Recruitment of extravascular fluid by hyperoncotic albumin.

BACKGROUND: Although hyperoncotic albumin may be used to recruit oedema, its effectiveness remains unclear. Therefore, this issue was studied during infusion experiments in healthy volunteers. METHOD: Fifteen healthy volunteers (mean age 31 years) received an infusion of 3 mL/kg of 20% albumin over 30 minutes. Their urinary excretion was recorded, and venous blood samples were taken to measure blood haemoglobin (Hb), haematocrit, colloid osmotic pressure as well as plasma albumin and sodium concentrations on 15 occasions over a period of 300 minutes. Plasma volume expansion was taken as the inverse of the fluid-induced dilution of venous plasma, as given by the blood Hb concentration. Mass balance calculations were used to estimate the mobilisation of fluid from the tissues. RESULTS: Maximum plasma volume expansion was reached 20 minutes after completing an infusion of 20% albumin. Urinary excretion was effectively increased, and the mobilised fluid from the tissues at 300 minutes amounted to 3.4 ± 1.2 mL for each infused mL of 20% albumin, of which 19% was of intracellular origin. The urinary excretion correlated strongly with the amount of recruited fluid (R2  = 0.87) and inversely with the plasma volume expansion (R2  = 0.53). CONCLUSION: The infusion of 20% albumin significantly increases the plasma volume by recruiting interstitial fluid. After completing the infusion, there is a delay of 20 minutes until maximum plasma dilution is reached, and the duration of the plasma volume expansion lasts far beyond 5 hours.

Distribution of crystalloid fluid changes with the rate of infusion: a population-based study.

BACKGROUND: Crystalloid fluid requires 30 min for complete distribution throughout the extracellular fluid space and tends to cause long-standing peripheral edema. A kinetic analysis of the distribution of Ringer's acetate with increasing infusion rates was performed to obtain a better understanding of these characteristics of crystalloids. METHODS: Data were retrieved from six studies in which 76 volunteers and preoperative patients had received between 300 ml and 2375 ml of Ringer's acetate solution at a rate of 20-80 ml/min (0.33-0.83 ml/min/kg). Serial measurements of the blood hemoglobin concentration were used as inputs in a kinetic analysis based on a two-volume model with micro-constants, using software for nonlinear mixed effects. RESULTS: The micro-constants describing distribution (k12) and elimination (k10) were unchanged when the rate of infusion increased, with half-times of 16 and 26 min, respectively. In contrast, the micro-constant describing how rapidly the already distributed fluid left the peripheral space (k21) decreased by 90% when the fluid was infused more rapidly, corresponding to an increase in the half-time from 3 to 30 min. The central volume of distribution (V(c)) doubled. CONCLUSION: The return of Ringer's acetate from the peripheral fluid compartment to the plasma was slower with high than with low infusion rates. Edema is a normal consequence of plasma volume expansion with this fluid, even in healthy volunteers. The results are consistent with the view that the viscoelastic properties of the interstitial matrix are responsible for the distribution and redistribution characteristics of crystalloid fluid.

Colloid osmotic pressure and extravasation of plasma proteins following infusion of Ringer's acetate and hydroxyethyl starch 130/0.4.

BACKGROUND: During fluid infusion therapy, plasma proteins are diluted and leak from the intravascular space, which alters the colloid osmotic pressure (COP) and potentially affects coagulation. We hypothesised that acetated Ringer's and starch solution, alone or in combination, influence these mechanisms differently. MATERIALS AND METHODS: On different occasions, 10 male volunteers were infused with 20 ml/kg acetated Ringer's and 10 ml/kg 6% hyroxyethyl starch 130/0.4 (Voluven(®) ) alone or in combination (first with starch solution followed by Ringer's solution). Blood samples were collected every 30-min for measurements of COP, blood haemoglobin, platelets, and plasma concentrations of albumin, immunoglobulins (IgG and IgM), coagulation factor VII (FVII), fibrinogen, cystatin C, activated partial thromboplastin time (APTT) and prothrombin international normalised ratio (PT-INR). Changes were compared with the haemoglobin-derived plasma dilution. RESULTS: The COP increased by 8.4% (SD 3) with starch and decreased by 26.2% (7.9) with Ringer's. These infusions diluted the plasma by 23.4% (5.3) and 18.7% (4.9) respectively. The COP changes in the combined experiment followed the same pattern as the individual infusions. Albumin and IgG changes in excess of the plasma dilution were very subtle. The intravascular contents of the IgM and platelets decreased, whereas FVII, fibrinogen and cystatin C increased. PT-INR increased by 1/3 of the plasma dilution, whereas changes in APTT did not correlate with the plasma dilution. CONCLUSIONS: The starch increased COP and only minor capillary leak occurred in healthy volunteers. The fluid-induced plasma dilution correlated with mild impairment of the extrinsic coagulation pathway but not of the intrinsic pathway.

