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  • 1.
    Andersson, Henrik
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska fakulteten.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Mandenius, Carl-Fredrik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska högskolan.
    Monitoring of troponin release from cardiomyocytes during exposure to toxic substances using surface plasmon resonance biosensing2010Ingår i: ANALYTICAL AND BIOANALYTICAL CHEMISTRY, ISSN 1618-2642, Vol. 398, nr 3, s. 1395-1402Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Troponin T (TnT) is a useful biomarker for studying drug-induced toxicity effects on cardiac cells. We describe how a surface plasmon resonance (SPR) biosensor was applied to monitor the release of TnT from active HL-1 cardiomyocytes in vitro when exposed to cardiotoxic substances. Two monoclonal human TnT antibodies were compared in the SPR immunosensor to analyse the TnT release. The detection limit of TnT was determined to be 30 ng/ml in a direct assay set-up and to be 10 ng/ml in a sandwich assay format. Exposure of the cardiomyocytes to doxorubicin, troglitazone, quinidine and cobalt chloride for periods of 6 and 24 h gave significant SPR responses, whereas substances with low toxicity showed insignificant effects (ascorbic acid, methotrexate). The SPR results were verified with a validated immunochemiluminescence method which showed a correlation of r(2)=0.790.

  • 2.
    Andersson, Henrik
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska fakulteten.
    Steel, Daniella
    Cellartis AB, Gothenburg, Sweden .
    Asp, Julia
    University of Gothenburg.
    Dahlenborg, Kerstin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska högskolan.
    Jonsson, Marianne
    University of Gothenburg.
    Jeppsson, Anders
    Sahlgrens University Hospital.
    Lindahl, Anders
    University of Gothenburg.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Sartipy, Peter
    Cellartis AB, Gothenburg, Sweden .
    Mandenius, Carl-Fredrik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska högskolan.
    Assaying cardiac biomarkers for toxicity testing using biosensing and cardiomyocytes derived from human embryonic stem cells2010Ingår i: JOURNAL OF BIOTECHNOLOGY, ISSN 0168-1656, Vol. 150, nr 1, s. 175-181Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Human embryonic stem cell (hESC) derived cardiomyocytes are in the present study being used for testing drug-induced cardiotoxicity in a biosensor set-up. The design of an in vitro testing alternative provides a novel opportunity to surpass previous methods based on rodent cells or cell lines due to its significantly higher toxicological relevance. In this report we demonstrate how hESC-derived cardiomyocytes release detectable levels of two clinically decisive cardiac biomarkers, cardiac troponin T and fatty acid binding protein 3, when the cardiac cells are exposed to the well-known cardioactive drug compound. doxorubicin. The release is monitored by the immuno-biosensor technique surface plasmon resonance, particularly appropriate due to its capacity for parallel and high-throughput analysis in complex media.

  • 3. Baryawno, Ninib
    et al.
    Sveinbjörnsson, Baldur
    Eksborg, Staffan
    Orrego, Abiel
    Segerström, Lova
    Öqvist, Carl Otto
    Holm, Stefan
    Gustavsson, Bengt
    Kågedal, Bertil
    Karolinska Institutet.
    Kogner, Per
    Johnsen, John Inge
    Tumor-growth-promoting cyclooxygenase-2 prostaglandin E2 pathway provides medulloblastoma therapeutic targets2008Ingår i: Neuro-Oncology, ISSN 1522-8517, E-ISSN 1523-5866, Vol. 10, nr 5, s. 661-674Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Prostaglandin E(2) (PGE(2)) has been shown to play important roles in several aspects of tumor development and progression. PGE(2) is synthesized from arachidonic acid by cyclooxygenases (COX) and prostaglandin E synthases (PGES) and mediates its biological activity through binding to the four prostanoid receptors EP(1) through EP(4). In this study, we show for the first time that medulloblastoma (MB), the most common malignant childhood brain tumor, expresses high levels of COX-2, microsomal prostaglandin E synthase-1, and EP(1) through EP(4) and secretes PGE(2). PGE(2) and the EP(2) receptor agonist butaprost stimulated MB cell proliferation. Treatment of MB cells with COX inhibitors suppressed PGE(2) production and induced caspase-dependent apoptosis. Similarly, specific COX-2 silencing by small interfering RNA inhibited MB cell growth. EP(1) and EP(3) receptor antagonists ONO-8713 and ONO-AE3-240, but not the EP(4) antagonists ONO-AE3-208 and AH 23848, inhibited tumor cell proliferation, indicating the significance of EP(1) and EP(3) but not EP(4) for MB growth. Administration of COX inhibitors at clinically achievable nontoxic concentrations significantly inhibited growth of established human MB xenografts. Apoptosis was increased, proliferation was reduced, and angiogenesis was inhibited in MBs treated with COX inhibitors. This study suggests that PGE(2) is important for MB growth and that therapies targeting the prostanoid metabolic pathway are potentially beneficial and should be tested in clinical settings for treatment of children with MB.  

  • 4.
    Bellanti, Francesco
    et al.
    Division of Pharmacology, Leiden/Amsterdam Centre Drug Research, Leiden, The Netherlands.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk kemi.
    Della Pasqua, Oscar
    Division of Pharmacology, Leiden/Amsterdam Centre Drug Research, Leiden, The Netherlands.
    Do pharmacokinetic polymorphisms explain treatment failure in high-risk patients with neuroblastoma?2011Ingår i: EUROPEAN JOURNAL OF CLINICAL PHARMACOLOGY, ISSN 0031-6970, Vol. 67, s. S87-S107Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Purpose Neuroblastoma is the most common extracranial solid tumour in childhood. It accounts for 15% of all paediatric oncology deaths. In the last few decades, improvement in treatment outcome for high-risk patients has not occurred, with an overall survival rate andlt;30-40%. Many reasons may account for such a low survival rate. The aim of this review is to evaluate whether pharmacogenetic factors can explain treatment failure in neuroblastoma. Methods A literature search based on PubMeds database Medical Subject Headings (MeSH) was performed to retrieve all pertinent publications on current treatment options and new classes of drugs under investigation. One hundred and fifty-eight articles wer reviewed, and relevant data were extracted and summarised. Results and conclusions Few of the large number of polymorphisms identified thus far showed an effect on pharmacokinetics that could be considered clinically relevant. Despite their clinical relevance, none of the single nucleotide polymorphisms (SNPs) investigated can explain treatment failure. These findings seem to reflect the clinical context in which anti-tumour drugs are used, i.e. in combination with multi-modal therapy. In addition, many pharmacogenetic studies did not assess (differences in) drug exposure, which could contribute to explaining pharmacogenctic associations. Furthermore, it remains unclear whether the significant activity of new drugs on different neuroblastoma cell lines translates into clinical efficacy, irrespective of resistance or myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN) amplification. Elucidation of the clinical role of pharmacogenetic factors in the treatment of neuroblastoma demands an integrated pharmacokinetic pharmacodynamic approach to the analysis of treatment response data.

