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A letter to the editor is presented related to Nephrocheck, an apparatus for early detection of acute kidney injury (AKI).

Investigates the prevalence of gallstone disease in Sweden. Identification on the symptoms of gallstone disease; Evaluation of differences between age groups; Capability of ethanol to form cholesterol gallstone.

Background: The latissimus dorsi (LD) flap is a commonly used method for breast reconstruction after mastectomy. In this study, we present a long-term follow-up and effects of refining surgery on patient satisfaction and quality of life after breast reconstruction with the extended LD flap, using the BREAST-Q questionnaire., Objectives: The aim of this study was to investigate the patient-reported long-term results after breast reconstruction with the extended LD myocutaneous flap., Methods: A retrospective cohort study of adult patients ( n = 101) who were operated on using the extended LD flap for breast reconstruction at the Linköping University Hospital during 1997 to 2012 was made using data retrieved from medical records. The patients were asked to complete the BREAST-Q questionnaire at 2 different postoperative time points., Results: Eighty-three patients replied to the first questionnaire, and 56 patients also replied to the second. Mean follow-up was 11.7 years. Higher age and living together with someone correlated to higher BREAST-Q results, while postoperative infection, bilateral LD flaps, smoking, and prior breast surgery had a negative impact on the results. Overall BREAST-Q results increased over time. No independent effect of refining surgery could be shown., Conclusions: Patient satisfaction after breast reconstruction with the LD flap as measured with the BREAST-Q questionnaire is high and in line with other studies. The overall satisfaction with the reconstruction method seems to increase with time, but no further increase in satisfaction after refining surgery could be established., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Aesthetic Society.)

Background: Elevated plasma concentrations of syndecan-1 and heparan sulfate in studies of trauma, sepsis, and major surgery are commonly assumed to indicate acute glycocalyx degradation. We explored a possible role of the kidneys for these elevations., Methods: Plasma and urine concentrations of syndecan-1, heparan sulfate, and biomarkers of inflammation were measured over 5 hours in 15 hospital patients treated for post-burn injury. The renal clearances of syndecan-1 and heparan sulfate (CL R ) were calculated and their influence on the plasma concentration predicted by simulation., Results: The urine/plasma concentration ratio was 0.9 (0.3-3.0) for syndecan-1 and 2.8 (2.0-4.3) for heparan sulfate. The CL R varied 250-fold for syndecan-1 and 10-fold for heparan sulfate. Multiple linear regression analysis showed that CL R for syndecan-1 was positively associated with the creatinine clearance (P < .0032) and the urine flow (P < .015). CL R for heparan sulfate increased with interleukin-6 (P < .003) and the urine flow (P < .01). Simulations suggested that a change in CL R from the mean of the highest 3 to the lowest three values would double plasma syndecan-1 within 4 hours and cause a 7-fold rise after 24 hours. A similar change in CL R for heparan sulfate would triple the plasma level within 24 hours, even if no increased shedding of the glycocalyx takes place., Conclusions: The renal elimination of syndecan-1 and heparan sulfate varied greatly. A change in kidney function, which is common after trauma and major surgery, might alone induce several-fold changes in their plasma concentrations., (© 2021 The Authors. Acta Anaesthesiologica Scandinavica published by John Wiley & Sons Ltd on behalf of Acta Anaesthesiologica Scandinavica Foundation.)

