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Biomedical subjects

A M Bakken

Publications and source records attributed to A M Bakken.

At least 19 recordsLinked to original sources
PubMed2003

No differences in colony formation of peripheral blood stem cells frozen with 5% or 10% dimethyl sulfoxide.

High-dose chemotherapy with autologous stem cell rescue usually requires cryopreservation of the cells. For several years, 10% dimethyl sulfoxide (DMSO) has been used as the standard cryoprotectant. Because DMSO infusion can lead to toxic clinical complications in a dose-related manner, we wanted to evaluate if reduction to 5% DMSO would be possible. We have compared colony formation in the myeloid, erythropoietic, and megakaryocyte lineages in peripheral blood progenitor cell (PBPC) samples cryopreserved in parallel with 5% and 10% DMSO. Twenty-seven PBPC samples from patients with malignant diseases were investigated after 3 months of cryopreservation in liquid N(2), and samples from 14 of these patients were investigated after 1 year. A significantly higher colony formation was demonstrated for colony-forming units-erythrocyte (CFU-E) and CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) both at 3 months and at 1 year in the 5% samples. For CFU-granulocyte-macrophage (GM) and CFU-megakaryocyte (Mk) no significant difference was demonstrated neither at 3 months nor at 1 year in samples frozen with 5% and 10% DMSO. Also, there was a statistically significant correlation between the CFU-total and CFU-Mk-total, indicating that the CFU-total might be used as an evaluation of megakaryocyte progenitors. Viability testing with the Trypan Blue exclusion test showed that cells cryopreserved in 5% DMSO had significantly higher viability than the cells cryopreserved in 10% DMSO. We conclude that 5% DMSO is at least as good for cryopreservation of small-volume PBPC samples as the conventional 10% DMSO, and our results suggest that the possibility of using 5% DMSO for cryopreservation of autologous PBPC grafts should be further investigated in clinical studies.

Bone Neoplasms↗
PubMed2002

Flow cytometric measurement of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases.

The number of viable precursor cells actually reinfused into patients after high-dose chemotherapy is one of the most clinically important variables determining graft success or failure. A modified, previously described flow cytometric method based on annexin V staining was therefore applied to assess the degree of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases. Twenty-two samples of unmanipulated cryopreserved PBPC concentrates were analyzed by flow cytometry. The samples were triple-stained with anti-CD34 PE, annexin V-FITC and actinomycin D, which enabled the separation of viable, early apoptotic and late apoptotic/necrotic CD34(+) precursor cells. Apotosis and necrosis were also measured in the total cell population of the concentrates. Eighty-one percent (range 49-97) of the CD34(+) cells were viable, while 7% (range 1-15) were early apoptotic and 12% (range 2-36) were late apoptotic/necrotic after freeze/thaw. There was no difference in apoptosis and necrosis in CD34(+) cells harvested from mildly pretreated patients with multiple myeloma and heavily pre-treated patients with non-Hodgkin's lymphoma. Apoptosis and necrosis were higher in the total mature cell population of the concentrates. Thirty-two percent (range 7-69) of the cells were apoptotic and 33% (range 12-60) were necrotic. We conclude that flow cytometric analysis of annexinV/actinomycin D binding in PBPC concentrates is a simple technique that can give additional information of the viability status of the cells post thaw. The present study confirms the relative robustness of human CD34(+) precursor cells concerning the freeze/thaw procedure, which are carried out in daily clinical practice.

Adult↗
PubMed1999

[Normal tissue radiation toxicity in radiotherapy of localized prostatic cancer].

Radical radiotherapy of prostate cancer has to balance tumour control against the risk of radiation injury of normal tissue. The normal tissue toxicity is the main dose-limiting factor, and consequently a limiting factor of the curative potential of prostate cancer by irradiation. The SOMA scale is a new toxicity scoring system that registers late side effects from the most important anatomical sites. The SOMA scale is internationally approved and facilitates a standardised evaluation of radiation toxicity. The scoring system is based on four main components: subjective, objective, management and analysis. The SOMA scoring system was applied to a group of 21 patients from Ullevaal Hospital who were given intentionally curative radiation therapy for localised prostate cancer. The SOMA scale scoring system appears to be a valuable tool in the evaluation of normal tissue toxicity. It may contribute to a new standard in quality assurance of radiation therapy of prostate cancer.

