Up-Regulation of Carnitine Transporters Helps Maintain Tissue Carnitine Levels in Carnitine Deficiency Induced by Pivalic Acid

Purpose. Pivalic acid (PVA) forms conjugates with endogenous carnitine and enhances its excretion. The purpose of this study is to determine whether tissue carnitine levels decrease in parallel with plasma levels in carnitine deficiency induced by PVA. Methods. PVA was orally administered to rats for 5 days. Carnitine levels in plasma, liver, kidney, muscle, and heart were monitored. The tissue uptake clearance (CL_uptake) was determined in vivoby the integration plot method. Hepatocytes were prepared from control and PVA-treated rats, and the uptake of L-carnitine was determined. Results. Plasma concentrations of L-carnitine decreased as a result of the enhanced carnitine elimination as pivaloylcarnitine (PCN) when rats were treated with PVA. However, L-carnitine concentrations in liver, muscle, and heart remained relatively constant during the study period. CL_uptake increased in liver and muscle and, thus, the rate of carnitine uptake from plasma into these tissues did not change even at low plasma concentrations. This helps maintain carnitine levels in these tissues. Up-regulation of carnitine transporters is suggested to be a mechanism for the increased CL_uptake. Conclusions. In the carnitine deficiency state induced by PVA, increased CL_uptake owing to up-regulation of carnitine transporters is suggested to help maintain carnitine levels in some tissues.     

反相高效液相色谱法测定人血清中丙戊酸钠浓度

目的 :测定人血清中丙戊酸钠浓度。方法 :以反相高效液相色谱法 ,甲醇 -水(70:30)为流动相 ,检测波长为248nm ;采用衍生化法 ,环己烷羧酸为内标。结果 :丙戊酸钠浓度在14 47～248 0μg/ml范围内呈现良好线性关系。结论 :该方法简便、准确 ,适合于临床血药浓度监测。     

快速柱前衍生-高效液相色谱法测定丙戊酸血药浓度

目的建立一种简便可行的快速柱前衍生-高效液相色谱法(HPLC)测定丙戊酸血药浓度。方法患者血清经正己烷提取后在C18柱上分析,流动相为甲醇-水(75∶25),柱温30℃,检测波长254 nm,流速1 mL.m in-1。结果丙戊酸在12.5~400μg.mL-1范围内线性关系良好,日内和日间RSD均小于10%(n=5)。结论本法简便、稳定,用于丙戊酸的血药浓度监测,效果良好。     

丙戊酸治疗躁狂症及其血药浓度的临床对照研究

目的探讨丙戊酸治疗躁狂症的临床疗效、血药浓度及药物不良反应。方法60例躁狂症患者分别接受丙戊酸钠缓释剂及丙戊酸镁治疗6周,并于服药后1,2,4,6周末使用高效液相色谱法检测丙戊酸血药浓度,以Bech—Rafaelsen躁狂量表（BRMS）和不良反应量表（TESS）观察临床疗效和药物不良反应。结果观察组有效率为80．00％,对照组有效率为76．67％,两组疗效无明显差异,有效病例的血药浓度平均为（77．58±14．45）μg·mL^-1,治疗窗范围为50～100μg·mL^-1之间。结论丙戊酸能有效治疗躁狂症,血药浓度监测有助于临床安全有效用药。     

影响丙戊酸钠血药浓度波动的因素及应对措施

目的 :探讨影响丙戊酸钠血药浓度波动的因素 ,提高丙戊酸钠血药浓度检测的有效性及可利用性。方法 :归纳总结影响丙戊酸钠血药浓度波动的因素 ,提出应对措施。结果 :服药时间、采血时间、监测时机、剂型及制剂质量、患者依从性、合并用药、患者生理和病理指标等均为影响丙戊酸钠血药浓度波动的因素。结论 :在癫痫患者长期服药治疗过程中 ,医师、患者、药师应互相沟通 ,建立监测档案 ,密切注意影响丙戊酸钠血药浓度波动的因素 ,以确保患者用药的安全、有效     

癫痫患者丙戊酸血药浓度监测及个体化给药

目的：为临床癫痫患者丙戊酸的合理用药提供参考。方法：采用荧光偏振免疫法对１２３ 例癫痫患者进行血药浓度监测,并对其结果及疗效进行分析总结。结果：达到丙戊酸有效血药浓度范围未有效控制的有１４ 例,占１１－４ ％ ;不在有效血药浓度范围而控制良好的有１１ 例,占８－９ ％ 。结论：丙戊酸的个体化给药必须综合考虑各方面的因素,不能仅仅以血药浓度为依据,并建议在适当情况下有必要监测游离丙戊酸血药浓度。     

