Vitamin C improves endothelium-dependent vasodilation in patients with non-insulin-dependent diabetes mellitus.

Endothelium-dependent vasodilation is impaired in humans with diabetes mellitus. Inactivation of endothelium-derived nitric oxide by oxygen-derived free radicals contributes to abnormal vascular reactivity in experimental models of diabetes. To determine whether this observation is relevant to humans, we tested the hypothesis that the antioxidant, vitamin C, could improve endothelium-dependent vasodilation in forearm resistance vessels of patients with non-insulin-dependent diabetes mellitus. We studied 10 diabetic subjects and 10 age-matched, nondiabetic control subjects. Forearm blood flow was determined by venous occlusion plethysmography. Endothelium-dependent vasodilation was assessed by intraarterial infusion of methacholine (0.3-10 micrograms/min). Endothelium-independent vasodilation was measured by intraarterial infusion of nitroprusside (0.3-10 micrograms/min) and verapamil (10-300 micrograms/min). Forearm blood flow dose-response curves were determined for each drug before and during concomitant intraarterial administration of vitamin C (24 mg/min). In diabetic subjects, endothelium-dependent vasodilation to methacholine was augmented by simultaneous infusion of vitamin C (P = 0.002); in contrast, endothelium-independent vasodilation to nitroprusside and to verapamil were not affected by concomitant infusion of vitamin C (P = 0.9 and P = 0.4, respectively). In nondiabetic subjects, vitamin C administration did not alter endothelium-dependent vasodilation (P = 0.8). We conclude that endothelial dysfunction in forearm resistance vessels of patients with non-insulin-dependent diabetes mellitus can be improved by administration of the antioxidant, vitamin C. These findings support the hypothesis that nitric oxide inactivation by oxygen-derived free radicals contributes to abnormal vascular reactivity in diabetes.

Outcomes of patients with chronic renal insufficiency in the bypass angioplasty revascularization investigation.

BACKGROUND: Although severe chronic kidney disease (CKD) is an independent predictor of mortality among patients with coronary artery disease, the impact of mild CKD on morbidity and mortality has not been fully defined. METHODS AND RESULTS: Morbidity and mortality for the 3608 patients with multivessel coronary artery disease enrolled in the Bypass Angioplasty Revascularization Investigation randomized trial and registry were compared on the basis of the presence and absence of CKD, defined as a preprocedure serum creatinine level of >1.5 mg/dL. Seventy-six patients had CKD. Patients with renal insufficiency were older and more likely to have a history of diabetes, hypertension, and other comorbidities. Among patients undergoing PTCA, patients with CKD had a greater frequency of in-hospital death and cardiogenic shock (P<0.05 and 0.01, respectively). There was a trend toward a larger proportion of patients with CKD experiencing angina at 5 years (P=0.079). Patients with CKD had more cardiac admissions (P=0.003 and <0.0001 for patients undergoing PTCA and CABG, respectively) and a shorter time to subsequent CABG after initial revascularization than patients without CKD (P=0.01). CKD was associated with a higher risk of death at 7 years, both of all causes (relative risk 2.2, P<0.001) and of cardiac causes (relative risk 2.8, P<0.001). CONCLUSIONS: CKD is associated with an increased risk of recurrent hospitalization, subsequent CABG, and mortality. This increased risk of death is independent of and additive to the risk associated with diabetes.

Vitamin C improves endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus.