  • 5. Berois, Nora
    et al.
    Blanc, Etienne
    Ripoche, Hugues
    Mergui, Xénia
    Trajtenberg, Felipe
    Cantais, Sabrina
    Barrois, Michel
    Dessen, Philippe
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Bénard, Jean
    Osinaga, Eduardo
    Raguénez, Gilda
    ppGalNAc-TI3: A new molecular marker of bone marrow involvement in neuroblastoma2006Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 52, nr 9, s. 1701-1712Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: To identify new molecular markers of bone marrow dissemination in human neuroblastoma (NB), we studied the transcriptome profiles of malignant neuroblasts established from the human MYCN-amplified IGR-N-91 model. Methods: This experimental model includes human neuroblastoma cells derived from & subcutaneous stage 4 disease, myocardium (Myoc) and bone marrow (BM) metastatic cells. Results: Gene expression profiles obtained with Agilent oligo microarrays revealed a set of 107 differentially expressed genes in the metastatic neuroblasts. This set included up-regulated genes involved in chemoresistance, cell motility, neuronal structure/signaling, and the recently characterized GALNT13 gene encoding a glycosyltransferase that initiates mucin-type O-glycosylation. Because the glycosylation process is involved in the progression of primary tumor to metastatic disease, we investigated whether the most strongly upregulated gene, GALNT13, might be a marker of bone marrow involvement in stage 4 NB patients. Importantly, in the BM of healthy adults no GALNT13 transcript was detected with analysis by quantitative (n = 3) and nested reverse transcription-PCR (n = 4) assays. In contrast, GALNT13 transcripts were detected in 23/23 cytologically involved BM samples obtained at diagnosis of stage 4 NB patients and in 5/27 cytologically noninvolved BM samples obtained from patients with stage 1-4 and 4S and treated stage 4 NB. The quantitative measurements of tyrosine hydroxylase (TH), ganglioside D2 synthase, dopa decarboxylase, and GALNT13 transcript values were compared in the same NB patients, and the results showed that GALNT13 expression was most highly correlated to poor clinical outcome at diagnosis. Conclusion: We propose ppGalNAc-T13 as a new informative marker for the molecular diagnosis of BM involvement and the follow-up of minimal residual disease in NB patients. © 2006 American Association for Clinical Chemistry.

  • 6.
    Björk, Per
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Lenner, Liselotte
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Persson, Birgitta
    Linköpings universitet, Institutionen för biomedicin och kirurgi. Linköpings universitet, Hälsouniversitetet.
    Inganäs, Olle
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska högskolan.
    Jonasson, Jon
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Molekylär och immunologisk patologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk patologi och klinisk genetik.
    Conjugated polythiophene probes target lysosome-related acidic vacuoles in cultured primary cells2007Ingår i: Molecular and Cellular Probes, ISSN 0890-8508, Vol. 21, nr 5-6, s. 329-337Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Conformation-sensitive optical probes for studying biological processes and structures are of great interest. The present work shows for the first time that conjugated polyelectrolyte (CPE) probes can be used for specific targeting of chromatin, nuclear and cytoplasmatic vesicles, and cytoskeletal components in a complex system of cultured cells. One of the probes could also be used for vital staining of live cells. When bound to different entities, the polythiophene derivative probes emitted light with different colors due to the unique spectral properties of these conformation sensitive probes. The physical pre-requisites for binding could also be exploited for characterization of the target. Unexpectedly, lysosome-related acidic vacuoles were targeted in cultured primary cells by both anionic, cationic, and zwitter-ionic polythiophene derivatives. Pre-treatment with Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, caused redistribution of the staining. The targeting of lysosome-related acidic vesicles could not be demonstrated in transformed cells (melanoma, neuroblastoma, and prostate cancer cell lines), indicating a difference in the localization, structure, accessibility, or quantity of the target in cultured normal cells as compared with the malignant cell lines. The chemical nature of the conjugated polyelectrolyte complex in the cytoplasmatic vacuoles remains elusive.

  • 7. Blomquist, L.
    et al.
    Dizdar, N.
    Linköpings universitet, Institutionen för biomedicin och kirurgi.
    Karlsson, M.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Ossowicki, H.
    Pettersson, A.
    Smeds, Staffan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Microdialysis of 5-S-cysteinyldopa from interstitial fluid in cutaneous human melanoma transplanted to athymic mice1991Ingår i: Melanoma Research, ISSN 0960-8931, Vol. 1, nr 1, s. 23-32Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Microdialysis was investigated as a tool for the determination of the extracellular concentration of the pigment metabolite 5-S-cysteinyldopa in human melanoma transplanted to athymic mice. Histology of the tumour with the microdialysis probes in situ showed no tissue damage. With probes equipped with polycarbonate membranes (20 kD) extraction (relative recovery) was approximately 50% at pH 4.0 and flow rates of 1 microliter/min, but at pH 7.0 recoveries were markedly lower, particularly from serum. In a first series of human melanomas transplanted to athymic mice low concentrations of 5-S-cysteinyldopa were detected in only two out of ten dialysates and were not detected in the other eight. Utilizing devices constructed for comparison of membrane characteristics in vitro we found about 4-fold higher recoveries with cuprophane and polyamide membranes than with polycarbonate membranes. Therefore newly constructed microdialysis probes (CMA/11) with cuprophane membranes were tested in vitro and gave recoveries of 38-48% from Ringer-Acetate solutions and 22-31% from serum, and the pH effects were low. When these probes were utilized in a second series of melanomas transplanted to athymic mice, 5-S-cysteinyldopa could easily be quantified in 10/10 experiments. A steady-state level of the dialysate 5-S-cysteinyldopa concentration was reached after 45 min.

  • 8.
    Dahlin, Lars-Göran
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Östergötlands Läns Landsting, Hjärtcentrum, Thorax-kärlkliniken.
    Kågedahl, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Olin, Christian
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Östergötlands Läns Landsting, Hjärtcentrum, Thorax-kärlkliniken.
    Rutberg, Hans
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Anestesiologi. Östergötlands Läns Landsting, Hjärtcentrum, Thorax-kärlkliniken.
    Svedjeholm, Rolf
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Östergötlands Läns Landsting, Hjärtcentrum, Thorax-kärlkliniken.
    An attempt to quantify the plasma levels of troponin-T and CK-MB after coronary surgery caused by release unrelated to permanent myocardial injury.2001Ingår i: Abstract 50th Annual meeting of the Scandinavian Association for Thoracic Surgery. June 14-16, 2001, Oslo, Norway,2001, 2001Konferensbidrag (Refereegranskat)
  • 9.
    Dahlin, Lars-Göran
    et al.
    Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Nylander, Eva
    Linköpings universitet, Institutionen för medicin och vård, Klinisk fysiologi. Linköpings universitet, Hälsouniversitetet.
    Olin, Christian
    Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Linköpings universitet, Hälsouniversitetet.
    Rutberg, Hans
    Linköpings universitet, Institutionen för medicin och vård, Anestesiologi. Linköpings universitet, Hälsouniversitetet.
    Svedjeholm, Rolf
    Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Linköpings universitet, Hälsouniversitetet.
    An attempt to quantify the plasma levels of troponin-T and CK-MB after coronary surgery caused by release unrelated to permanent myocardial injuryManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Background: Release of biochemical markers of myocardial injury unrelated to permanent myocardial damage has been claimed to explain a major proportion of elevations seen after cardiac surgery. However, little is known about the magnitude of this unspecific release. The aim of this study was to shed light on this issue by serial measurements in patients without permanent myocardial injury after coronary surgery.

    Methods: The unique release kinetics of troponin-T were employed to identify patients with no or minimal permanent myocardial injury. 302 patients undergoing CABG procedures (employing cardiopuhnonary bypass, crystalloid cardioplegia and retransfusion of shed mediastinal blood) were studied.

