Lack of usefulness of radiographic screening for pulmonary disease in asymptomatic HIV-infected adults. Pulmonary Complications of HIV Infection Study Group.

OBJECTIVE: To determine the use of chest radiographs in the screening of asymptomatic adults infected with the human immunodeficiency virus (HIV). METHODS: A prospective, multicenter study of the pulmonary complications of HIV infection in a community-based cohort of persons with and without HIV infection. The subjects included 1065 HIV-seropositive subjects without the acquired immunodeficiency syndrome at the time of enrollment: 790 homosexual men, 226 injection drug users, and 49 women with heterosexually acquired infection. Frontal and lateral chest radiographs were performed at 3-, 6-, and 12-month intervals, CD4 lymphocyte measurements at 3- and 6-month intervals, tuberculin and mumps skin tests at 12-month intervals, and medical histories and physical examinations at 3- and 6-month intervals. Pulmonary diagnoses that occurred within 2 months following each radiograph were analyzed and correlated with the radiographic results. RESULTS: Evaluable screening chest radiographs (5263) were performed in HIV-seropositive subjects while they were asymptomatic; of these, 5140 (98%) were classified as normal and 123 (2%) as abnormal. A new pulmonary diagnosis was identified within 2 months following a screening radiograph in 55 subjects. Only 11 of these subjects had abnormal radiographs; the sensitivity of the radiograph was 20%. The sensitivity was similarly low at baseline, within each transmission category, and in subjects whose CD4 lymphocyte counts were less than 0.2 x 10(9)/L (200/microL). The types of pulmonary diseases that occurred were similar in the subjects with normal and abnormal screening radiographs. CONCLUSION: Screening chest radiography in asymptomatic HIV-infected adults is unwarranted because the diagnostic yield is low.

Single-photon emission computed tomography and positron-emission tomography assays for tissue oxygenation.

Radiotherapy prescription can now be customized to target the major mechanism(s) of resistance of individual tumors. In that regard, functional imaging techniques should be exploited to identify the dominant mechanism(s). Tumor biology research has identified several mechanisms of tumor resistance that may be unique to radiation treatments. These fall into 3 broad areas associated with (1) tumor hypoxic fraction, (2) tumor growth rate, (3) and the intrinsic radiosensitivity of tumor clonogens. Imaging research has markers in various stages of development for quantifying relevant information about each of these mechanisms, and those that measure tumor oxygenation and predict for radioresistance are the most advanced. Positron-emission tomography (PET) measurement of oxygen 15 has yielded important information, particularly about brain tissue perfusion, metabolism, and function. Indirect markers of tumor hypoxia have exploited the covalent binding of bioreductive intermediates of azomycin-containing compounds whose uptakes are inversely proportional to intracellular oxygen concentrations. Pilot clinical studies with single-photon emission computed tomography (SPECT) and PET detection of radiolabeled markers to tumor hypoxia have been reported. Recently, other studies have attempted to exploit the reduction properties of both technetium and copper chelates for the selective deposition of radioactive metals in hypoxic tissues. A growing number of potentially useful isotopes are now available for labeling several novel chemicals that could have the appropriate specificity and sensitivity. Preclinical studies with "microSPECT" and "microPET" will be important to define the optimal radiodiagnostic(s) for measuring tissue oxygenation and for determining the time after their administration for optimal hypoxic signal acquisition. Radiolabeled markers of growth kinetics and intrinsic radiosensitivity of cells in solid tumors are also being developed. We conclude that radiation oncology is uniquely positioned to benefit from functional imaging markers that identify important mechanisms of tumor radioresistance, since several strategies for overcoming these individual mechanisms have already been identified.

Preclinical assessment of hypoxic marker specificity and sensitivity.