Transport of Amino Acid Esters and the Amino-Acid-Based Prodrug Valganciclovir by the Amino Acid Transporter ATB0,+

PURPOSE: The purpose of this study was to analyze the transport of amino acid esters and the amino-acid-based prodrug valganciclovir by the Na(+)/Cl(-)-coupled amino acid transporter ATB(0,+). METHODS: The interaction of amino acid esters and valganciclovir with the cloned rat ATB(0,+) was evaluated in a mammalian cell expression system and in the Xenopus oocyte expression system. RESULTS: In mammalian cells, expression of ATB(0,+) induced glycine uptake. This uptake was inhibited by valine and its methyl, butyl, and benzyl esters. The benzyl esters of other neutral amino acids were also effective inhibitors. Valganciclovir, the valyl ester of ganciclovir, was also found to inhibit ATB(0,+)-mediated glycine uptake competitively. Exposure of ATB(0,+)-expressing oocytes to glycine induced inward currents. Exposure to different valyl esters (methyl, butyl, and benzyl), benzyl esters of various neutral amino acids, and valganciclovir also induced inward currents in these oocytes. The current induced by valganciclovir was saturable with a K0.5 value of 3.1+/-0.7 mM and was obligatorily dependent on Na+ and Cl-. The Na+:Cl-:valganciclovir stoichiometry was 2 or 3:1:1. CONCLUSIONS: Amino acid esters and the amino-acid-based prodrug valganciclovir are transported by ATB(0,+). This shows that ATB(0,+) can serve as an effective delivery system for amino acid-based prodrugs.     

EMIT和FPIA测定丙戊酸、甲氨喋呤和环孢霉素A血药浓度的比较研究

目的比较均相酶放大免疫分析法(EMIT)和荧光偏振免疫分析法(FPIA)测定丙戊酸(VPA)、甲氨喋呤(MTX)和环孢霉素A(CsA)血药浓度结果,并评价两种测定方法所得结果的相关性。方法收集患者服药后的血样,采用EMIT和FPIA同时测定VPA、MTX和CsA血药浓度;以FPIA测定值为X,以EMIT测定值为Y,进行线性回归,评价其相关性。结果所得线性回归方程如下:YVPA=1.894 1+1.190 3X,r=0.983;YMTX=0.099 24+1.136X,r=0.992;YCsA=1.146 5+0.846 1X,r=0.971。EMIT测定血浆中VPA血药浓度较FPIA高11.2μg.mL 1,EMIT测定MTX血药浓度较FPIA高0.22μmol.L 1,EMIT测定CsA血药浓度较FPIA低20.6 ng.mL 1,差异有统计学意义(P<0.05)。结论 EMIT与FPIA测定VPA、MTX和CsA血药浓度差异具有统计学意义,在治疗药物监测中应予以注意。     

Surface binding and uptake of cadmium (Cd2+) by LLC-PK1 cells on permeable membrane supports

Recent studies have shown that Cd²⁺ has relatively specific damaging effects on cell-cell junctions in the renal epithelial cell line, LLC-PK₁. The objective of the present studies was to examine the surface binding and uptake of Cd²⁺ by LLC-PK₁ cells in relation to the disruption of cell-cell junctions. LLC-PK₁ cells on Falcon Cell Culture Inserts were exposed to CdCl₂ containing trace amounts of¹⁰⁹Cd²⁺ from either the apical or the basolateral compartments, and the accumulation of¹⁰⁹Cd²⁺ was monitored for up to 8 h. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that the cells accumulated 3–4 times more Cd²⁺ from the basolateral compartment than from the apical compartment. The accumulation of Cd²⁺ from the basolateral compartment occurred in two phases: a rapid, exponential phase that occurred in 1–2 h and coincided with a decrease in transepithelial resistance, and a slower, linear phase that continued for 6–8 h. The Cd²⁺ that accumulated during the rapid phase was easily removed by washing the cells in EGTA, indicating that most of it was bound to sites on the cell surface. By contrast, most of the Cd²⁺ that accumulated during the slower phase could not be removed by EGTA, indicating that it had been taken up by the cells. Additional studies showed that the rapid phase of Cd²⁺ accumulation was enhanced when Ca²⁺ was present at low concentrations (0.1 mM), and was greatly reduced when Ca²⁺ was present at high concentrations (10 mM). These results suggest that Cd²⁺ damages the junctions between LLC-PK₁ cells by interacting with Ca²⁺-sensitive sites on the basolateral cell surface.     