    Results: 90 patients were found to have normalized troponin-T levels no later than the fourth postoperative day indicating that early elevation of biochemical markers was explained almost purely by unspecific release. In this subgroup troponin-T (2.03±1.36 µg/L; range 0.35-8.99 µg/L) peaked at the 3 hour recording and CK-MB (28.3±10.7 µg/L; range 11.9-86 µg/L) peaked at the 8 hour recording after unclamping the aorta.

    Conclusions: A substantial early release of CK-MB and troponin-T occurred in patients with no or minimal permanent myocardial injury after CABG. The time frame when unspecific release was most pronounced is frequently studied to evaluate myocardial protective strategies or to compare different treatment modalities. Also, differences in unspecific release of biochemical markers can be expected depending on type of surgical procedure or coronary intervention. Therefore, further efforts to hring clarity about diagnostic pitfalls are warranted to prevent inappropriate comparisons and to improve our assessment of myocardial damage in association with revascularisation procedures.

  • 10.
    Dahlin, Lars-Göran
    et al.
    Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Nylander, Eva
    Linköpings universitet, Institutionen för medicin och vård, Klinisk fysiologi. Linköpings universitet, Hälsouniversitetet.
    Olin, Christian
    Linköpings universitet, Institutionen för medicin och vård, Klinisk fysiologi. Linköpings universitet, Hälsouniversitetet.
    Rutberg, Hans
    Linköpings universitet, Institutionen för medicin och vård, Anestesiologi. Linköpings universitet, Hälsouniversitetet.
    Svedjeholm, Rolf
    Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Linköpings universitet, Hälsouniversitetet.
    Early Identification of Permanent Myocardial Damage after Coronary Surgery is Aided by Repeated Measurements of CK-MB2002Ingår i: Scandinavian Cardiovascular Journal, ISSN 1401-7431, E-ISSN 1651-2006, Vol. 36, nr 1, s. 35-40Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Objective - ECG diagnosis of myocardial infarction after cardiac surgery is associated with major pitfalls and enzyme diagnosis is interfered by unspecific elevation unrelated to permanent myocardial injury. Sustained release of troponin-T is a marker of permanent myocardial injury if renal function is maintained. However, early identification of perioperative myocardial infarction is desirable and therefore the usefulness of creatine kinase monobasic (CK-MB) kinetics to detect myocardial injury early after coronary surgery was investigated.

    Design - Two hundred and eighty-six patients undergoing coronary surgery were studied with respect to release of enzymes and troponin-T preoperatively and postoperatively 3 and 8 h after unclamping the aorta, and every morning postoperative days 1-4.

    Results - CK-MB peak was found at 3 h ( n = 145), 8 h ( n = 103) and 16-20 h after unclamping ( n = 38). Depending on when the CK-MB peak was recorded different demographic and perioperative characteristics were found. A sustained release of troponin-T was characteristic for the group with the CK-MB peak at 16-20 h after unclamping.

    Conclusion - If CK-MB is measured only once it may be advisable to do it on the first postoperative morning as these measurements provided the best discrimination between patients with and without sustained elevation of troponin-T. However, repeated sampling provides additional information that aids in the early identification of permanent myocardial injury particularly in patients with borderline elevations of CK-MB.

  • 11.
    Dahlin, Lars-Göran
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Östergötlands Läns Landsting, Hjärtcentrum, Thorax-kärlkliniken.
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Nylander, Eva
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Klinisk fysiologi. Östergötlands Läns Landsting, Hjärtcentrum, Fysiologiska kliniken.
    Olin, Christian
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi.
    Rutberg, Hans
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Anestesiologi. Östergötlands Läns Landsting, Hjärtcentrum, Thorax-kärlkliniken.
    Svedjeholm, Rolf
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Thoraxkirurgi. Östergötlands Läns Landsting, Hjärtcentrum, Thorax-kärlkliniken.
    Unspecific elevation of plasma troponin-T and CK-MB after coronary surgery2003Ingår i: Scandinavian Cardiovascular Journal, ISSN 1401-7431, E-ISSN 1651-2006, Vol. 37, nr 5, s. 283-287Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Objective - Biochemical markers of myocardial injury are frequently elevated after cardiac surgery. It is generally accepted that release unrelated to permanent myocardial damage explains a proportion of these elevations. However, little is known about the magnitude and temporal characteristics of this diagnostic noise. One way to address this issue would be to study a group without permanent myocardial injury. Design - The unique release kinetics of troponin-T (permanent myocardial injury causes a sustained release of structurally bound troponin) were used to identify patients with no or minimal permanent myocardial injury. Blood was sampled from patients undergoing coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) before surgery, 3 and 8 h after unclamping the aorta, and each morning until postoperative day 4, for analysis of enzymes and troponin-T. From 302 consecutive patients a subgroup was identified that fulfilled the following criteria: (a) normalized troponin-T levels =postoperative day 4, (b) no ECG changes indicating myocardial injury. Results - Seventy-seven patients fulfilled the criteria above and in this subgroup troponin-T (2.08 ▒ 1.42 ╡g/ 1, range 0.35-8.99 ╡g/l) peaked at the 3 h recording and creatine kinase monobasic (CK-MB) (28.6 ▒ 11.3 ╡g/l, range 11.9-86.0 ╡g/l) peaked at the 8 h recording after unclamping the aorta. Conclusion - Substantial early elevations of plasma CK-MB and troponin-T occurred in patients with no or minimal permanent myocardial injury after CABG. Unspecific release was most pronounced during the timeframe that is usually studied to evaluate myocardial protective strategies or to compare revascularization procedures.

  • 12.
    Dizdar (Dizdar Segrell), Nil
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Neurologi. Linköpings universitet, Hälsouniversitetet.
    Granerus, Ann-Kathrine
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Geriatrik. Linköpings universitet, Hälsouniversitetet.
    Hannestad, Ulf
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kullman, Anita
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Ljungdahl, Å.
    Department of Neurology, Huddinge Hospital, Stockholm.
    Olsson, Jan-Edvin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Neurologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Närsjukvården i centrala Östergötland, Neurologiska kliniken.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    L-dopa pharmacokinetics studied with microdialysis in patients with Parkinson's disease and a history of malignant melanoma1999Ingår i: Acta neurologica Scandinavica, ISSN 0001-6314, Vol. 100, nr 4, s. 231-237Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    OBJECTIVES: The pharmacokinetics of free L-dopa in blood and tissue of five parkinsonian patients with malignant melanoma was studied with microdialysis. In one case the effect of L-dopa treatment on 5-S-cysteinyldopa and the melanoma was studied. Gastric emptying and its effects on free L-dopa in blood were also investigated in one of the patients.

    METHODS: Five patients were given 100 mg L-dopa with 25 mg benserazide. Blood and dialysates from the circulation and fatty tissue were collected for analysis. [13C]-Octanoic breath test was used for analyzing gastric half-emptying time.

    RESULTS: Four of the patients had similar pharmacokinetic patterns for L-dopa and a significant (P < 0.05) increase of serum 5-S-cysteinyldopa occurring 30 min after L-dopa intake. Delayed L-dopa peaks and slow gastric half-emptying time were found in 1 patient. A dose-dependent increase of 5-S-cysteinyldopa occurred but no melanoma metastases were seen during long-term L-dopa therapy.

    CONCLUSION: L-dopa therapy increases 5-S-cysteinyldopa levels but does not seem to cause progress of melanomas. Gastric emptying impacts L-dopa pharmacokinetics.

