Donald L. Kaminski

Substrates of human hepatic cytochrome P450 3A4

Cytochrome P450 isozyme 3A4 (CYP3A4) is a major isozyme in the human liver and is known to metabolize a larger variety of xenobiotics and endogenous biochemicals. The identities of CYP3A4 substrates are summarized here. A total of 32 chemicals belonging to different structural classes have been evaluated and found to be substrates for CYP3A4. The metabolic pathways for these substrates include N-oxidation, C-oxidation, N-dealkylation, O-dealkylation, nitro-reduction, dehydration, and C-hydroxylation. While the major experimental system used to elucidate the role of CYP3A4 in the metabolic transformation of these substrates is the human liver microsome system, cultured human hepatocytes and yeast/cultured cells genetically engineered to express CYP3A4 are also employed by the different investigators. The common approaches to identify the role of CYP3A4 are also summarized, which include correlation of metabolic activity of the substrates studied with those for known CYP3A4-catalyzed substrates, correlation of activity with CYP3A4 content, inhibition of activity with CYP3A4 specific antibodies, inhibition of activity with known CYP3A4 substrates and inhibitors, induction of activity with CYP3A4 inducers and demonstration of activity with purified CYP3A4 enzyme.

Primary human hepatocytes as a tool for the evaluation of structure—activity relationship in cytochrome P450 induction potential of xenobiotics: evaluation of rifampin, rifapentine and rifabutin

In our laboratory, primary human hepatocytes are being investigated as an in vitro experimental system for the evaluation of pharmacokinetic drug-drug interactions. Our study here represents the first reported study that directly compares the cytochrome P450 isozyme 3A (CYP3A) induction potential of three antimicrobials derived from rifamycin B, namely, rifampin, rifapentine and rifabutin. Two endpoints of CYP3A activity, testosterone 6 beta-hydroxylation and midazolam 1-hydroxylation have been used. Results obtained with hepatocytes from four different human donors show consistently that rifampin and rifapentine are potent inducers of CYP3A, while a significantly lower induction potential is observed for rifabutin. The relative induction potency of the three antimicrobials (rifampin > rifapentine >> rifabutin) is consistent with the available human in vivo data. For CYP1A measured as ethoxyresorufin O-deethylase activity, CYP2C8/9 measured as tolbutamide 4-hydroxylation activity, CYP2D6 measured as dextromethorphan O-demethylation, and AZT glucuronidation, there is either no effect or, where induction is found to be statistically significant in these other endpoints, the maximum induction values are consistently < 100% of the control. Our results suggest that CYP3A is the major CYP induced by these rifamycin B derivatives. These studies illustrate the application of human hepatocytes in the evaluation of the structure-activity relationships in CYP induction for this class of chemicals and as an in vitro screen for drug-drug interaction potential via CYP induction.

Hypercarbia during carbon dioxide pneumoperitoneum

Patients with cardiopulmonary insufficiency undergoing laparoscopic surgery with carbon dioxide (CO2) pneumoperitoneum may retain CO2 resulting in clinically significant respiratory acidosis. A canine model of pulmonary emphysema induced by papain inhalation was utilized to evaluate the respiratory effects of both CO2 and helium pneumoperitoneum. Prior to papain inhalation and 5 and 8 weeks after initial treatment under general anesthesia, mechanical ventilation was adjusted to maintain the end-tidal CO2 (ETCO2) at 40 mm Hg during baseline and pneumoperitoneum physiologic monitoring periods. Utilizing an analysis of variance, hemodynamic and respiratory physiologic parameters were compared. In this canine model, all dogs demonstrated consistent hypercarbia during CO2 pneumoperitoneum prior to papain treatments, but CO2 retention was significantly increased in the emphysematous state. The occurrence of hypercarbia during CO2 pneumoperitoneum may be underestimated by ETCO2 monitoring as was revealed by an increased PaCO2 (arterial carbon dioxide pressure)-ETCO2 gradient with an increasing time interval between papain exposure and period of physiologic monitoring. Irrespective of the pulmonary condition of the dog, helium pneumoperitoneum did not produce any hypercarbic or acidic changes when compared with the concomitant baseline period of dogs prior to the induction of pneumoperitoneum, thus suggesting that helium pneumoperitoneum may be a reasonable alternative in patients at risk for CO2 retention.