Protection of renal tubular cells against the cytotoxicity of cadmium by glycine

Glycine treatment is reported to protect against the nephrotoxicity of cadmium (Cd) in rats. The purpose of the present study was to explore the mechanism of this protection using a renal epithelial cell line, LLC-PK(1). The cells were incubated with 10-30 microM Cd in serum-free DMEM and cytotoxicity was evaluated by LDH leakage into the incubation medium. Under these conditions, 20 and 30 microM Cd concentrations were cytotoxic. As compared to the non-Cd-exposed cells, the LDH release was elevated more than six-fold in cells exposed to 30 microM Cd for 24h. Co-treatment with 5-50mM glycine was cytoprotective in a concentration-dependent manner. Prior treatment with 50 mM glycine for 16 h, or co-treatment for 24h, reduced LDH leakage due to 30 microM Cd exposure by 60 and 66%, respectively. Co-incubation with 50 mM alanine was also protective but only about half as effective as with glycine. During the first 4h, prior to the onset of any significant cell membrane damage, the Cd-exposed cells accumulated 0.55 microg Cd/mg protein. Glycine pre-treatment or co-treatment reduced Cd accumulation by about one-quarter or one-half, respectively. To delineate the mechanism of glycine's effect on Cd accumulation, the efflux of Cd was studied after a 30 min pulse exposure. The results suggested that pre-treatment reduced Cd accumulation by increasing its efflux from the cells. In contrast, co-treatment reduced Cd efflux, suggesting that the co-treatment lowered Cd accumulation by suppressing its uptake. When co-incubated, Cd and glycine formed a complex that was apparently responsible for the marked reduction in Cd uptake. It is concluded that, regardless of the mode of treatment, glycine is cytoprotective against Cd and that it may do so by lowering the intracellular Cd burden.     

Differential effects of fumonisin B1 on cell death in cultured cells: the significance of the elevated sphinganine

Fumonisins are specific inhibitors of ceramide synthase in sphingolipid metabolism. An alteration in sphingolipid metabolism as a result of fumonisin exposure is related to cell death (Yoo et al., 1992). The objective of this study was to investigate whether elevated free sphinganine levels are related to the sensitivity of cultured cells to fumonisin exposure. Fumonisin B1 elevated the intracellular free sphinganine concentraions in both LLC-PK1 and Chinese hamster ovary (CHO) cells. However, CHO cells are resistant to fumonisin cytotoxicity at 50 microM, while LLC-PK1 cells are sensitive at concentrations greater than 35 microM. The intracellular concentration of free sphinganine in LLC-PK, cells treated at 50 microM fumonisin B1 for 72 h was approximately 1450 pmol/mg protein relative to the 37 pmol observed in the control culture. Under the same conditions, the population of apoptotic cells in the 50 M fumonisin B1-treated culture was approximately 37% of the total compared to 12% in the control. The caspase III-like activity after 72 h in the 50 microM fumonisin B1-exposed culture increased to approximately 50 pmol/mg protein/hr compared to 6 pmol/mg protein/hr in the control. L-cycloserine, a serine palmitoyltransferase inhibitor, reduced the fumonisin B1-stimulated caspase III-like activity down to the control level. Under the same culture conditions, the intracellular concentration of free sphinganine after L-cycloserine plus fumonisin B1 treatment was 140 pmol/mg protein compared to 1450 pmol/mg protein in fumonisin B1 alone. The intracellular concentration of free sphinganine in CHO cells treated with 50 microM fumonisin B1 for 72 h was approximately 460 pmol/mg protein, indicating that the mass amount of elevated free sphinganine in the CHO cells was about 32% of that in LLC-PK1 cells. Adding exogenous sphinganine to the CHO cells along with 50 microM fumonisin B1 treatment for 72 h caused both necrosis and apoptosis. In conclusion, the elevated endogenous sphinganine acts as a contributing factor to the fumonisin-induced cell death.     