  • 13.
    Dizdar (Dizdar Segrell), Nil
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Neurologi. Linköpings universitet, Hälsouniversitetet.
    Kullman, Anita
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Comparison of N-acetylcysteine and l-2-oxothiazolidine-4-carboxylate as cysteine deliverers and glutathione precursors in human malignant melanoma transplants in mice2000Ingår i: Cancer chemotherapy and pharmacology, ISSN 0344-5704, Vol. 45, nr 3, s. 192-198Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Purpose: Glutathione is an important cellular compound which affects detoxification of electrophiles and may have direct or indirect effects on pigment formation. It is therefore of importance to study interstitial concentrations in melanoma tissue while decreasing its formation with an enzyme inhibitor and increasing its amount with cysteine deliverers. Method: Glutathione formation was inhibited by intraperitoneal (i.p.) injection of BSO. N-Acetylcysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) were then given i.p. to subgroups of the animals. Intratumoral microdialysis was performed during BSO treatment, during BSO treatment combined with NAC or OTC and after discontinuation of BSO but ongoing NAC or OTC treatment. Results: Glutathione formation was inhibited during BSO treatment. The dialysate concentrations of both glutathione and cysteine decreased during concomitant treatment with BSO and NAC or OTC. Recovery of the amounts of the two compounds was seen in both groups after discontinuation of BSO treatment. In the NAC group we also observed an acute increase in dialysate concentrations of cysteine after NAC injection. The 5-S-cysteinyldopa concentrations were unaffected by variations in glutathione and cysteine concentrations. Conclusions: 5-S-Cysteinyldopa in melanoma is not formed from glutathione in vivo to any appreciable extent. The intracellular amount of cysteine is probably not a limiting factor for cysteinyldopa formation. It seems that both NAC and OTC can be used as cysteine deliverers to melanoma cells in vivo to produce recovery of glutathione levels after synthesis inhibition by BSO treatment.

  • 14.
    Dizdar (Dizdar Segrell), Nil
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Neurologi. Linköpings universitet, Hälsouniversitetet.
    Kullman, Anita
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Årstrand, Kerstin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Effects on interstitial glutathione, cysteine and 5-S-cysteinyldopa of buthionine sulphoximine in human melanoma transplants1997Ingår i: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 7, nr 4, s. 322.-328Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Using microdialysis of human melanoma transplants in athymic mice we have shown that interstitial glutathione levels decreased during treatment with buthionine sulphoximine (BSO) and recovered after cessation of treatment. The cysteine concentrations also decreased, while 5-S-cysteinyldopa tended to increase during BSO treatment. Restoration of the glutathione levels was not seen after either N-acetylcysteine (NAC) or L-2-oxothiazolidine-4-carboxylate (OTC) injections, given on the third day of BSO treatment. These results were to be expected since NAC and OTC were given during the BSO treatment, and BSO is a specific and potent inhibitor of glutathione synthesis. Cysteine levels, however, increased after the NAC injection but remained unaltered after the OTC injection, while 5-S-cysteinyldopa remained unaltered after both the NAC and the OTC injections.

  • 15.
    Dizdar (Dizdar Segrell), Nil
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Neurologi. Linköpings universitet, Hälsouniversitetet.
    Kullman, Anita
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Norlander, Björn
    Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet.
    Olsson, Jan-Edvin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Neurologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Närsjukvården i centrala Östergötland, Neurologiska kliniken.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Human pharmacokinetics of L-3,4-dihydroxyphenylalanine studied with microdialysis1999Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 45, nr 10, s. 1813-1820Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Intravenous and subcutaneous microdialysis was performedto compare the free concentrations and pharmacokinetics of L-3,4-dihyroxyphenylalanine(L-dopa) in blood and tissue in healthy subjects and in patientswith Parkinson disease.

    Methods: Nine healthy volunteers and 10 patients with Parkinson disease, stage 1.5–2 according to the Hoehn-Yahr rating scale, took part of the study. In the patient group subcutaneous microdialysis and ordinary blood sampling were performed, whereas in the control group intravenous microdialysis was also performed. Microdialysis samples were collected in fractions of 15 min. The first two fractions were collected for analysis of basal concentrations. A blood sample was also taken. The patients were then given one tablet of Madopar® (100 mg of L-dopa and 25 mg of benserazide),and the microdialysis was continued for another 210 min. Bloodsamples were obtained at 30-min intervals.

    Results: The serum samples gave a significantly higher meanarea under the curve (AUC; 491 ± 139 µmol ·min/L) than that for intravenous dialysates (235 ± 55.3µmol · min/L), suggesting a protein binding of50%. The L-dopa concentrations from the subcutaneous dialysatesmatched those from the intravenous dialysates, indicating rapiddistribution of L-dopa to the tissues.

    Conclusions: Parkinsonian patients in early stages of the disease have a pharmacokinetic pattern of free L-dopa similar to that of healthy subjects. Comparison of AUCs from microdialysis with ordinary serum analysis revealed data indicating significant protein binding. Microdialysis is a suitable and easily applied tool in pharmacokinetic studies.

  • 16.
    Dizdar (Dizdar Segrell), Nil
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Neurologi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Smeds, Staffan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Årstrand, Kerstin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    A high-sensitivity fluorometric high-performance liquid chromatographic method for determination of glutathione and other thiols in cultured melanoma cells, microdialysis samples from melanoma tissue, and blood plasma.1991Ingår i: Melanoma Research, ISSN 0960-8931, Vol. 1, nr 1, s. 33-42Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A high-performance liquid chromatographic method with fluorometric detection is described which is suitable for determination of glutathione in small samples. Reduced glutathione (GSH) and total glutathione obtained as GSH after reduction with glutathione reductase is derivatized with N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM) and subjected to chromatography. The detection limit for the GSH-DACM derivative was 5-10 fmol/injection, and analytical recovery was quantitative. The method is suitable for determination of both reduced and total glutathione in samples from microdialysis of melanoma tumours, and cysteine can be quantified in the same chromatogram. Application is shown also for glutathione determinations in cultured melanoma cells, melanoma homogenates and plasma.

  • 17.
    Dizdar (Dizdar Segrell), Nil
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Årstrand, Kerstin
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi.
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Analysis of L-dopa in human serum2002Ingår i: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 33, nr 5, s. 1000-1002Artikel i tidskrift (Refereegranskat)
  • 18.
    Farnebäck, Malin
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Håkansson, Annika
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Onkologi. Linköpings universitet, Hälsouniversitetet.
    Håkansson, Leif
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Onkologi. Linköpings universitet, Hälsouniversitetet.
    Gustafsson, Bertil
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk patologi och cytologi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Expression of tumor associated transcripts in malignant melanoma metastases: with methodological aspectsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Several antigens expressed in malignant melanoma are involved in the immunological surveillance of the tumor. The mRNAs of these antigens, especially tyrosinase, have also been used in the detection of minimal residual disease in the blood of melanoma patients. We have therefore analyzed the expression of tyrosinase, TRP-1, TRP-2, MART-1/Melan-A, MAGE-A3, MAGE-A12, S-100 and GD2 synthase by real-time PCR in snap frozen sections from 28 regional and systemic metastases. Treatment with cisplatinum, dacarbazine and interferon-α2b was given to 15 patients before surgery. The transcript concentrations varied widely between individual metastases. However, in general the pigment-related transcripts and that of S-100 were found at higher levels than those of MAGE-A3, MAGE-A12 and GD2 synthase. Significant correlations (p<0.001) were found between tyrosinase and MART-1/Melan-A and between MAGE-A3 and MAGE-A12. TRP-2 and GD2 synthase were both influenced by the treatment, TRP-2 expression was increased in the metastases from treated patients, whereas GD2 synthase expression was decreased. Furthermore, a decrease in tyrosinase expression was found in metastases without tumor-infiltrating lymphocytes. ln this work normalization by the section area contra normalization by housekeeping genes was also evaluated and similar results were obtained with both methods.

