the anti-atherosclerotic effect of PLF-PC versus PLF.The levels of nitric oxide,endothelial nitric oxide synthase,intracellular adhesion molecule-1,reactive oxygen species,superoxide dismutase,tumor necrosis factor-αand nuclear factor-κB were observed via assay kits.RESULTS:A total of 31 compounds were identified in PLF.PLF-PC showed better anti-atherosclerotic power compared with PLF,moreover,enzyme linked immuneosorbent assay analysis showed that the PLF-PC may effect on endothelial dysfunction and atherosclerosis via antioxidant-related mechanisms.CONCLUSIONS:Our findings elucidated that PLF-PC significantly enhanced the PLF’s efficacy on atherosclerosis.

Effects of Electroacupuncture on the Rats with Neuropathic Pain and EAAs in Spinal Cord

Objective： To observe the impact of electroacupuncture on the pain threshold and the content of excitatory amino acids in the spinal cord of the rats with neuropathic pain to discuss the mechanism of electroacupuncture in treating neuropathic pain. Methods： The rats were randomly divided into a control group （n=10）, a sham surgery group （n=10） and a surgery group （n=30） to take spared nerve injury （SNI） surgery of sciatic nerve. The successful SNI model rats in the surgery group were randomly divided into a model group （n=10）, an electroacupuncture group （n=10） and a sham electroacupuncture group （n=10）. The mechanical pain threshold （MPT） and heat pain threshold （HPT） were measured on the preoperative day, postoperative day 7, postoperative day 9 and the day after the sixth acupuncture treatment. The electroacupuncture was carried out at Huantiao （GB 30） and Weizhong （BL 40） starting from the postoperative day 10 to reflect the impact on the mechanical pain threshold and heat pain threshold. The pre-column deriverization and HPLC fluorescence detection as well as microdialysis method were adopted to test the content of EAAs in spinal cord of rats. Results： The SNI surgery decreased the mechanical pain threshold of rats significantly. The glutamate （Glu） and aspartic acid （Asp） in the microdialysis solution of rats in the model group were higher than in the control group and sham surgery group at the same time point （P〈0.05）. The contents of Glu and Asp in the microdialysis solution of rats in the electroacupuncture group and sham electroacupuncture group decreased significantly （P〈0.05）, comparing with model group at the same time point （except the Glu of the sham electroacupuncture group at the second time point）. The electroacupuncture reduced the mechanical allodynia markedly. Conclusion： The mechanism of electroacupuncture treating neuropathic pain may relate with the decrease of excitatory amino acids in spinal cord.

Effect of oral administration of vitamin C on human aqueous humor ascorbate concentration

AIM:To study oral administration of vitamin C on human aqueous humour ascorbate concentration.METHODS:High performance liquid chromatography(HPLC)coupled with electrochemical detector(BCD)was used.The effect of oral administration of various doses of ascorbic acid,0(control),1.0,1.5,2.0,3.0,and 5.0 g,on its concentration in aqueous humour,obtained from volunteer cataract patients was studied.RESULTS:The concentration of ascorbic acid in aqueous humour of control group without administration of vitamin-C tablet or drug containing ascorbic acid was(254±119)mg·L^(-1).This study revealed that the administration of 2.0 g of.ascorbic acid saturate the aqueous humour and further increase in the dose(3.0 g and 5.0 g)did not increase its concentration in aqueous humour,although its concentration was increased in plasma.CONCLUSION:Oral administration of 2.0 g of Vc is sufficient to saturate the aqueous humour where it may be helpful in controlling the intra-ocular pressure.

Prognostic significance of clinicopathologic parameters in gastrointestinal stromal tumors:a study of 156 cases

The biological behavior of gastrointestinal stromal tumors(GISTs)are highly variable.To invest-igate the biological behavior of GIST,we collected 83 cases of gastric and 62 cases of small intestinal GIST from the Department of Pathology of the Chinese PLA General Hospital.The parameters include age,primary tumor location,tumor diameter,mitotic index,tumor necrosis,risk assessment,clinical stage and the c-kit exon 11 muta-tion.All these were analyzed in 105 cases along with the follow-up data and tested by log rank and COX hazard proportional model.We find that the average age of gast-ric GIST was 55.4 years.Of the 62 cases that were fol-lowed up,17 cases had metastasis or recurrence and the 5-year survival rate was(66.51±17.06)%.For the small intestinal GIST,the average age was 50.6 years and 43 cases were followed up.Of these,22 cases had meta-stasis or recurrence and the 5-year survival rate was(61.76±18.30)%.Small intestinal GIST was more fre-quently associated with metastasis and tumor relapse than gastric GIST(x^(2)=56.131,P=0.013).For gastric GIST,patients younger than 50 years(P=0.046),the advanced clinical stage(P=0.0001),the large tumor diameter(P=0.0001),a high mitotic index(P=0.0001),necrosis(P=0.0001)and a high risk grade(P=0.004)were all correlated with a lower survival rate.The COX hazard proportional model revealed that advanced clinical stage(P=0.001),large tumor size(P=0.001),a high mitotic index(P=0.002)and the high risk grade(P=0.018)indi-cated a poorer prognosis in gastric GIST.For small intest-inal GIST,necrosis(P=0.036)and advanced clinical stage(P=0.010)were associated with lower survival rates and the clinical stage was shown to be an independent prognostic indicator.A total of 25 cases harbored muta-tions in c-kit exon 11.The frequency of c-kit mutation was 32%and 22.5%for gastric and small intestinal GIST,respectively.In gastric GIST,the mutated c-kit was pre-dominant in patients older than 50 years of age.But in the small intestinal GIST,the mutated c-kit was predominant in the age group of 40-49 years.In conclusion,for gastric GIST,clinical stage,tumor size,mitotic index,and risk grade are the prognostic indicators.For small intestinal GIST,necrosis and clinical stage are the prognostic indi-cators.Small intestinal GIST are more aggressive than gastric GIST.The occurrence of c-kit mutation may cor-relate with the age of patients.