Hemodynamic effects of S-nitrosocysteine, an intravenous regional vasodilator.

BACKGROUND: S-nitrosocysteine is a carrier form of nitric oxide that can be delivered intravenously. S-nitrosocysteine is rapidly metabolized by plasma (half-life = 2-3 seconds), forming nitric oxide and cysteine. With its short half-life and potent vasodilatory properties, S-nitrosocysteine may be useful as a pulmonary vasodilating agent in cases of postoperative and chronic pulmonary hypertension. OBJECTIVE: Our objective was to determine the hemodynamic properties of S-nitrosocysteine on the pulmonary and systemic circulations to assess its potential utility as a pulmonary vasodilatory agent. METHODS: Eleven adult swine were anesthetized. Thermodilution (Swan-Ganz; Baxter International, Inc, Deerfield, Ill) and arterial catheters were inserted. Flow probes were placed around the coronary, renal, superior mesenteric, and iliac arteries. Incremental infusion doses of S-nitrosocysteine (5-80 nmol. kg(-1). min(-1)) were delivered into the right atrium. Cardiac output, right and left heart pressures, heart rate, Pao(2), and iliac, renal, coronary, and mesenteric blood flow rates were recorded at baseline and at each infusion dose of S-nitrosocysteine. RESULTS: Low-dose S-nitrosocysteine infusion decreased mean pulmonary artery pressure (15%, P =.013) without a significant reduction in mean systemic artery pressure. Higher dose infusions produced further dose-dependent declines in pulmonary vascular resistance and measurable reductions in systemic vascular resistance (P =.01). At an S-nitrosocysteine dosage of 40 nmol. kg(-1). min(-1), there was a significant reduction in renal (P <.001) and mesenteric (P =.003) blood flow but no change in iliac (P >.2) or coronary (P >.2) blood flow. Cardiac output remained constant up to infusion rates of 40 nmol. kg(-1). min(-1) (P >.2). Doses higher than 5 nmol. kg(-1). min(-1) resulted in a substantial dose-dependent reduction in Pao(2) (P <.001), suggesting dilation of atelectatic areas of the lung. CONCLUSION: S-nitrosocysteine is a potent vasodilatory agent capable of overcoming the hypoxic vasoconstrictive response of the lung. Our results suggest it may prove useful as a pulmonary vasodilatory agent at low doses. Higher dose infusions reduce mean systemic pressure and lead to compensatory reductions in renal and mesenteric blood flow without a decrease in cardiac output.

Nitrosothiol quantification in human plasma.

A high-pressure liquid chromatography (HPLC) assay for measuring picomole quantities of nitrosothiol in biological samples was developed. The assay utilizes the catalytic reduction of nitrosothiol by mercuric cation (Hg2+). Released nitrogen oxide reacts with sulfanilamide (SA) and N-(1-napthyl)ethylenediamine (NNED) to form a stable azo dye. The azo dye is then separated from N-(1-napthyl)ethylenediamine and quantified by reversed-phase HPLC. In addition to nitrosothiol, nitrite and atmospheric nitrogen oxides are sources of nitrogen oxide that react with the reagents, SA and NNED, to form the azo dye. Therefore, a reference sample, which includes the nitrosothiol sample and all reagents except Hg2+, is utilized for the subtraction of nitrite and atmospheric nitrogen oxides which "contaminate" the nitrosothiol sample and reagents. This method is a sensitive (approximately 3 pmol; approximately 10(-1) microM) and accurate means to measure nitrosothiol concentration in biologic samples.

Risk, diagnosis and management of prosthetic valve endocarditis: a review.