Non-invasive blood haemoglobin and plethysmographic variability index during brachial plexus block.

BACKGROUND: Plethysmographic measurement of haemoglobin concentration ([Formula: see text]), pleth variability index (PVI), and perfusion index (PI) with the Radical-7 apparatus is growing in popularity. Previous studies have indicated that [Formula: see text] has poor precision, particularly when PI is low. We wanted to study the effects of a sympathetic block on these measurements. METHODS: Twenty patients underwent hand surgery under brachial plexus block with one Radical-7 applied to each arm. Measurements were taken up to 20 min after the block had been initiated. Venous blood samples were also drawn from the non-blocked arm. RESULTS: During the last 10 min of the study, [Formula: see text] had increased by 8.6%. The PVI decreased by 54%, and PI increased by 188% in the blocked arm (median values). All these changes were statistically significant. In the non-blocked arm, these parameters did not change significantly. CONCLUSIONS: Brachial plexus block significantly altered [Formula: see text], PVI, and PI, which indicates that regional nervous control of the arm greatly affects plethysmographic measurements obtained by the Radical-7. After the brachial plexus block, [Formula: see text] increased and PVI decreased.

Non-invasive monitoring of blood haemoglobin for analysis of fluid volume kinetics.

BACKGROUND: A commercially available pulse oximeter that reports blood haemoglobin (Hb) concentration is evaluated. This study considers whether this device can provide serial Hb data that would be sufficiently reliable for volume kinetic analysis of infusion fluids. METHODS: Forty infusions of 5 or 10 ml/kg of acetated Ringer's solution were given over 15 min in 10 healthy volunteers. Hb was measured on 17 different occasions over 120 min using the Radical 7 pulse oximeter and compared with the result of invasive blood sampling (control). A one-volume kinetic model was applied to each data series. The pulse oximeter also reported the perfusion index (PI). RESULTS: The median deviation between the 680 invasive and non-invasive Hb samples (the accuracy) was 1.6% and the absolute median deviation (precision) was 4.6%. Between-subject factors explained half of the variation in the difference between non-invasive vs. invasive sampling. Ten of the 40 non-invasive series of Hb values were discarded from kinetic analysis due to poor quality. The remaining 30 series showed a smaller distribution volume for the infused fluid when kinetic analysis was based on the non-invasive method (3.0 vs. 5.3l; P<0.001). This was due to co-variance with the PI, which exaggerated the decrease in Hb caused by the infusions. The non-invasive method might provide useful kinetic data at the group level, but individual curves deviated too much from the invasive data to be reliable. CONCLUSIONS: Non-invasive measurement of the Hb concentration during volume loading could not provide useful kinetic data for individuals.

Identification of Helicobacter pylori DNA in human cholesterol gallstones.

