Carnitine and glucuronic acid conjugates of pivalic acid

The [1-14C]pivaloyloxyethyl ester of methyldopa administered to man and cynomolgus monkeys resulted in the elimination in the urine of 14C-pivalic acid metabolites. Pivaloyl glucuronide and pivaloyl carnitine were identified as the major radioactive urinary metabolites in monkey urine and human urine, respectively. N.m.r. analysis indicated that pivaloyl carnitine had a cyclic structure. Although the role of carnitine is in the transport of fatty acids across mitochondrial membranes, it may also function in the conjugation of carboxylic acid xenobiotics in humans.     

Conventional and laser induced fluorescence detection of glucuronic acid conjugates after derivatization and liquid chromatographic separation

Pre-chromatographic derivatisation of the carboxylic acid function of glucuronic acid conjugates is a suitable method for the selective and ultra-sensitive analysis of these compounds in urine and plasma samples. This goal is achieved by applying an indirect derivatisation procedure and laser induced fluorescence detection with a homemade detection system equipped with a continuous-wave argon-ion laser. The minimum detectable amounts for the analytes, after derivatisation, are about 3 amol using the fluorescein fluorophore. In comparison with conventional induced fluorescence detection a gain in sensitivity of over four orders of magnitude is obtained.     

Isolation, purification, and structure identification of glucuronic acid conjugates of propranolol and alprenolol and their ring-hydroxylated metabolites

The purpose of this study was to develop a procedure for the isolation, purification, and structure identification of glucuronic acid conjugates of propranolol and alprenolol and their active metabolites, 4-hydroxypropranolol and 4-hydroxyalprenolol. As both aliphatic and aromatic glucuronides may be formed from 4-hydroxypropranolol and 4-hydroxyalprenolol, the structure identification of these conjugates has to be based on the intact conjugates. Using DEAE-Sephadex anion-exchange chromatography, milligram quantities of these glucuronides were isolated from urine of dogs pretreated with propranolol and alprenolol, respectively. A high degree of purification was achieved by reversed-phase HPLC. Structure identification of the methyl ester-trimethylsilyl derivatives was accomplished by electron impact GC/MS. Only one 4-hydroxypropranolol glucuronide was found, having the glucuronic acid linked to the aromatic hydroxyl group. For 4-hydroxyalprenolol glucuronide, however, two structures were identified, one with the glucuronic acid linked to the aromatic hydroxyl group, and the other with the glucuronic acid linked to the aliphatic hydroxyl group of the side chain. Using this analytical approach, 4-hydroxypropranolol glucuronide was identified in the urine of patients on propranolol therapy. In man, as in the dog, only the aromatic glucuronide was found, i.e. containing an unsubstituted beta-blocking side chain.     

The formation of β-glucuronidase resistant glucuronides by the intramolecular rearrangement of glucuronic acid conjugates at mild alkaline pH

It is well known to carbohydrate chemists that substituted sugars may undergo facile rearrangement involving the migration of the aglycone from —OH to adjacent —OH. Despite the importance of glycoside conjugates, notably involving glucuronic acid, in the metabolism of xenobiotics, drug metabolism workers have neglected this phenomenon. The potential rearrangement of glucuronides from the biosynthetic C-1 isomers to other positional and stereo-isomers is important, since only 1-O-substituted β-d-glucosiduronates are substrates for β-glucuronidase, which is commonly used to identify such conjugates. The intramolecular rearrangement of clofibryl glucuronide has been studied over the pH range 5.2–8.6, by enzymic hydrolysis with β-glucuronidase, and by HPLC. The amount of clofibric acid released from the conjugate by β-glucuronidase falls with increasing pH of preincubation above pH 7.4, and this is accompanied by the appearance of three new peaks, each containing both clofibric and glucuronic acids, in the HPLC traces of the incubation mixtures. Similar experiments with three ether glucuronides, those of p-nitrophenol, phenolphthalein and 7-hydroxycoumarin, did not show any conversion to β-glucuronidase resistant forms. The phenomenon of intramolecular rearrangement of ester glucuronides must be considered whenever β-glucuronidase is used in the analysis of conjugates of carboxylic acids.     

Enterohepatic recycling of phenolphthalein,morphine, lysergic acid diethylamide (LSD) and diphenylacetic acid in the rat Hydrolysis of glucuronic acid conjugates in the gut lumen

1. Biliary elimination in female Wistar albino rats 3h after i.p. injection of [³H]phenolphthalein, [³H]morphine, ¹⁴C-LSD and [¹⁴C]diphenylacetic acid was 90%, 45%, 75% and 57% respectively, predominantly as glucuronides.

2. Infusion of 3 h bile from the previous experiments into the duodena of bile-duct-cannulated animals demonstrated enterohepatic circulation, amounting in 24 h to 85%, 41%, 28% and 66% of the infused doses of the conjugates of phenolphthalein, morphine, LSD and diphenylacetic acid respectively.