Aged↗
PubMed1999

The swirling phenomenon in stored platelets is influenced by their endogenous serotonin.

The presence of the swirling phenomenon is useful to define platelet concentrates that are suitable for transfusion. If it is possible to identify donor-related factors which are related to persisting swirling during storage, it is possible to select platelet donors. Endogenous platelet serotonin content is stable and easily measured and related to agonist-induced serotonin secretion. During a 3-month period, the swirling in 825 single donor platelet concentrates was controlled before issue. Endogenous serotonin, % serotonin release and swirling were tested in 21 concentrates with poor or no swirling during storage. Sixty-three concentrates were randomly selected from the routinely prepared platelet concentrates and were routinely tested with the same analyses on days 1 and 7. To evaluate an obvious effect of endogenous serotonin on the swirling phenomenon, eight platelet concentrates prepared from buffy coat, each from four donors, were divided. One part was stored in the presence of 8.5 micromol serotonin L-1, and analysed as the control concentrates. The endogenous serotonin content in the 'low- swirling' concentrates was significantly lower than in the control group (P < 0.001). PCO2 and pH were significantly lower, and PO2 and MPV significantly higher than in the controls. In the control group, swirling after 7 days was significantly correlated with serotonin release. In the eight buffy-coat concentrates enriched in endogenous serotonin, both swirling and the percentage serotonin release were improved after storage for 10 days, compared with nonenriched concentrates. This study suggests that endogenous serotonin content and serotonin release are factors that may be of significance concerning preservation of the swirling phenomenon in platelet concentrates during storage.

Blood Donors↗
PubMed1999

Benzalkonium chloride interferes with energy production, secretion and morphology in human blood platelets.

Benzalkonium chloride (BC) is a bactericidal compound used as a topical antiseptic and as a preservative in various products for local treatment, e.g., eye and nose drops. BC is toxic to human cells, including those of the respiratory mucosa. Few studies have, however, focused on what cellular functions BC interferes with. The effects of BC were studied on washed human blood platelets in vitro . Cellular energy production as well as secretion were studied. Incubation of platelets with BC resulted in rapid swelling and toxic morphological changes. After incubation with BC oxidation of [1-14C] palmitate was inhibited, and both lactate dehydrogenase and endogenous serotonin were spontaneously released. Thrombin-induced secretion of serotonin was strongly reduced after BC exposure. Histological changes with increased size, spherical form, decreased numbers of pseudopodia, loss of an intact continuous tubulus system and reduced number of granules were found by scanning and transmission electron microscopy. It is concluded that the toxic effects of BC are because of interference with membrane function and energy production.

Journal Article↗
PubMed1994

The activities of acyl-CoA hydrolase in lysate and subcellular fractions of human blood platelets in relation to activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase.

The activities of acyl-CoA hydrolase (EC 3.1.2.2.) and acyl-CoA:1-acyl- lysophospholipid acyltransferase (EC 2.3.1.23) have been studied in subcellular fractions of human platelets. The acyl-CoA:1-acyl-lysophospholipid acyltransferase activity was higher in the 'dense-tubular-system-enriched' fraction than in the 'light-mitochondrial' fraction, using endogenously acyl-CoAs formed from labelled fatty acids, ATP, CoA and various lysophospholipids. No activity was found in the 'particle-free' fraction. No difference in specificities was observed between the incorporation of various fatty acids into different lysoPLs in the subcellular fractions compared with the platelet lysates. Generally, arachidonic, linoleic and eicosapentaenoic acids were better substrates for the acyl-CoA:1-acyl-lysophospholipid acyltransferases than oleic, docosahexaenoic and palmitic acids. The opposite was observed with the acyl-CoA hydrolase activity, palmitoyl-CoA was the substrate giving the highest activity, and eicosapentaenoyl-CoA and arachidonoyl-CoA the lowest. About 85% of the hydrolase activity was detected in the 'particle-free' fraction, with each of the six acyl-CoA derivatives tested.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1992

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase(s) in human platelets.