  • 19.
    Farnebäck, Malin
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kullman, Anita
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Stability of blood samples for analysis of tumour cell transcriptsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    When mRNA expression from tumor cells is analyzed in blood, one of the greatest problems is to keep the samples stable during transfer to the laboratory. Improved systems for stabilization of transcripts therefore have to be developed.

    Blood was collected in PAXgene™ Blood RNA Tubes and spiked with melanoma and neuroblastoma cells. The stability of tumor transcripts at room temperature was tested after 1, 2, 3, 5, 7, and 9 days. PAXgene tubes were also frozen at -20 °C and -70 °C for 14 days. Total RNA was extracted by PAXgene™ Blood RNA Kit and mRNA was extracted by the GenoPrep Direct mRNA Kit. Tyrosinase, MAGE, tyrosine hydroxylase, and GD2 synthase mRNA was quantified by real-time PCR. The stability of these transcripts was compared with the stability in ACD tubes when RNA extraction was performed with QIAamp RNA Blood Mini Kit.

    The yields of transcripts from the PAXgene tubes using the PAXgene™ Blood RNA Kit varied from 37% to 49% of the yields from ACD tubes. When mRNA was extracted with the GenoPrep Direct mRNA Kit the yields increased to 67-121%. Stability tests showed significant decreases of all the transcripts in the PAXgene tubes after two days at room temperature. After seven days, 10% of the initial transcript concentrations remained, whereas 32% remained in the ACD tubes. However, in the frozen PAXgene tubes the transcript levels were unchanged for 14 days.

    We conclude that the PAXgene tube is suitable when the sample is stored at -20 °C or -70 °C and mRNA is extracted using GenoPrep Direct mRNA Kit.

  • 20.
    Fyrberg, Anna
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet.
    Peterson, Curt
    Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Onkologiska kliniken US.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk kemi.
    Lotfi, Kourosh
    Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk farmakologi.
    Induction of fetal hemoglobin and ABCB1 gene expression in 9-β-D-arabinofuranosylguanine-resistant MOLT-4 cells2011Ingår i: Cancer Chemotherapy and Pharmacology, ISSN 0344-5704, E-ISSN 1432-0843, Vol. 68, nr 3, s. 583-591Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    PURPOSE: To characterize resistance mechanisms to the nucleoside analog 9-β-D-arabinofuranosylguanine (AraG) in the T-cell acute lymphoblastic leukemia cell line MOLT-4 and its AraG-resistant variant.

    METHODS: A gene expression microarray analysis was performed, as well as gene expression and enzyme activity measurements of key enzymes in the activation of AraG. Cytotoxicity of AraG and cross-resistance to other compounds were evaluated using a standard cytotoxicity assay.

    RESULTS: Gene expression microarray analysis revealed that fetal hemoglobin genes and the multidrug resistance ABCB1 gene, encoding the drug efflux pump P-gp, were the most highly upregulated genes in the resistant cells, while genes traditionally associated with nucleoside analog resistance were not. Fetal hemoglobin and ABCB1 induction can be due to global DNA hypomethylation. This phenomenon was studied using AraG during a period of 4 weeks in MOLT-4 cells and the lung adenocarcinoma cell line A549, leading to up-regulation of hemoglobin gamma and ABCB1 as well as DNA hypomethylation. Inhibiting P-gp in the AraG-resistant MOLT-4 cells led to decreased proliferation, reduced hemoglobin expression, and highly induced ABCB1 expression.

    CONCLUSIONS: We show that AraG can cause hypomethylation of DNA and induce the expression of the fetal hemoglobin gamma gene and the ABCB1 gene. We speculate that the induction of ABCB1/P-gp may occur in order to help with excretion of hemoglobin degradation products that would otherwise be toxic to the cells, and we present data supporting our theory that P-gp may be linked to the induction of hemoglobin.

  • 21.
    Håkansson, Annika
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Onkologi. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Onkologiska kliniken US.
    Håkansson, Leif
    Gustafsson, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Patologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk patologi och klinisk genetik.
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Bcl-2 monitoring in malignant melanoma2004Ingår i: Hospital Pharmacy, ISSN 0018-5787, E-ISSN 1945-1253, s. 46-47Artikel i tidskrift (Övrigt vetenskapligt)
  • 22. Ifversen, Marianne RS
    et al.
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Christensen, Lisa D
    Rechnitzer, Catherine
    Petersen, Bodil L
    Heilmann, Carsten
    Comparison of immunocytochemistry, real-time quantitative RT-PCR and flow cytometry for detection of minimal residual disease in neuroblastoma2005Ingår i: International Journal of Oncology, ISSN 1019-6439, Vol. 27, nr 1, s. 121-129Artikel i tidskrift (Refereegranskat)
  • 23.
    Johansson, Malin
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Pisa, Eva
    AB Sangtec Medical, Bromma, Sweden..
    Törmänen, Vuokko
    AB Sangtec Medical, Bromma, Sweden..
    Årstrand, Kerstin
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Quantitative analysis of tyrosinase transcripts in blood2000Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 46, nr 7, s. 921-927Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Tyrosinase is an enzyme unique to pigment-forming cells. Methods using this transcript for detection of melanoma cells in blood have given divergent results. Quantitative analytical procedures are therefore needed to study the analytical performance of the methods.

    Methods: Mononucleated cells were isolated by Percoll centrifugation. RNA was isolated by each of three methods: UltraspecTM-II RNA isolation system, FastRNATM GREEN Kit, and QIAamp RNA Blood Mini Kit. cDNA was synthesized using random hexamer primers. A tyrosinase-specific product of 207 bp was amplified by PCR. As an internal standard (and competitor) we used a 207-bp cDNA with a base sequence identical to the tyrosinase target except for a 20-bp probe-binding region. The PCR products were identified by 2,4-dinitrophenol (DNP)-labeled probes specific for tyrosinase (5′DNP-GGGGAGCCTTGGGGTTCTGG-3′) and internal standard (5′DNP-CGGAGCCCCGAAACCACATC-3′) and quantified by ELISA.

    Results: The calibration curves were linear and had a broad dynamic measuring range. A detection limit (2 SD above zero) of 48 transcripts/mL of blood was obtained from a low control. The analytical imprecision was 50% and 48% at concentrations of 1775 and 17 929 transcripts/mL (n = 12 and 14, respectively). With the cell line SK-Mel 28 added to blood and RNA extracted with the Ultraspec, Fast RNA, and QIAamp RNA methods, we found (mean ± SD) 1716 ± 1341, 2670 ± 3174, and 24 320 ± 5332 transcripts/mL of blood. Corresponding values were 527 ± 497, 2497 ± 1033, 14 930 ± 1927 transcripts/mL of blood when the cell line JKM86-4 was added. One high-risk patient was followed by repeated analysis of tyrosinase transcripts in blood. The melanoma marker 5-S-cysteinyldopa in serum and urine was within reference values, but tyrosinase mRNA was slightly increased (120–168 transcripts/mL of blood). The tyrosinase mRNA increased to 1860 transcripts/mL concomitant with the increase in 5-S-cysteinyldopa; later a spleen metastasis was found.