Background: Preclinical experiments show that an inflammatory reaction causes degradation of the endothelial glycocalyx layer and accelerated capillary leakage of albumin and fluid. The hypothesis in the present study was that elevated plasma concentrations of glycocalyx degradation products are associated with greater capillary leakage in humans., Methods: This open clinical trial involved administration of an intravenous infusion of 20% albumin at 3 mL/kg over 30 minutes to 15 postburn patients who showed an activated inflammatory response. Blood samples and urine were collected for 300 minutes. The plasma concentrations of 2 biomarkers of glycocalyx degradation-syndecan-1 and heparan sulfate-were measured at 0, 60, and 300 minutes and compared to the capillary leakage of albumin and fluid obtained by mass balance calculations and population kinetic analysis., Results: Patients were studied at 7 days (median) after a burn injury that covered 15% (maximum 48%) of the body surface area. The median plasma syndecan-1 concentration was 71 (25th-75th percentiles, 41-185) ng/mL. The 2 patients with highest values showed 2279 and 2395 ng/mL (normal 15 ng/mL). Heparan sulfate concentrations averaged 915 (673-1539) ng/mL. The infused amount of albumin was 57 (48-62) g, and 6.3 (5.1-7.7)% of that leaked from the plasma per hour.Linear correlation analysis of the relationship between the 10logarithm of the mean syndecan-1 and the albumin leakage showed a slope coefficient of -1.3 (95% confidence interval [CI], -3.6 to 1.0) and a correlation coefficient of -0.33 (P = .24). The kinetic analysis revealed that syndecan-1 served as a statistically significant covariate to the albumin leakage, but the relationship was inverse (power exponent -0.78, 95% CI, -1.50 to -0.05; P < .02). Heparan sulfate levels did not correlate with the capillary leakage of albumin or fluid in any of the analyses., Conclusions: A raised plasma concentration of syndecan-1 alone cannot be extrapolated to indicate increased capillary leakage of albumin and fluid., Competing Interests: Conflicts of Interest: See Disclosures at the end of the article., (Copyright © 2020 International Anesthesia Research Society.)

Background: Early treatment of severe hyperglycemia involves large shifts of body fluids that entail a risk of hemodynamic instability. We studied the feasibility of applying a new electrolyte equation that estimates the degree of volume depletion and the distribution of infused 0.9% saline in this setting., Methods: The new equation was applied to plasma and urinary concentrations of sodium and chloride measured before and 30 min after a 30-min infusion of 1 L of 0.9% saline on two consecutive days in 14 patients with severe hyperglycemia (mean age 50 years). The extracellular fluid (ECF) volume was also estimated based on the volume dilution kinetics of chloride., Results: On day 1, the baseline ECF volume amounted to 11.5 L. The saline infusion expanded the ECF space by 160 mL and the intracellular fluid space by 375 mL. On day 2, the ECF volume was 15.5 L, and twice as much of the infused fluid remained in the ECF space. The chloride dilution kinetics yielded baseline ECF volumes of 11.6 and 15.2 L on day 1 and day 2, respectively. No net uptake of glucose to the cells occurred during the two 1-h measurement periods despite insulin administration in the intervening time period., Conclusions: The electrolyte equation was feasible to apply in a group of hyperglycemic patients. The ECF space was 3 L smaller than expected on admission but normal on the second day. Almost half of the infused fluid was distributed intracellularly.

The Revised (or "Extended") Starling principle is based on highly controlled laboratory-based frog and rodent experiments and remains a hypothesis awaiting clinical validation. A key point is that the endothelial glycocalyx layer moves the oncotic gradient from being between the plasma and the interstitium to between the plasma and a virtually protein-free space between the glycocalyx and the endothelial cell membrane, which dramatically changes the prerequisites for fluid absorption from tissue to plasma. However, many experimental and clinical observations in humans agree poorly with the new microcirculatory proposals. The most troubling aspect of the explanation regarding the role of the glycocalyx in the Revised Starling principle is the effective reabsorption of fluid by skeletal muscle when the capillary filtration pressure is acutely reduced. Other issues include the plasma volume effects of hypertonic saline, iso-oncotic and hyper-oncotic albumin, fluid distribution during cardio-pulmonary bypass, and the virtually identical capillary leakage of plasma and albumin despite marked inflammation found in our fluid therapy studies. The Revised Starling principle deals mainly with steady-state conditions, but the circulatory system is highly dynamic. Second to second vasomotion is always operational and must be considered to understand what we observe in humans., (© 2020 The Authors. Acta Anaesthesiologica Scandinavica Foundation published by John Wiley & Sons Ltd on behalf of Acta Anaesthesiologica Scandinavica Foundation.)

