The storage defects in grey platelet syndrome and alphadelta-storage pool deficiency affect alpha-granule factor V and multimerin storage without altering their proteolytic processing.

Among proteins stored in alpha-granules, multimerin and factor V share unusual features: they bind to each other, are proteolysed to unique forms and are stored eccentrically in alpha-granules. These unique features of their processing led us to study these proteins in alpha delta storage pool deficiency (alphadelta-SPD) and grey platelet syndrome (GPS, alpha-SPD), two conditions known to impair alpha-granule protein storage. Platelet factor V and multimerin were severely reduced in GPS, whereas they ranged from reduced to normal in alphadelta-SPD. The platelet levels of factor V and multimerin in these disorders indicated multimerin deficiency was not predictive of platelet factor V deficiency, although it reduced the amount of multimerin associated with platelet factor V. In GPS only, the defect in storing proteins was associated with increased multimerin and multimerin-factor V complexes in plasma. Like normal platelets, GPS and alphadelta-SPD platelets contained factor V mainly in granules. Platelet factor V and multimerin were proteolysed to normal platelet forms in GPS and alphadelta-SPD platelets, indicating that these conditions preserve some aspects of normal alpha-granule protein processing. Although we found factor V can be stored in platelets deficient in multimerin, our data indicate that multimerin storage influences the point at which multimerin binds factor V.

Ultrastructural alterations during the critical phase of reperfusion: a stereological study in buffer-perfused isolated rat hearts.

The present study focuses on myocardial ultrastructural alterations during the early phase of reperfusion. Isolated buffer-perfused rat hearts were exposed to standard perfusion (control group,n = 10); 60 min of global ischemia (n = 10); 60 min of global ischemia followed by 2 min of reperfusion (n = 10); or 60 min of global ischemia followed by 10 min of reperfusion (n = 10). The hearts were perfusion-fixed for electron microscopy, and ultrastructural evaluation was performed using stereological technique in order to obtain an estimate of the volume fraction and absolute volume of different tissue components. EFFECT OF ISCHEMIA: Neither the ventricular nor the myocytic volume differed significantly from the respective control values. Both the myocytic mitochondrial volume (135+/-8 vs control 89+/-6 microl) and the volume of myocytic clear space (35+/-6 vs control 10+/-2 microl) were significantly increased. The capillary volume (22+/-4 vs control 58+/-6 microl) and the volume of the capillary lumen (15+/-3 vs control 48+/-5 microl) were significantly decreased. The volume of the capillary wall, however, was not altered after exposure to ischemia (7+/-3 vs control 10+/-1 microl). ADDITIVE EFFECT OF ISCHEMIA AND REPERFUSION: Both the ventricular volume (755+/-28 vs control 600+/-32 microl) and the myocytic volume (396+/-24 vs control 287+/-16 microl) were significantly increased after 10 min of reperfusion. EFFECT OF REPERFUSION: The ischemic-induced myocytic mitochondrial swelling and increase of clear space were not reinforced during reperfusion. Furthermore, the volume of the capillary lumen and the capillary wall did not alter significantly in the groups exposed to reperfusion compared to the ischemic hearts. In conclusion, stereological evaluation did not reveal significant aggravation of ischemic-induced myocardial injury during the early phase of reperfusion.

The initial phase of myocardial reperfusion is not associated with aggravation of ischemic-induced ultrastructural alterations in isolated rat hearts exposed to prolonged global ischemia.