    Conclusions: The results obtained with different RNA extraction methods illustrate the importance of quantitative methods for validation of methods. The use of QIAamp RNA improved the extraction efficiency considerably. Data from a case study suggest the assay is suitable in the follow-up of patients with high risk of developing metastases.

  • 24.
    Johansson, Malin
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Takasaki, A.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lenner, Liselotte
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Årstrand, Kerstin
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Quantitative relationships between pigment-related mRNA and biochemical melanoma markers in melanoma cell lines2002Ingår i: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 12, nr 3, s. 193-200Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The use of reverse transcription polymerase chain reaction (RT-PCR) analysis of melanoma-specific transcripts for the identification of circulating melanoma cells has shown very variable results in different studies on melanoma patients. We have therefore developed quantitative methods to study both analytical and biological variations as possible causes of this phenomenon. Pigment-related and S-100β transcripts were quantified in 12 different melanoma cell lines and related to the amounts of 5-S-cysteinyldopa, pigment and S-100B protein. A real-time PCR method was used and the results were expressed as absolute number of transcripts per cell. Tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2 and MART-1/Melan-A mRNA varied from undetectable (< 10-4 transcripts/cell) to 103 transcripts/cell, i.e. by a factor > 107 in the different cell lines. S-100β mRNA varied from 2.8 to 165 transcripts/cell, i.e. by a factor of 60. Tyrosinase, TRP-1 and TRP-2 mRNA correlated significantly with the amount of 5-S-cysteinyldopa, an intermediate pigment metabolite (P < 0.001, P < 0.001 and P < 0.01, respectively). The amount of S-100β mRNA correlated significantly with the amount of S-100B protein (P < 0.001). No cross-correlations were seen between the pigment-related and S-100-related analytes. We conclude that one reason behind the negative results of RT-PCR measurement of pigment-related mRNA may be that these transcripts are not always expressed in the particular cells present in the patient's blood. Furthermore, variation in the expression of the order of 107 must have great impact on the diagnostic sensitivity. Measurement of S-100β mRNA would be more sensitive, but the use of this transcript is hampered by its presence in the blood cells. © 2002 Lippincott Williams & Wilkins.

  • 25.
    Johansson, Malin
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Årstrand, Kerstin
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Håkansson, Annika
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Onkologi. Linköpings universitet, Hälsouniversitetet.
    Lindholm, C
    Department of Oncology, Ryhov County Hospital, Jönköping, Sweden.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction2000Ingår i: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 10, nr 3, s. 213-222Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several studies have evaluated the use of polymerase chain reaction (PCR) amplification of tyrosinase mRNA to detect melanoma cells in blood. However, contradictory results have been obtained from different groups. We therefore have developed and validated a quantitative PCR method for tyrosinase and tyrosinase-related protein-2 (TRP-2) mRNA. An important methodological finding was that high concentrations of reverse transcriptase or RNA sample inhibited the following PCR. This could be abolished by dilution of the cDNA sample before the PCR. Standard curves with a linear range over at least five logs were obtained with dilutions of melanoma cell cDNA. Controls (RNA and cDNA) consisting of melanoma cells (1000/ml) added to blood were analysed repeatedly over 3 months, resulting in means between 880 and 1074 AU/ml. The RNA controls were stable, whereas the cDNA controls, as well as the calibrators, showed a tendency to change over time. The variation in the RNA controls was 25% for tyrosinase and 22% for TRP-2. Seven stage III-IV melanoma patients were tested for tyrosinase and TRP-2 transcripts in blood drawn from a peripheral vein and from a Port-a-cath. Tyrosinase mRNA was found in three patients (0.8-12.4 AU/ml). For TRP-2, the same amount was found in the patients as in healthy donors. No differences were seen between blood from a peripheral vein and from the Port-a-cath. We here present fast and sensitive methods for the quantification of tyrosinase and TRP-2 mRNA in blood.

  • 26. Johnsen, J I
    et al.
    Segerstrom, L
    Orrego, A
    Elfman, L
    Henriksson, M
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Eksborg, S
    Sveinbjornsson, B
    Kogner, P
    Inhibitors of mammalian target of rapamycin downregulate MYCN protein expression and inhibit neuroblastoma growth in vitro and in vivo2008Ingår i: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 27, nr 20, s. 2910-2922Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mammalian target of rapamycin (mTOR) has been shown to play an important function in cell proliferation, metabolism and tumorigenesis, and proteins that regulate signaling through mTOR are frequently altered in human cancers. In this study we investigated the phosphorylation status of key proteins in the PI3K/AKT/mTOR pathway and the effects of the mTOR inhibitors rapamycin and CCI-779 on neuroblastoma tumorigenesis. Significant expression of activated AKT and mTOR were detected in all primary neuroblastoma tissue samples investigated, but not in non-malignant adrenal medullas. mTOR inhibitors showed antiproliferative effects on neuroblastoma cells in vitro. Neuroblastoma cell lines expressing high levels of MYCN were significantly more sensitive to mTOR inhibitors compared to cell lines expressing low MYCN levels. Established neuroblastoma tumors treated with mTOR inhibitors in vivo showed increased apoptosis, decreased proliferation and inhibition of angiogenesis. Importantly, mTOR inhibitors induced downregulation of vascular endothelial growth factor A (VEGF-A) secretion, cyclin D1 and MYCN protein expression in vitro and in vivo. Our data suggest that mTOR inhibitors have therapeutic efficacy on aggressive MYCN amplified neuroblastomas. © 2008 Nature Publishing Group All rights reserved.

  • 27.
    Kechagias, Stergios
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för kardiovaskulär medicin. Linköpings universitet, Medicinska fakulteten. Östergötlands Läns Landsting, Hjärt- och Medicincentrum, Magtarmmedicinska kliniken.
    Dernroth, Dženeta Nezirević
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten.
    Blomgren, Anders
    Skåne University Hospital, Lund.
    Hansson, Therese
    Skåne University Hospital, Lund.
    Isaksson, Anders
    Skåne University Hospital, Lund.
    Walther, Lisa
    Skåne University Hospital, Lund.
    Kronstrand, Robert
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Medicinska fakulteten. Natl Board Forens Med, Dept Forens Genet & Forens Toxicol, Linkoping, Sweden.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk kemi.
    Nystrom, Fredrik H
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för kardiovaskulär medicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Endokrinmedicinska kliniken.
    Phosphatidylethanol Compared with Other Blood Tests as a Biomarker of Moderate Alcohol Consumption in Healthy Volunteers: A Prospective Randomized Study.2015Ingår i: Alcohol and Alcoholism, ISSN 0735-0414, E-ISSN 1464-3502, Vol. 50, nr 4, s. 399-406Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    AIM: It is generally agreed that traditional alcohol biomarkers lack in sensitivity to detect hazardous alcohol consumption. The present study was undertaken to evaluate the ability of phosphatidylethanol (PEth) and traditional alcohol markers to detect moderate alcohol consumption and to distinguish between moderate alcohol consumption and abstinence.

