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Metformin, an antihyperglycemic, is widely used in the treatment of type 2 diabetes mellitus (DM). A rare, but important complication associated with this drug is the development of lactic acidosis: Overall mortality of lactic acidosis is approximately 50%. Certain subsets of patients taking metformin are at greater risk of developing lactic acidosis. This report discusses the development of metformin-associated lactic acidosis in four older adults admitted to an institution during a 2-month period, treatments, and outcomes. We recommend an aggressive treatment strategy of hemodialysis followed by peritoneal dialysis, continuous bicarbonate infusion, and tight glucose control. We review the cautions and contraindications of metformin use for the treatment of type 2 DM and report an educational plan for residents and staff instituted to improve drug complication awareness and reduce mortality.

Blood loss in patients undergoing radical prostatectomy may be substantial. In a randomized, prospective study, we assessed two methods of reducing the need for allogeneic blood transfusion with regard to efficacy and costs. Sixty patients undergoing retropubic radical prostatectomy were allocated randomly to one of three groups. In group 1 (n = 20), acute normovolaemic haemodilution (ANH) was initiated after induction of anaesthesia; autologous blood 15 ml kg-1 was withdrawn and replaced by colloid solutions (gelatin) to maintain haemodynamic stability. In group 2 (n = 20), controlled hypotension was established using sodium nitroprusside (target mean arterial pressure (MAP) approximately 50 mm Hg). Group 3 (n = 20), without manipulations, served as a control group. Troponin T (TnT), a sensitive marker for myocardial ischaemia, and various coagulation variables were measured in the perioperative period. Packed red blood cells (PRBC) were given when haemoglobin concentration was less than 7 g dl-1. Cost calculations did not include hospital overhead costs or staff costs. In the ANH group, mean 1278 (SD 150) ml of autologous blood were withdrawn. Significantly more volume was infused in the ANH patients (gelatin 2450 (550) ml) than in the two other groups. Coagulation data (platelet count, activated partial thromboplastin time (aPTT), fibrinogen, antithrombin III (AT III), D-dimers) did not differ significantly between the three groups. The hypotension group had significantly lower blood loss (1260 (570) ml), whereas the ANH (1820 (680) ml) and control group (1920 (590) ml) did not differ significantly. Patients in the hypotension group needed significantly less PRBC (total 14 units; 75% of patients did not need PRBC) than the ANH (total 21 units; 55% of patients did not need PRBC) or control patients (total 28 units; 40% of patients did not need PRBC). Total costs were lowest in the hypotension group (41% less than in the control patients) (P < 0.05). We conclude that the use of hypotension during radical prostatectomy resulted in approximately 40% reduction in total transfusion costs. ANH was less effective and more costly than controlled hypotension.

Objective: The survival of compromised skin flaps depends on neovascularization for their nutrition and metabolic waste removal. Our study investigated the effectiveness of angiogenic factors in accelerating peripheral neovascularization and in increasing skin flap viability., Design: We elevated pedicled dorsal skin flaps on the ears of 23 New Zealand white rabbits, and the vascular pedicle was ligated to achieve partial flap necrosis. Fifteen flaps were treated with 0.1 micrograms/mL vascular permeability factor, and eight flaps were treated with 1.0 micrograms/mL tumor necrosis factor alpha. The ear flaps that were not treated with growth factor functioned in each rabbit as a normal saline control., Main Outcome Measures: The viability of skin flaps was observed visually and was measured by Cartesian planimetry using templates. Neovascularization was documented by microangiography and by histologic analysis of the flaps., Results: Although the angiogenic factors accelerated neovascularization, increased flap survival was demonstrated only in those animals treated with vascular permeability factor that was supplied by an absorbable gelatin sponge., Conclusion: This experimental model, despite different levels of controls, contains multiple variables, including the use of an absorbable gelatin sponge, seroma formation, bioactivity of the angiogenic factors, optimal dosages and dosimetry, the need for a "blinded" format, and the validity of the histologic analysis. Additional investigation must be done and the experimental model itself must be improved before these apparently positive results may be accepted as clinically useful.

