沉默调节蛋白6-谷胱甘肽巯基转移酶融合蛋白的克隆表达

目的 构建含有人沉默调节蛋白6(hSIRT6)基因的重组原核表达载体,获得高效表达hSIRT6蛋白的基因工程菌,以及较高产量的SIRT6蛋白.方法 取205 ng HEK293细胞总RNA逆转录后合成的cDNA为模板,聚合酶链反应(PCR)扩增hSIRT6的编码序列,并克隆至原核表达载体pGEX-4T-3中,构建重组的质粒pGEX-4T-3/SIRT6;将重组质粒pGEX-4T-3/SIRT6转化感受态细菌BL21( DE3),在含100 mg/L氨苄西林的LB平板37℃培养过夜,以pGEX-4T-3空载体作为对照,在相同条件下转化并异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达谷胱甘肽巯基转移酶(GST)蛋白.产物经SDS-PAGE电泳及Western blot鉴定.结果 PCR产物大小及双酶切鉴定证明所克隆的基因是hSIRT6,DNA测序进一步证实与GeneBank序列完全一致.获得高表达及纯化的SIRT6-GST融合蛋白,该可溶蛋白的相对分子质量为72×103,经Western blot鉴定证实为目的蛋白.结论 成功构建了基因重组体pGEX-4T-3/hSIRT6,并制备出可溶性SIRT6-GST融合蛋白.     

DOC-1基因的原核表达载体构建及其重组蛋白的表达纯化

目的：克隆DOC-1基因、构建原核表达载体并纯化出其重组蛋白。方法：从人胎脑组织中提取总RNA，经逆转录一聚合酶链式反应（RT—PCR）扩增DOC-1的CDS序列，再通过基因重组技术将该基因片段依次克隆到pMO18-T和pGEX-4T-1载体中，构建融合表达载体pGEX-4T-1-DOC-1，经酶切、测序鉴定后，用该重组质粒转化E．coliBL21，用IPTG诱导表达，Glutathlone Sepharose 4B柱亲和层析纯化重组蛋白。结果：电泳证实RT-PCR扩增产物与预期目的基因DOC-1长度一致，测序结果与GenBank公布的DOC-1基因序列完全一致，IPTG诱导后经SDS—PAGE电泳分析表明，在相对分子质量38000左右出现新的蛋白表达条带，经亲和层析柱纯化后得到高纯度的GST-p12重组蛋白。结论：成功构建了pGEX-4T-1-DOC-1原核表达载体，表达并纯化出GST-p12重组蛋白，为进一步研究p12^DOC-1蛋白打下了实验基础。     

淋病奈瑟菌外膜蛋白PorB基因的克隆及原核表达

目的:构建淋病奈瑟菌外膜蛋白PorB的融合表达载体,并在原核系统中表达,获得基因重组蛋白,为进一步研究淋病奈瑟菌外膜蛋白的致病作用和免疫保护性提供基础。方法:根据PorB已知序列设计一对引物,用PCR方法从淋病奈瑟菌标准菌株NG 29403基因组DNA中扩增出PorB基因,与原核表达质粒pGEX-4T-1构建重组子pGEX-4T-PorB,并将重组质粒转化大肠埃希菌BL21(DE3)感受态细胞。重组子经酶切、PCR鉴定和核酸序列分析正确后,异丙基硫代半乳糖苷(IPTG)诱导重组蛋白的表达,用SDS-PAGE及Western印迹进行鉴定。结果:限制性核酸内切酶酶切鉴定、PCR和核酸序列分析表明,扩增出了淋病奈瑟菌外膜蛋白1 047 bp的PorB基因,成功构建了重组质粒pGEX-4T-PorB,经SDS-PAGE及Western印迹分析显示重组质粒pGEX-4T-PorB在大肠埃希菌中得到了高效融合表达。结论:成功构建pGEX-4T-PorB原核表达载体,并在原核系统中得到了高效表达。     

国人瘦素基因原核重组表达载体的构建及鉴定

目的 构建并鉴定瘦素基因原核重组表达载体.方法 以RT-PER法自人脂肪组织制备目的 基因,应用DNA重组技术,克隆至pMD18T载体与pGEX-4T-1原核表达载体,转化大肠杆菌DH5α,EcoR I+Xho I双酶切及测序鉴定.结果 扩增得到520 bp目的 基因并构建原核重组表达载体pGEX-4T-1-leptin.双酶切电泳见520bp目的 条带,测序结果与GenBank收录序列一致.结论 成功构建原核表达载体pGEX-4T-1-leptin,为leptin功能研究及进一步的临床应用提供了条件.     