    METHODS: Forty-four subjects, 32 females and 12 males, were included in the study. They were randomized to alcohol abstention or to alcohol consumption. Female participants consumed 150 ml of red wine (equivalent to 16 g of alcohol) per 24 h and the male participants double the amount. The study lasted for 3 months. Blood samples were drawn at the start and at the end of the study period. Blood samples were analysed for PEth, carbohydrate-deficient transferrin (CDT), mean corpuscular volume (MCV), γ-glutamyltransferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

    RESULTS: ROC curves for the various biochemical markers were plotted in order to assess their ability to discriminate between abstention and moderate daily consumption of alcohol. PEth and CDT were the only markers with AUROCs significantly higher than 0.5, and PEth was detected in all participants randomized to alcohol consumption.

    CONCLUSION: PEth was the only marker that could detect moderate intake and the present results also indicate that PEth probably can distinguish moderate alcohol consumption from abstinence.

  • 28.
    Kronstrand, Robert
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Rättskemi. Linköpings universitet, Hälsouniversitetet.
    Förstberg-Peterson, Sophie
    Linköpings universitet, Institutionen för medicin och hälsa, Rättskemi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Ahlner, Johan
    Linköpings universitet, Institutionen för medicin och hälsa, Rättskemi. Linköpings universitet, Hälsouniversitetet.
    Larsson, Göran
    Linköpings universitet, Institutionen för medicin och hälsa, Rättskemi. Linköpings universitet, Hälsouniversitetet.
    Codeine Concentration in Hair after Oral Administration Is Dependent on Melanin Content1999Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 45, nr 9, s. 1485-1494Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Analysis of drugs in hair has been used on a qualitative basis to estimate earlier exposure to drugs. Clinical applications are rare because of the lack of dose–response relationships in the studies performed to date, and questions remain regarding the mechanisms of drug incorporation into hair. Several human studies have shown differences in drug accumulation between pigmented and nonpigmented hair. However, the melanin concentration in hair was not determined and correlated to the amount of drug incorporated.

    Methods: Nine human subjects were given codeine as a single oral dose, and plasma codeine concentrations were determined for 24 h, using gas chromatography–mass spectrometry. Hair samples were obtained weekly for a month. Total melanin, eumelanin, and codeine were measured quantitatively in hair samples by spectrophotometry, HPLC, and gas chromatography–mass spectrometry, respectively.

    Results: There was an exponential relationship between codeine and melanin concentrations in hair, (r2 = 0.95 with total melanin and r2 = 0.83 with eumelanin). After normalizing the results by the area under the curve for codeine in plasma, we obtained r2 = 0.86 for codeine vs total melanin and r2 = 0.90 vs eumelanin.

    Conclusions: Our results stress the importance of melanin determination when measuring drugs in hair. We postulate that analysis of drug concentration in hair may be worthwhile in the monitoring of drug compliance if the results are normalized for melanin content.

  • 29. Kärnell, R
    et al.
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Lindholm, C
    Nilsson, B
    Årstrand, Kerstin
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi.
    Ringborg, U
    The value of cysteinyldopa in the follow-up of disseminated malignant melanoma2000Ingår i: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 10, nr 4, s. 363-369Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In a series of 92 patients with malignant melanoma, clinical stage III or IV, both 5-S-cysteinyldopa (5SCD) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) were measured in urine during chemotherapy. A total of 434 urine specimens were analysed. The sensitivity of 5SCD for the detection of stage III-IV melanoma was 83%, while the corresponding sensitivity of 6H5MI2C was 52%. Fifty per cent of patients with one metastatic site had increased 5SCD excretion, while all patients with four or more metastatic sites had increased excretion. A significant correlation was found between 5SCD decrease and clinical regression (P < 0.001) and between 5SCD increase and clinical progression (P < 0.001). Corresponding correlations were not found for 6H5MI2C. Increments in 5SCD excretion (median 269 ╡mol/mol creatinine) were seen for 83% of the occasions when clinical progression was recorded, and decrements in 5SCD excretion (median 145 ╡mol/mol creatinine) were seen for 85% of the occasions when clinical regression was seen. During clinical 'stable disease' increases in 5SCD excretion were seen in 59% and decreases in 41%. The median value of 5SCD changes for stable disease was 7.0 ╡mol/mol creatinine, indicating a chemical marker stability in many cases. We recommend the use of 5SCD in urine as a valuable, reliable and simple biochemical marker to use in the clinical follow-up of melanoma patients with advanced disease.

  • 30.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Biologiska variabler i blod och urin - könsskillnader av stor betydelse för sjukdomsdiagnostik2010Ingår i: Genus och kön inom medicin- och vårdutbildningar / [ed] Barbro Wijma, Goldina Smirthwaite, Katarina Swanberg, Lund: Studentlitteratur AB , 2010, 1, s. 497-516Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [sv]

    Kvinnor och män är delvis lika, delvis olika. Det innebär att kvinnor och män både har behov av likadan behandling och av behandling som är anpassad till det egna könets förutsättningar. Denna antologi belyser kvinnors och mäns förutsättningar och behov inom en rad olika medicinska områden och tar upp både biologiska och sociala faktorer som påverkar hälsa och behandling. Den behandlar även den roll som kön spelar inom vårdens arbetsliv samt hur köns- och genusperspektiv kan integreras inom olika typer av medicin- och vårdutbildningar. Ett av bokens teman är våld, kränkningar och diskriminering, och inom ramen för detta behandlas några av de olika maktordningar som kommer till uttryck vid behandlingar inom hälso- och sjukvården. Antologin har en stor spännvidd när det gäller ämnen och författare. Förhoppningsvis ska den bredd som antologin uppvisar, leda fram till frågeställningar där läsaren utmanar sina förgivettaganden inom både genusvetenskap och mer traditionell medicin samt väcka nya frågor: Om könet snarare ses som en konstruktion än en fysisk realitet - kan då kvinnor lika gärna äta mediciner som är utprovade på män och opereras med metoder och verktyg anpassade till mäns fysiologi? Å andra sidan - hur objektiv är den naturvetenskapligt inriktade medicinska forskningen egentligen om man börjar granska den utifrån frågeställningar om perspektivval och genus? Antologin vänder sig till lärare på utbildningar inom medicin, hälsa och vård. Andra målgrupper är studenter på sådana utbildningar, vårdpersonal och en intresserad allmänhet.

  • 31.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Detecting Minimal Residual Disease in Neuroblastoma: Still a Ways to Go2009Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 55, nr 7, s. 1268-1270Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    n/a

  • 32.
    Kågedal, Bertil
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Farnebäck, Malin
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Håkansson, Annika
    Department of Oncology, University Hospital MAS and Lund University, Malmö, Sweden.
    Gustafsson, Bertil
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk patologi och cytologi. Linköpings universitet, Hälsouniversitetet.
    Håkansson, Leif
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Onkologi. Linköpings universitet, Hälsouniversitetet.
    How useful are housekeeping genes?: variable expression in melanoma metastases2007Ingår i: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 45, nr 11, s. 1481-1487Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: There is a certain difference in opinion regarding the optimal choice of housekeeping genes used as normalization factors in gene expression analysis. We have therefore examined the suitability of three housekeeping genes, hypoxanthine phosphoribosyl transferase, β-glucuronidase and β2-micro-globulin, for normalization of expression data from melanoma metastases.

    Methods: The expression of the three housekeeping genes was quantified by quantitative reverse transcription PCR in snap-frozen sections from 44 melanoma metastases, of which 19 were from patients treated with cisplatinum, dacarbazine and interferon-α2b.

