MUG-Indole法快速检定大肠杆菌方法介绍

本文用MUG-Indole法(快速检定大肠杆菌新方法,以下简称M-I法)与药典法对20个供试品进行对比实验,实验结果两种方法基本一致,且M-I法在时间性、准确性、灵敏性、特异性等方面明显优于药典法。     

药品微生物限度检查中大肠杆菌检查特例

目的:对疑似大肠杆菌的菌株进行检测。方法:MUG Indole法和药典法。结果:MUG Indole法简便、快速、准确,优于药典法。结论:检查大肠菌群比检查大肠杆菌更能说明药品中污染控制菌的情况。     

用MUG-Indole法及部颁法对大肠杆菌比较鉴定的研究

对16种药品36批次进行大肠杆菌部颁法及MUG-Indole法检验比较,证明两法相符率高,且MUG-Indole法具有快速、简便、准确、无主观性的优点.     

用MUG-Indole法及部颁法对大肠杆菌比较鉴定的研究

对16种药品36批次进行大肠杆菌部颁法及MUG-Indole法检验比较,证明两法相符率高,且MUG-Indole法具有快速、简便、准确、无主观性的优点.     

药品微生物限度检查中大肠杆菌检查特例

本文对实验工作中发现的一例疑似大肠杆菌的菌株作了常规生化试验和MUG-Indole快速检验,结果发现:该菌株在标准规定的检验时间内观查结果则完全符合非典型大肠杆菌的生化反应,为检出大肠杆菌,但MUG-Indole试验却均为阴性.若将柠檬酸盐利用试验时间延至144小时,则生化反应变为－＋－＋.     

快速检定大肠杆菌法在医院制剂质控中的应用

目的:对我院自制的20种内服制剂做了大肠杆菌的快速检定.方法:用MUG-Indole(M-I)法对制剂中的大肠杆菌进行检定.结果:自然污染的20种制剂中16种样品M-I结果为阴性,4种样品M-I结果不一致;20种制剂的阳性对照试验结果均显阳性.结论:16种制剂对M-I法均无影响,4种制剂M-I结果不一致,需进一步做IMViC生化试验检定.     

盐酸马普替林有关物质检查商榷

目的:对《中国药典》2020年版二部盐酸马普替林有关物质的检查方法提出意见和建议.方法:根据《中国药典》2020年版方法(TLC法)对可能出现的检验结果进行分析,存在结论无法判定的情况.为此建立了HPLC法作为有关物质检查的方法.结果:HPLC法通过计算杂质峰面积,结论能明确判定.结论:HPLC法解决了结论无法判定的情...     

VitC片溶液颜色检查方法的探讨

目的 探讨影响VitC片溶液颜色检查的因素。方法 ①采用药典法，延长检测时间，观察检测结果；②用0．1％的碳酸钠溶液和0.05％的硼砂溶液作为VitC片溶液颜色检查的溶解介质，同药典法检测。结果 药典法检查Vitc片溶液颜色结果受检查时间影响较大，用0.1％的碳酸钠溶液或用0.05％的硼砂溶液作为溶解介质时，结果较为稳定。结论建议：①药典法检查vitC片溶液颜色时，应限定检查时间在20min内；②采用0.1％的碳酸钠溶液或用0. 05％的硼砂溶液作为溶解介质。     

药品在被少量大肠杆菌污染而造成漏检的有关问题的探讨

目的:探讨药品在被少量大肠杆菌污染后,按中国药典现行方法检验是否存在漏检可能,建立了在该种情况下可行的大肠杆菌检查的方法.方法:对8个品种人为污染少量大肠杆菌后,按中国药典检查.结果:药品在被少量大肠杆菌污染后,按药典法大肠杆菌阳性检出率为0～66%,改进法为100%.结论:药品在被少量大肠杆菌污染后,按中国药典现行方法检验存在漏检可能.     

口服药品中大肠菌群的检查

目的：比较口服药品中大肠杆菌与大肠菌群的检出率。方法：按中国药典2000年版微生物限度检查法和最大可能数法(MPN)进行检查。结果：40批样品中均未检出大肠杆菌，其中有6批检出大肠菌群，检出率为15％。结论：检查大肠菌群比大肠杆菌更能反映药品受污染的程度。     

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics. III: mechanisms for sinusoidal efflux of 4-methylumbelliferone sulfate

1. To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions. 2. The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL(eff)) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate. 3. Under Cl(-)-depleted conditions, the CL(eff) of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl(-) might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL(eff) of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl(-) is involved in the sinusoidal efflux of 4MUS. 4. The effect of glutathione was examined. CL(eff) of 4MUG was not affected by the additional glutathione, but CL(eff) of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG. 5. Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.     