地西他滨联合丙戊酸钠促胃癌MGC-803细胞nm23-H1基因表达及机制研究

目的 探讨地西他滨(DCA)和丙戊酸钠(VPA)联用对胃癌细胞株MGC-803的作用及对non-metastasis 23-H1基因(nm23-H1)的表达调控的影响。方法 DCA 1.5 及3.0 μmol·L^-1,VPA 1.5 mmol·L^-1,DCA 1.5 μmol·L^-1+VPA 1.5 mmol·L^-1,DCA 3.0 μmol·L^-1+VPA 1.5 mmo·L^-1作用MGC-803细胞72 h。Annexin V/PI法检测细胞凋亡,实时荧光定量PCR检测nm23-H1 mRNA表达,焦磷酸测序法检测nm23-H1启动子上随机选取的两个CpG岛位点甲基化状态。结果 VPA 1.5 +DCA 1.5联合用药组[早期：(33.58±3.88)%,晚期：(31.52±4.20)%]和VPA 1.5 +DCA 3.0联合用药组[早期：(42.61±4.23)%,晚期：(38.01±3.86)%]凋亡率均高于其相应单药组,差异具有统计学意义(P<0.01)。nm23-H1 mRNA在VPA 1.5 +DCA 1.5联合用药组(1.84±0.46)和VPA 1.5 +DCA 3.0联合用药组(2.88±0.42)的表达水平均高于其相应单药组,差异具有统计学意义(P<0.01)。VPA 1.5 +DCA 1.5联合用药组[位点1：(53.50±3.39)%,位点2：(51.17±2.71)%]和VPA 1.5 +DCA 3.0联合用药组[位点1：(41.17±2.14)%,位点2：(39.83±2.56)%]nm23-H1启动子两位点甲基化阳性率均低于其相应单药组,差异具有统计学意义(P<0.01)。VPA 1.5 mmol·L^-1、VPA 1.5+DCA 1.5、VPA 1.5+DCA 3.0这3组的HDAC酶活性均低于正常对照、DCA 1.5 μmol·L^-1、DCA 3.0 μmol·L^-1任一组,差异具有统计学意义(P<0.01)。结论 DCA联合VPA能显著上调nm23-H1基因的表达,其机制与启动子上的甲基化水平降低和去乙酰化酶活性降低有关。     

Effects of Arbekacin and Vancomycin on Release of Lactate Dehydrogenase and Fragmentation of DNA in LLC-PK1 Kidney Epithelial Cells

Pharmaceutical Research -     

丙戊酸钠血药浓度与抗癫作用的相关性

目的：了解丙戊酸钠血药浓度与疗效的关系.方法：采用荧光偏振免疫仪测定丙戊酸钠血药浓度.结果：有效血药浓度为50～100 μg&#8226;mL 1,在此浓度范围抗癫作用较好,低于此范围抗癫作用较差,高于此范围可增加不良反应.结论：丙戊酸钠血药浓度与疗效有较好的相关性.     

HPLC-ESI-MS/MS同时测定癫痫患儿血浆中丙戊酸、苯巴比妥和托吡酯的药物浓度

目的建立基于高效液相色谱串联质谱技术(HPLC-ESI-MS/MS)同时测定癫痫患儿血浆中丙戊酸、苯巴比妥和托吡酯浓度的方法,用于癫痫患儿的血药浓度监测及结果分析。方法样品用有机溶剂沉淀蛋白法进行前处理,分别以丙戊酸-D6、托吡酯-D12、苯巴比妥-D5为内标,采用Phenomenex Kinetex^?EVO C₁₈色谱柱(2.1 mm×50 mm,2.6μm),以0.07%甲酸-1 mmol乙酸铵-水(A)和甲醇(B)作为流动相进行梯度洗脱,流速为0.5 mL·min^-1。质谱检测系统采用电喷雾离子源负离子模式,多离子反应监测模式扫描。考察该方法的专属性、定量下限(LLOQ)、标准曲线、残留效应、稀释效应、准确度、精密度、基质效应和稳定性。结果丙戊酸在0.6～120μg·mL^-1线性关系良好(r=0.998 9),LLOQ为0.6μg·mL^-1;苯巴比妥在0.25～50μg·mL^-1线性关系良好(r=0.999 1),LLOQ为0.25μg·mL     