肾癌肿瘤相关抗原G250/MN/CAIX基因的克隆、表达及鉴定

目的 报道G250／MN／CA Ⅸ基因的克隆，蛋白表达及鉴定。方法 从肾癌组织中提取总RNA，采用RT—PCR法扩增G250／MN／CA Ⅸ全长cDNA，将获得的cDNA片段插入pET22b（＋）表达载体，转化BL-21大肠杆菌中，以IPTG诱导进行蛋白表达，SDS—PAGE凝胶电泳观察结果，实验验证。结果 克隆的G250／MN／CA Ⅸ基因经测序证实序列正确，构建重组质粒pET22b（＋）／G250并在原核系统中表达G250／MN／CA Ⅸ重组蛋白，该蛋白具有较好的抗原性和特异性。结论 成功完成G250基因克隆，为在G250蛋白纯化基础上进行抗体制备和G250功能研究提供了实验依据。     

人前列腺干细胞抗原的克隆及表达

目的 克隆人前列腺干细胞抗原(PSCA)编码基因,并在大肠杆菌中表达PSCA蛋白。方法 从培养的人前列腺癌细胞株中提取总RNA,反转录成cDNA,通过聚合酶链反应(PCR)方法扩增PSCA编码基因,克隆至pMD181-T载体,经DNA测序证实后亚克隆至谷胱甘肽巯基转移酶(GST)融合表达载体pGEX-5X-1,重组质粒转化大肠杆菌,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达PSCA/GST融合蛋白。结果 从培养的人前列腺癌细胞株中扩增出编码PSCA的cDNA(241 bp),序列测定证实与 Genebank上登录的序列一致;成功构建了GST融合表达载体 pGEX-5X-1/PSCA;表达了PSCA/GST融合蛋白(34.5×10~3)。结论 成功克隆了人PSCA基因,构建了GST融合表达载体并表达了 PSCA/GST蛋白。     

在大肠杆菌中融合表达重组羧肽酶抑制剂

利用PCR技术扩增得到编码土豆羧肽酶抑制剂（carboxypeptidase inhibitor from potato，CPI）活性多肽基因，克隆于表达载体pGEX-4T-1的多克隆位点中，得到重组质粒pGCPI，测序后将重组质粒转化到受体菌Escherichia coli BL21中。重组菌经IPTG诱导表迭，超声波破菌后，通过谷胱甘肽sepheroseFF亲和层析柱一步纯化得到融合蛋白，纯度大于979，6。融合蛋白经凝血酶切割并分离除去谷胱甘肽-S-转移酶后得到纯度大于98％的重组CPI，ELISA鉴定产物具有免疫原性，体外检测表明其促进纤溶的生物功能与天然CPI无明显差异。     

MGP基因的重组及在大肠杆菌中的表达

目的重组骨质疏松候选基因基质Gla蛋白（MGP）基因使其蛋白在大肠杆菌中高效表达。方法利用反转录聚合酶链反应（RT—PCR）从正常人肺组织总RNA中扩增出MGP基因cDNA序列，与克隆pGEM—Teasy载体相连，测序为完整的编码序列后与表达载体pTrcHisB构建重组体，转化入大肠杆菌Top10后用IPTG诱导，Western bloting证实蛋白表达。结果克隆至pGEM-Teasy载体及pTreHisB载体中的MGP基因cDNA序列与基因库完全一致。转入大肠杆菌后经IPTG诱导有蛋白的表达，Western bloting证实诱导后2、3、4h蛋白的表达量显著增加。结论成功重组的人MGP基因，重组体在大肠杆菌内能成功高效地表达。IPTG诱导后蛋白的表达为时间依赖性。     