BACKGROUND: The gallbladder mucosa secretes hydrogen ions and is covered by mucus. The environmental conditions for bacterial colonization are similar to those in the stomach. Gallbladder stones often contain DNA from enteric bacteria, but no compelling evidence demonstrates that Helicobacter spp. have been present. The aim of this study was to establish bacterial DNA profiles in cholesterol gallstones with special reference to Helicobacter pylori. METHODS: Cholesterol gallstones from 20 patients were subjected to polymerase chain reaction, bacterial profiling by temporal temperature gradient gel electrophoresis, automated DNA sequencing, and Southern blot analysis using a Helicobacter sp. specific primer. A nested ureI-PCR assay was used to discriminate between gastric and non-gastric H. pylori. RESULTS: TTGE, partial 16S rDNA sequencing, and hybridization analysis revealed the presence of DNA presumably representing a mixed bacterial flora in cholesterol gallstones, including H. pylori in the gallstone centres in 11 out of 20 patients. In three cases, the urel-PCR assay revealed non-gastric H. pylori. CONCLUSIONS: These data support the presence of DNA from a mixed bacterial population, including H. pylori in cholesterol gallstones, reflecting either that H. pylori is an indigenous part of a flora in the stone-containing gallbladder or, alternatively, that H. pylori colonization in the biliary tract predisposes to cholesterol gallstone formation.

Utility of an intervention scoring system in documenting effects of changes in burn treatment.

The consequences of the introduction of a program of consistent use of topical antimicrobials and early aggressive excision of deep burn wounds by utilizing a comprehensive, computerized patient registry/therapeutic intervention scoring system, were investigated. Prospectively, the clinical course, mortality, outcome and hospital costs were compared for the year preceding (89 patients) and the 4 years following (226 patients) the introduction of the new treatment program. It was found that mortality decreased from 10.1 to 4.6% after change in therapy (P<0.001), despite an increase in mean burn extent. The length of hospital stay per % burn surface area declined from 1.2 to 1.0 days (P<0.001). The number and complexity of therapeutic interventions and the associated costs, also declined. Patients in the new treatment program had a better level of physical and psychosocial function at follow up. In conclusion, the introduction of a program of consistent use of topical antimicrobials and early, aggressive surgical excision was associated with an improved outcome at lesser cost. The combined registry-intervention scoring system permits ready analysis of results using data entered on a daily, near-real time basis.

Glutathione S-transferase T1 and M1 genotypes in normal mucosa, transitional mucosa and colorectal adenocarcinoma.

Gene codings for glutathione S-transferase T1 (GSTT1) and M1 (GSTM1) are polymorphic in humans with null genotypes present in approximately 20 and 50%, respectively. A significant excess of homozygous null GSTT1 and GSTM1 genotypes has been demonstrated among individuals with certain types of cancers. This finding suggests that GSTT1 and GSTM1 may play a role in tumour susceptibility. However, reports concerning colorectal cancer susceptibility are controversial. In the present study, we used a multiplex polymerase chain reaction (PCR) approach to identify and analyze simultaneously the genotypes of both the genes in 99 patients with colorectal cancer and 109 healthy controls. Compared with the control group, a significant excess of homozygous null genotypes for GSTT1 was found in normal mucosa among the cancer patients, but not for GSTM1. Both genes were more frequently deleted in tumours than in corresponding normal mucosa. Furthermore, GSTT1 null genotype in tumour tissue, was significantly related to old age and to poor differentiation of tumours. GSTM1 null genotype in tumour was more frequent in the rectal tumours compared with tumours of left colon and right colon. Our results suggest that individuals with GSTT1 null genotype may be genetically predisposed for an increased risk of developing colorectal cancer. Allele loss in tumour tissue, which reflects genetic instability, may be considered as a marker for evaluating clinico-pathological characteristics of the cancer patients.

Prevalence of gallstone disease in a Swedish population sample. Relations to occupation, childbirth, health status, life style, medications, and blood lipids.

BACKGROUND: There are only a few Swedish studies on the prevalence of gallstone disease in selected age groups, and none including possible risk factors. METHODS: Of a population sample of 1200 individuals (age, 35-85 years), 857 participated in the study. The study subjects were asked to answer a questionnaire about potential risk factors (occupation, childbirth, life style, and so forth), symptoms, and quality of life. Cholecystectomy had previously been done in 115 subjects, leaving 742 for ultrasound examination of the gallbladder. RESULTS: The prevalence of gallstone disease increased with age, and at 75 years or more, 53% of the women and 32% of the men either had gallstones or had previously undergone cholecystectomy (32% and 13%, respectively). When comparing subjects with and without gallstones, there were no differences with regard to any variable, including blood lipid levels. The odds ratio of previous cholecystectomy was increased in subjects with an occupation requiring no specific education and reduced in subjects using wine or spirits every week. The odds ratio of abdominal pain was increased after previous cholecystectomy. Women in this group also experienced a lower quality of life. CONCLUSIONS: The age and sex distribution of gallstone disease was in the order of the magnitude seen in other Scandinavian countries. None of the studied variables differed between subjects with and without gallstones. Subjects previously operated on with cholecystectomy did worse with regard to symptoms and quality of life.