PURPOSE: In the search for a sensitive, accurate, and noninvasive technique for quantifying human tumor hypoxia, our laboratory has synthesized several potential radiodiagnostic agents. The purpose of this study was to assess and compare the hypoxic marking properties of both radioiodinated and Tc-99m labeled markers in appropriate test systems which can predict for in vivo activity. MATERIALS AND METHODS: Preclinical assessment of hypoxic marker specificity and sensitivity employed three laboratory assays with tumor cells in vitro and in vivo. Radiolabeled marker uptake and/or binding to whole EMT-6 tumor cells under extremely hypoxic and aerobic conditions was measured and their ratio defined hypoxia-specific factor (HSF). Marker specificity to hypoxic tumor tissue was estimated from its selective avidity to two rodent tumors in vivo, whose radiobiologic hypoxic fractions (HF) had been measured. The ratios of % injected dose/gram (%ID/g) of marker at various times in EMT-6 tumor tissue relative to that in the blood and muscle of scid mice were used to quantify hypoxia-specific activity. This tumor in this host exhibited an average radiobiologic HF of approximately 35%. As well, nuclear medicine images were acquired from R3327-AT (HF approximately =15%) and R3327-H (no measurable HF) prostate carcinomas growing in rats to distinguish between marker avidity due to hypoxia versus perfusion. RESULTS: The HSF for FC-103 and other iodinated markers were higher (5-40) than those for FC-306 and other Tc-99m labeled markers. The latter did not show hypoxia-specific uptake into cells in vitro. Qualitative differences were observed in the biodistribution and clearance kinetics of the iodinated azomycin nucleosides relative to the technetium chelates. The largest tumor/blood (T/B) and tumor/muscle (T/M) ratios were observed for compounds of the azomycin nucleoside class in EMT-6 tumor-bearing scid mice. These markers also showed a 3-4 x higher uptake into R3327-AT tumors relative to the well-perfused R3327-H tumors. While both FC-306 and CERETEC rapidly distributed at unique concentrations to different tissues, their avidity to EMT-6 and R3327-AT tumors did not correlate with tumor HF. CONCLUSIONS: The halogenated azomycin nucleosides with the lowest lipid/water partition coefficient values were found to yield the optimal hypoxia-specific signal in these animal tumors. Our Tc-99m-labeled azomycin chelates showed little or no hypoxia-specific uptake and had in vivo biodistribution and clearance kinetics similar to those of CERETEC, a perfusion agent with no known hypoxic binding activity.

The localization of indium-111-leukocytes, gallium-67-polyclonal IgG and other radioactive agents in acute focal inflammatory lesions.

A variety of radioactive agents, injected directly intravenously have demonstrated foci of inflammation by gamma camera imaging, avoiding the in vitro preparation of labeled leukocytes. This study sought to find out if any of these agents mimicked the biodistribution in abscesses and non-target organs of labeled mixed leukocyte suspensions. Eight different agents were compared with 111In-oxine labeled leukocytes in an acute soft tissue E. coli abscess and an acute arthritic lesion in 24 dogs one day after intravenous administration. These included 67Ga-citrate, human and canine polyclonal immunoglobulin (IgG), rabbit anti-dog polyclonal IgG, serum albumin, monoclonal antibody TNT-1 F(ab')2 against nuclear antigens, 57Co-porphyrin and serum albumin nanocolloid. None of these agents achieved abscess concentrations approaching those obtained with labeled leukocytes, and their abscess/blood and abscess/muscle concentration ratios were considerably lower. No statistically significant differences were found between the different radiolabeled proteins evaluated. The abscess concentration of 99mTc-nanocolloid was much lower than that of other agents, and the results with the oldest agent, 67Ga-citrate, were disappointing in these acute experiments.

Marking hypoxia in rat prostate carcinomas with beta-D-[125I]azomycin galactopyranoside and.