新基因BC047440原核表达载体构建及其重组蛋白的表达纯化

目的 构建肝癌相关新基因BC047440原核表达载体,表达并纯化出其重组蛋白.方法 从HepG2细胞中提取总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增BC047440cDNA,克隆进质粒pMD19-T,转化E.coliDH5α,测序正确后,酶切目的基因片段,插入质粒pET-28a(+),转化E.coliBL21,IPTG诱导蛋白表达,Ni-NTA柱亲和层析纯化重组蛋白.结果 电泳证实RT-PCR扩增产物与预期目的基因BC047440长度一致.RT-PCR纯化产物与载体pMD19-T连接后,经蓝白斑筛选,挑出5个白色菌落做酶切鉴定,其中2个菌落被证实为阳性克隆.测定其基因序列,结果显示与Genbank公布的BC047440基因序列完全一致.将BC047440cDNA插入质粒pET-28a(+),转化E.coliBL21,培养过夜后,挑选7个白色菌落做酶切鉴定,证实均为阳性克隆.IPTG诱导其中一个克隆表达蛋白,SDS-PAGE电泳分析表明,在相对分子质量23000左右出现新的蛋白表达条带,诱导4h后表达蛋白量最多,占菌体总蛋白的22.3%.将诱导3h的菌体超声破碎后,Ni柱亲和层析,50和125mmol/L咪唑洗脱液SDS-PAGE电泳显示出清晰的单一条带.结论 成功构建BC047440基因原核表达载体,表达并纯化出重组蛋白,为深入研究其临床应用打下基础.     

重组人中性粒细胞弹性蛋白酶的制备及鉴定

目的:利用基因工程技术制备纯化的重组人中性粒细胞弹性蛋白酶(HNE),为进一步制备HNE的相应抗体和建立精液HNE的检测方法奠定基础。方法:利用HNE的特异引物从人外周血粒细胞中获得HNEmRNA,并将其cDNA克隆入质粒pGEX-2T中以获得重组质粒pGEX-2T/HNE。重组质粒经PCR、双酶切和基因测序鉴定后转入感受态大肠埃希菌DH5α中,并用异丙基β-D-硫代半乳糖苷(IPTG)诱导表达重组融合蛋白GST/HNE。重组融合蛋白经凝血酶裂解后获得重组HNE,并经谷胱甘肽琼脂糖珠纯化后获得纯化的重组HNE。结果:成功制备重组表达质粒pGEX-2T/HNE,并转化入大肠埃希菌DH5α中。经IPTG在18℃过夜诱导后成功获得重组融合蛋白GST/HNE的表达。经凝血酶裂解和谷胱甘肽琼脂糖珠纯化后成功获得纯化的重组蛋白HNE。结论:纯化的重组HNE的获得为进一步制备HNE的相应抗体和建立精液HNE的检测方法奠定了基础。     

Vasopressor hormone response following mesenteric traction during major abdominal surgery

Background : We investigated the vasopressor hormone response following mesenteric traction (MT) with hypotension due to prostacyclin (PGI₂) release in patients undergoing abdominal surgery with a combined general and epidural anesthesia. Methods : In a prospective, randomized, placebo-controlled study we administered 400 mg ibuprofen (i.v.) in 42 patients scheduled for abdominal surgery. General anesthesia was combined with epidural anesthesia (T4-L1). Before as well as 5, 15, 30, 45, and 90 min after MT we recorded plasma osmolality, hemodynamics and measured 6-keto-PGFlα (stabile metabolite of PGI₂), TXB₂ (stabile metabolite of thromboxane A₂) active renin, and arginine vasopressin (AVP) plasma concentrations by radioimmunoassay. Catecholamine levels were assessed by high-pressure liquid chromatography (HPLC) with electrochemical detection. Results : Following MT, arterial hypotension occurred along with a substantial PGI₂ release. This was completely abolished by ibuprofen administration. Although plasma levels of 6-keto-PGF_1α (1133 (708) vs. 60 (3) ng/L, median (median absolute deviation), P=0.0001, placebo vs. ibuprofen) remained significantly elevated, blood pressure was restored within 30 min after MT in the placebo group. At the same point in time plasma concentrations of TXB² (164 (87) vs. 58 (1) ng/L, P=0.0001), epinephrine (46 (33) vs. 14 (6) ng/L, P=0.001), AVP (41 ± (18) vs. 12 (7) ng/L, P=0.0004), and active renin (27 (12) vs. 12 (4) ng/L, P = 0.001) were significantly higher in placebo-treated patients. Conclusion : Under combined general and epidural anesthesia arterial hypotension following MT due to endogenous PGI₂ release is associated with enhanced release of AVP, active renin, epinephrine and thromboxane A₂, presumably contributing to hemodynamic stability within 30 min after MT.     