MUG─Indole快速检定大肠杆菌方法研究

本文报道用ＭＵＧ－Ｉｎｄｏｌｅ检定大肠杆菌方法，在２４ｈ内能将９４％～９７％的大肠杆菌检出。对ＭＵＧ阳性、Ｉｎｄｏｌｅ阴性或ＭＵＧ阴性、Ｉｎｄｏｌｅ阳性的菌株，辅以ＥＭＢ琼脂或麦康凯琼脂平板分离，挑选可疑菌落，做ＩＭＶｉＣ试验，使假阳性和假阴性降到最低水平。本法比传统的生化试验检定大肠杆菌法快速、简便、准确、无主观性。     

Differential disposition of intra-renal generated and preformed glucuronides: studies with 4-methylumbelliferone and 4-methylumbelliferyl glucuronide in the filtering and nonfiltering isolated perfused rat kidney

Objectives This study was designed to investigate the renal disposition of 4‐methylumbelliferone (4MU) and 4‐methylumbelliferyl glucuronide (4MUG) to characterise the contribution of excretion and metabolic clearance to total clearance in the kidney. Methods The isolated perfused kidney (IPK) from the male Sprague–Dawley rat was used in filtering and non‐filtering mode to study the renal disposition of 4MU, renally generated 4MUG and preformed 4MUG. Perfusate and urine (filtering IPK only) was collected for up to 120 min and 4MU and 4MUG in perfusate and urine were determined by HPLC. Analytes were also measured in kidney tissue collected at 120 min. Non‐compartmental analysis was used to derive pharmacokinetic parameters. Key findings The concentration of 4MU in perfusate declined with a terminal half‐life of approximately 120 min following administration to the filtering IPK and nonfiltering IPK. There was a corresponding increase in the concentration of 4MUG. Metabolic clearance of 4MU accounted for 92% of total renal clearance. After bolus dosing of preformed 4MUG in the perfusion reservoir of the filtering IPK, the perfusate concentration declined with the terminal half‐life of approximately 260 min. The renal excretory clearance of preformed 4MUG accounted for 96% of total renal clearance. 4MU was extensively metabolized by glucuronidation in the filtering and nonfiltering IPK, and the total renal clearance of 4MU was far greater than its renal excretory clearance. This indicated that glucuronidation was the major elimination pathway for 4MU in the kidney. Conclusions The data confirmed an important role for the kidney in the metabolic clearance of xenobiotics via glucuronidation and signalled the lack of impact of impaired glomerular filtration on renal drug metabolism.     

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics. iii: mechanisms for sinusoidal efflux of 4-methylumbelliferone sulfate

1.?To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions.

2.?The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL_eff) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate.

3.?Under Cl^?-depleted conditions, the CL_eff of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl^? might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL_eff of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl^? is involved in the sinusoidal efflux of 4MUS.

4.?The effect of glutathione was examined. CL_eff of 4MUG was not affected by the additional glutathione, but CL_eff of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG.

5.?Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.     

The important role of Bcrp (Abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in mice

The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(-/-), Abcg2(-/-), and Abcb1a(-/-)/Abcb1b(-/-) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also approximately 35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (> or =37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse Mrp2.     

Determination of the kinetics of rat UDP-glucuronosyltransferases (UGTs) in liver and intestine using HPLC

Uridinediphosphoglucuronosyl transferases (UGTs) are a group of membrane bound proteins which catalyze the transfer of glucuronic acid from UDP-glucuronic acid to a wide variety of xenobiotics and drug molecules enabling them to be eliminated. The major UGT isoforms found in the rat are 1A1, 1A6, 2B1 and 2B12. Conventional methods for the assay of glucuronides (GLs) include TLC, extraction and colorimetry or quantification of the aglycone, liberated after hydrolyzing the GL with beta-glucuronidase. However these techniques cannot distinguish between isomeric GLs or GLs of multiple acceptor site substrates. Therefore the purpose of this study was to develop simple and sensitive HPLC methods for the direct and simultaneous analysis of the GL(s) and their aglycones without the drawbacks of the conventional methods. The three classical substrates we chose were 4-methylumbelliferone (4MU), testosterone (TES) and 8-hydroxyquinoline (8HOQ) representing UGT isoforms 1A6, 2B1 and 2B12 of the rat family, respectively. Here we report the validated HPLC conditions, for the detection and separation of 4-methylumbelliferone glucuronide (4MUG), testosterone glucuronide (TESG) and 8-hydroxyquinoline glucuronide (8HOQG) and their aglycones in incubation media containing male Sprague-Dawley rat liver and intestinal microsomal preparations. The separations were achieved on a Zorbax SB-CN column (150 x 4.6 mm, 5 micron). The analysis time for the separation of TES, 8HOQ and 4MU and their glucuronides were 17, 12 and 30 min, respectively. The methods showed excellent linearity (r2 > 0.99) over the concentration ranges tested (0.25-5.0 nmoles of TESG; 0.125-18.75 nmoles of 8HOQG and 0.125-12.5 nmoles of 4MUG), good precision and accuracy (RSD<2.5%). Inter-day variability studies (n = 3) showed no significant difference between the regression lines obtained on the three days. Recoveries were good ( > 90%) at all three points (low, mid-point, high) of the standard curve. The limits of detection were 0.125, 0.1 and 0.1 nmole for TESG, 8HOQG and 4MUG. respectively. The above methods were used to estimate kinetic parameters such as Vmax and Km for the GLs of the three substrates in both liver and intestinal tissue preparations and the values were comparable with previously reported results. UGT2B1 was found primarily in the liver while UGTs 1A6 and 2B12 were present in comparable amounts in both tissues.     