UNLABELLED: The purpose of this study was to determine, with a rodent tumor model, if microelectrode measurements of unmodulated tumor oxygenation predict for the avidity of hypoxic markers to tumor tissue. METHODS: The rapidly growing, anaplastic variant of the Dunning rat prostate carcinoma cell line (R3327-AT) was implanted subcutaneously on the upper backs of Fischer X Copenhagen rats. Approximately 100 measurements of PO2 were obtained from tumors of 5-10 g in animals that were restrained and then subjected to different anesthetic procedures. Values of median PO2 (in mm Hg) and percentage of measurements <5 mm Hg obtained from individual tumors were used to define tumor oxygenation status. The radiodiagnostic hypoxic markers beta-D-iodinated azomycin galactopyranoside (IAZGP) and [99mTc]HL-91 were simultaneously administered to 26 animals whose tumor oxygen levels had been measured. Six hours after marker administration, the animals were killed; tumor, blood, and muscle tissues were sampled; and percentage injected dose per gram (%ID/g*), tumor/blood ratio (T/B), and tumor/muscle ratio (T/M) parameters were determined. Parameters of marker avidity to individual tumors were linearly correlated with microelectrode measurements of tumor oxygenation to determine the significance of inverse associations. RESULTS: The median PO2 of 41 tumors varied from 2.0 to 20.9 mm Hg, with an average value of 7.5 +/- 1.4 mm Hg. Six tumors had unusually high values; that is, >10 mm Hg, and when these were excluded from the analysis, the average median PO2 of the remaining 35 was 4.3 +/- 0.7 mm Hg. When electrode measurements of tumor oxygenation were obtained under conditions of halothane anesthesia with the animals breathing O2, carbogen, or air, median PO2 values increased significantly (P = 0.001). When animals were deeply anesthetized by intraperitoneal injection of ketamine-xylazine, median PO2 values were not significantly different (P = 0.13) from those obtained while the animals were restrained and breathing air. There was no inverse correlation of significance between the electrode measurements of median PO2 and the avidity of beta-D-IAZGP nor [99mTc]HL-91 in this tumor model. The range of median PO2 values in these tumors was at least 3 mm Hg, and the range of hypoxic marker avidity was less than twofold. CONCLUSION: These data demonstrate that microelectrode measurements of rat tumor oxygenation did not correlate with the avidity of the two hypoxic markers, at least in this tumor model. The larger dynamic range of tumor oxygen measurements obtained with microelectrodes might be biased to low values by their necrotic fractions, the zones within solid tumors that contain dead cells and debris that will not be labeled by bioreducible hypoxic markers. Hypoxic marker avidity to individual tumors will have to be validated by other assays that can predict for their radiosensitivity.

N,N'-bis(S-benzoylmercaptoacetamido) ethylenediamine and propylenediamine ligands as renal function imaging agents. I. Alternate synthetic methods.

A new method was developed to synthesize tetradentate ligands containing the N,N'-bis(S- benzoylmercaptoacetyl ) ethylenediamine and propylenediamine moieties (DADS compounds). Methods are also represented with which to synthesize some of the positional isomers of the above compounds. These isomers represent a new class of compounds. A total of 21 different compounds were prepared. These will be used in an effort to establish a relationship between structure and renal imaging properties.

Technetium-99m DADS complexes as renal function and imaging agents: II. Biological comparison with iodine-131 hippuran.

To find a 99mTc agent with a high renal extraction efficiency similar to [131]hippuran, 21 analogs of DADS were labeled and evaluated. Preliminary screening by serial gamma camera imaging in rabbits showed that most analogs had a higher liver uptake and/or slower renal clearance, than hippuran. Three agents (P-DADS, AP-DADS, and CAP-DADS), exhibiting a relatively rapid blood clearance and lower liver uptake in the rabbit, were studied in greater detail in comparison with hippuran and CO2-DADS-A, the best analog to date. In the rat, the plasma clearance of the four DADS analogs was slower than that of hippuran. In rats with tubular damage induced by cisplatin, the difference in renal retention at 1 hr compared to controls was much greater with hippuran than with the DADS compounds. In the dog, there was marked hepatic retention of the four DADS compounds. In volunteers, serial posterior images obtained with these [99mTc]DADS complexes showed significant hepatic as well as renal activity. The 1-hr plasma clearance and urinary excretion were much lower than with simultaneously injected hippuran. Although these 99mTc agents are satisfactory for imaging the kidneys, they closely mimic the biodistribution of hippuran only in the rabbit, and not in the rat, dog, or man.