Background: The pectoralis major flap, which is usually harvested bilaterally, is considered a workhorse flap in the reconstruction of sternal defects. After a median sternotomy for open heart surgery, 1%-3% of patients develop deep infection and dehiscence of the sternal wound, some of which will eventually require reconstructive surgery. Our aim was to describe the clinical feasibility and associated complications of the unilateral pectoralis major advancement flap in the reconstruction of sternal defects., Methods: A retrospective analysis of all adult patients who were operated on using a unilateral pectoralis major flap for reconstruction of the chest wall at the Linköping University Hospital during 2008-18 was made using data retrieved from medical records., Results: Forty-three patients had reconstructions with unilateral pectoralis major flaps. Three flaps failed completely, and another 10 patients developed complications that required further operation. The factors that were independently associated with loss of the flaps and complications were: older age, male sex, the number of different antibiotics used, and a long duration of treatment with negative wound pressure. Fewer wound revisions before the reconstruction resulted in more complications. The factors that were independently associated with prolonged time to complete healing were emergency reoperation after the initial operation and complications after reconstruction., Conclusion: The unilateral pectoralis major advancement flap has proved to be a useful technique in the reconstruction of most sternal defects after sternal wound infection in older patients. There is, however, need for a follow-up study on a larger number of procedures to evaluate the long-term outcome compared with other methods of sternal reconstruction.

Background: Ringer's solution might be used to treat volume depletion (extracellular dehydration) and hemorrhage, but there is no integrated view of how these fluid balance disorders influence the kinetics of the infused volume., Methods: Acute dehydration (mean 1.7 L) was induced by repeated doses of furosemide (5 mg) in 10 healthy male volunteers, and 0.5 L and 0.9 L of blood was withdrawn in random order on different occasions in another 10 male volunteers, just before administration of Ringer's acetate solution. Infusions performed in the normovolemic state served as controls. Measurements of blood hemoglobin and urinary excretion were used to create volume kinetic profiles that were analyzed using mixed-effects modeling software., Results: Infusions over 15 to 30 min showed a marked distribution phase during which the plasma volume transiently increased by 50% to 75% of the administered volume. Dehydration and hemorrhage accelerated redistribution but retarded the elimination; the half-life of the infused fluid increased from 36 to 51 min (mean) from 1 L of dehydration and to 95 min from 1 L of hemorrhage. Extravascular accumulation decreased with the dehydration volume and increased with the hemorrhage volume.Simulations show that 60% as much Ringer is needed to replace volume depletion amounting to 1 L as compared with hemorrhage over a 2-h period. A continued but slower drip after the initial fluid resuscitation prevents rebound hypovolemia., Conclusions: Furosemide-induced dehydration and blood withdrawal in normotensive volunteers had modest effects on the Ringer's acetate kinetics. Urinary excretion was inhibited more by hemorrhage than by dehydration.

Aims: A population kinetic model was developed for the body fluid shifts occurring when 20% albumin is given by intravenous infusion. The aim was to study whether its efficacy to expand the plasma volume is impaired after major surgery., Methods: An intravenous infusion of 3 mL/kg 20% albumin over 30 minutes was given to 15 volunteers and to 15 patients on the 1 st day after major open abdominal surgery. Blood samples and urine were collected during 5 hours. Mixed-effect modelling software was used to develop a fluid volume kinetic model, using blood haemoglobin and urine excretion the estimate body fluid shifts, to which individual-specific covariates were added in sequence., Results: The rise in plasma albumin expanded the plasma volume in excess of the infused volume by relocating noncirculating fluid (rate constant k 21 ), but it also increased losses of fluid from the kinetic system (k b ). The balance between k 21 and k b maintained the rise in plasma albumin and plasma volume at a virtual steady-state for almost 2 hours. The rate constant for urinary excretion (k 10 ) was slightly reduced by the preceding surgery, by a marked rise in plasma albumin, and by a high preinfusion urinary concentration of creatinine. The arterial pressure, body weight, and plasma concentrations of C-reactive protein and shedding products of the endothelial glycocalyx layer (syndecan-1, heparan sulfate, and hyaluronic acid) did not serve as statistically significant covariates., Conclusions: There were no clinically relevant differences in the kinetics of 20% albumin between postoperative patients and volunteers., (© 2019 The British Pharmacological Society.)