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferases (EC 2.3.1.23) have been studied in human platelet lysates by using endogenously formed [14C]acyl-CoA from [14C]fatty acid, ATP and CoA in the presence of 1-acyl-lysophosphatidyl-choline (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) or -inositol (lysoPI). Linoleic acid as fatty acid substrate had the highest affinity to acyl-CoA:1-acyl-lysophospholipid acyltransferase with lysoPC as variable substrate, followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 10(9) platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. EPA and AA were incorporated into all lyso-forms. Linoleic acid was also incorporated into lysoPE at a high rate, but less into lysoPS and lysoPI. DHA was incorporated into lysoPC and lysoPE, but only slightly into lysoPI and lysoPS. Whereas incorporation of all fatty acids tested was maximal for lysoPC and lysoPI at 200 and 80 microM respectively, maximal incorporation needed over 500 microM for lysoPE and lysoPS. The optimal concentration for [14C]fatty acid substrates was in the range 15-150 microM for all lysophospholipids. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost as if lysoPI or lysoPE were not added to the assay medium.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1992

The activity and subcellular distribution of the peroxisomal enzyme acyl-CoA oxidase in human blood platelets.

The peroxisomal enzyme acyl-CoA oxidase is localized in the 'dense-tubular-system-enriched fraction', probably identical with the endoplasmic reticulum, in human blood platelets. This localization is strongly different from the localization of catalase which seems to be a cytosolic enzyme, in agreement with Marcus, Zucker-Franklin, Safir & Ullman [(1966) J. Clin. Invest. 45, 14-28]. A localization of acyl-CoA oxidase in the endoplasmic reticulum seems to be in good accordance with the important role of peroxisomes in the metabolism of prostaglandins, as recently demonstrated by Diczfalusy, Kase, Alexson & Bjørkhem [(1991) J. Clin. Invest. 88, 978-984].

Acyl-CoA Oxidase↗
PubMed1991

Identity between palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase in human platelet?

Apparent Km values have been determined for the substrates ATP, CoA and fatty acids for the long-chain acyl-CoA synthetase (EC 6.2.1.3) reaction in lysates of human blood platelets. The apparent Km for ATP was higher for saturated fatty acids (C12:0 to C18:0) than for unsaturated acids (C18:1 to C22:6). Other apparent Km values were very similar for all long-chain fatty acids tested. Palmitic acid inhibited the formation of [14C]arachidonoyl-CoA, and arachidonic acid inhibited the formation of [14C]palmitoyl-CoA, with [14C]arachidonate or [14C]palmitate respectively as substrate. After chromatography of Triton X-100-extracted platelet protein in several systems (hydroxyapatite, DEAE-Sepharose, Sephacryl S-200 HR, CoA-Sepharose, Sephadex G-100 and AcA 34), both arachidonoyl-CoA synthetase and palmitoyl-CoA synthetase activities were eluted together in the various protein peaks, and with approximately the same ratio of activities in all peaks. After some purification steps (DEAE-Sepharose and Sephacryl S-200 HR), the acyl-CoA synthetase activity was up to 37 nmol/min per mg of protein with [14C]palmitate as substrate, and up to 116 nmol/min per mg of protein with [14C]arachidonate as substrate. The purification was respectively about 8- and 10-fold. The results indicate that palmitoyl-CoA (or unspecific) synthetase and arachidonoyl-CoA (or specific) synthetase are in fact the same enzyme, in agreement with previously reported results from this laboratory.

Blood Platelets↗
PubMed1991

Fatty acids in human platelets and plasma. Fish oils decrease sensitivity toward N2 microbubbles.