OBJECTIVES: We sought to determine whether the antioxidant vitamin C improves endothelium-dependent vasodilation of forearm resistance vessels in patients with insulin-dependent diabetes mellitus. BACKGROUND: Endothelium-dependent vasodilation is impaired in patients with diabetes mellitus. Oxidatively mediated degradation of endothelium-derived nitric oxide contributes to abnormal endothelium-dependent vasodilation in animal models of diabetes mellitus. METHODS: The study group included 10 patients with insulin-dependent diabetes mellitus and 10 age-matched control subjects. Forearm blood flow was determined by venous occlusion plethysmography. Endothelium-dependent vasodilation was assessed by intraarterial infusion of methacholine (0.3 to 10 microg/min). Endothelium-independent vasodilation was assessed by intraarterial infusion of nitroprusside (0.3 to 10 microg/min). Forearm blood flow dose-response curves were determined for each drug infusion before and during concomitant infusion of vitamin C (24 mg/min). RESULTS: In diabetic subjects, endothelium-dependent vasodilation was augmented by the concomitant infusion of vitamin C (p = 0.001). Endothelium-independent vasodilation was not affected by the concomitant infusion of vitamin C (p = NS). In control subjects, vitamin C infusion did not affect endothelium-dependent vasodilation (p = NS). CONCLUSIONS: Vitamin C selectively restores the impaired endothelium-dependent vasodilation in the forearm resistance vessels of patients with insulin-dependent diabetes mellitus. These findings indicate that nitric oxide degradation by oxygen-derived free radicals contributes to abnormal vascular reactivity in humans with insulin-dependent diabetes mellitus.

Immediate and long-term outcome of intracoronary stent implantation for true bifurcation lesions.

OBJECTIVES: The aim of this study was to evaluate the immediate and long-term outcome of intracoronary stent implantation for the treatment of coronary artery bifurcation lesions. BACKGROUND: Balloon angioplasty of true coronary bifurcation lesions is associated with a lower success and higher complication rate than most other lesion types. METHODS: We treated 131 patients with bifurcation lesions with > or =1 stent. Patients were divided into two groups; Group (Gp) 1 included 77 patients treated with a stent in one branch and percutaneous transluminal coronary angioplasty (PTCA) (with or without atherectomy) in the side branch, and Gp 2 included 54 patients who underwent stent deployment in both branches. The Gp 2 patients were subsequently divided into two subgroups depending on the technique of stent deployment. The Gp 2a included 19 patients who underwent Y-stenting, and Gp 2b included 33 patients who underwent T-stenting. RESULTS: There were no significant differences between the groups in terms of age, gender, frequency of prior myocardial infarction (MI) or coronary artery bypass grafting (CABG), or vessels treated. Procedural success rates were excellent (89.5 to 97.4%). After one-year follow-up, no significant differences were seen in the frequency of major adverse events (death, MI, or repeat revascularization) between Gp 1 and Gp 2. Adverse cardiac events were higher with Y-stenting compared with T-stenting (86.3% vs. 30.4%, p = 0.004). CONCLUSIONS: Stenting of bifurcation lesions can be achieved with a high success rate. However, stenting of both branches offers no advantage over stenting one branch and performing balloon angioplasty of the other branch.

The influence of abciximab use on clinical outcome after aortocoronary vein graft interventions.

OBJECTIVES: The purpose of this study was to evaluate the effect of abciximab use on clinical outcome in aortocoronary vein graft interventions. BACKGROUND: Although large randomized trials have demonstrated a significant benefit of abciximab use in the setting of percutaneous coronary interventions, there is relatively little data with respect to the use of this agent in percutaneous vein graft interventions. METHODS: Three hundred and forty-three patients were identified; 210 undergoing vein graft intervention without abciximab and 133 patients with abciximab. RESULTS: There were differences in baseline clinical and angiographic characteristics between the two groups; advanced age, unstable angina, older vein grafts and thrombus containing lesions were relatively common in both groups. Angiographic and procedural success rates were similar with or without the use of abciximab (89% vs. 92%, p = 0.15, and 85% vs. 91%, p = 0.12, respectively). The in-hospital composite end point of death/Q-wave myocardial infarction (QWMI)/repeat revascularization was similar between the two groups. Utilizing statistical modeling to adjust for baseline differences between the groups, abciximab use did not influence the cumulative long-term composite end point of death/MI/repeat revascularization. CONCLUSIONS: This study demonstrates that in this relatively high-risk population undergoing aortocoronary vein graft interventions, the administration of abciximab periprocedurally does not appear to reduce major adverse clinical events.