Background: Injury (shedding) of the endothelial glycocalyx layer, which alters local blood flow and microvascular permeability, is assessed by measuring components of this layer in circulating blood. The influence of renal function on their concentrations is unknown., Methods: Plasma and urine concentrations of three shedding products (syndecan-1, hyaluronic acid, and heparan sulfate) and creatinine were measured over 5 hours in 15 healthy volunteers and 15 postoperative patients; this guaranteed a spread of kidney functions. Renal clearances were calculated., Results: Low renal clearances of syndecan-1 (mean 3.5 mL/min) and hyaluronic acid (0.8 mL/min) correlated inversely with the 6-fold variability in the plasma concentrations of these substances (r = - 0.45 and- 0.49). Low creatinine clearance correlated inversely (r = - 0.60) and plasma creatinine directly (r = 0.52) with the two-fold variability in heparan sulfate, which was the only shedding substance that also correlated with C-reactive protein (r = 0.51) and, therefore, showed higher concentrations after surgery., Conclusions: The present explorative study suggests that a 6-fold variability in the plasma concentrations of three commonly measured endothelial shedding products can be understood by the kidney's ability to excrete them. This finding has implications when interpreting results of studies where shedding is assessed.

Background: Reconstruction of complex defects in the lower leg is a challenge. Although microvascular free tissue transfer is a popular technique, experience and available resources limit its use. Furthermore, free tissue transfer is not always required in the reconstruction of small lower leg defects, as many of them can be reconstructed with local alternatives such as an extensor digitorum brevis flap (EDB). Our aim was to describe our experience of the last 20 years with the EDB as a local muscle flap to cover small complex lower leg defects to establish its clinical feasibility and to document its associated complications., Methods: All adult patients who were operated with EDB flap reconstruction of the lower limb during 1997-2017 at the Department of Hand and Plastic Surgery, Linköping University Hospital, were included in this retrospective study., Results: Of 64 patients operated, only 7 had total flap failure, and the rate of complete success was 73% (47/64). Most of the skin defects were associated with fractures or complications thereof and were located in the ankle region, the dorsum of the foot, and the distal third of tibia or even the proximal tibia. Defects in the malleolar region and coexisting cardiovascular condition were factors associated with flap loss (either partial or total)., Conclusion: The pedicled EDB-flap has, in our hands, proved to be a versatile and safe reconstructive option in the reconstruction of small defects in the lower leg and foot. Long-time follow-up is, however, recommended.

Background: Volume kinetic fluid turnover of three beverages was investigated for the purpose of estimating their rates of absorption and elimination as well as their maximum effect on the blood volume. The results were then used to simulate the effects of ingesting different combinations of these fluids., Method: Ten healthy volunteers ingested 0.5 L of tap water, lemonade (90 g/L carbohydrates) and isotonic saline (9 g/L) on different occasions. Venous blood samples for measurement of the blood haemoglobin (Hb), haematocrit and glucose concentrations were collected on 10 occasions over 2 h. A kinetic model based on haemoglobin dilution and urinary excretion was used to estimate the rate of absorption, the blood volume expansion over time, and the rate of elimination. Obtained kinetic data was used to simulate combinations of the three beverages in order to reach a predetermined goal of a 1:1 hydration of the blood volume and peripheral tissues over 6 h., Results: Tap water had the fastest absorption but primarily hydrated peripheral tissues. Maximum hydration was reached after 17 min. Lemonade effectively expanded the blood volume and was absorbed and excreted at a high rate. The maximum hydration from isotonic saline occurred 60 min after ingestion. Slow excretion could make it possible to use saline to prolong the effects of the other two beverages., Conclusions: It is possible to use the kinetic model to evaluate fluid turnover and compartmental distribution. Composition and timing of fluid intake can be calculated mathematically to meet predetermined goals of hydration and distribution., Trial Registration: NCT01360333 Date of registration: 05/23/2011.