Platelet aggregation induced by N2 microbubbles (simulating microbubbles developed during deep diving) was measured in seven volunteers before and after intake of ethyl-eicosapentaenoate (-EPA, 3.5 g/day) and ethyl-docosahexaenoate (-DHA, 2.5 g/day) for 2 wk. The relative content of arachidonic acid (AA) decreased in platelets from all individuals, whereas the content of EPA and DHA increased. The decrease of AA was almost identical with the increase of EPA plus DHA. In plasma the AA content was unchanged, while EPA and DHA increased. The N2 microbubble-induced aggregation showed a significant negative correlation with the DHA content both in platelets and in plasma. Less aggregation was also observed with high EPA content in platelets or plasma. A significant correlation between AA content in platelets and aggregation was seen. Intake of marine oils may be beneficial to divers under deep diving and to patients during extracorporeal circulation, because this may reduce the microbubble-induced aggregation.

Adult↗
PubMed1990

Thrombin-induced serotonin release as an in vitro indicator of the functional integrity of stored platelets.

Measurements of endogenous serotonin and the thrombin-induced serotonin release reaction in platelet concentrates were compared with other methods of quality control: particle counting and pH and gas analyses. A discrepancy between the serotonin release reaction and the other data was observed after seven days of storage. The decline in the release reaction was not predictable from pH measurements or the number of platelets or leukocytes in the concentrate. There was a significant positive correlation between endogenous serotonin content and the thrombin-induced serotonin secretion after seven days of storage. We conclude that the current methods for routine control of platelet concentrates may not ensure the quality of the product after it has been stored for seven days. The thrombin-induced serotonin release may provide a valuable addition to the battery of tests available.

Blood Gas Analysis↗
PubMed1989

Identical subcellular distribution of palmitoyl-CoA and arachidonoyl-CoA synthetase activities in human blood platelets.

Fractionation of human blood platelets showed that palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase activities had an identical distribution among subcellular fractions. The activity was highest with arachidonic acid as substrate in all fractions, with an enzyme activity of 50 nmol/min per mg of protein, in a 'dense-tubular-system'-enriched fraction. The ratio activities with arachidonate and palmitate as substrates was about 1.5 in all fractions. Heat inactivation did not distinguish between arachidonoyl-CoA synthetase and a palmitoyl-CoA synthetase. On the other hand, heat inactivation indicated two pools of long-chain acyl-CoA synthetases: one in a mitochondria- and one in the dense-tubular-system-enriched fraction.

Blood Platelets↗
PubMed1986

Adenine nucleotides in cultured brain cells after exposure to methyl mercury and triethyl lead.

To obtain more information on the biochemical background, of methyl mercury (MeHg) and triethyl lead (Et3Pb) toxicity, the doses which were needed to alter the cellular content of adenine nucleotides were compared to doses which induced morphological alterations. Three-week-old cultures of brain cells from new-born rat were incubated for 5-60 min. with 28, 16, and 8 X 10(-6) M MeHg or with 3 X 10(-6) M Et3Pb in the nutrition medium. MeHg at 28 and 16 X 10(-6) M simultaneously changed the cell shape and reduced both the cellular content of adenine nucleotides and the relative level of ATP. In cells treated with 16 X 10(-6) M MeHg, a cellular increase in inosine and hypoxanthine and an unaltered content of adenine nucleotides in the nutrition medium, points to an intracellular degradation of adenine nucleotides. Incubation with 8 X 10(-6) M MeHg during the first hour of incubation, did not change the content of adenine nucleotides, however, MeHg induced a slowly progressing degeneration of cellular extensions. 3 X 10(-6) M Et3Pb induced morphological alterations but no changes in cellular nucleotide levels. The results show that morphological alterations occur in MeHg and Et3Pb treated cultures at concentrations of the compounds which are too low to change the content and composition of cellular adenine nucleotides.