Mercury induces in vivo and in vitro secretion of interleukin-1 in mice.

Macrophages from SJL and DBA mice incubated with mercuric chloride (HgCl2) in vitro for 24-72 h secreted an increased amount of interleukin 1 (IL-1) to the supernatant compared with control-incubated macrophages, as determined by a sensitive thymocyte proliferation assay. The increase of IL-1 activity showed a highly significant dose-response relationship, being close to that in controls at 10(-8) M, and maximal after incubation with 10(-5)-10(-6) M HgCl2 in both strains. At optimal concentrations of HgCl2 the IL-1 activity started to increase after 6 hrs incubation and reached a maximum after 48 h. Incubation with concentrations of HgCl2 higher than 10(-5) M resulted in a severely reduced IL-1 activity, which correlated with a reduced cell viability. Extracts of HgCl2-incubated macrophages representing cell-bound IL-1 showed no increase in IL-1 activity, irrespective of the concentration or incubation time. Topical application of HgCl2 in a mixture of acetone-olive oil on the external ear of SJL mice induced a dose- and time-dependent increase in IL-1 activity. A maximal increase was seen after application of 1% HgCl2 for 24 h with lower IL-1 activity after 48 and 72 h. Application of 5%, but not 1% or 0.1%, slightly increased the IL-1 activity in the contralateral ear treated with acetone-olive oil only, as compared with the activity in ears from animals given no mercury treatment, suggesting a systemic effect by application of 5% HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Human postburn oedema measured with the impression method.

The course of tissue swelling in human non-injured skin after burn injury was investigated with a non-invasive impression method that measures force and tissue fluid translocation during mechanical compression of the skin. Time-dependent changes in the fluid translocation and the interstitial-pressure related to impression force were measured on 11 occasions, during 3 weeks, in seven patients postburn. A mathematical model was fitted to the impression force curves and the parameters of the model depicted the time-dependent compartmental fluid shift in the postburn generalized oedema. Tissue fluid translocation increased significantly (P < 0.05) up to a maximum value after 6 days postburn and declined thereafter. This indicated a continuous increase in the generalized postburn oedema for the first 6 days postburn. Impression force at 3 weeks postburn was significantly lower (P < 0.001) as compared with the half-day postburn value, indicating an increased tissue pressure during the first days postburn. Parameter analysis indicated a flux of water-like fluid from the vasculature to the interstitial space during the first 6 days postburn. The spread of the values registered between different measurement sites was, however, large.

Visualization of iron in cultured macrophages: a cytochemical light and electron microscopic study using autometallography.

The objective of this study was to develop a sensitive cytochemical method for the visualization of iron, both at light microscopical (LM) and at electron microscopical (EM) levels, in glutaraldehyde-fixed cultured cells with reasonable morphological preservation. The method is based on autometallography (also called the sulfide silver method or the Timm technique). Gold, silver, and various metal sulfides have previously been shown to act as catalysts for cellular silver deposition from a physical developer (autometallography). In our modification of this cytochemistry, a high pH is used during the initial sulfidation step to guarantee adequate levels of sulfide ions to generate enough Fe(II or III) sulfide. Since this procedure may cause severe cellular distortion, we initially stabilize the cultured cells by a glutaraldehyde fixation. We have compared our new high pH, high S2- LM and EM variety of autometallography with other modifications of this technique that have previously been used for LM and EM demonstration of easily sulfidated heavy metals, such as zinc. Cultured mouse macrophages were examined for the localization of reactive metals following endocytosis of ferritin or inorganic Fe(III) iron. Ag-precipitates, presumed to indicate the presence of iron, were predominantly found within secondary lysosomes of the acidic vacuolar apparatus. The relation of the Ag-precipitates to iron was proven by the fact that iron-exposed cells showed a much reduced amount of silver precipitates after subsequent exposure to deferoxamine a potent iron chelator. Moreover, control macrophages neither exposed to iron nor to ferritin showed only a low normal lysosomal content--and a few extralysosomal sites--of reactive substances, believed to be iron.