The present study focuses on the qualitative and sequential development of myocardial ultrastructural changes during the first 10 min of reperfusion in isolated rat hearts exposed to 60 min of global ischemia. The frequency of and the association between ultrastructural changes were examined by semiquantitative morphometry using the micrograph as unit. In each micrograph the subcellular components of the myocytes (sarcolemma, mitochondria, myofilaments and nucleus) and the endothelial cells were evaluated and graded as slightly, moderately, or severely altered. Ischemia alone induced moderate to severe ultrastructural alterations. The myocytes revealed sarcolemmal disattachment or rupture. The myocytic mitochondria had a clear matrix with abundant broken cristae and amorphous matrix densities. The myofilamental pattern was irregular or even disrupted, and most nuclei had reduced density and showed margination of chromatin. The endothelium showed vacuolization, rupture of the plasma membrane, and extracellular accumulation of cellular debris. During the first 2 min of reperfusion severe ultrastructural alterations were partly reversed. After 10 min of reperfusion both the frequency and grade of myocardial ultrastructural alternations were similar to that observed after ischemia. Cristal adhesions occurred predominately during reperfusion and were associated with moderately and severely altered myocytic mitochondrial alterations. In conclusion, the results showed that ischemic-induced ultrastructural alterations were transiently improved upon reperfusion. With exception of the development of cristal adhesions, the acute phase of reperfusion was not associated with additional ultrastructural changes in isolated buffer-perfused rat hearts exposed to prolonged ischemia.

[Laparoscopic cholecystectomy]. / Laparoskopisk kolecystektomi.

The appropriateness of a laparoscopic approach to cholecystectomy in community hospital settings has been questioned. To address this issue a prospective study of outcomes of laparoscopic cholecystectomies performed during a three year period at Telemark Community Hospital was undertaken. There were 229 procedures performed by five surgeons. 24 (10.5%) of the attempted laparoscopic cholecystectomies were converted to open cholecystectomies. The average hospital stay after laparoscopic cholecystectomy was 3.0 days (SD = 2.6). Minor intraoperative complications (gall bladder perforation, gall bladder bed bleeding) occurred in 43% of the laparoscopic procedures. There were nine cases (4.4%) of major intraoperative complications which included laceration of the common bile duct (n = 4, one discovered during surgery), ileal perforation (n = 1) and laceration of the liver (n = 4). The frequency of postoperative complications after laparoscopic cholecystectomy was 8.8%. Bile peritonitis was observed in three patients, of whom one died. There were no significant differences in intra- and postoperative complications between the surgeons performing the operations. The present results support the argument that laparoscopic cholecystectomies can be performed safely and effectively in community hospitals.

Low concentrations of hydrogen peroxide improve post-ischaemic metabolic and functional recovery in isolated perfused rat hearts.

The aim of the present study was to test the hypothesis that low concentrations of hydrogen peroxide (H2O2) have a beneficial effect on post-ischaemic myocardial recovery. Functional and metabolic measurements were performed in isolated buffer-perfused rat hearts exposed to 30 min perfusion with 0 (control group A), 25, 50, 100 or 200 microM H2O2 or 30 min global ischaemia followed by 30 min reperfusion with 0 (control group B), 25, 50 or 100 microM H2O2. Catalase (200 U/ml) was added as scavenger during reperfusion with 25 microM H2O2. Non-ischaemic perfusion: All concentrations of H2O2 induced an immediate vasodilatation, which was maintained in the 50 microM group, but it was followed by vasoconstriction in the 100 and 200 microM group. Left ventricular developed pressure (LVDP) was significantly increased at the end of perfusion in the 50 microM group compared to the control group. Exposure to 100 and 200 microM H2O2 significantly decreased LVDP and increased end-diastolic pressure. ATP was reduced in the 100 microM group. Post-ischaemic perfusion: Exposure to 25 microM H2O2 caused improved coronary flow during the first 20 min of reperfusion compared to the control group (accumulated coronary flow; 235.5 +/- 10.8 v 172.7 +/- 8.6 ml). LVDP was significantly higher in the 25 microM group compared to the control (59.8 +/- 10.2 v 22.1 +/- 7.3 mmHg), and end-diastolic pressure was significantly lower (32.1 +/- 19.6 v 78.8 +/- 2.2 mmHg) at the end of reperfusion. Improved recovery was not observed in the group exposed to 25 microM H2O2 plus catalase. Treatment with 25 microM H2O2 caused significantly improved recovery of tissue ATP and creatine phosphate. In conclusion, the present study showed that exposure to 25 microM H2O2 improved post-ischaemic recovery in hearts subjected to global ischaemia.

Reversible ultrastructural alterations in the myocytic mitochondria of isolated rat hearts induced by oxygen radicals.