Prosthetic valve endocarditis (PVE) emerged approximately 37 years ago when the first human heart valve replacements were performed. PVE can be classified as 'early' or 'late' with the pathophysiology and etiologic organisms varying between the two subgroups. The incidence of PVE ranges up to 0.5% per patient-year for mechanical mitral valves and up to 1.0% per patient-year for other valves. The clinical presentation is similar to that of native valve endocarditis, with fever being the most prevalent sign. Diagnosis is based on a constellation of clinical signs and symptoms as well as echocardiographic evaluation of the valve and perivalvular tissues. An algorithm is set forth for diagnosis and management of patients with suspected PVE based on our personal experience and the published literature. Indications for surgery, the surgical approach and methods of PVE prophylaxis and prevention are discussed.

Functional outcome after surgical treatment of esophageal perforation.

BACKGROUND: The functional results after treatment of intrathoracic esophageal perforations have been poorly documented. METHODS: A retrospective review of 42 patients who underwent treatment of intrathoracic esophageal perforation associated with benign esophageal disease was performed. RESULTS: Of 42 patients treated for esophageal perforation, 25 underwent primary repair, 15 underwent esophagectomy and reconstruction, 1 underwent cervical esophagostomy and drainage followed by esophageal resection, and 1 had drainage alone followed by primary repair. Among the patients treated with primary repair, at least one additional operation was required in 13 patients. Of the 15 patients treated with esophagectomy and reconstruction, none required further operative treatment. Follow-up averaged 3.7 years, and of the 36 survivors available for follow-up, 18 (50%) required at least one esophageal dilation postoperatively, and 3 (8.3%) have required regular dilations. Subjectively, 19 of 36 patients (53%) indicate that their swallowing is better than before perforation, it was the same in 12 (33%), and worse in 4 (11%). CONCLUSIONS: In conclusion, approximately one third of patients surviving primary repair of esophageal perforations have continued difficulty with swallowing, which often requires esophageal dilations or esophageal reconstructive procedures, or a combination of both. Optimal long-term results are achieved when primary repair is performed in patients with motor disorders or a "normal" esophagus. Esophagectomy is a better option in those patients with strictures or diffuse esophageal disease.

Infective endocarditis: ten-year review of medical and surgical therapy.

BACKGROUND: Infective endocarditis is a complex disease process. Optimal outcome often requires both medical and surgical expertise. The need for and timing of surgical intervention is controversial and continues to evolve in parallel to advancements in diagnosis and treatment. Our experience with the treatment of infective endocarditis is reviewed herein. METHODS: A retrospective review was compiled of 140 consecutive patients who fulfilled the modified von Reyn criteria for the diagnosis of endocarditis between January 1982 and April 1992. RESULTS: Patient characteristics, symptoms, and risk factors are described. Follow-up averaged 3.5 +/- 0.8 years and totaled 491 patient-years. New York Heart Association functional class at presentation had a significant influence on survival (p < 0.0001). Long-term survival was significantly greater (p = 0.036) in patients treated medically/surgically than those treated with medical therapy alone (75% versus 54% at 5 years). Medical treatment of aortic and prosthetic endocarditis was associated with higher mortality (58% and 67%, respectively) when compared with combined medical/surgical treatment (28% and 38%, respectively). Among the survivors, New York Heart Association class at follow-up was better (p < 0.0001) in the medical/surgical group (1.05 +/- 0.04) versus the medical treatment group (1.70 +/- 0.14). CONCLUSIONS: Combined medical/surgical treatment for infective endocarditis is associated with improved survival. Patients with aortic or prosthetic endocarditis are identified as subgroups that benefit most from surgical intervention. Valvular dysfunction incited by the infective process is an important factor that should be weighed carefully in the therapeutic decision.

New concepts in the pathophysiology of oxygen metabolism during sepsis.

Sepsis is an intriguing pathological condition associated with many complex metabolic and physiological alterations. In this review a novel hypothesis in the pathophysiology of oxygen metabolism during sepsis is explored. It is proposed that the hypermetabolic response to sepsis results from enhanced reactive oxygen generation by phagocytes. Reactive oxygen detoxification by host enzyme systems subsequently leads to alterations in oxidative metabolism. The similarities between the metabolic consequences of reactive oxygen metabolism and the metabolic changes observed during sepsis are outlined. A unified concept is presented to help explain the pathophysiological changes in oxygen metabolism during sepsis.