Activities of the anti-oxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase were studied in rat tissues to determine the ability of detergents both to solubilize the enzymes and also to stabilize enzyme activity. Rat brain, heart and liver were homogenized in 0.1M KCl, 0.1% sodium dodecyl sulfate, 0.1% lubrol, or 0.1% cetyl-trimethylammonium bromide. In general lubrol was more effective than the other solutions in solubilizing GPx and catalase. Lubrol and 0.1M KCl were equally effective in solubilizing SOD. The highest enzyme activities were (1) SOD: 2484 ng/mg (brain), 2501 ng/mg (heart), and 5586 ng/mg (liver); (2) GPx: 224 mU/mg (brain), 1870 mU/mg (heart), and 7332 mU/mg (liver); (3) catalase: 2.8 mU/mg (brain), 10.6 mU/mg (heart), and 309 mU/mg (liver). While cetyl trimethylammonium bromide is marginally better than sodium dodecyl sulfate in solubilizing active enzyme, neither ionic detergent has any advantage over lubrol or 0.1M KCl. For catalase and GPx, enzyme activity loss with time is biphasic. After initial, rapid activity loss (1-5 days for GPx and 7-10 days for catalase) the differences noted among the homogenizing solutions disappear and very little if any activity loss is noted over the next 2-3 weeks. For catalase and GPx, only baseline enzyme activity from t = 0-3 weeks is found in the most chaotropic solution, 0.1% sodium dodecyl sulfate while biphasic activity loss is most pronounced in 0.1% lubrol. These results may indicate active GPx and catalase species stabilized by a lipid-like environment. Correlating in vitro catalase or GPx measurements with in vivo anti-oxidative protection may underestimate tissue defences.

Membrane abnormalities and a shortened life span are closely associated with the progressive cardiomyopathy of dystrophic hamsters. In the present work we investigate whether this membrane damage is associated with changes in the primary membrane defences (the anti-oxidative enzymes). We measured the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH.Px), and catalase (CAT) in hearts of normal and cardiomyopathic (CHF 147) hamsters, aged 17 days to 12 months. In normal hearts all the enzyme activities follow a U-shaped curve: unweaned animals have 20-40% higher enzyme activities and 11-month-old hamsters 50-160% higher activities than adolescent or adult hamster hearts. Changes in this age-related pattern of enzyme activities are seen in dystrophic hearts in all but the 17-20-day-old animals. At 30 days of age and older, GSH.Px activities are decreased and SOD and CAT activities increased in cardiomyopathic hamsters compared to normal animals. SOD, while elevated, seems less affected than GSH.Px and CAT as the disease progresses. The changes in both absolute activities and ratio of activities of the anti-oxidative enzymes parallel the changes in the cardiomyopathic pathology. This work supports the view that the progressive cardiomyopathy of CHF 147 hamsters may be associated with changes in primary membrane defenses.

Hyperthermia induces the synthesis of the 71-kDa heat-shock protein (heat-shock response) in all rat tissues, including heart. We examined whether induction of the heat-shock response alters the response of isolated hearts to ischemia and reperfusion. Anesthetized male rats were pretreated with 15 min of hyperthermia (42 degrees C) and then recovered for 0, 24, 48, 96, or 192 h. Hearts were isolated from control and hyperthermia-treated rats and retrogradely perfused. Greatest recovery occurred in 48-h postheat-shock hearts; after 30 min of reperfusion there was a 38, 62, and 62% recovery of force, +dF/dt, and -dF/dt, respectively, and 17, 36, and 30% recovery, respectively, for the control hearts. Creatine kinase efflux during reperfusion was reduced by 75% for 24-h postheat-shock hearts. The antioxidative enzyme catalase was increased 24, 48, and 96 h posthyperthermia. Treatment of rats with 3-amino-1,2,4-triazole (1 g/kg body wt), which irreversibly inactivates catalase, 30 min before isolation of hearts, abolished the hyperthermia-induced enhancement of postischemic recovery. These results show a strong relationship between the acquisition and decay of the enhanced postischemic ventricular recovery and the hyperthermic induction of the heat-shock response indicated by the accumulation of heat-shock protein HSP71 (mol mass 71 kDa) and the increase in catalase activity.