Impact of physician experience on triage of emergency room patients with acute chest pain at three teaching hospitals.

PURPOSE: To determine whether the experience of the physician (as measured by postgraduate training level or time during the academic year) who performs the initial evaluation affects the triage of patients with acute chest pain. PATIENTS AND METHODS: Prospective data on the presenting clinical features, initial triage, final diagnosis, and complications were collected for 7,857 patients who presented to the emergency rooms of three teaching hospitals, including 1,118 (14%) with acute myocardial infarction (AMI), 2,477 (32%) with acute ischemic heart disease (AIHD) (i.e., AMI or unstable angina), and 335 (4%) with major complications. The experience of the evaluating physicians, who were in their first three postgraduate years in 93% of cases, was measured in three ways: (1) postgraduate training level, (2) month during the academic year, and (3) number of patients with acute chest pain previously evaluated. Multivariate logistic regression analyses that adjusted for hospital site and 20 clinical variables estimated the odds ratios for admission to the coronary care unit (CCU) and hospital associated with each incremental increase in physician experience. RESULTS: With more experience (as measured by postgraduate training level or time during the academic year), the sensitivity of physicians for admitting patients with AMI, AIHD, or major complications to the hospital increased. For example, each incremental increase in postgraduate training level carried a 1.4 increase in the adjusted odds ratio for admission of a patient with AIHD to the hospital (p less than 0.05), corresponding to an increase in the probability of admission from 93% to 97%. However, increasing physician experience was also associated with an elevated false-positive rate in admitting patients without these diagnoses to the CCU and hospital. Thus, each incremental increase in postgraduate training level carried a 1.2 increase in the adjusted odds ratio for admission of a patient without AIHD to the CCU and hospital (p less than 0.005), corresponding to an increase in the probability of admission from 34% to 47%. By receiver operating characteristic curve (ROC) regression analyses, these changes in triage patterns were consistent with movement along a single ROC curve, rather than a shift to a new or better ROC curve. CONCLUSIONS: As the experience of the physician who performed the initial evaluation increased, there was a lower threshold for admitting all patients with and without AMI, AIHD, or major complications to the CCU and hospital without a detectable improvement in diagnostic accuracy.

Long-term survival of emergency department patients with acute chest pain.

To evaluate the long-term prognosis of patients with acute chest pain, prospective clinical data and long-term follow-up data (mean 30.1 +/- 9.4 months) were collected for 1,956 patients who presented to the emergency department of an urban teaching hospital with this chief complaint. During follow-up of the 1,915 patients who were discharged alive from the emergency department or hospital, there were 113 (6%) cardiovascular deaths. No differences were detected in the post-discharge cardiovascular survival rates after 3 years of experience with patients who were discharged from the emergency department with a known prior diagnosis of angina or myocardial infarction (89%) and patients who had been admitted and found to have acute myocardial infarction (85%), angina (87%), or other cardiovascular diagnoses (87%). Patients who were discharged from either the hospital or the emergency department without cardiovascular diagnoses had an excellent prognosis. Multivariate Cox regression analysis identified 5 independent correlates of cardiovascular mortality after discharge: age, prior history of coronary disease, ischemic changes on the emergency department electrocardiogram, congestive heart failure and cardiogenic shock. These findings indicate that the postdischarge cardiovascular mortality of patients with chest pain who are discharged from the emergency department with a known history of coronary disease is similar to that of admitted patients with angina or myocardial infarction. These data suggest that the same types of prognostic evaluation strategies that have been developed for admitted patients with ischemic heart disease should also be considered when such patients present to the emergency department but are not admitted.

Evaluation of long-term survival after successful percutaneous coronary intervention among patients with chronic renal failure.

We studied the long-term outcomes after percutaneous coronary intervention in dialysis patients and in patients with chronic renal failure (CRF) (serum creatinine > or = 3.0 mg/dl). All-cause mortality at 1 year was 2.9% for the control group, 16.2% for the group with CRF, and 14.1% for dialysis patients. Cardiac mortality at 1 year was 1.9% for ther control group, 15.2% for the group with CRF, and 10.0% for dialysis patients.