3. Pretreatment with antibiotics to suppress intestinal microflora decreased this enterohepatic recirculation to 22%, 86% and 21% in 24h for phenolphthalein, morphine and diphenylacetic acid glucuronides respectively. Antibiotic pretreatment did not influence the absorption and re-excretion of infused doses of the free aglycones, thus demonstrating the importance of bacterial β-glucuronidase hydrolysis of the biliary conjugates.

4. The extent of intestinal absorption of the aglycones after bacterial β-glucuronidase hydrolysis of the conjugates is related to their lipid-solubility as estimated by octan-1-o1:0·1 M phosphate buffer partition ratios (P-values).

5. The persistence of compounds in the enterohepatic circulation is determined by the faecal and urinary elimination of the circulating compounds. Faecal elimination is governed by the extent of intestinal absorption of the circulating compounds, which is influenced by the efficacy of intestinal hydrolysis of the conjugates and the relative lipophilicity of the aglycones released.     

尿苷二磷酸葡萄糖醛酸转移酶与内源性物质的相互作用

尿苷二磷酸葡萄糖醛酸转移酶(UGT)是人体重要的II相代谢酶,代谢药物的同时也代谢许多重要的内源性物质,如胆红素、甲状腺激素、雌激素、雄激素、胆汁酸和5-羟色胺等。该酶对许多内源性物质的代谢是灭活和清除这些内源性物质的关键步骤,能够防止内源性物质累积引发的毒性反应,或及时终止内源性激素的信号防止肿瘤的发生。然而,内源性物质对UGT酶也会产生影响,特别是在一些生理病理条件下,某些内源性物质能够抑制UGT酶活性,影响其参与的代谢反应。将就内源性物质和UGT酶的相互作用做一综述,以引起人们对UGT酶和内源性物质相互作用的关注。关键词:药物代谢;尿苷二磷酸葡萄糖醛酸转移酶;内源性物质     

酸性多糖中的葡萄糖醛酸与中性糖的含量测定

目的对酸性多糖中的葡萄糖醛酸 (GA)与中性糖的含量进行测定。方法将葡萄糖和GA配成一系列混合糖 ,比较硫酸咔唑法和间羟联苯法测定GA含量的准确度 ;分别建立葡萄糖和GA对硫酸蒽酮的标准曲线以扣除GA对中性糖测定的干扰。结果葡萄糖的存在对用硫酸咔唑法测定GA有干扰 ,而用间羟联苯法测定几无干扰 ;另外 ,GA的存在对用硫酸蒽酮法测定中性糖有一定干扰。结论应采用间羟联苯法测定酸性多糖中的GA含量 ,同时扣除GA在硫酸蒽酮法中的干扰以得到中性糖的含量。     

Monooxygenase reactions glucuronic acid and sulphate conjugation in isolated hepatocytes

Freshly isolated hepatocytes can be used to identify some of the key features determining the rates of xenobiotic metabolism and the nature of the metabolites which are formed. Our findings suggest that the intrinsic activities of cytochtome P-450 reductase and/or cytochrome b₅ reductase glucuronyl transferase(s) and sulphotransferase(s) are probably the key factors determining the rate of formation of phenols and their subsequent conjugation rather than the levels of cofactors or rates of substrase accessability and excretion.Other uses of hepatocytes, e.g. to investigate the selective activation of certain cytochrome P-450 activities and to characterise species differences in drug metabolism are briefly discussed.     

Possible mechanism for species difference on the toxicity of pivalic acid between dogs and rats

In a high dose toxicity study of pivalic acid (PA), PA caused skeletal muscle disorder in dog, and a significant increase of pivaloyl carnitine (PC) was observed in canine muscle, but not in rat muscle. In order to understand species difference of the toxicity of PA, we compared the in vitro metabolism of PA among dog, rat and rabbit, especially focussing on the carnitine conjugate. Canine muscle showed low, but significant carnitine conjugating activity, while that of rat was negligible. Canine kidney mitochondria had significant activity in the pivaloyl CoA synthesis (7 nmol/mg protein/h), but muscle mitochondria showed only trace activity. Both kidney and muscle mitochondria displayed similar carnitine acyltransferase activity (2-3 nmol/mg protein/h) towards pivaloyl CoA. On the other hand, with respect to the activity of carnitine acyltransferase in the reverse direction using PC as substrate, canine muscle mitochondria showed higher activity than that of kidney mitochondria. This means that PC is not the final stable metabolite, but is converted easily to pivaloyl CoA in canine muscle. These results suggest one of the possible mechanisms for canine selective muscle disorder to be as follows. Only canine muscle can metabolize PA to its carnitine conjugate slowly, but significantly. In canine muscle, PC is not the final stable metabolite; it is easily converted to pivaloyl CoA. As carnitine conjugation is thought to be the only detoxification metabolic route in canine muscle, under certain circumstances such as carnitine deficiency, the risk of exposure with toxic pivaloyl CoA might increase and the CoASH pool in canine muscle might be exhausted, resulting in toxicity in canine muscle.     