A Teaspoon Pump for Pumping Blood with High Hydraulic Efficiency and Low Hemolysis Potential

Abstract: Virtually all blood pumps contain some kind of rubbing, sliding, closely moving machinery surfaces that are exposed to the blood being pumped. These valves, internal bearings, magnetic bearing position sensors, and shaft seals cause most of the problems with blood pumps. The original teaspoon pump design prevented the rubbing, sliding machinery surfaces from contacting the blood. However, the hydraulic efficiency was low because the blood was able to "slip around" the rotating impeller so that the blood itself never rotated fast enough to develop adequate pressure. An improved teaspoon blood pump has been designed and tested and has shown acceptable hydraulic performance and low hemolysis potential. The new pump uses a nonrotating "swinging" hose as the pump impeller. The fluid enters the pump through the center of the swinging hose; therefore, there can be no fluid slip between the revolving blood and the revolving impeller. The new pump uses an impeller that is comparable to a flexible garden hose. If the free end of the hose were swung around in a circle like half of a jump rope, the fluid inside the hose would rotate and develop pressure even though the hose impeller itself did not "rotate"; therefore, no rotating shaft seal or internal bearings are required.     

Microspheres Based Detoxification System: A New Method in Convective Blood Purification

Abstract: A variety of protein-bound or hydrophobic substances, accumulating as a result of pathologic conditions such as exogenous or endogenous intoxications, are removed poorly by conventional detoxification methods because of low accessibility (hemodialysis), insufficient adsorption capabilities (hemosorption), low efficiency (peritoneal dialysis), or economic limitations (high-volume plasmapheresis). Combining advantages of existing methods with microspheric technology, a module-based system was designed. Major operating parameters of the latter can be modified to allow for adjustment to individual clinical situations. An extracorporeal blood circuit including a plasmafilter is combined with a secondary high-velocity plasma circuit driven by a centrifugal pump. Different microspheric adsorbers can be combined in one circuit or applied in sequence. Thus, a prolonged treatment can be tailored using specially designed selective adsorber materials. Comparing this system with existing methods (high-flux hemodialysis, molecular adsorbent recycling system), results from our in vitro studies and animal experiments demonstrate the superior efficiency of substance removal.     

Tissue perfusion in relation to cardiac output during continuous positive-pressure ventilation and administration of propranolol or verapamil

Background : Our objective was to determine whether administration of propranolol or verapamil modifies the hemodynamic adaptation to continuous positive-pressure ventilation (CPPV), in particular the regional distribution of cardiac output (CO).
Methods : General hemodynamics and regional blood flows assessed by microsphere technique (15 (μm) were recorded in 16 anesthetized pigs during spontaneous breathing (SB) and CPPV with 8 cm H₂O end-expiratory pressure (CPPV8) before and after intravenous administration of propranolol (0.3 mg · kg⁻¹ followed by 0.15 mg · kg⁻¹ · h⁻¹, n=8) or verapamil (0.1 mg · kg⁻¹ followed by 0.3 mg · kg⁻¹ · h⁻¹, n=8).
Results : CPPV8 depressed CO by 25% without shifts in its relative distribution with the exception of a noteworthy increase in adrenal perfusion. Propranolol increased arterial blood pressure, and due to a fall in heart rate, CO dropped by 25%. The kidneys and, to a lesser extent, the splanchic region and central nervous system received increased fractions of the remaining CO at the expense of skeletal muscle flow. Similar patterns were seen during SB and CPPV8 such that the combination of propranolol and CPPV8 depressed CO by 50%. The circulatory effects of verapamil were less evident but myocardial perfusion tended to increase.
Conclusions : The combination of propranolol or verapamil with CPPV does not result in any specific hemodynamic interaction in anesthetized pigs, except that the combined effect of propranolol and CPPV may severely reduce CO.     