Influence of coronary thrombus on outcome of percutaneous coronary angioplasty in the current era (the Mayo Clinic experience).

Earlier studies documented an increased risk of percutaneous coronary intervention (PCI) in patients with angiographic evidence of thrombus. With newer antiplatelet agents and stents, it is not known whether thrombus is a risk factor after PCI. This study examines whether outcome of PCI in patients with thrombus has improved, and whether thrombus is associated with adverse outcome after PCI in the current era. This single-institution retrospective analysis of PCI in 7,184 patients was divided into 2 periods: group I, 1990 to 1995 (n = 3,640), and group II, 1996 to 1999 (n = 3,544). The groups were subdivided according to the presence or absence of angiographic thrombus before PCI. We compared the outcome of PCI for patients with and without thrombus in group II. A comparison was made in the 2 groups in patients with angiographic thrombus. Procedural success improved in group II compared with group I patients with thrombus (93% vs 88%, p <0.001). There was significant reduction in abrupt closure in the recent era in patients with thrombus (4% vs 7%, p = 0.01). In group II, procedural success remained lower in patients with (93% vs 96%) than without thrombus (p <0.001). After adjusting for the significant univariate characteristics of group II patients, thrombus remained an independent predictor of Q-wave infarction (odds ratio 3.78; 95% confidence interval [CI], 1.8 to 8.0; p <0.0013) and the composite end point of death, Q-wave infarction, and emergency bypass surgery (odds ratio 2.37; 95% CI 1.4 to 4.1; p = 0.002). There was a trend toward increased in-hospital death among patients with thrombus (odds ratio 2.06; 95% CI 0.9 to 4.8; p = 0.09). The 1-year outcome after successful PCI was similar for those with and without thrombus. Despite improvement in the outcome of patients with thrombus undergoing PCI in recent years, thrombus is still an independent predictor of adverse in-hospital outcomes after PCI.

NADPH-oxidising activity in lens and erythrocytes in diabetic and nondiabetic patients with cataract.

Levels of lens aldose reductase, aldehyde dehydrogenase activity, and erythrocyte NADPH-oxidising (or glyceraldehyde reductase) activity were determined in 17 diabetic and 16 nondiabetic patients undergoing cataract extraction. Lens aldose reductase and aldehyde dehydrogenase activities were significantly lower in diabetics than in nondiabetics. Both enzymes showed significant inverse correlations with grouped HbA1c and fasting blood glucose levels. By contrast, erythrocyte NADPH-oxidising activity showed a significant positive correlation with grouped HbA1C. It is suggested that a direct effect of the glycaemic status on the lens enzymes is masked by a loss of enzymes secondary to the development of cataract. It is not yet possible to say whether erythrocyte NADPH-oxidising activity can be used to monitor aldose reductase activity in the lens or other tissues in clinical trials of aldose reductase inhibitors.

Bovine lens aldehyde dehydrogenase. Purification and preliminary characterization.

Cytoplasmic aldehyde dehydrogenase from bovine lens was purified to apparent homogeneity by using ion-exchange and affinity chromatography. Sedimentation-equilibrium ultracentrifugation, gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis show that the enzyme is a dimer of Mr 114000, with subunits of Mr 57000. The enzyme does not dissociate into monomers in the presence of Ca2+ or Mg2+. The enzyme has a pI of 5.0, an activation energy of 35.1kJ/mmol and a pK value of 8.6 with acetaldehyde as substrate. The enzyme is a prolate ellipsoid with a Stokes radius of 4nm. Progesterone, deoxycorticosterone and chlorpropamide inhibited enzyme activity, and this inhibition may play a role in cataract formation in patients maintained on systemic corticosteroids and in tablet-dependent diabetics.

Bovine lens aldehyde dehydrogenase. Kinetics and mechanism.

Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.

A coupled enzyme assay for isopenicillin N synthetase.

