| 1.ObjectiveTo explore the absorption,distribution and metabolism of arsenicin vivo of patients with myelodysplastic syndromes(MDS)in treatment of Qinghuang Powder.This article is also aim to provide the basis for study the cellular pharmacokinetics and mechanism of Qinghuang Powder in vivo.2.Research Contents2.1 Analysis of arsenic speciation in vivo of of MDS patients in treatment of Qinghuang Powder combined with Chinese herbs for reinforcing shen and strengthening pi.Detection and analysis of four kinds of arsenic speciation(AsIII、AsV、MMA、DMA)in urine and plasma of 15 patients with MDS who were treated by Qinghuang powder with Chinese herbs for reinforcing shen and strengthening pi through the technology of Atomic fluorescence spectrometry combined with high performance liquid chromatography(AFS-HPLC),and compared the concentrations of arsenic metabolites with different therapeutic effects in those patients.Explore the absorb regularity and metabolism characteristics of Qinghuang powder in vivo.2.2 Analysis of absorption and distribution of arsenic in bone marrow and peripheral blood of MDS patients in treatment of Qinghuang Powder combined with Chinese herbs for reinforcing shen and strengthening piDetection and analysis of the differences of arsenic concentration of whole blood,plasma,red cells and nucleated cell respectively both in bone marrow and peripheral blood of 20 patients with MDS who were treated by Qinghuang powder with Chinese herbs for reinforcing shen and strengthening pi through the technology of microwave digestion combined with Atomic fluorescence spectrometry(HG-AFS),and explore distribution character and target organ of arsenic in blood system of those patients.2.3 Comparison of dissolution of arsenic between Qinghuang powder and other arsenic agents in artificial gastrointestinal fluidComparison of dissolution among As2S2,realgar and Qinghuang powder in artificial gastrointestinal fluid,and explore the absorption characteristics and influencing factors of Qinghuang powder in gastrointestinal fluid.Research the solubility difference of chemical arsenic(As2S2)and Chinese medicine compound which containing arsenic(Qinghuang powder).3.Research method3.1 Observation of clinical casesAccording to the research content,recruited the MDS patients who met the inclusion criteria,evaluation of the clinical efficacy and detection of the biological samples(blood,bone marrow,urine)of those patients,and compared the result by the method of statistics.3.2 Microwave Digestion MethodMicrowave digestion is a method to rapidly dissolve samples using strong acid/strong base in a closed space through microwave,high temperature and high pressure.In this study,addition of 0.5ml samples contained arsenic,1ml nitric acid and a magnetic rotor into the digestion instrument for dissolving.3.3 Atomic fluorescence spectrometry(AFS)Atomic fluorescence spectrometry(AFS)is a method of measuring the wavelength and intensity of atomic arsenic fluorescence lines by the interaction between hydrides with samples.3.4 High performance liquid chromatography(HPLC)High performance liquid chromatography(HPLC)used mobile phase as the carrier and adopted high-pressure infusion system to pump arsenic with different polarity into chromatographic column which with fixed phase,each component of arsenic species were separated and different curves were formed.In this process,the standard curve were configured according to the species of arsenic3.5 The test method by simulating gastrointestinal environmentDetection and comparison of the dissolution among different arsenics by simulatie gastrointestinal environment used artificial gastrointestinal fluid in 37℃ constant temperature box oscillations for 4 hours.4.Result4.1 The analysis of arsenic species in MDS patients with the treatment of Qinghuang powder combined with Chinese herbs for reinforcing shen and strengthening pi.4.1.1 Comparisons of four arsenic species in urineFour arsenic species(AsIII、DMA、MMA、As V)were detected in urine of 15 patients with MDS in treatment of Qinghuang powder.The concentrations of AsⅢ、DMA、MMA、As V were 