When isolated rat heart mitochondria are subject to xanthine/xanthine oxidase generated free radicals, nmol quantities of ADP are phosphorylated to ATP. This effect is proportional to xanthine oxidase concentration, and is relatively independent of ADP concentration. Exogenous superoxide dismutase partially suppresses the phosphorylation. Micromolar concentrations of iron salts completely eliminate the phosphorylation. Catalase has no effect. The likely electron source, then, is superoxide radicals. The reduced minus oxidised spectra of superoxide-bombarded mitochondria show that superoxide enters the electron transport chain by reducing cytochrome c and complex IV. Mitochondria retain their ability to phosphorylate ADP in more traditional ways under the experimental conditions described. Superoxide under physiological conditions in vivo may be a source of electrons for the oxidative phosphorylation of ADP.

The ultraviolet (uv) difference spectra of M(II)-apocarbonic anhydrase at pH 5-9 are reported. For Zn(II) at all pH's and Co(II) at pH greater than or equal to 7.65 identical protein difference spectra are seen and a positive 300 nm feature is interpreted as consistent with interaction of a metal-bound hydroxyl with a Trp chromophore near the active site. Hg(II), Cu(II), and Cd(II) do not provoke a positive 300 nm band even at alkaline pH (although a Cd(II) spectral band at 300 nm becomes less negative, i.e., more like the holoenzyme with increasing pH) and the 280-292 nm spectral region is generally different from that of Zn(II) and high pH Co(II). A specific orientation of M-OH and, hence, an ordered solvent structure in the enzyme site is implied for enzyme activation. Ni(II) appears to bind to the vacated zinc site slowly, at low pH, in a manner similar to zinc. At higher pH's Ni(II) may be displaced toward a Tyr residue in the active site of apocarbonic anhydrase.

The interaction of lead (Pb2+) ions with apocarbonic anhydrase is studied by u.v. difference spectroscopy. The Pb2+ results are compared with those of Zn2+ interacting with apocarbonic anhydrase. The results of both metals interacting with apoenzyme are related to the difference spectra of Pb2+ and Zn2+ with Trp and Tyr model compounds. The interaction of Pb2+ with apocarbonic anhydrase containing an acetylated tyrosine residue is examined. Evidence is presented that Pb2+ is located in the active site cavity of apocarbonic anhydrase, but displaced from the zinc His ligands, interacting with Tyr 7. An attempt is made to rationalize the metal-chromophore micro-environment based on analysis of the 270-295 nm u.v. region of the difference spectra.

In cells, hyperthermia induces synthesis of heat-shock proteins and the acquisition of thermotolerance. Thermotolerant cells are resistant to subsequent oxidative stress. In this study, heat-shocked hearts were examined for evidence of protection during ischemia and reperfusion. Rats were exposed to 15 minutes of 42 degrees C hyperthermia. Twenty-four hours later their hearts were isolated and perfused and the contractility examined during and after ischemic perfusion. No protection was observed during ischemic perfusion. However, upon reperfusion heat-shocked hearts had recovery of contractility within 5 minutes of reperfusion, while control hearts showed no contractility at this time. Throughout 30 minutes of reperfusion heat-shocked hearts had significantly improved recovery of contractile force, rate of contraction and rate of relaxation. Creatine kinase release, associated with reperfusion injury, was significantly reduced from a high of 386.8 +/- 78.9 mU/min/g heart wt for controls to 123.7 +/- 82.9 mU/min/g heart wt for heat-shocked hearts at 5 minutes of reperfusion. Following 30 minutes of reperfusion, ultrastructural examination revealed less damage of mitochondrial membranes in the heat-shocked hearts. Further biochemical investigations revealed that the antioxidative enzyme, catalase, was significantly increased to 137 +/- 12.7 U/mg protein in the heat-shocked hearts while the control value was 64.8 +/- 8.3 U/mg protein. Hyperthermic treatment, which induces the heat-shock response, may be therapeutic for salvaging ischemic myocardium during reperfusion, through a mechanism involving increased levels of myocardial catalase.