The development of a coupled enzyme assay for the determination of isopenicillin N synthetase activity in purified extracts from Cephalosporium acremonium was described. Isopenicillin N formed from its precursor, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), by the synthetase was hydrolyzed by beta-lactamase I to the corresponding penicilloic acid. Automatic titration of the acid with standard sodium hydroxide delivered by a pH-stat gave a continuous plot of product formed vs time. This assay has been used in kinetic studies and to determine the effects of pH, ionic strength, and temperature on the enzyme's activity.

Bovine lens aldehyde dehydrogenase: activity and non-linear steady-state kinetics.

Bovine lens aldehyde dehydrogenase is located predominantly in the cortical and nuclear regions, although the specific activity is highest in the epithelial cells. A novel two-step procedure has been used to purify aldehyde dehydrogenase from bovine lens to homogeneity. A comparison using published assay methods for aldehyde dehydrogenases showed that the dimeric lens enzyme had the highest specific activity of any cytoplasmic aldehyde dehydrogenase, although the kcat value was not exceptional. Computer curve-fitting showed that the minimum degree of the rate equation with propionaldehyde and acetaldehyde as substrates was 2:2. The relationship (a2 X b1)/(a1 X b2) was used to show the marked effect of temperature, and to a lesser extent pH, on the non-linear steady-state kinetics. These results indicate that the rate-determining step at low aldehyde concentrations (probably aldehyde binding) is accelerated by increasing temperature to a much greater degree than the rate-determining step at high aldehyde concentration (probably NADH release).

Beta-lactam biosynthesis in a gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154.

The occurrence, localization, and extraction of isopenicillin N-synthase (IPNS) were investigated in the gram-negative low-level beta-lactam producer Flavobacterium sp. strain SC 12.154, which forms deacetoxycephalosporin and excretes the cephabacin 7-formamidocephalosporin. IPNS was detected with anti-IPNS antibodies raised against the Cephalosporium acremonium enzyme. The flavobacterium enzyme, whose molecular mass (38 kilodaltons) and cofactor requirements resemble those of the fungal and Streptomyces enzymes, is formed at the transition from growth to the stationary phase. It was extracted into the polyethylene glycol phase of a polyethylene glycol-Ficoll-dextran three-phase system and was purified by quaternary aminoethyl ion-exchange chromatography, gel filtration, covalent chromatography on cystamine-Sepharose, and fast-protein liquid chromatography on Mono Q. The enzyme was characterized with respect to sulfhydryl requirement, inhibition by disulfides and metal ions, pH and temperature dependence, and stimulation by polyethylene glycol and low Triton X-100 concentrations, as well as by several amino acids, including alpha-aminoadipic acid and cysteine. The Km for alpha-aminoadipyl-cysteinyl-D-valine was 0.08 mM. An inactive membrane-associated form of IPNS was detected together with a beta-lactamase active on isopenicillin N. The system has been suggested as a model for the study of endogenous functions of beta-lactams in bacteria.

Erythrocyte aldehyde dehydrogenase, plasma chlorpropamide concentrations and the chlorpropamide alcohol flush.

Erythrocyte aldehyde dehydrogenase activity (EALDH) was measured in 21 diabetics on long-term chlorpropamide therapy. Median EALDH was 0.362 units, range 0.108 to 0.750 units and correlated neither with previously assessed chlorpropamide alcohol flushing nor with coincident plasma or erythrocyte chlorpropamide concentration. The hypothesis that genetic or permanently acquired reduction in EALDH correlates with CPAF status was not supported. There was no concentration-related inhibition of the enzyme by prevailing plasma or erythrocyte chlorpropamide.

Vitamin C improves endothelium-dependent vasodilation in forearm resistance vessels of humans with hypercholesterolemia.