288.88±181.9μg/L,996.41±615.33μg/L,325.16±209.74μg/L and 9.34±8.21 μg/L respectively.The concentrations of arsenic in urine from high to low were:DMA>MMA、AsⅢ>AsⅤ.The concentrations of DMA were significantly higher than that of MM A and AsIII(P=0.001,P=0.001),and there were no significant differences between MMA with AsIII(P=0.645).The concentrations of AsIII were significantly higher than that in As V(P=0.001).4.1.2 Comparisons of arsenic concentrations in urine with different doses of Qinghuang powder.The concentrations of AsIII、DMA、MMA、As V in urine of patients with high dose Qinghuang powder were 349.37±256.85μg/L、1482.11±345.13μg/L、540.04±160.5μg/L、9.07±4.27μg/L respectively,and the concentrations of those in patients with normal doses Qinghuang powder were 296.24± 158.4μg/L、937±605.66pg/L、291.33±184.11μg/L、10.23±9.41 μg/L respectively.All the concentrations of four arsenic species in high dose group were higher than those in normal dose group,but there were no significant differences(P=0.697、P=0.052、P=0.073、P=0.938)4.1.3 Comparisons of four arsenic species between plasma with urine9 out of 15 patients were detected the arsenic species both in plasma and urine.In the urine,the concentrations of AsⅢ、DMA、MMA、As V were 285.4±142.01μg/L、991.39±751.47μg/L、310.06±219.24pg/L、14.04±9.21 μg/L respectively.In the plasma,the concentrations of AsⅢ、DMA、MMA、As V were 2.8±2.12μg/L、7.29±4.68μg/L、2.67±2.24μg/L、0.38±0.42μg/L respectively.The concentrations of DMA were significantly higher than that of AsIII in plasma(P=0.026),and there were no significant difference between MMA with AsⅢ in plasma(P=0.971).The concentrations of AsⅢ in plasma were significantly higher than that of As V(P=0.001).The concentrations of arsenic in plasma from high to low were:DMA>AsIII(MMA)>AsⅤ.4.1.4 Comparisons of arsenic concentrations in urine with different clinical efficacy in patients with Qinghuang powderThe concentrations of AsIII、DMA、MMA、As Ⅴ in urine of patients with hematological improvement after treated with Qinghaung powder were 411.12±163.4μg/L、972.32±730.35μg/L、335.16±244.2μg/L、12.49±10.61μg/L respectively,and the concentrations of AsⅢ、DMA、MMA、As V in those patients without hematological improvement were 158.38± 107.52μg/L、894.88±535.66μg/L、238.74±167.15μg/L、7.52±4.39μg/L respectively.The concentrations of AsⅢ in patients without hematological improvement were significantly higher than the patients with hematological improvement(P=0.003).4.1.5 Comparisons of arsenic concentrations in plasma with different clinical efficacy in patients with Qinghuang powderThere were no significant differences about the concentrations of AsⅢ、DMA、MMA、AsV between the patients in hematological improvement group with the patients in no hematological improvement group(P=0.886、P=0.443、P=0.6、P=0.773)4.1.6 Comparisons of the value about DMA/AsIII of the patients both in plasma and urine with different clinical efficacy.In the urine,the average DMA/AsⅢ value of the patients with hematological improvement were higher than those of patients without hematological improvement(6.34±1.66 vs 2.72±3.77,P=0.004)In the plasma,there were no significant differences of the average DMA/AsIII value between the patient in hematological improvement group with the patient in no hematological improvement group(3.46±0.44 vs 2.54±1.97,P=0.286).4.2 Research of absorption and distribution of arsenic in bone marrow and peripheral blood of MDS patients with the treatment of Qinghuang powder4.2.1 Comparisons of total arsenic concentrations between bone marrow with peripheral blood of MDS patientsThe concentrations of total arsenic in bone marrow were no significant differences with those in peripheral blood of MDS patients after the treatment by Qinghuang powder for 6 months(47.23±21.67 vs 41.27 ±14.48μg/L,P=0.51).4.2.2 Comparisons of plasma arsenic concentrations between bone marrow with peripheral blood of MDS patients13 out of 20 patients were detected plasma arsenic concentrations both in bone marrow and peripheral blood,the arsenic concentrations of plasma both in bone marrow and peripheral blood were no significant differences(18.22±12.97 vs 17.35±11.31 