Bariatric Surgery in Some "Central and East-European (former Eastern bloc)"Countries - Current Status and Prediction for the Next Millennium

Background: Obesity is increasing globallly, including in the formerly "Eastern Bloc" countries. Methods: A survey was made of obesity and bariatric surgery. Results: In the 8 East and Central European countries studied, with total population 300 million, roughly 43% of the population was overweight (BMI 25-30), 23% obese (BMI > 30), with about 15 million people morbidly obese (BMI > 40). From 0-10 morbidly obese individuals/100,000/year undergo bariatric surgery. Conclusion: Most countries were found to provide inadequate treatment for obesity.The majority of the morbidly obese are not treated effectively. However, health-care awareness of obesity and bariatric surgeons are slowly increasing.     

Volatile anaesthetics reduce adhesion of blood platelets under low-flow conditions in the coronary system of isolated guinea pig hearts

Background : Inhibitory effects of volatile anaesthetics on platelet aggregation have been demonstrated in several studies. However, the influence of volatile anaesthetics on intracoronary platelet adhesion has not been elucidated so far.
Methods : Isolated hearts of guinea pigs were perfused with buffer in the absence or presence of volatile anaesthetics (0.5 and 1 MAC) at constant coronary flow rates of 5 ml/min for 25 min, then 1 ml/min for 30 min and again 5 ml/min for 10 min. Before, during and after low-flow perfusion, a bolus of human platelets was applied into the coronary system. To simulate thrombogenic conditions, 0.3 U/ml human thrombin was infused during low-flow perfusion and reperfusion. The number of platelets sequestered to the endothelium was calculated from the difference between coronary in- and output of platelets. The myocardial production of lactate and consumption of pyruvate and coronary perfusion pressure were also determined.
Results : At a flow rate of 5 ml/min only about 3% of the applied platelets did not emerge from the coronary system, in any group. In contrast, 13.1±1.2% (mean±SEM) of infused platelets became adherent in low-flow perfusion in the control group without anaesthetic. The adherence was reduced with each 1 MAC isoflurane (to 6.2±1.2%), sevoflurane (to 4.4±0.9%) or halothane (to 3.2±1.5%) (each P <0.05 vs. control). Volatile anaesthetic, 0.5 MAC, did not inhibit platelet adhesion to a statistically significant extent in any case. Perfusion pressure and metabolic parameters were not statistically different between the control and the hearts exposed to anaesthetics.
Conclusion : Volatile anaesthetics in a concentration of 1 MAC can reduce the adhesion of platelets in the coronary system under reduced flow conditions. This action does not arise from vasodilation or inhibition of ischaemic stress.     

Synergistic interaction between the effects of propofol and midazolam with fentanyl on phrenic nerve activity in rabbits

Background: It has been shown that the depressive effects of both propofol and midazolam on consciousness are synergistic with opioids, but the nature of their interactions on other physiological systems, e. g. respiration, has not been fully investigated. The present study examined the effect of propofol and midazolam alone and in combination with fentanyl on phrenic nerve activity (PNA) and whether such interactions are additive or synergistic. Methods: PNA was recorded in 27 anaesthetised and artificially ventilated rabbits. In three groups, propofol, fentanyl and midazolam were administered intravenously in incremental doses to construct dose-response curves for the depressant effects of each one on PNA. In another two groups, the effect of pretreatment with either fentanyl 1 μg · kg^?1 i. v. or midazolam 0.05 mg · kg^?1 i. v. on the effects of propofol and fentanyl respectively on PNA were studied. Results: Propofol and fentanyl caused a dose-dependent depression of PNA with complete abolition at the highest total doses of 16 mg · kg^?1 i. v. and 32 μg · kg^?1 i. v., respectively. In contrast, midazolam in incremental doses to a total of 0.8 mg · kg^?1 reduced mean PNA by 63%, but approximately 12% of PNA remained at a total dose as high as 6.4 mg · kg^?1. The mean ED₅₀s, calculated from dose-response curves, were 5.4 mg · kg^?1, 3.9 μg · kg^?1 and 0.4 mg · kg^?1 for propofol, fentanyl and midazolam, respectively. Initial doses of either fentanyl 1 μg · kg^?1 i. v. or midazolam 0.05 mg · kg^?1 i. v. acted synergistically with subsequent doses of either propofol or fentanyl to abolish PNA at total doses of 8 mg · kg^?1 and 8 μg · kg^?1, respectively. Conclusion: Fentanyl has a synergistic interaction with both propofol and midazolam on PNA and hence potentially on respiration.     