The present study focuses on reversible mitochondrial ultrastructural alterations in myocardial myocytes that correspond or accompany reversible metabolic depression observed after oxygen radical exposure. The myocytic mitochondrial membranes and matrix of isolated Langendorff-perfused rat hearts were examined by semiquantitative morphometry using the electron micrograph as unit. The hearts were exposed to either standard perfusion (group A), 10 min of oxygen radicals together with superoxide dismutase and catalase followed by 35 min of recovery (group B), 10 min of oxygen radicals alone (group C), or 10 min of oxygen radicals followed by 35 min of recovery (group D). Mitochondrial ultrastructural alterations were detected in only a few micrographs in groups A and B. The frequency of micrographs with mitochondrial ultrastructural alterations was 69% in group C and 62% in group D. In the group exposed to 10 min of oxygen radicals without recovery (group C) condensed pentalaminar membranous profiles arranged in parallel, interpreted to be closely adhering cristae, were detected in the intracristal compartment of myocytic mitochondria in 50% of the micrographs. The cristal adhesions were associated with other mitochondrial ultrastructural changes. Cristal adhesions were not present in group A or B, and were rarely found in the group exposed to 10 min of oxygen radicals followed by 35 min of recovery (group D). Thus, the cristal adhesions appear to be reversible alterations caused by exposure to oxygen radicals.

Mepacrine protects the isolated rat heart during hypoxia and reoxygenation--but not by inhibition of phospholipase A2.

Mepacrine (quinacrine) has in a number of studies been shown to protect the heart from ischemic injury, a protection commonly claimed to be due to inhibition of phospholipase A2. The aim of the present study was to investigate the effect of mepacrine 1 microM on isolated, buffer perfused rat hearts subjected to 60 min hypoxia and 30 min reoxygenation. We also wanted to clarify whether any cardioprotective effect was due to inhibition of phospholipase A2 or to other effects of the drug. Mepacrine led to a substantial fall in left ventricular developed pressure (LVDP) and coronary flow (CF) during normoxic perfusion. Treated hearts showed less increase in LVEDP and less fall in LVDP during the hypoxic period, and significantly fewer hearts stopped beating compared to untreated controls. Release of CK during hypoxia and reoxygenation was reduced in treated hearts compared to controls (19.9 +/- 3.8 vs. 73.1 +/- 13.3 IU, p < 0.05). Lipid analyses of the myocardium showed a significant increase in the total amount of non esterified fatty acids (NEFA) in both untreated and mepacrine treated hypoxic hearts compared to normoxic controls, but to a significantly lower level in the mepacrine treated hearts. However, contribution of polyunsaturated NEFAs to total NEFAs did not differ between the groups. Also, neither total amount of fatty acids nor amount of polyunsaturated fatty acids obtained from the 2-position of the phospholipid fraction differed between the treated and untreated groups. In an enzyme assay, mepacrine 1 microM did not inhibit phospholipase A2 activity. We conclude that in our model mepacrine protects the heart from hypoxic injury, but probably by another mechanism than inhibition of phospholipase A2 induced membrane damage.

[Scientific misconduct and medical research in Norway]. / Uredelighet i medisinsk og helsefaglig forskning i Norge.

To evaluate the occurrence of scientific misconduct in medical research in Norway, 274 randomly selected Norwegian scientists were asked to complete a questionnaire on their knowledge and experience of deviations from the Medical Research Council's professional standard. Altogether 215 (80%) completed the questionnaire. Knowledge of severe misconduct was reported by 22% while 3% reported occurrence of falsification or fabrication of data within their own research group. Ten percent of the respondents knew of plagiarism, while 58% confirmed experience of less severe misconduct, most frequently misleading authorship. Nine percent had themselves contributed to one or more incidents of misconduct. Occurrence of misconduct was not related to area of research. The gap between admitting own misconduct and knowledge of misconduct by others was striking. According to 54% of the respondents, organized ethical education for young scientists would be an effective way of preventing misconduct. Altogether, the present data show that efforts are needed to prevent misconduct in medical research.

Phospholipid peroxidation after 60 min of global ischaemia and 10 min of reperfusion. A study in the isolated rat heart.