Use of autologous umbilical artery and vein for vascular reconstruction in the newborn.

Autologous umbilical artery and vein were evaluated as vascular conduits in newborn lambs. Eight newborn lambs were delivered transabdominally under sterile conditions at term. The umbilical artery and vein were dissected from the cord and stored in culture media. On the same day, each lamb underwent bilateral superficial femoral artery transection and reconstruction. Nine arteries were reconstructed with autologous umbilical vein interposition grafts, five with umbilical artery interposition grafts, and two by primary native artery anastomosis. After the birth weight of the lambs quadrupled (37 to 45 days), they were killed and all grafts and anastomoses were examined grossly and histologically. At the conclusion of the study, both native artery anastomoses (2/2) were patent. Five umbilical vein (5/9) and two umbilical artery (2/5) autografts were also widely patent. Patent autografts retained an intact endothelium supported by a viable media. The nonpatent autografts had become atrophic remnants displaying histologic signs of early closure. Graft failures are attributed to the extreme vasoactive nature of the umbilical vessels. These preliminary results suggest that umbilical vessels may be useful as a vascular autograft if the vasoactive nature of these vessels can be overcome during the immediate perioperative period.

Role of reactive O2 in phagocyte-induced hypermetabolism and pulmonary injury.

Activated phagocytes possess an enormous capacity for O2 consumption via NADPH oxidase. NADPH oxidase partially reduces O2, forming superoxide (O2-). Host enzymes rapidly complete O2- reduction to H2O, leaving little trace of its prior existence. Our objectives were to estimate the magnitude of whole body phagocyte respiration and determine the contribution of NADPH-derived O2- to the ensuing phagocyte-induced pulmonary injury. These objectives were accomplished using specific inhibitors of NADPH oxidase, diphenyl iodonium (DPI) and di-2-thienyl iodonium (DTI). Guinea pigs received intravenous injections of DPI (3.5 mg/kg), DTI (7.5 mg/kg), or vehicle followed by phorbol myristate acetate (PMA). Phagocyte activation by PMA immediately increased whole body respiration from 13.6 to 16.1 ml O2.kg-1.min-1 (P < 0.05). DPI and DTI completely blocked the increase in respiration induced by PMA injection (P < 0.05). Baseline respiration was unchanged by the NADPH oxidase inhibitor alone. Likewise, there was no effect on the respiration of isolated heart and kidney mitochondria from animals receiving the inhibitor with or without PMA. DPI attenuated the pulmonary injury induced by PMA. DPI attenuated the pulmonary injury induced by PMA. The ratio of lung water weight to dry weight was lower (6.4 +/- 0.3 vs. 8.3 +/- 0.6) and arterial PO2 was higher (86 +/- 9 vs. 56 +/- 6 Torr) in animals receiving DPI plus PMA than in those receiving PMA alone. In conclusion, phagocyte activation in vivo increased total body respiration by approximately 18%. The burst in respiration is attributed to the phagocyte respiratory burst in which NADPH oxidase partially O2 to O2-.(ABSTRACT TRUNCATED AT 250 WORDS)

Purine efflux from transplanted human cardiac allografts. Correlation with graft function.