Influence of dopexamine hydrochloride on haemodynamics and regulators of circulation in patients undergoing major abdominal surgery

Background: Catecholaminergic support is often used to improve haemodynamics in patients undergoing major abdominal surgery. Dopexamine is a synthetic vasoactive catecholamine with beneficial microcirculatory properties. Methods: The influence of perioperative administration of dopexamine on cardiorespiratory data and important regulators of macro- and microcirculation were studied in 30 patients undergoing Whipple pancreaticduodenectomy. The patients received randomized and blinded either 2 μg · kg^?1 · min^?1 of dopexamine (n=15) or placebo (n=15, control group). The infusion was started after induction of anaesthesia and continued until the morning of the first postoperative day. Endothelin-1 (ET-1), vasopressin, atrial natriuretic peptide (ANP), and catecholamine plasma levels were measured from arterial blood samples. Measurements were carried out after induction of anaesthesia, 2 h after onset of surgery, at the end of surgery, 2 h after surgery, and on the morning of the first postoperative day. Results: Cardiac index (CI) increased significantly in the dopexamine group (from 2.61±0.41 to 4.57±0.78 1 · min^?1 · m^?2) and remained elevated until the morning of the first postoperative day. Oxygen delivery index (DO₂I) and oxygen consumption index (VO₂I) were also significantly increased in the dopexamine group (DO₂I: from 416±91 to 717±110 ml/m² · m²; VO₂I: from 98±25 to 157±22 ml/m² · m²), being significantly higher than in the control group. pHi remained stable only in the dopexamine patients, indicating adequate splanchnic perfusion. Vasopressive regulators of circulation increased significantly only in the untreated control patients (vasopressin: from 4.37±1.1 to 35.9±12.1 pg/ml; ET-1: from 2.88±0.91 to 6.91±1.20 pg/ml). Conclusion: Patients undergoing major abdominal surgery may profit from prophylactic perioperative administration of dopexamine hydrochloride in the form of improved haemodynamics and oxygenation as well as beneficial influence on important regulators of organ blood flow.     

Systemic analgesia adapted to the children's condition

A concept of balanced analgesia using nonsteroidal anti-inflammatory drugs (NSAIDs), paracetamol (acetaminophen), opioids, and corticosteroids can also be used in patients with pre-existing illnesses. NSAIDs are the most effective treatment for acute pain of moderate intensity in children; however, these drugs should be avoided in patients at increased risk for serious side effects, e.g. patients with renal impairment, bleeding tendency, or extreme prematurity. NSAIDs can be given with minimal risks to the younger child with mild to moderate asthma, and, in these patients, the use of steroids can be encouraged; in addition to their antiemetic and analgesic action, a beneficial effect on asthma symptoms can be expected. In the non-intubated child with cerebral trauma, exaggerated sedation caused by opioids and increased bleeding tendency caused by NSAIDs must be avoided. In neonates and small infants, the oral administration of sucrose or glucose is helpful to minimize pain reaction during short uncomfortable interventions.     

A comparison of the inhibitory effects of four volatile anaesthetics on the metabolism of chlorzoxazone, a substrate for CYP2E1, in rabbits

Background: Halothane inhibits in vitro and in vivo activity of cytochrome P-450 (CYP) 2E1. There are several fluorinated volatile anaesthetics besides halothane, and most of them are defluorinated by CYP2E1. It is unclear whether other fluorinated anaesthetics inhibit the in vivo activity of CYP2E1.
Methods: We compared the inhibitory effects of therapeutic concentrations of four inhalational anaesthetics, halothane, enflurane, isoflurane, and sevoflurane, on chlorzoxazone metabolism in rabbits receiving artificial ventilation.
Results: All four inhalational anaesthetics decreased arterial blood pressure and increased plasma chlorzoxazone concentration. However, no significant differences in the plasma chlorzoxazone concentration were found between the four anaesthetics. The estimated chlorzoxazone clearance increased after beginning inhalation with all four agents, but no significant difference in clearance was noted between agents.
Conclusions: At therapeutic concentrations, the in vivo inhibitory effect on chlorzoxazone metabolism was similar for all four inhalational anaesthetics examined, even though their chemical characteristics and extent of hepatic metabolism differ considerably.