中枢神经系统疾病状态下脑中L-型氨基酸转运体1功能与表达的相关研究进展

L-型氨基酸转运体1在脑中尤其在脑微血管内皮细胞中高表达,它能将大型中性氨基酸转运到脑中以维持中枢神经系统的正常功能。一些中枢神经系统疾病与脑内氨基酸紊乱有关,且伴随脑中L-型氨基酸转运体1功能与表达的改变。L-型氨基酸转运体1功能和表达的改变可能会影响内源性底物在脑中的稳态,进而促进或延缓疾病的发展过程;同时也可能影响底物药物的疗效或毒性。对中枢神经系统疾病状态下脑中L-型氨基酸转运体1功能与表达的相关研究进展进行总结,旨在为中枢神经系统疾病进一步研究及相关药物研发提供思路与参考。     

The Enantiomers of the Valproic Acid Analogue 2-n-Propyl-4-pentynoic Acid (4-yn-VPA): Asymmetric Synthesis and Highly Stereoselective Teratogenicity in Mice

The teratogenic activities of R( +)- and S( – )-2-n-propyl-4-pentynoic acid (R and S-4-yn-VPA), the enantiomers of the highly teratogenic valproic acid (VPA) analogues (±)-4-yn-VPA, were investigated in mice. The enantiomers were prepared via asymmetric synthesis, each in three steps employing the chiral auxiliaries (4R,5S)-4-methyl-5-phenyl-2-oxazolidinone and S-4-benzyl-2-oxazolidinone. The determination of the absolute configurations and the optical purities is described. R( + )-4-yn-VPA contained 7%, and S( – )-4-yn-VPA 8%, of the respective antipodes. The aqueous solutions of the sodium salts of R- and S-4-yn-VPA were administered as single i.p. injections during early organogenesis in the mouse (day 8 of gestation) using the induction of exencephaly as the teratological end point. Dose/exencephaly curves indicated that S-4-yn-VPA is 7.5 times more teratogenic than its antipode, 1.9 times more teratogenic than (±)-4-yn-VPA and 3.9 times more teratogenic than the parent drug VPA. In contrast, the neurotoxicity (maternal toxicity) of the 4-yn-VPA enantiomers was found to be independent of the stereo-chemical configuration and lower than achieved after VPA administration. Due to its low neurotoxicity and highly Stereoselective neural tube-inducing activity, S-4-yn-VPA should prove an important tool for the investigation of molecular mechanism of the teratogenic action in this class of compounds; R-4-yn-VPA could act as the negative control in these studies.     

HPLC法测定丙戊酸血清浓度的衍生化条件考察

目的：考察丙戊酸的衍生化条件,并建立测定丙戊酸血清浓度的方法。方法：血样采用α-溴苯乙酮衍生化,用高效液相色谱（HPLC）法进行测定。以丙戊酸衍生物产率为指标,采用单因素法考察反应温度、时间、三乙胺用量、衍生化试剂用量对丙戊酸衍生化的影响。结果：丙戊酸完全衍生化的条件为衍生化试剂用量400-600μg、三乙胺用量10～20μL、在45-60℃条件下反应20-60min。丙戊酸血药浓度在5．0-200．7μg·mL-1范围内线性关系良好（r=0．9999）,方法回收率为99．80％～100．43％,日内与日间RSD≤4．45％。结论：衍生化试剂用量、三乙胺用量、反应温度和时间等对丙戊酸衍生化产率有较大的影响,在丙戊酸血清浓度测定中应加以控制。     

Decrease in Aflatoxin M1 Concentration in Milk during Cholesterol Removal by Application of β-Cyclodextrin

Approximately one-third of humankind is chronically exposed to the carcinogenic aflatoxin M₁ contained in milk. As β-cyclodextrin is frequently used in the food industry, its effect on aflatoxin M₁ concentration was investigated during cholesterol removal from milk due to the similarity among the physicochemical properties of aflatoxin M₁ and cholesterol. Moreover, the elimination of cholesterol using β-cyclodextrin has been successfully applied in many studies without any substantial effect on the quality of the treated milk. Therefore, milk samples were spiked with aflatoxin M₁ within the range from 0.20 to 2.00 µg/kg, and cholesterol removal was carried out by 2.0% (w/w) β-cyclodextrin addition, as this concentration is enough for the sufficient removal of cholesterol. It was found that the mean cholesterol concentration decreased by 92.3%, while the aflatoxin M₁ concentration decreased to 0.53 ± 0.04 µg/kg, i.e., by 39.1% after treatment (n = 2). This mitigation procedure itself is easy and inexpensive and thus is fully applicable with a high potential for complete decontamination of aflatoxin M₁ milk. This method will therefore considerably improve the food safety issues associated with aflatoxin M₁ presence in milk and dairy products.