Introduction: The turnover of Ringer´s solutions is greatly dependent on the physiological situation, such as the presence of dehydration or anaesthesia. The present study evaluates whether the kinetics is affected by previous infusion of colloid fluid., Methods: Ten male volunteers with a mean age of 22 years underwent three infusion experiments, on separate days and in random order. The experiments included 10 mL/kg of 6% hydroxyethyl starch 130/0.4 (Voluven™), 20 mL/kg of Ringer's acetate, and a combination of both, where Ringer´s was administered 75 minutes after the starch infusion ended. The kinetics of the volume expansion was analysed by non-linear least- squares regression, based on urinary excretion and serial measurement of blood haemoglobin concentration for up to 420 minutes., Results: The mean volume of distribution of the starch was 3.12 L which agreed well with the plasma volume (3.14 L) estimated by anthropometry. The volume expansion following the infusion of starch showed monoexponential elimination kinetics with a half-life of two hours. Two interaction effects were found when Ringer´s acetate was infused after the starch. First, there was a higher tendency for Ringer´s acetate to distribute to a peripheral compartment at the expense of the plasma volume expansion. The translocated amount of Ringer´s was 70% higher when HES had been infused earlier. Second, the elimination half-life of Ringer´s acetate was five times longer when administered after the starch (88 versus 497 minutes, P<0.02)., Conclusions: Starch promoted peripheral accumulation of the later infused Ringer´s acetate solution and markedly prolonged the elimination half-life., Trial Registration: ClinicalTrials.gov: NCT01195025.

Background: Patients admitted to surgery may be dehydrated, which is difficult to diagnose except when it is severe (>5% Gl116 of the body weight). We hypothesized that modest dehydration can be detected by kinetic analysis of the blood hemoglobin concentration after a bolus infusion of crystalloid fluid., Methods: Four series of experiments were performed on 10 conscious, healthy male volunteers. Separated by at least 2 days, they received 5 or 10 mL/kg acetated Ringer's solution over 15 minutes. Before starting half of the IV infusions, volume depletion amounting to 1.5 to 2.0 L (approximately 2% of body weight) was induced with furosemide. The elimination clearance and the half-life of the infused fluid were calculated based on blood hemoglobin over 120 minutes. The perfusion index and the pleth variability index were monitored by pulse oximetry after a change of body position., Results: Dehydration decreased the elimination clearance of acetated Ringer's solution [median (25th-75th percentile)] from 1.84 (1.23-2.57) to 0.53 (0.41-0.79) mL/kg/min (Wilcoxon matched-pair test P < 0.001) and increased the half-life from 23 (12-37) to 76 (57-101) minutes (P < 0.001). The smaller infusion, 5 mL/kg, fully discriminated between experiments performed in the euhydrated and dehydrated states, whereas the urinary excretion provided a less-reliable indication of hydration status. Dehydration decreased the perfusion index but did not affect the pleth variability index., Conclusion: Dehydration amounting to 2% of the body weight could be detected from the elimination clearance and the half-life of an infusion of 5 mL/kg Ringer's solution.