BACKGROUND: Endothelium-dependent vasodilation is impaired in humans with hypercholesterolemia. Oxidative degradation of endothelium-derived nitric oxide plays a major role in endothelial dysfunction in animal models of hypercholesterolemia. To assess whether this mechanism is relevant to humans, we studied the effect of vitamin C, an antioxidant, on vasodilator function in forearm resistance vessels of patients with hypercholesterolemia. METHODS AND RESULTS: We studied 11 hypercholesterolemic and 12 healthy control subjects. Forearm blood flow was determined by venous occlusion plethysmography. Endothelium-dependent vasodilation was assessed by intra-arterial infusion of methacholine (0.3 to 10 micrograms/min). Endothelium-independent vasodilation was measured by intra-arterial infusion of nitroprusside (0.3 to 10 micrograms/min) and verapamil (10 to 300 micrograms/min). Forearm blood flow dose-response curves were determined for each drug before and during coadministration of vitamin C (24 mg/min). In hypercholesterolemic subjects, endothelium-dependent vasodilation to methacholine was augmented by coinfusion of vitamin C (P = .001); in contrast, endothelium-independent vasodilation to nitroprusside and verapamil were not affected by coinfusion of vitamin C (P = .8 and P = .3, respectively). In control subjects, vitamin C administration did not alter endothelium-dependent vasodilation (P = .2). CONCLUSIONS: We conclude that vitamin C improves endothelium-dependent vasodilation in the forearm resistance vessels of patients with hypercholesterolemia. These findings suggest that nitric oxide degradation by oxygen-derived free radicals contributes to abnormal vascular reactivity in hypercholesterolemic humans.

Purification of isopenicillin N synthetase.

Isopenicillin N synthetase was extracted from Cephalosporium acremonium and purified about 200-fold. The product showed one major protein band, coinciding with synthetase activity, when subjected to electrophoresis in polyacrylamide gel. An isopenicillin N synthetase from Penicillium chrysogenum was purified about 70-fold by similar procedures. The two enzymes resemble each other closely in their Mr, in their mobility on electrophoresis in polyacrylamide gel and in their requirement for Fe2+ and ascorbate for maximum activity. Preliminary experiments have shown that a similar isopenicillin N synthetase can be extracted from Streptomyces clavuligerus.

Acute hyperglycemia attenuates endothelium-dependent vasodilation in humans in vivo.

BACKGROUND: Endothelial function is impaired in patients with diabetes mellitus. However, the factors contributing to this defect are currently unknown. Hyperglycemia attenuates endothelium-dependent relaxation in normal rabbit arteries in vitro and rat arterioles in vivo. Accordingly, this study examined the effect of acute hyperglycemia on endothelium-dependent vasodilation in nondiabetic humans in vivo. METHODS AND RESULTS: Endothelium-dependent vasodilation was assessed through brachial artery infusion of methacholine chloride both before and during 6 hours of local hyperglycemia (300 mg/dL) achieved by intra-arterial infusion of 50% dextrose. Forearm blood flow was determined by plethysmography. In a group of 10 subjects, there was a trend toward attenuated methacholine-mediated vasodilation during hyperglycemia compared with euglycemia (P=.07 by ANOVA; maximal response, 13.3+/-2.8 versus 14.7+/-1.5 mL x min(-1) x 100 mL(-1), respectively). In these subjects, the systemic serum insulin levels increased significantly during the dextrose infusion (P<.001). To eliminate the confounding vasoactive effects of insulin, the protocol was repeated during systemic infusion of octreotide (30 ng x kg(-1) x min(-1)) to inhibit pancreatic secretion of insulin. In these subjects (n=10), hyperglycemia significantly attenuated the forearm blood flow response to methacholine (P<.01 by ANOVA; maximal response, 16.9+/-2.5 before versus 12.7+/-1.8 mL x min(-1) x 100 mL(-1) during hyperglycemia). Methacholine-mediated vasodilation was not attenuated by an equimolar infusion of mannitol (P>.40), nor did hyperglycemia reduce endothelium-independent vasodilation to verapamil (P>.50). CONCLUSIONS: Acute hyperglycemia impairs endothelium-dependent vasodilation in healthy humans in vivo. This finding suggests that elevated glucose may contribute to the endothelial dysfunction observed in patients with diabetes mellitus.

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.