Adenine Nucleotides↗
PubMed1985

Effects of isoproterenol on adenine nucleotide catabolism in cat hearts with acute regional ischaemia.

Previous studies have demonstrated that beta-adrenergic agonists cause extension of ischaemic lesions in the myocardium. In the present study the effects of isoproterenol on adenine nucleotide metabolism were investigated in cat hearts with regional ischaemia following permanent coronary occlusion for 45 min. Adenine nucleotides and their metabolites were measured by high performance liquid chromatography and regional myocardial blood flow was measured by 15-microns radiolabelled microspheres in a total of 255 paired tissue samples. Compared with untreated control cats, ATP and the energy charge were more reduced in ischaemic myocardium of isoproterenol-treated cats. Increased amounts of the degradation products inosine and hypoxanthine/xanthine were also found in these regions. These results could be ascribed to increased cardiac performance caused by isoproterenol, which augments the imbalance between energy production and oxygen supply in ischaemic myocardium.

Adenosine Diphosphate↗
PubMed1985

Liquid chromatography of serotonin and adenine nucleotides in blood platelets, illustrated by evaluation of functional integrity of platelet preparations.

In this relatively rapid liquid-chromatographic procedure the endogenous serotonin in blood platelets is quantified by fluorometry. We used this experimental procedure to estimate the platelet-release reaction as an indicator of the functional integrity of stored platelets. a decrease in the platelet-release reaction more sensitively indicates platelet changes during storage than do alterations in total ATP concentration or adenylate energy charge. The described method for quantifying serotonin is convenient enough for routine use in blood banks.

Adenine Nucleotides↗
PubMed1984

Effects of timolol on adenine nucleotide catabolism in cat hearts with acute regional ischaemia.

The effects of the beta-adrenergic blocking agent timolol on cardiac function and adenine nucleotide metabolism were investigated in cats with 45 min of acute ischaemia following coronary occlusion. Regional myocardial blood flow was measured by 15 micron radiolabelled microspheres, and adenine nucleotides and their degradation products were measured by high performance liquid chromatography in 16 tissue regions in each cat. During ischaemia, cats treated with timolol exerted a reduced cardiac performance as judged from reduced heart rate and cardiac contractility; consequently preocclusion tissue blood flow was reduced compared with a control group of cats. The ATP concentration was less severely depleted in ischaemic regions of the left ventricle in cats treated with timolol. The energy charge was reduced to a lesser extent in ischaemic regions, and in intermediate flow ranges the contents of the inosine and hypoxanthine/xanthine were reduced compared with untreated cats. These results indicate that the beta-adrenergic blocking agent timolol might protect the myocardium within a 45 min ischaemic period. The mechanism for this effect is most probably related to the reduction in overall cardiac performance.

Acute Disease↗
PubMed1984

Relationship between myocardial adenine nucleotide catabolism and tissue blood flow rate in experimental ischaemia.

A method was developed for tissue preservation and evaluation of the adenine nucleotide metabolism in small samples of myocardium after 45 min of ischaemia. Ischaemia was produced by coronary artery occlusion in anaesthetized cats. Adenine nucleotides and their metabolites were measured by isocratic liquid chromatographic systems which allow quantitative analysis of the nucleotides and their metabolites inosine, hypoxanthine and xanthine in biopsies of 5-20 mg tissue. Regional myocardial blood flow was measured in the tissue surrounding the biopsies by the distribution of 15 micron radiolabelled microspheres. In central ischaemic regions the ATP level was approximately 1 mumol/g wet weight, whereas in normally perfused myocardium the ATP level was approximately 5 mumol/g tissue. In tissue with intermediate flow values, intermediate ATP levels were found. Energy charge, which summarizes all adenine nucleotide concentrations, was reduced from 0.88 to 0.50, and the molar concentrations of inosine, hypoxanthine and xanthine increased in ischaemic tissue. We conclude that this method provides reliable characterization of the local cellular energy status in cat hearts with ischaemic regions.

Adenine Nucleotides↗