μg/L,P=0.95).4.2.3 The ratio of plasma arsenic concentration to total arsenic concentration in peripheral bloodThe median concentrations of arsenic of plasma in peripheral blood were 14.19(10.2,19.1)μg/L,the median concentrations of total arsenic in peripheral blood were 39.52(34.18,48.4)μg/L.The median percentage of plasma arsenic to total arsenic in peripheral blood was 41.3%(21.68%,61.64%).4.2.4 The ratio of arsenic concentration in nucleated cells to mature red blood cells of bone marrowThe arsenic concentration in nucleated cells were significant higher than that in mature red blood cells of bone marrow(P=0.001).The median ratio of arsenic concentration in nucleated cells to mature red blood cells of bone marrow were 113.64(52.86,169).4.3 Comparison of dissolution of arsenic between Qinghuang powder and other arsenic agents in artificial gastrointestinal fluid4.3.1 Comparison of dissolution among three agents contained arsenic in the artificial gastric fluidThe dissolved concentrations of arsenic among As2S2,realgar and Qinghuang powder in the artificial gastric fluid were 3.93±1.54μg/L、29.28± 1.77pg/L、51.3±6.23μg/L respectively.The concentrations of arsenic from high to low were:Qinghuang powder>realgar>As2S2(P=0.027).4.3.2 The influence of dissolution with different times of Qinghuang powder in the artificial gastric fluidThe dissolved arsenic concentrations of Qinghuang powder in the artificial gastric fluid on the first,second,third and fourth hour were 31.24±8.64μg/L、42.78±0.47μg/L、44.95±5.59μg/L、51.3±6.23μg/L respectively.The concentrations were increased gradually over times,but there were no significant differences of concentrations with different hours4.3.3 Comparison of dissolution among three agents contained arsenic in the artificial intestinal fluidThe dissolved concentrations of arsenic among AS2S2,realgar and Qinghuang powder in the artificial intestinal fluid were 1.72±0.38μg/L、21.64±3.22μpg/L、38.6±3.61μg/L respectively.The concentrations of arsenic from high to low were:Qinghuang powder>realgar>AS2S2(P=0.031).4.3.4 The influence of dissolution with intestinal bacteria of Qinghuang powder in the artificial intestinal fluidThe dissolved arsenic concentrations of Qinghuang in artificial intestinal fluid powder were significant higher than that in artificial intestinal fluid combined with triple viable bacteria(38.6±3.61μg/L vs24.93±2.52μg/L,P=0.049)4.3.5 Comparison of dissolution among three agents contained arsenic in the artificial gastric and intestinal fluidThe dissolved concentrations of realgar in the artificial gastric fluid were significant higher than that in artificial intestinal fluid(P=0.033).but there were no significant differences of dissolved concentrations of As2S2,Qinghuang powder between the artificial gastric fluid with artificial intestinal fluid(P=0.094、P=0.067).5.Conclusion5.1 After the treatment of Qinghuang powder in the patients with MDS,the arsenic metabolites(AsⅢ、AsV、MMA、DMA)could be detected both in plasma and urine.The content of DMA was the highest and the content of AsV was the lowest,the content of MMA and AsIIIwere similar.Compared with the patients in the hematological improvement group,the concentrations of AsIII were higher in urine,the concentrations of DMA and DMA/AsⅢ were lower in patients without hematological improvement.5.2 The concentrations of total arsenic and plasma arsenic between bone marrow with peripheral blood were similar,but plasma weren’t the main distribution sites of arsenic in vivo.Compared with RBC,the arsenic had higher affinity for nucleated cells.5.3 A small part of arsenic among As2S2,realgar and Qinghuang powder could be dissolved in gastrointestinal tract,and the dissolved concentrations were different among three arsenic agent.The solubility of indigo with realgar was higher than that with realgar alone,and the solubility of realgar was higher than that with As2S2.Compatibility of traditional Chinese medicine and intestinal bacteria are important factors for affecting the dissolution of arsenic in artificial gastrointestinal fluid. |
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