Purine efflux from transplanted human cardiac allografts was investigated as a potential biochemical correlate to graft preservation and eventual function. Coronary sinus effluent from 14 allografts was sampled at 1, 5, 10, 15, 20, and 25 minutes after reperfusion. The plasma fraction from each sample was analyzed for hypoxanthine, xanthine, urate, inosine, and adenosine by high-performance liquid chromatography. Total organ preservation time, aortic crossclamp and bypass times, and initial cardiac index off bypass were recorded. An inotropic score was calculated from the dosages of inotropic agents each recipient required immediately after transplantation. Inosine and adenosine were not detectable in the coronary sinus effluent at any time during reperfusion. Hypoxanthine concentration rose sevenfold (p < 0.001) 1 minute after reperfusion. Xanthine concentration peaked later at 5 minutes after reperfusion, a twofold increase (p < 0.02). As reperfusion continued, hypoxanthine and xanthine concentrations returned toward baseline levels. The rise in coronary sinus xanthine concentration provides evidence for hypoxanthine degradation by xanthine oxidase during the immediate reperfusion period. The extent of hypoxanthine efflux correlated with total graft ischemic time (p < 0.05), inotropic score (p < 0.005), and the time from crossclamp release to cessation of bypass (p < 0.01). Hypoxanthine efflux can be used as a sensitive and objective biochemical indicator of graft preservation and immediate function.

Microassay of decarboxylation reactions in cultured cells.

The currently described methods for determination of decarboxylation reaction rates in cultured cells require large quantities of cells and often involve cell manipulation prior to assay. We describe a simple microassay for the rapid measurement of various decarboxylation reaction rates in intact cultured cells. The assay is based on the traditional measurement of 14CO2 generated from 14C-labeled substrates. Key to the method is a novel modification of the standard petri dish. Pyruvate dehydrogenase, branched chain alpha-ketoacid dehydrogenase, alpha-ketoglutarate dehydrogenase, and ornithine decarboxylase activities were determined in adult cardiomyocyte cultures containing only 0.1-0.5 mg of protein per culture dish. Efficiency of 14CO2 collection ranged between 94 and 100%. Pharmacological enhancement or inhibition of pyruvate dehydrogenase activity was easily detected in the culture system. This new method simplifies the measurement of various decarboxylation reaction rates in cultured cells and allows rapid, reproducible measurements to be made on small numbers of cells without perturbation of the culture conditions or the cells themselves.

Chronic hyperdynamic sepsis in the rat. II. Characterization of liver and muscle energy metabolism.

Sepsis was induced in male rats by injections of live Escherichia coli No. 4 (or E. coli No. 3) and Bacteroides fragilis organisms into a preformed subcutaneous abscess. Body weight, food and water intake, and cardiac output were measured daily. After 1, 2, or 3 weeks, animals were sacrificed, and blood, liver, and muscle were collected for measurements of plasma glucose and carnitine, mitochondrial respiratory activity, mitochondrial cytochrome concentrations, and tissue adenine nucleotides. Compared with sham controls, no significant differences were found in state 3 respiratory activities of liver mitochondria isolated from rats with moderate (no weight loss, cardiac output increased to 150% of control) or severe (0.5% weight loss/day, cardiac output increased to 200% of control) sepsis at any time. After 1 week of severe, but not moderate, sepsis, pyruvate-supported respiration in muscle mitochondria was significantly decreased, while branched-chain ketoacid and beta-hydroxybutyrate-supported respiration remained unchanged. After 2 weeks of severe, but not moderate, sepsis, beta-hydroxybutyrate and branched-chain ketoacid oxidation increased severalfold; pyruvate utilization remained depressed. Severe or moderate sepsis did not uncouple mitochondrial respiration at any time. Total muscle carnitine concentration was significantly decreased after long-term but not short-term severe sepsis. Severe short-term sepsis caused a significant increase in liver short-chain acyl and total carnitines. Muscle energy charge was unaltered by either moderate or severe sepsis. These results represent the first demonstration of sepsis-induced fuel shifts at the mitochondrial level in muscle: Severe hyperdynamic sepsis is characterized by the reduced ability of muscle mitochondria to utilize pyruvate with a simultaneous increase in branched-chain ketoacid and ketone body utilization. These changes were not observed in liver mitochondria.

Mechanism of peroxide-induced cellular injury in cultured adult cardiac myocytes.