Substrate recognition by a family of uracil-DNA glycosylases: UNG,MUG, and TDG

In response to continuous hydrolytic and oxidative DNA damage, cells of all organisms have a complex network of repair systems that recognize, remove, and rebuild the injured sites. Damaged pyrimidines are generally removed by glycosylases that must scan the entire genome to locate lesions with sufficient fidelity to selectively remove the damage without inadvertent removal of normal bases. We report here studies conducted with a series of base analogues designed to test mechanisms of base recognition suggested by structural studies of glycosylase complexes. The oligonucleotide series examined here includes 5-halouracils with increasing substituent size and purine analogues placed opposite the target uracil with hydrogen, amino, and keto substituents in the 2- and 6-positions. The glycosylases studied here include Escherichia coli uracil-DNA glycosylase (UNG), E. coli mismatch uracil-DNA glycosylase (MUG), and the Methanobacterium thermoautotrophicum mismatch thymine-DNA glycosylase (TDG). The results of this study suggest that these glycosylases utilize several strategies for base identification, including (1) steric limitations on the size of the 5-substituent, (2) electronic-inductive properties of the 5-substituent, (3) reduced thermal stability of mispairs, and (4) specific functional groups on the purine base in the opposing strand. Contrary to predictions based upon the crystal structure, the preference of MUG for mispaired uracil over thymine is not based upon steric exclusion. Furthermore, the preference for mispaired uracil over uracil paired with adenine is more likely due to reduced thermal stability as opposed to specific recognition of the mispaired guanine. On the other hand, TDG, which exhibits modest discrimination among various pyrimidines, shows strong interactions with functional groups present on the purine opposite the target pyrimidine. These results provide new insights into the mechanisms of base selection by DNA repair glycosylases.     

Characterization of the substrate specificity of a human 5-hydroxymethyluracil glycosylase activity.

The oxidation of pyrimidine 5-methyl groups, derived from either thymine or 5-methylcytosine, can generate 5-hydroxymethyluracil (HmU) in DNA. An activity from HeLa cells that removes 5-hydroxymethyluracil (HmU) from DNA has been partially purified and characterized using a battery of oligonucleotides containing modified bases. This partially purified activity preferentially removes HmU mispaired with guanine. The HmU repair activity also acts on uracil and fluorouracil but not 5-substituted uracil derivatives with halogens larger than fluorine. However, neither mispaired thymine nor ethenocytosine are substrates. HmU is readily removed when paired with guanine, hypoxanthine (deoxyinosine), and purine (deoxynebularine), but not from single-stranded substrates. Upon the basis of these substrate preferences, we conclude that (1) the mispaired HmU repair activity is distinct from previously reported glycosylases including UDG, TDG, MUG, and SMUG1 activities, (2) the binding pocket is highly selective for the 5-hydroxymethyl group, and (3) the preference for mispaired HmU derives from reduced thermal stability of the mispair, as opposed to selective recognition of the mispaired guanine residue in the opposing DNA strand.     

Synthesis and characterization of oligonucleotides containing 5-chlorocytosine

Recent studies have shown that reactive chlorine species, derived from myeloperoxidase-mediated inflammation responses, can modify DNA bases, generating 5-chloropyrimidines. The chlorinated adducts could be mutagenic or perturb DNA-protein interactions; however, the biological significance of these adducts is as yet unknown. We report here a method for the synthesis of 5-chlorocytosine- (ClC-) containing oligonucleotides that will be used in subsequent biochemical and biophysical studies to determine the consequences of pyrimidine chlorination. The ClC-phosphoramidite synthon is obtained by chlorination of 2'-deoxyuridine followed by conversion to the O(4)-ethyl analogue. The amino group needed to form the corresponding cytosine derivative is added by displacement of the O(4)-ethyl group during ammonia deprotection. A battery of methods, including mass spectrometry, has been used to characterize oligonucleotides containing ClC. Following oligonucleotide synthesis and deprotection, only trace amounts of the deamination product 5-chlorouracil can be detected by enzymatic cleavage of duplex oligonucleotides with the mispaired uracil glycosylase, MUG. In contrast to previous reports, we find that ClC is more stable in DNA than anticipated. Approximately 20% ClC is lost under standard formic acid hydrolysis conditions (88% formic acid, 140 degrees C, 30 min), while only 5% is recovered as 5-chlorouracil (ClU).