Acridine orange-mediated photodamage to cultured cells.

Photosensitization mediated by the lysosomotropic, weakly basic dye acridine orange (AO) was studied on cultured J-774 cells. The phototoxicity was found to be potentiated by elevated oxygen tension and reduced at low oxygen tension. Moreover, cell cultures pre-exposed to the singlet oxygen scavenger sodium azide showed pronounced protection against the loss of viability induced by AO and blue light. AO-mediated photosensitization was neither increased by pre-exposure of cell cultures to ferric chloride or the catalase-inhibitor aminotriazole nor decreased by exposure to deferoxamine. These observations suggest that type II (singlet oxygen-mediated) reactions predominate over type I reactions (radical-mediated). A rapid and pronounced decrease in lysosomal cathepsin L activity (up to 60%) was observed after an initial 10 min irradiation, indicating the lysosomal compartment to be an early target. This irradiation time did not, however, result in any substantial loss of viability. Levels of cytosolic lactate dehydrogenase were unaffected even after 30 min irradiation, indicating that neither cytosol nor plasma membrane is a primary target of the AO-mediated photodamage. Glutathione depletion by pre-exposure to buthionine-S,R-sulfoximine (BSO) much enhanced the sensitivity of J-774 cells to AO-mediated photosensitization, indicating a protective role for thiol-containing compounds against AO-mediated photodamage.

Effect of reactive oxygen species on lysosomal membrane integrity. A study on a lysosomal fraction.

Using a lysosome-enriched "light mitochondrial" fraction of a rat liver homogenate, the effects of the reactive oxygen species hydrogen peroxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme, N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was assayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membrane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of about 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-dependent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a lag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indicate that neither superoxide radicals nor hydrogen peroxide are by themselves damaging to lysosomes. Available catalytically active iron in Fe(II) form, however, allows reactions yielding powerful oxidative species--probably hydroxyl radicals formed via Fenton reactions--to take place inducing peroxidation of the lysosomal membranes resulting in dissipation of the proton-gradient and leakage of their enzyme contents.

H2O2-mediated damage to lysosomal membranes of J-774 cells.

The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal integrity were investigated in a model system of cultured, established, macrophage-like J-774 cells. The cells were found to rapidly degrade added hydrogen peroxide, withstanding concentrations < or = 250 microM without cell death; however, all tested concentrations (100-500 microM) substantially decreased cellular ATP to approximately the same degree. Concentrations of hydrogen peroxide > or = 500 microM resulted in a pronounced and rapid decrease in cell viability preceded by the loss of lysosomal integrity, as judged by the relocalization of acridine orange, a lysosomotropic weak base, in pre-labelled cells. Hydrogen peroxide-induced relocalization of acridine orange and cell death were either enhanced or much prevented, according to if the cells were initially allowed to endocytose ferric iron or the specific iron-chelator deferoxamine, respectively. Depletion of ATP, however, was not associated with the loss of lysosomal integrity and viability regardless of iron or deferoxamine pretreatment. Pre-exposure to E-64, an inhibitor of lysosomal thiol proteases, resulted in the reduction of both lysosomal membrane damage and cell death. The results are interpreted as indicating (i) generation of hydroxyl radicals within the secondary lysosomal compartment due to the occurrence of reactive ferrous iron, leading to (ii) peroxidative alterations of the lysosomal membrane resulting in (iii) loss of lysosomal membrane integrity with dissipation of the proton gradient and leakage of lysosomal contents, including hydrolytic enzymes, into the cell sap.

Alloxan cytotoxicity involves lysosomal damage.