Reactive oxygen species contribute to the tissue injury seen after reperfusion of ischemic myocardium. We propose that toxicity originates from the effect that mitochondrial peroxide metabolism has on substrate entry into oxidative pathways. To support our contention, cultured adult rat cardiomyocytes were incubated with physiological concentrations of peroxide. The cellular extract and incubation medium were analyzed for adenine nucleotides and purines by reverse-phase high-pressure liquid chromatography. Cellular glutathione efflux was determined by enzymatic analysis of the incubation medium. Pyruvate dehydrogenase (PDH) activity was determined in the cultured myocytes as well as in freshly isolated cardiac mitochondria using [1-C14]pyruvate. Extracellular glutathione rose 3.3-fold in response to small doses of peroxide (approximately 108 nmol/mg protein). Likewise, small quantities of peroxide reduced total cellular adenine nucleotides to 50-60% of control values with only a modest (0.95-0.91) reduction in energy charge [ATP + 1/2 ADP)/(ATP + ADP + AMP]. Peroxide-treated myocytes selectively release inosine and adenosine, as only these two purine degradation products were detected in the incubation medium. The most dramatic response was a peroxide dose-dependent inhibition of PDH activity in cultured myocytes as well as freshly isolated mitochondria; just 65 and 30 nmol peroxide/mg protein induced a 50% reduction in cellular and mitochondrial PDH activity, respectively. In conclusion, physiological quantities of peroxide potently inhibit PDH in cultured cardiomyocytes and isolated cardiac mitochondria. PDH inhibition blocks the aerobic oxidation of glucose and inhibits the oxidative phosphorylation of ADP, which in turn leads to cellular adenine nucleotide degradation.

Open versus laparoscopic cholecystectomy: an initial analysis.

Laparoscopic cholecystectomy is a new procedure in the armamentarium of the general surgeon. Its utility was investigated by comparison to open cholecystectomy in terms of procedure time, complications, hospital stay, and total hospital cost. Procedure time was approximately 200% longer with a higher incidence of intraoperative stone and bile spillage (17%) in the laparoscopic group. Hospital stay was reduced by 60% using the laparoscopic technique. No difference in total hospital cost existed between the two groups. The learning curve had an affect on hospital costs, which will decrease as more experience is gained with this procedure. Although laparoscopic cholecystectomy, at least initially, has no cost advantage over open cholecystectomy, laparoscopic cholecystectomy may be preferred by patients seeking shorter hospital stays and presumably shorter total recovery time.

Importance of spontaneous alpha-ketoacid decarboxylation in experiments involving peroxide.

The potential role of spontaneous alpha-ketoacid decarboxylation as a source of interference in experiments involving peroxide was investigated. The assay of pyruvate dehydrogenase activity in isolated renal mitochondria was employed as an example. Spontaneous peroxide-induced pyruvate decarboxylation competed significantly with enzymatic decarboxylation at peroxide concentrations greater than 50 microM. Corrected values for enzymatic decarboxylation could be obtained by subtracting spontaneous decarboxylation rates from rates obtained in the presence of mitochondria. At higher peroxide concentrations (greater than 200 microM), reaction product accumulates (acetoacetate) to levels which may have regulatory effects on mitochondrial metabolism. The divalent cations, Ca2+ and Mg2+, both accelerate spontaneous peroxide-induced pyruvate decarboxylation while other components of the assay medium had an inhibitory effect on the reaction. The results are discussed in relation to the currently accepted reaction mechanism. Investigators who perform experiments involving reactive oxygen species should be familiar with this often overlooked reaction.

Effect of peroxide, sodium, and calcium on brain mitochondrial respiration in vitro: potential role in cerebral ischemia and reperfusion.