Measuring hypoxia and predicting tumor radioresistance with nuclear medicine assays.

Tumor cells at low oxygen tension are relatively radioresistant. The hypoxic fraction of individual tumors before, during and after radiotherapy is likely to have prognostic value but its diagnosis still awaits an accurate and acceptable assay. The recent indications that hypoxia can also induce the expression of specific genes and promote a more aggressive tumor phenotype makes its diagnosis even more important. Over 15 years ago, misonidazole, an azomycin-based hypoxic cell radiosensitizer, was found to link covalently to cellular molecules at rates inversely proportional to intracellular oxygen concentration. The use of bioreducible markers to positively label zones of viable hypoxic cells within solid tumors and to predict for tumor radioresistance was proposed. Several hypoxic markers have now been identified and their selective binding within tumors has been measured by both invasive and non-invasive assays. Research from our laboratory has emphasized both mechanistic and preclinical studies associated with nuclear medicine procedures for measuring tumor hypoxia and predicting tumor radioresistance. This report updates radiation oncologists about the status of nuclear medicine hypoxic marker research and development as of mid-1997. While several potential imaging agents have been identified, their testing and validation in appropriate human tumors will require focused research efforts by individual academic departments and, possibly, by clinical trials performed through cooperative groups. Since the prediction of hypoxia in individual tumors could strongly impact radiotherapy treatment planning, the radiation oncology research community is best positioned to execute the validation studies associated with these markers.

Discriminate use of electrocautery on the median sternotomy incision. A 0.16% wound infection rate.

Between June 1978 and June 1989, superficial or deep mediastinitis (or both) developed in only five (0.16%) of 3118 consecutive patients. All patients studied underwent cardiac procedures through a median sternotomy and survived more than 7 postoperative days. The surgical team disciplined itself to divide presternal soft tissues with a scalpel and used electrocautery for pinpoint hemostasis only. This 0.16% infection rate was statistically significantly lower than those in 28 previously published studies (Pearson's chi 2 test, p less than 0.05). Twenty-four predisposing factors were evaluated by Fisher's exact test. Among these only an operating time longer than 3 hours is related to sternotomy infections (p = 0.0208), and this effect was not a strong one. Statistical evidence strongly suggests that discriminate use of electrocautery is a major reason for the lowest median sternotomy infection rate reported to date.

Lack of efficacy of intrapleural bupivacaine for postoperative analgesia following thoracotomy.

Intrapleural bupivacaine has been reported to be effective for analgesia following cholecystectomy and thoracic surgery. Twenty patients who had a posterolateral thoracotomy were studied in a randomized, double-blind, placebo-controlled fashion. Patients were assigned to receive intrapleural administration of either 0.5 percent bupivacaine or saline solution every 4 h for 12 doses postoperatively, as well as narcotic analgesics as needed for additional pain control. Pain was assessed using a visual analogue scale. Narcotic analgesic use, duration of hospitalization, and the development of complications were recorded. There were nine evaluable patients who received bupivacaine, and ten patients who received placebo. The age, sex, and type of operation were similar in the two groups, and the procedures were performed by the same two surgeons. The mean pain score at 24 h postoperatively was 5.8 +/- 0.8 in the bupivacaine group and 6.0 +/- 0.6 in the placebo group. At 48 h, the scores were 4.6 +/- 0.8 in the bupivacaine group and 5.1 +/- 0.9 in the placebo group. The mean dose of morphine sulfate or equianalgesic dose of meperidine during the first 24 h was 13.9 +/- 3.7 mg in the bupivacaine group and 12.6 +/- 1.8 mg in the placebo group, and during the next 24 h it was 40.0 +/- 13.4 mg in the bupivacaine group and 38.0 +/- 9.2 mg in the placebo group. The mean duration of hospitalization was 12.8 +/- 3.2 days in the bupivacaine group and 12.1 +/- 2.9 days in the placebo group. Two patients who received bupivacaine and three patients who received placebo had development of pneumonia or atelectasis postoperatively. There was no statistically significant difference in any parameter between those who received bupivacaine and those who received placebo. Thus, there was no subjective or objective clinical benefit of this method of postoperative analgesia compared with placebo following posterolateral thoracotomy.