Aim: To contrast the effects of various modifications of body fluid volumes on thirst as reported by healthy volunteers., Methods: Ten male volunteers aged between 19 and 37 years (mean 22 years) underwent four experiments each, which comprised infusion of 400-800 mL of acetated Ringer's solution and intake of 600 mL of tap water. Half of the experiments were preceded by volume depletion (median 1.7 L) with furosemide. A visual analogue scale (0-100 mm) was used to assess perceived thirst during each experiment., Results: Volume depletion (P < 0.001) and tap water (P < 0.03) both affected thirst by 13 mm per L of fluid, whereas spontaneous diuresis and infusion of Ringer's acetate did not significantly change the thirst rating (multiple regressions). More detailed analyses showed that the volume depletion increased the median (25th-75th percentiles) thirst rating from 28 mm (21-43) to 59 mm (46-72, P < 0.001) while no change occurred in those who were only slightly thirsty (< 30 mm) before the volume depletion began. Ringer's solution alleviated thirst in those who were very thirsty, but tended to increase thirst in the volunteers who were not thirsty before the infusion. Similarly, hydration with tap water decreased thirst (by 24 mm, P < 0.04) in those who were thirsty (> 60 mm) while the others reported no change., Conclusion: The change in thirst rating during volume depletion, administration of Ringer's acetate, and ingestion of tap water were all dependent on the thirst rating obtained when the manipulation of the body fluid volume was initiated.

Background: Medical implants often fail as a result of so-called foreign body reactions during which inflammatory cells are recruited to implant surfaces. Despite the clinical importance of this phenomenon, the mechanisms involved in these reactions to biomedical implants in humans are not well understood. The results from animal studies suggest that both fibrinogen adsorption to the implant surface and histamine release by local mast cells are involved in biomaterial-mediated acute inflammatory responses. The purpose of this study was to test this hypothesis in humans., Methods: Thirteen male medical student volunteers (Caucasian, 21-30 years of age) were employed for this study. To assess the importance of fibrinogen adsorption, six volunteers were implanted with polyethylene teraphthalate disks pre-coated with their own (fibrinogen-containing) plasma or (fibrinogen-free) serum. To evaluate the importance of histamine, seven volunteers were implanted with uncoated disks with or without prior oral administration of histamine receptor antagonists. The acute inflammatory response was estimated 24 hours later by measuring the activities of implant-associated phagocyte-specific enzymes., Results: Plasma coated implants accumulated significantly more phagocytes than did serum coated implants and the recruited cells were predominantly macrophage/monocytes. Administration of both H1 and H2 histamine receptor antagonists greatly reduced the recruitment of macrophages/monocytes and neutrophils on implant surfaces., Conclusion: In humans--as in rodents--biomaterial-mediated inflammatory responses involve at least two crucial events: histamine-mediated phagocyte recruitment and phagocyte accumulation on implant surfaces engendered by spontaneously adsorbed host fibrinogen. Based on these results, we conclude that reducing fibrinogen:surface interactions should enhance biocompatibility and that administration of histamine receptor antagonists prior to, and shortly after, medical device implantation should improve the functionality and longevity of medical implants.

Sutured anastomoses of small vessels are considered difficult to learn. Mechanical anastomosis systems allow a more rapidly performed anastomosis. In order to compare the process of learning to perform sutured and mechanical microvascular anastomoses, two surgeons, with limited microvascular experience, performed 30 aortic and 30 femoral vein anastomoses in 30 Wistar rats. The methods compared were conventional suture, vascular closure system (VCS) and microvascular anastomatic coupler system (MAC). There were no inter-surgeon differences regarding patency or time to perform anastomoses. The average time to perform a suture anastomosis was 39 min (patency 80%). Anastomoses with the VCS system took 24 min (patency 25%), whereas the MAC couplers took 13 min to perform (patency 95%). There was a significant learning effect with sutures, but no obvious reduction in time to perform MAC coupler or VCS clip anastomoses was seen. MAC couplers were easiest to use, and allowed us to perform rapid anastomoses with high patency., ((c) 2005 Wiley-Liss, Inc. Microsurgery 25:596-598, 2005.)

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.

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)

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.

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.

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.

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.

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.

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.

Reflocheck, a new microprocessor-regulated apparatus determining blood sugar levels was tested in six laboratories. In the range of 20-450 mg/dl the apparatus gave precise results (co-efficient of variance 0.8%-6.4%), in good agreement with results by the hexokinase method. Reflocheck is an accurate and reliable instrument for the measuring of blood glucose levels.

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)