Mitochondrial pyruvate-supported respiration was studied in vitro under conditions known to exist following ischemia, i.e., elevated extramitochondrial Ca2+, Na+, and peroxide. Ca2+ alone (7-10 nmol/mg) decreased state 3 and increased state 4 respiration to 81 and 141% of control values, respectively. Sodium (15 mM) and/or tert-butyl hydroperoxide (tBOOH; up to 2,000 nmol/mg protein) alone had no effect on respiration; however, Na+ or tBOOH in combination with Ca2+ dramatically altered respiration. Respiratory inhibition induced by Ca2+ and tBOOH does not involve pyruvate dehydrogenase (PDH) inhibition since PDH flux increased linearly with tBOOH concentration (R = 0.96). Calcium potentiated tBOOH-induced mitochondrial NAD(P)H oxidation and shifted the redox state of cytochrome b from 67 to 47% reduced. Calcium (5.5 nmol/mg) plus Na+ (15 mM) decreased state 3 and increased state 4 respiratory rates to 55 and 202% of control values, respectively. Sodium- as well as tBOOH-induced state 3 inhibition required mitochondrial Ca2+ uptake because ruthenium red addition before Ca2+ addition negated the effect. The increase in state 4 respiration involved Ca2+ cycling since ruthenium red immediately returned state 4 rates back to control values. The mechanisms for the observed Ca2(+)-, Na(+)-, and tBOOH-induced alterations in pyruvate-supported respiration in vitro are discussed and a multifactorial etiology for mitochondrial respiratory dysfunction following cerebral ischemia in vivo is proposed.

NADH-linked substrate dependence of peroxide-induced respiratory inhibition and calcium efflux in isolated renal mitochondria.

Peroxide-induced state 3 respiratory inhibition and Ca2+ efflux in isolated renal mitochondria exhibited a NADH-linked substrate dependence. ADP-stimulated respiratory rates in the presence of various concentrations of tert-butyl hydroperoxide (tBOOH, 0-1000 nmol/mg protein) were determined using glutamate, beta-hydroxybutyrate, or pyruvate as substrates. Pyruvate-driven respiration was most sensitive to inhibition (Ki approximately equal to 75 nmol of tBOOH/mg protein) followed by beta-hydroxybutyrate and glutamate (Ki approximately equal to 150 nmol of tBOOH/mg protein for each). Calcium (5-10 nmol/mg protein) potentiated tBOOH-induced respiratory inhibition using all three substrates. Mitochondrial Ca2+ efflux, induced by tBOOH, was most pronounced with pyruvate as substrate. Glutamate prevented Ca2+ efflux while the efflux rate with beta-hydroxybutyrate was intermediate between glutamate and pyruvate. The substrate-dependent pattern of tBOOH-induced NAD(P)H (NADH plus NADPH) and cytochrome b oxidation was similar to that seen for respiratory inhibition and Ca2+ efflux suggesting that NAD(P)H may be a common factor in both responses. Low tBOOH concentrations inhibited pyruvate dehydrogenase flux while higher concentrations enhanced pyruvate dehydrogenase flux and activation. The results are discussed in relation to currently proposed theories of reactive oxygen-induced respiratory inhibition, Ca2+ efflux, and reperfusion injury.

Perinatal development of heart, kidney, and liver mitochondrial antioxidant defense.

Development of the mitochondrial antioxidant defense system was studied to assess its potential role in the newborn mammal's tolerance to oxidative challenge and to gain insight into the fetal adaptation to a relatively hyperoxic adult environment. Isolated heart, kidney, and liver mitochondria from fetal, newborn, and adult guinea pigs were used. In situ function of the antioxidant enzymes was estimated in mitochondrial suspensions after the addition to selenite or tert-butyl hydroperoxide by determining NAD(P)H oxidation rates spectrophotometrically at 340-375 nm. Kidney and liver mitochondria from newborn animals were less susceptible to selenite and tert-butyl hydroperoxide-induced NAD(P)H oxidation. The pattern of change, however, varied widely with tissue type. Kidney mitochondria displayed the largest change with a 3- to 4-fold increase in rate from the fetal to adult period. NAD(P)H oxidation rates in intact mitochondria did not correlate consistently with glutathione reductase and peroxidase activities in sonicated mitochondria suggesting in situ regulation by other endogenous factors. Immediately after birth, mitochondrial glutathione reductase and peroxidase activities dropped 38-50% and 50-70%, respectively, in all tissues studied. Total glutathione content of heart and liver mitochondria did not change with age. Adult kidney mitochondrial glutathione, however, declined to 24% of fetal values. Mitochondrial superoxide dismutase activity increased 150-300% from the fetal to the adult period in all tissues studied. Perinatal changes in the mitochondrial antioxidant system and their relationship to mitochondrial calcium metabolism are discussed in terms of the newborn's resistance to oxidative stress.