Carotid and aortic arch endarterectomy using hypothermic arrest with coronary bypass.

A 67-year-old man with symptomatic bilateral carotid artery obstructions and a large, friable atheromatous plaque of the transverse aortic arch required coronary artery bypass grafting for severe triple-vessel disease. An endarterectomy of the transverse arch and a left carotid endarterectomy were performed using deep hypothermic circulatory arrest concomitant with quadruple coronary artery bypass grafting. Recovery was uneventful. Hypothermic circulatory arrest provides adequate protection for this combined procedure and may eliminate cerebral embolization.

Surgical closure of the patent ductus arteriosus in the neonatal intensive care unit.

Efficacy of surgical closure versus indomethacin for treatment of patent ductus arteriosus in symptomatic neonates is an ongoing controversy. In recent years, surgical closure has been performed in the neonatal intensive care unit rather than the operating room in some centers, creating further controversy. In a retrospective study of the charts of 115 sequential patent ductus arteriosus surgical closures performed in the neonatal intensive care unit in premature infants, we found no surgical morbidity or mortality. Ninety-nine of these infants of less than 33 weeks gestational age were evaluated for various factors that might influence outcome. All were operated on within 72 hours of diagnosis, with an extra-pleural approach and metal clips used for closure of the ductus. All infants were extubated at an average of 33 weeks in each age group studied unless they had underlying severe bronchopulmonary dysplasia. We conclude that surgical closure of the symptomatic patent ductus arteriosus in neonates is safe and 100% effective, with none of the reported complications of indomethacin therapy, and should be the treatment of choice in neonates aged less than 33 weeks (gestational age) at birth with symptomatic patent ductus arteriosus. Closure performed in the neonatal intensive care unit eliminates transport risks and is ultimately safer and easier than transport to an operating room.

Lymphocytic interstitial pneumonitis and nonspecific interstitial pneumonitis.

Nonspecific interstitial pneumonitis (NIP) and lymphocytic interstitial pneumonitis (LIP), with or without lymphocytic alveolitis, are poorly understood pulmonary complications of HIV infection. These disorders probably represent a spectrum of lymphoproliferative processes that overlap, rather than distinct illnesses. The clinical presentation, radiographic findings, and physiologic abnormalities in NIP and LIP are not specific and therefore require a biopsy for definitive diagnosis. The clinical course of these illnesses is generally favorable, even without specific treatment.

Enhanced ventricular recovery from high dose glucose, insulin and potassium with cardiopulmonary bypass support prior to cardioplegic arrest.