Using UV absorption spectroscopy, first derivative spectroscopy, and UV difference spectroscopy, the active site of human superoxide dismutase is probed. First derivative spectra (dA/d lambda versus lambda) show the HESOD spectrum to be a composite of Phe and Trp absorbance. The 278 and 288 nm Trp absorbance peaks are sensitive to solvent polarity. A 5-10% decrease in these peaks accompanies copper removal from the active site indicating greater solvent access to Trp in the apoenzyme than the holoenzyme. A Trp UV difference peak at 305-310 nm documents the presence or absence of copper at the active site, and documents also the movement of a nonbridging copper-binding His (His 46 or 120) when HESOD is inhibited by azide or when the copper moiety is reduced. Trp absorbances indicate that neither cyanide nor KCl inhibition affects the Cu(II)-His bonds. Phe UV absorbance is increased by the presence of copper at the active site and increased further by the addition of cyanide or azide. Neither Trp nor Phe responds to the presence of zinc in the active site. A molecular graphics program, FRODO, shows Trp and the four Phe residues lying in an approximate ring around the active site of HESOD and thus excellently placed to report on active site perturbations.

Lead-substituted bovine carbonic anhydrase is investigated and the return to the holoenzyme form with exchange of Pb2+ by Zn2+ is followed by uv difference spectroscopy and by esterase activity methods. Equimolar amounts of Pb2+ added to apocarbonic anhydrase release one hydronium ion per molecule below pH 6. Above this pH there is a net gain of hydronium ions by the enzyme, due to Pb(OH)+----Pb(OH2)2+, when the metal is bound within the active site of the enzyme molecule. The reduced hydrolysis by lead when it is bound to the enzyme is relevant to the theory of Zn2+ hydrolysis as a mechanism for carbon dioxide hydration by the holoenzyme and to the idea of an altered pKhydrolysis when Zn2+ is bound in the enzyme active site cavity. Lead appears to be bound to a His residue in the active site and to interact with a Tyr residue nearby. The Tyr interaction is disrupted by a high concentration of chloride ions, (also by lower concentrations of cyanide ions), but such anions do not displace lead from the enzyme. At pH 8.0 the buffer-free exchange of Pb2+ by Zn2+ is found to be consistent with a second-order process with an effective beta = (95 +/- 7) M-1 sec-1. Thus lead is more rapidly replaced by zinc than is Mn2+ or VO2+, whose replacement kinetics have been reported by others. Comparison of esterase-activation and spectral curves with second-order models shows that the effective beta is both large and buffer dependent, indicating that a proton transfer process or buffer anion effects may be rate limiting in the buffer-free case.

The effect of phenylmercury and methylmercury on rat liver glutathione peroxidase (GSH X Px) is investigated and compared with that of Hg(II) and with some previously reported results for Cd(II). Analysis of the kinetics of metal binding to the enzyme gives apparent inhibition rate constants: kc = 9.7 mM-1 min-1 for all three mercury compounds and 75 mM-1 min-1 for CdCl2. Glutathione (0.2 mM) protects the enzyme from metal inhibition, decreasing the apparent inhibition rate constants (kc) by 3.6 times for mercury compounds and 4.4 times for CdCl2. KI for the three mercury compounds is found to be 53 microM. It is unexpected that the same value of KI exists for all three forms of mercury studied and that inhibition of the enzyme by the metals is a relatively slow process. For Cd(II) the value of KI is 8.5 microM. It is suggested that inhibition of GSH X Px enzyme activity by cadmium, mercury, and organic mercury salts may not be due to simple complexation of the active site selenium moiety but may be due to a slower process, e.g., an alteration of the enzyme tertiary or quaternary structure.