Ischemic colitis: spectrum of disease and outcome.

BACKGROUND The aim of this study was to identify risk factors, clinical characteristics, and outcome of patients with colon ischemia. METHODS A 10-year (1992-2002) retrospective study was undertaken. Patients were identified from computerized hospital discharge information. Patient variables were entered into a computerized database and analyzed. RESULTS One hundred twenty-nine patients were identified. The mean age was 66 years (range, 29-98 years); 47% were male. Forty-three patients (33%) had chronic renal failure; 73 patients (57%) were receiving vasoactive drugs, and 72 patients (56%) had atherosclerosis. Fifty-four of 129 patients (42%) had ischemic colitis in-hospital. Fifty-six of 129 patients (43%) had melena; 49 of 56 patients (88%) survived. Forty-three of 129 patients (33%) had an acute abdomen; 22 of 43 patients (51%) died. Seventy of 129 patients (54%) were treated nonoperatively initially; the condition of 17 of 70 patients (24%) required surgery. Of 76 patients who were treated operatively, 31 patients (41%) died. Eleven patients at operation had ischemia without colon infarction or perforation; 5 of these patients (45%) died. The overall mortality rate was 29% (37/129 patients). CONCLUSION Ischemic colitis is associated with chronic renal failure and atherosclerosis. Patients commonly have an acute abdomen. The absence of colonic infarction does not ensure a favorable outcome. Patients who are felt to be candidates for nonoperative therapy have significant mortality rates. Mortality rates remain high, despite treatment.

Isolation and culturing of hepatocytes from human livers

Using a modified collagenase digestion procedure, we were successful in the isolation of viable hepatocytes from human liver surgical wastes, unused transplantable livers, and diseased livers from transplant recipients. The modified procedures for isolation involved collagenase perfusion of a sample of limited size (<50 g) with only one cut surface, perfusion via only one blood vessel, the use of an appropriate amount of collagenase, as well as manual “massaging” of the liver sample during critical stages of the collagenase perfusion. Using this modified procedures, we had close to 100% success rate in the isolation of viable hepatocytes from the human liver samples.

Acute surgical emergencies in inflammatory bowel disease

BACKGROUND Acute surgical emergencies in patients with inflammatory bowel disease may carry a substantial morbidity, but fortunately today, a low mortality. The aim of this review is to delineate the treatment of acute surgical emergencies that occur in patients with ulcerative colitis and Crohns disease. METHODS Suitable English language reports were identified using PubMed search. RESULTS Inflammatory bowel disease can present in numerous ways as an acute surgical emergency. These include toxic colitis, hemorrhage, perforation, intra-abdominal masses or abscesses with sepsis, and intestinal obstruction. Toxic colitis and perforation are best managed with intestinal resection and fecal diversion. Hemorrhage in ulcerative colitis initially requires colectomy with rectal preservation and ileostomy. In Crohns disease hemorrhage is often focal and localization and segmental resection are performed. Intra-abdominal abscesses should initially be attempted by computed tomography-guided percutaneous drainage followed subsequently by definitive resection. Perianal disease requires abscess drainage with minimal tissue trauma. Intestinal obstruction should be initially managed nonoperatively, with surgery reserved for complete obstruction or intractability. CONCLUSIONS Acute surgical emergencies in patients with inflammatory bowel disease are rare and can have a high morbidity. With a multidisciplinary approach, morbidity can be reduced and patients can have a rapid return and improved quality of life.

2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes

We report here a novel observation that 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) induced predominantly cytochrome P4501A1 (CYP1A1) in rat hepatocytes and predominantly CYP1A2 in human hepatocytes. As part of our research program to evaluate species-differences in response to CYP inducers, we studied the effects of TCDD on CYP1A activity, protein, and gene expression in primary cultures of rat and human hepatocytes. TCDD was found to induce CYP1A activity, measured as ethoxyresorufin-O-deethylase (EROD) activity, in both rat and human hepatocytes. TCDD induction of EROD activity in human hepatocytes (2-5 fold of concurrent solvent control), was significantly lower than that found in rat hepatocytes ( 20-fold of concurrent solvent control). Two structural analogs of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF), were also evaluated. As observed for TCDD, human hepatocytes consistently showed a lower response than rat hepatocytes. As most TCDD-related effects are believed to be mediated via binding of the TCDD-Ah receptor (AhR) complex to DNA, nuclear AhR levels were measured in rat and human hepatocytes after TCDD treatment. We found that the nuclear AhR levels in TCDD-treated rat hepatocytes were approximately 4 times higher than found in TCDD-treated human hepatocytes. However, the estimated binding affinity of [3H]TCDD to nuclear AhR from rat hepatocytes was similar. The species difference in response to TCDD was further evaluated by analysis of CYP1A1 and CYP1A2 mRNA levels using Northern analysis, and P4501A1 and 1A2 protein levels using Western immunoblotting. Results showed that, at both gene expression and protein levels, TCDD induced predominantly CYP1A1 in rat hepatocytes and CYP1A2 in human hepatocytes.