The concept that a beneficial preconditioning effect in ventricular recovery exists using high dose glucose (0.5 mg/kg), insulin (0.3 unit/kg), and potassium (0.2 mmol/kg) (GIK) with 20 min of normothermic cardiopulmonary bypass support (CPB) prior to 60 min of cardioplegic arrest (CA) was tested using 32 mongrel dogs divided into four subset test groups. Group 1 was given GIK and 20 min of CPB prior to CA, Group 2 was given GIK systemically over 10 min but no CPB prior to CA, Group 3 underwent 20 min of CPB without GIK and Group 4 was the control group with no GIK and no CPB assist. To focus specifically on in vivo ventricular recovery, dP/dT (mmHg/sec), developed pressure (dP) (mmHg), and segmental shortening (SS) (%) were measured prior to CPB, then 15, 30, 60, and 90 min after weaning from CPB, while left atrial pressure was kept constant. The average dP/dT (% recovery) at 60 min in Group 1 was 1,454 (122%) and significantly higher (P < or = 0.05) than Groups 2: 1,189 (99%), 3: 1,027 (79%) and 4: 1,030 (82%). Developed pressure at 90 min (% recovery) in Group 1, 88 (111%) was also better than Groups 3, 74 (86%), and 4, 72 (87%) (P < or = 0.05). Segmental shortening (% recovery) at 30 min was better in Group 1 (94%) than in Groups 2 (59%), 3 (73%) and 4 (68%). We conclude that GIK added to 20 min of CPB support prior to cardioplegic arrest enhances post CPB ventricular recovery and weaning from CPB.

Assessment of perfusion toward the aortic valve using the new dispersion aortic cannula during coronary artery bypass surgery.

When there is an echocardiographic diagnosis of severe mobile atherosclerotic plaque in the aortic arch or descending aorta, perfusion toward the aortic arch during cardiopulmonary bypass may create a high risk of embolic neurologic injury. Other perfusion methods, such as cannulation of the femoral or axillary arteries, are not always possible, due to atherosclerosis. The ascending aorta may be an alternative site for perfusion, since it is less frequently diseased. We assessed a new technique of perfusion toward the aortic valve using a new cannula designed for this purpose (Dispersion aortic cannula). Our study included 100 consecutive patients, 72 men and 28 women, with an average age of 68 +/- 1.0 years (range, 39-89 years). There were no complications related to insertion of the cannula or perfusion. The ascending aorta could be cross-clamped and side-clamped without perfusion problems. Three deaths occurred; none was related to the cannulation technique. No intra-operative stroke occurred. Two patients suffered neurologic events, one on day 1 and the other on day 6; both had been fully alert after surgery. Perfusion toward the aortic valve appears to be safe and hemodynamically effective. This cannulation technique appears to be an acceptable alternative to present methods. Comparative studies will be needed to determine whether this alternative technique is effective in patients with severe aortic arch disease.

Screening and noninvasive testing for pulmonary disease.

Although detection of preclinical disease using screening tests is desirable if earlier treatment improves outcome, the available data show no morbidity or mortality benefit using chest radiography, sputum analysis for Pneumocystis carinii and acid-fast bacilli, or serial measurements of single-breath carbon monoxide diffusing capacity to detect pulmonary disease in asymptomatic persons with HIV. It seems prudent to evaluate asymptomatic patients periodically with a careful history and focused physical examination and to perform only those tests that are likely to alter the plan of management such as tuberculin skin testing, CD4 lymphocyte measurement, and measurement of viral burden. Noninvasive diagnostic studies including chest radiography, arterial blood gas analysis, induced or expectorated sputum analysis, exercise testing, and nuclear scans should be performed if new pulmonary symptoms are observed. The use of these tests should be guided by the clinical presentation and followed, when necessary, by the appropriate invasive studies.

Bacterial pneumonia.