Assignment of the 750 MHz 1H NMR resonances from a mixture of transacylated ester glucuronic acid conjugates with the aid of oversampling and digital filtering during acquisition

Many drugs containing carboxylic acid functional groups are metabolised in vivo to ester glucuronides (1-O-acyl-β-d-glucopyranuronates) and, of these, a number show a propensity to undergo internal isomerisation via a transacylation process, causing the carboxylic acid moiety to migrate successively to the 2-, 3- and 4-positions of the glucuronic acid. These products may be responsible, through reactions with plasma proteins, for some of the allergenic side effects in a number of non-steroidal anti-inflammatory drugs. It is important to understand those properties of the drug molecules which facilitate this reaction, and to this end we have studied the transacylation product formation and reaction kinetics in a series of aryl carboxylic acid glucuronides using NMR spectroscopy. However, the resulting ¹H NMR spectra are very complex with much resonance overlap, and recourse to spectral simplification processes is necessary. Here, improvement in spectral resolution by oversampling and digital filtering to restrict the detection range of the spectrometer, thus yielding improved digital resolution, is demonstrated. The approach has been applied to the assignment of a mixture of transacylated ester glucuronides of 2-trifluoromethylbenzoic acid through the use of a two-dimensional ¹H-¹H TOCSY experiment.     

壳聚糖-巯基醋酸偶合物的制备及结构分析

目的制备一种新型生物粘附材料-壳聚糖-巯基醋酸偶合物,并对其结构进行分析。方法以N-羟基丁二酰亚胺(NHS)作催化剂,采用新的合成工艺制备壳聚糖-巯基醋酸偶合物,并测定了偶合物中巯基的含量,同时对偶合物进行了元素分析和红外光谱测定。结果制备得到了巯基含量为1034μmol.g-1的壳聚糖-巯基醋酸偶合物,阐明了该材料的结构特征。结论新的合成工艺可行,材料的结构可用红外光谱来表征。     

熊果酸氨基酸偶联物的合成及保肝活性

目的 设计合成熊果酸 28 位氨基酸偶联物并测定其保肝活性。方法 以熊果酸为起始原料,首先将 3 位羟基进行酯保护,28 位羧基与保护的氨基酸反应,然后脱保护基,得到偶联物。利用肝细胞培养和小鼠急性肝损伤 CCl₄、ConA 筛选模型分别测定偶联物的体外、体内保肝活性。结果与结论 得到 3 个未见报道的化合物,其结构经核磁共振谱和质谱确定。水溶性测定结果显示 3 个偶联物的水溶性很低,保肝试验结果显示 3 个偶联物无明显的保肝活性。     

HPLC测定注射用葡醛酸钠的含量及有关物质

目的　建立测定注射用葡醛酸钠中葡醛酸钠含量及其有关物质的检查方法。方法　采用HPLC法HypersilC1 8色谱柱,1 0mmol·L-1 磷酸盐缓冲液(6mmol·L-1 NaH2 PO4 4mmol·L-1 Na2 HPO4) -甲醇(98∶2 ,磷酸调pH 6 )为流动相,检测波长2 2 5nm ,流速0 .8ml·min-1 。结果　葡醛酸钠在0 .1～2mg·ml-1 范围内,峰面积与浓度线性关系良好(r=0 .9999) ,高、中、低3种浓度的平均加样回收率为1 0 0 .0 %～1 0 0 .3%,RSD为0 .4 4 %～0 .82 %,有关物质检查的限量为1 .0 %。结论　所用方法准确、简便、快速,适用于注射用葡醛酸钠的质量控制。     

Ascorbic acid in thyroidectomized rats. II) Ascorbic acid status of the storage tissues and hepatic biosynthesis of glucuronic acid

Depletion of ascorbic acid from adrenals, brain and epididymis along with loss in weight were noticed in the state of thyroidectomy. This decrease appears to be due to an effect of thyroidectomy on the membrane integrity since the membrane bound sialic acid was found to be significantly lowered in these tissues as a consequence of the elevated activity of sialidase. Thyroidectomy was also found to cause an adverse effect on the activities of hepatic UDP-glucuronyl transferase and beta-glucuronidase with no alteration in UDP-glucose dehydrogenase.     

Synthesis of fenoprofen and gemfibrozil styrene-maleic acid copolymer conjugates

Two types of polymer-drug conjugates were synthesized starting from styrene-maleic acid anhydride copolymer (SMA). Fenoprofen and gemfibrozil were chosen as model drugs because of their short plasma half lives. Both drugs were first converted to their 2-aminoethylamides, which possess free amino groups capable of reacting with SMA anhydride rings. By modifying the degree and type of substitution, lipophilic and hydrophilic conjugates were obtained. Drug loading in the conjugates was between 17 and 47%.