Spontaneous and shock-associated ischemic colitis

Ischemic changes in the colon that progress to gangrene present diagnostic and therapeutic difficulties associated with poor survival. During the past 10 years, 36 patients with colonic ischemia were treated. Two clinical groups were evident. The spontaneous ischemic colitis group (type I) included 17 patients who were well before the onset of gastrointestinal symptoms. The cause of type I ischemic colitis is not apparent; it is attributed to occlusive or nonocclusive ischemia. The other group (type II) included 19 patients who developed ischemia of the colon associated with shock secondary to various disease processes. Radiographic evidence of distended bowel correlated well with full-thickness necrosis as determined clinically or pathologically. Twenty-one patients died, for a mortality rate of 58 percent. While full-thickness gangrene was fatal in 71 percent of the patients, mucosal necrosis only was associated with an 88 percent survival rate. The results of treatment should improve with an increased awareness of ischemic colitis, earlier appropriate operative intervention, and more appropriate use of ileostomy and colostomy.

Prospective comparison of helium versus carbon dioxide pneumoperitoneum

BACKGROUND During prolonged laparoscopic operations with carbon dioxide (CO2) pneumoperitoneum (PP), hypercapnia with significant acidosis has been reported to occur in some patients with pulmonary dysfunction. An alternate inert insufflation gas like helium (He) could avoid this problem. METHODS This prospective, IRB-approved study compared the cardiopulmonary response in 20 patients with both CO2 and He PP. With the minute ventilation held constant, baseline arterial blood gases and ventilatory and cardiac parameters were obtained after anesthetic induction but prior to CO2 PP. All values were repeated at 20 to 30 and 40 to 60-minute intervals after the insufflation of CO2 PP, then again during He PP. Values were compared by a paired t test analysis. RESULTS Patients experienced significant hypercapnia during CO2 PP when compared with baseline arterial blood gases, but all values returned to baseline levels during He PP. CONCLUSIONS He PP is an effective alternative to CO2 PP for a laparoscopic cholecystectomy avoiding CO2 retention and subsequent acidosis. Carbon dioxide retention may be dangerous in patients with pulmonary dysfunction who undergo laparoscopy.

Comparison of Double Indicator Thermodilution Measurements of Extravascular Lung Water (EVLW) with Radiographic Estimation of Lung Water in Trauma Patients

The availability for clinical use of a simple reliable method of determining extravascular lung water (EVLW) provided us with the opportunity to evaluate the accuracy of the plain chest roentgenogram in estimating EVLW in patients with severe trauma. Twelve patients who sustained blunt trauma in motor vehicular accidents were studied. Interval measurements of EVLW were made utilizing the thermal-green dye, double-indicator dilution technique and the results compared in a blind manner to estimation of lung water content on standard chest roentgenograms. The results indicate a significant correlation was present when EVLW was compared to roentgenographic density grade. Considerable overlap existed in quantitative EVLW content between radiographic grades except when extensive consolidation was present on the chest radiograph which was associated with EVLW measurements of greater than 10 ml/kg. The random comparison of a change in measured EVLW with a change in radiographic density indicated that there were opposite changes in 23% of the comparisons. However, in selected patients with progressive improvement (decrease) in EVLW or gradual deterioration (increase) in EVLW over time there was good correlation between improvement or deterioration in the chest radiographic appearance with the decrease or increase in EVLW. There is some ability to ascertain in trauma patients interstitial fluid accumulation from chest radiographs, but attempts at quantitation would be fraught with considerable error.