Bacterial pneumonia is significantly more common in persons who are HIV-infected than in the general population and is most common among injection drug users and in persons with advanced HIV disease and immunosuppression. The clinical features of bacterial pneumonia are similar to those in HIV-seronegative persons, but bacteremia is more common. When a pathogen is identified, Streptococcus pneumoniae is consistently the most common, occurring in 20% to 70% of cases. Haemophilus influenzae, Staphylococcus aureus, Escherichia coli, and other gram-negative organisms are mainly responsible for the remainder of bacterial pneumonia episodes in the United States, Central Africa, Australia, and England. In some studies, Chlamydia pneumoniae was recognized as a common cause in persons with early HIV disease, whereas Pseudomonas aeruginosa is recognized as a community- and hospital-acquired lower respiratory tract pathogen in patients with severe immunosuppression. Although antimicrobial therapy is frequently empiric, it should be tailored to the severity of illness, local prevalence of infections, resistance patterns, or when an etiologic agent is identified. The treatment response is similar in patients with and without HIV infection, but bacterial pneumonia may accelerate the progression of HIV disease. Preventative measures include use of the polyvalent pneumococcal vaccine, especially early in the course of HIV infection, when it is most likely to be effective. The incidence of bacterial pneumonia is also reduced in HIV-seropositive persons who use trimethoprim-sulfamethoxazole to prevent Pneumocystis carinii pneumonia.

Pulmonary complications of HIV infection.

With changes in epidemiology and the application of newer treatment and prophylactic regimens, the types of pulmonary diseases that occur in HIV-infected persons are changing. New ways to assess the progression of HIV disease and new antiretroviral treatments are available. Increased survival is often coupled with worsening immunosuppression. Overall mortality from Pneumocystis carinii pneumonia is declining, but mortality from bacterial pneumonia and mycobacterial disease is increasing. Infections with unusual and resistant organisms are also increasing. Patients with severe immunosuppression are susceptible to fungal, viral, and neoplastic pulmonary disease.

Pneumococcal infections in HIV-infected adults.

Bacterial pneumonia, specifically pneumococcal infection, is a frequent cause of morbidity and mortality in persons infected with human immunodeficiency virus (HIV). It causes morbidity directly and possibly progression of HIV infection. The clinical presentation and response to therapy are usually similar to that of patients without HIV infection, although radiographic presentations may be atypical. There is a higher incidence of invasive disease and extrapulmonary disease, and mortality may be increased in HIV-infected patients. HIV infection impairs the host response to pneumococcus in a variety of ways. Colonization with Streptococcus pneumoniae may be prolonged for reasons that are incompletely understood. Concern about the rising prevalence of resistant pneumococcal strains is increasing, but the clinical relevance is uncertain. At least 90% of the strains that cause invasive disease are present in the 23-valent pneumococcal vaccine. The response to vaccination declines as immunodeficiency progresses; however, the potential benefit to responders is great and the risk is minimal. Therefore, this vaccine is recommended for all HIV-infected persons.

Interaction of certain cationic dyes with the respiratory chain of rat liver mitochondria.

Cationic dyes of the cyanine type have been observed to specifically inhibit NAD-linked respiration in rat liver mitochondria, with 50% inhibition occurring at about 0.2 mumol/g of mitochondrial protein. The dyes show no effect on succinate oxidation or coupled phosphorylation in the range which completely inhibits NADH oxidation. This specific inhibition was found with all cyanine dyes tested, but, with the possible exception of pyronin B, was not observed with other cationic dyes such as safranine O, rhodamine dyes, or with simple derivatives of the quinaldinium ring structure. The inhibition was observed to be both time- and concentration-dependent, with the half-time for full inhibition determined to be on the order of 15 to 30 s at 25 degrees C. Furthermore, the inhibition was totally dependent on the energization of the mitochondrial membrane by either substrate oxidation or the presence of ATP. The explanation of the energy dependence of the inhibition by cyanine dyes as the simple requirement for energy-linked dye concentration within the mitochondria is not supported by the relatively slow onset of inhibition as compared with the very rapid rate of dye uptake observed. Furthermore, inhibition of energy-requiring, succinate-linked NAD reduction in submitochondrial particles was observed to be inhibited by dyes, while fumarate reduction by NADH was found to be inhibited significantly only in the presence of ATP in addition to the dye. The energy requirement for electron transport inhibition in submitochondrial particles indicates that energy-dependent accumulation of the dyes by mitochondria cannot alone explain the energy requirement for the cyanine dye inhibition of NADH oxidation.