The cytotoxic effects of alloxan are not understood in any great detail, although they are considered to involve reactions mediated by oxygen-derived free radicals. These reactive species may form extra-or intracellularly following alloxan reduction, and result in cell damage through a number of complex interactions with a variety of macromolecules. The purpose of the present study was to elucidate further the early intracellular effects of alloxan on a model system of macrophage-like cells in culture. Addition of alloxan (15 mM), without reducing agents, to the medium surrounding the cells (phosphate-buffered saline, PBS, 37 degrees C, pH 7.4) resulted in rapid lysosomal damage (disappearance of the proton gradient over the membrane) followed by severe cellular degeneration (swelling and blebbing) and 50% cell death (trypan blue dye exclusion test) within fifty min. Cells pretreated with the gamma-glutamyl cysteine synthetase-inhibiting agent BSO, to decrease levels of intracellular glutathione, showed enhanced sensitivity to alloxan. The results are interpreted as indicating the cytotoxicity to result from intracellular formation of superoxide radicals, hydrogen peroxide and hydroxyl radicals, the latter within secondary lysosomes containing trace amounts of reactive iron (inducing Fenton reactions). The ensuing lysosomal membrane damage may result in leakage of lysosomal hydrolases and further cellular degeneration.

Effects of alloxan and reducing agents on macrophages in culture.

The diabetogenic effect of the quinonoid compound alloxan is not understood in detail although it supposedly involves reactions mediated by alloxan and oxygen radicals. These reactive species may form extra- or intracellularly and cause cell damage through a variety of complex interactions with several macromolecules. The purpose of this study was to elucidate early (less than or equal to 60 min) effects of alloxan and reducing agents (cysteine and ascorbic acid) on cultured macrophages, as assayed by the trypan blue dye exclusion test and the sensitive fluorescein diacetate and propidium iodide (FDA/PI) double staining technique. During the reactions between alloxan and reducing agents, oxygen was consumed as a sign of superoxide anion radical formation. When alloxan alone was added to two different culture media without serum, oxygen was still consumed, indicating formation of oxygen radicals due to the occurrence of reducing substances in cell culture media. This finding demonstrated the necessity of performing further studies in solutions without reducing capacity, e.g. in phosphate-buffered saline. The experiments showed that exposure of normal and malignant macrophages to alloxan and reducing substances resulted in rapidly occurring plasma membrane damage and ensuing cell death. Separate addition of catalase, desferrioxamine or superoxide dismutase resulted in evident, slight and no protection, respectively. The combinations of (i) catalase and desferrioxamine, and (ii) catalase, desferrioxamine and superoxide dismutase, however, inhibited cell damage in a pronounced and complete way, respectively. The results are interpreted as indicating cell damage due to the extracellular formation of hydrogen peroxide and hydroxyl radicals. The latter in close proximity to the cells and acting on the plasma membrane, while the former, after diffusing into the cell, may have several intracellular targets. The FDA/PI technique proved its value as a quantifiable method for the evaluation not only of cell death but also of cell damage with computer-based fluorometry.

Photooxidative damage to lysosomes of cultured macrophages by acridine orange.

Cultured cells accumulate acridine orange (AO), which is a weak basic dye and a photosensitizer, in lysosomes and other acidic compartments. During exposure to blue light, AO-loaded macrophages show decreasing red granular fluorescence and increasing green diffuse fluorescence. This is hypothesized to represent peroxidative damage to lysosomal membranes resulting in an impaired proton gradient with deprotonation of the AO to its uncharged form and subsequent leakage of the dye. Further damage to the lysosomal membranes will result in release of lytic enzymes from the lysosomal compartment into the cytosol, leading to degeneration and finally cell death. The survival of AO-loaded and light-exposed macrophages is controllable by varying the exposure times to blue light. Inhibition of lysosomal proteases by E-64 results in increased cell survival after AO and blue light-mediated damage, indicating a role of proteolytic enzymes in this type of damage. Morphological analysis shows 'rounding up' with formation of retraction fibrils and pronounced plasma membrane blebbing. The formation of autophagic vacuoles is an early and pronounced event. After protease inhibition, however, all these phenomena are inhibitable to a considerable degree. We have thus directed photooxidative damage selectively to lysosomal membranes and their contents. This technique will allow further detailed studies of the role of lysosomes in degeneration-regeneration processes.

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction.

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)