Potential role of mitochondrial calcium metabolism during reperfusion injury.

Ischemia-reperfusion injury has been associated with intracellular H2O2 and superoxide radical production from accumulated hypoxanthine (HX) and xanthine oxidase (XO). The effect of H2O2 and superoxide radical on mitochondrial Ca2+ efflux was characterized in isolated renal mitochondria using a HX-XO system. Mitochondria were suspended in buffered medium containing 200 microM HX. Extramitochondrial Ca2+ was monitored kinetically at 660-685 nm using the Ca2+ indicator arsenazo III. After preloading mitochondria with 18-25 nmol Ca2+/mg protein, addition of XO to the medium caused a rapid oxidation of mitochondrial NAD(P)H followed by Ca2+ release. Ca2+ efflux was attributed to mitochondrial metabolism of H2O2 because efflux could be prevented with catalase but not superoxide dismutase. The Ca2+ efflux rate (r = 0.995) and lag time to Ca2+ efflux (r = 0.987) both correlate well with the NAD(P)H oxidation rate. Exogenous ATP prevents Ca2+ efflux in a dose-dependent fashion (Km = 35 microM ATP) without affecting NAD(P)H oxidation; ATP plus oligomycin, however, had no effect. The protective effect of ATP on Ca2+ efflux was diminished by ruthenium red (RR). XO-induced Ca2+ efflux increased state 4 respiration 148% via a futile Ca2+ cycle involving the Ca2+ uniport. The increase in state 4 respiration could be reversed with RR (alpha less than 0.001) or ATP (alpha less than 0.01); ATP plus oligomycin, however, had no effect. The results are discussed in relation to the oxygen free radical theory of reperfusion injury.

Selenite-induced NAD(P)H oxidation and calcium release in isolated mitochondria: relationship to in vivo toxicity.

The effects of selenite on the mitochondrial NAD(P)H/NAD(P) ratio and calcium pool are described. Small quantities of selenite can 1) oxidize mitochondrial NAD(P)H and 2) induce calcium release from isolated mitochondria. Reduced NAD(P)H within intact mitochondria was monitored kinetically using the wavelength pair, 340-375 nm. NAD(P)H oxidation rates at various concentrations of selenite were calculated. Mitochondria from older animals can oxidize NAD(P)H faster than those of younger animals; maximum selenite-induced oxidation rates correlate well with age of the animal in both kidney (r = 0.920) and liver (r = 0.839) mitochondria, the oxidation rates in the adult (liver 15.4, kidney 34.8 nmol/min/mg of protein) being 3-5 times the rates in the 1- to 2-day-old newborn (liver 2.8, kidney 10.3 nmol/min/mg protein). Calcium fluxes within mitochondrial suspensions were monitored kinetically using the calcium indicator, Arsenazo III, and the wavelength pair, 660-685 nm. Susceptibility to selenite-induced calcium release is age dependent, the mitochondria of older animals being more susceptible. Incubation time required to induce calcium release was 77 +/- 30 sec in the adult compared to 406 +/- 25 sec at the age of 0-4 days in the newborn. The bimodal toxic manifestations of selenite in vivo are discussed in view of the age-dependent differences in selenite metabolism at the cellular level.