Peroxidation of polyunsaturated fatty acids in cell membranes is thought to be a crucial factor in the cascade leading to reperfusion damage in the myocardium. However, some studies also describe increased lipid peroxidation in ischaemic tissue. The present study therefore examines phospholipid peroxidation after 60 min of global ischaemia and during the initial phase of reperfusion in isolated Langendorff-perfused rat hearts. Lipids were extracted from these hearts and separated into phospholipid, triglyceride and non-esterified fatty acid fractions. The phospholipid fraction was hydrolysed with phospholipase A2, and reverse-phase high performance liquid chromatography of the fatty acids derived from the phospholipids was performed. Peroxidized polyunsaturated fatty acids were separated from unchanged fatty acids and amounts of monohydroxy or monohydroperoxy isomers were quantified by measuring conjugated dienes by UV absorption (235 nm). Phospholipids from ischaemic as well as free-radical-exposed tissue contained increased levels of peroxidized polyunsaturated fatty acids (20.7 +/- 2.4 and 20.5 +/- 2.3 respectively, v 11.8 +/- 1.4 units/mg dry weight in controls). After 2-10 min of reperfusion, a significant increase in phospholipid peroxidation was no longer detected (12.5 +/- 1.2 units/mg). The amount and the composition of non-esterified fatty acids were examined by gas chromatography. Ischaemia significantly increased both the amount of non-esterified fatty acids (1.5 +/- 0.8 v 4.9 +/- 1.8 nmol/mg dry wt) as well as the percentage composed of arachidonic acid (3.4 +/- 3.2% v 7.4 +/- 1.4%). Fatty acid levels remained elevated during reperfusion (5.5 +/- 1.9 nmol/mg and 7.0 +/- 1.4%). In conclusion, our results have demonstrated that prolonged ischaemia alone caused phospholipid peroxidation as well as accumulation of non-esterified arachidonic acid. There was no sign of further phospholipid peroxidation during reperfusion.

Ultrastructural changes in the myocardial myocytic mitochondria: crucial step in the development of oxygen radical-induced damage in isolated rat hearts?

The present study focuses on the sequential development of myocardial ultrastructural changes produced by oxygen radicals. Isolated rat hearts were perfused with oxygen radicals, generated by hypoxanthine and xanthine oxidase, for 5 and 10 min followed by a 35-min recovery period. The frequency of, and the association between, ultrastructural changes were examined by semiquantitative morphometry using the micrograph as unit. In each micrograph sarcolemmal, myocytic mitochondrial and myofilamental alterations were observed and graded as slight, moderate or severe. The myocytic nucleus and the endothelial cells were scored as normal or altered. Five min group: Among the cellular organelles examined, the myocytic mitochondria showed the highest frequency of alteration (in 15.3% of the micrographs). Among the grades of myocytic mitochondrial ultrastructural changes, slight alterations predominated (12.5%). Slight myocytic mitochondrial alterations were not significantly associated with the occurrence of ultrastructural changes of other cellular organelles. Endothelial ultrastructural alterations were sparse (1.5%). Ten min group: The frequency of altered organelles was greater when compared to the 5 min group. The myocytic mitochondria were still the most frequently altered component (61.7%), and myocytic mitochondrial ultrastructural alterations of all grades were strongly associated with the occurrence of other myocytic ultrastructural changes. In conclusion, the present study showed that myocytic mitochondrial changes predominated after both 5 and 10 min of oxygen radical exposure followed by recovery. In the 5 min group slight myocytic mitochondrial changes appeared independent of other myocardial changes, but in the 10 min group, however, myocytic mitochondrial changes were strongly associated with other myocardial ultrastructural changes. These results indicate that myocytic mitochondria are especially vulnerable to oxygen radicals, and further that myocytic mitochondrial ultrastructural changes may be a crucial step in the development of oxygen radical-induced myocardial damage.

Lipid peroxidation and membrane damage of the heart.

Biological membranes separate the cells from the environment and control flow of molecules and information between the cell and its surroundings. In addition, the cellular energy supply is dependent on membrane functions. The present chapter deals with membrane lipid peroxidation and how this process might influence the physiology and pathophysiology of the myocardium.