    Results: The expression of each housekeeping gene varied considerably between the different metastases. Histopathological examination of the tissue sections revealed variation in the amount of tumor cells in the tissue, necrosis, varying degrees of lymphocyte infiltration, and lymph node remnants. Based on this examination, 16 biopsies were omitted from further analysis because they had cracked, contained empty or necrotic areas, or were dominated by lymph node tissue. Even in sections with more than 90% tumor cells, a wide variation in the expression of the three housekeeping genes was found. The amount of lymphatic infiltrate in the tumors can have an effect on the expression of housekeeping genes in the meta-stases, whereas treatment did not seem to influence the expression.

    Conclusions: We conclude that the choice of housekeeping genes can have great impact on the normalization of specific genes in melanoma metastases. Furthermore, in the analysis of mRNA expression in tumor tissue, microscopic examination is of great importance to evaluate the integrity and cellular composition of the biopsy.

  • 33.
    Kågedal, Bertil
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Kullman, Anita
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi.
    Lenner, Liselotte
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi.
    Träger, Catarina
    Kogner, Per
    Farnebäck, Malin
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi.
    Pterin-dependent tyrosine hydroxylase mRNA is not expressed in human melanocytes or melanoma cells2004Ingår i: Pigment Cell Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 17, nr 4, s. 346-351Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pterin-dependent tyrosine hydroxylase has been described to occur occasionally in melanocytes. It is therefore important to quantify the mRNA of this enzyme in pigment cells to understand whether this enzyme can take an active part in pigment formation. A real-time reverse transcription-polymerase chain reaction method was used to quantify tyrosine hydroxylase mRNA in melanocytes and melanoma cells. The calibrator was obtained by amplification of a segment of cDNA from tyrosine hydroxylase mRNA, which included the target thus allowing enumeration of the number of transcripts per cell. In melanocytes (n = 3), tyrosine hydroxylase mRNA ranged from non-detectable to 0.000492 transcripts/cell and in melanoma cells from non-detectable to 0.005340 transcripts/cell. In neuroblastoma cells, the median tyrosine hydroxylase mRNA number was 0.4 transcripts/cell (range 0.02-25 transcripts/cell). The amount of tyrosine hydroxylase mRNA in the pigment cells was far less than the mRNA concentrations of four melanocyte-specific proteins measured in the same melanocytes and melanoma cells. We conclude that on the average less than 1 of 1000 melanocytes and melanoma cells contains at least one tyrosine hydroxylase mRNA molecule. Consequently, in 999 of 1000 cells translation into the corresponding enzyme protein cannot occur because of the lack of an mRNA template. Thus, in these cells there is no pterin-dependent tyrosine hydroxylase that can contribute to pigment formation by producing priming amounts of L-dopa for proper function of tyrosinase.

  • 34.
    Kågedal, Bertil
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Lindblad, Eva
    Bjuremark, Anna
    Linköpings universitet, Filosofiska fakulteten. Linköpings universitet, Institutionen för beteendevetenskap, Avdelningen för studier av vuxenutbildning, folkbildning och högre utbildning, VUFo.
    Kompetensutveckling i forskargrupper. Ett av arbetslivsfonden stött projekt1995Rapport (Övrigt vetenskapligt)
  • 35.
    Kågedal, Bertil
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi.
    Lindqvist, Maria
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för molekylär och klinisk medicin.
    Farnebäck, Malin
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi.
    Lenner, Liselotte
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Experimentell hematologi.
    Peterson, Curt
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Onkologiska kliniken US.
    Failure of the PAXgene™ Blood RNA System to maintain mRNA stability in whole blood2005Ingår i: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 43, nr 11, s. 1190-1192Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In multicentre studies of malignant and inflammatory diseases, whole blood, cell or tissue samples are often collected for analyses of gene expression to predict or monitor treatment effects. For correct analysis, sample stability during handling and transport is crucial. In developing the logistics for multicentre studies in malignant melanoma and inflammatory bowel disease, we found poor stability of a number of transcripts using the PAXgene™ Blood RNA System, which was advertised to maintain RNA stability for several days at room temperature. The results indicate that general statements on sample stability are not reliable and have to be verified for the specific transcripts of interest. © 2005 by Walter de Gruyter. Berlin.

  • 36.
    Landberg, Eva
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk kemi.
    Åström, Eva
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk kemi.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Disialo–trisialo bridging of transferrin is due to increased branching and fucosylation of the carbohydrate moiety2012Ingår i: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 414, s. 58-64Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background

    Carbohydrate deficient transferrin (CDT) is used for detection of alcohol abuse and follow-up. High performance liquid chromatography (HPLC) of transferrin glycoforms is highly specific for identification of alcohol abuse, but unresolved disialo- and trisialotransferrin glycoforms sometimes makes interpretation difficult. The cause of this phenomenon is unknown, cannot be explained by genetic variants of transferrin, but seems to be associated with liver disease.

    Methods

    Nineteen serum samples showing di–tri bridging when analyzed by HPLC were collected. Transferrin was purified by affinity chromatography, and N-linked oligosaccharides were released enzymatically. The N-glycans were further analyzed by high performance anion-exchange chromatography with pulsed amperometric detection and MALDI-TOF mass spectrometry.

    Results

    The HPLC-analysis showed three different types of glycoform patterns. The N-glycans from fifteen samples showed patterns with increased number of triantennary structures containing one or two fucose residues. One sample contained an increased amount of triantennary glycans without fucose. Three samples showed a glycosylation pattern similar to normal transferrin.

    Conclusions

    The di–tri bridging phenomenon was associated with alterations in transferrin glycosylation in the majority of cases. Transferrin contained a higher extent of triantennary and often fucosylated N-linked oligosaccharides. These results may be important in future diagnostic approaches to liver diseases.

  • 37.
    Magnusson, Karin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Appelqvist, Hanna
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Cieslar-Pobuda, Artur
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet. Institute of Automatic Control, Silesian University of of TechnologyGliwice, Poland.
    Wigenius, Jens
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska högskolan. Carl Zeiss AB, Sweden.
    Karlsson, Thommie
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet. Application Specialist Confocal Microscopy at Leica MicrosystemsIL, United States.
    Los, Marek Jan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet. Department of Pathology, Pomeranian Medical UniversitySzczecin, Poland.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk kemi.
    Jonasson, Jon
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Diagnostikcentrum, Klinisk patologi och klinisk genetik.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Differential vital staining of normal fibroblasts and melanoma cells by an anionic conjugated polyelectrolyte2015Ingår i: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 87, nr 3, s. 262-272Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblasts, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was nontoxic, photoinduced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photoinduced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be used to study cancer-related biological and pathological processes.

  • 38.
    Magnusson, Karin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Appelqvist, Hanna
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Cieślar-Pobuda, Artur
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Bäck, Marcus
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten.
    Jonasson, Jon
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Diagnostikcentrum, Klinisk patologi och klinisk genetik.
    Los, Marek J.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Nilsson, Peter R.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    An imidazole functionalized pentameric thiophene displays different staining patterns in normal and malignant cells2015Ingår i: Frontiers in Chemistry, E-ISSN 2296-2646, Vol. 3, artikel-id 58Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Molecular tools for fluorescent imaging of cells and their components are vital for understanding the function and activity of cells. Here, we report an imidazole functionalized pentameric oligothiophene, p-HTIm, that can be utilized for fluorescent imaging of cells. p-HTIm fluorescence in normal cells appeared in a peripheral punctate pattern partially co-localized with lysosomes, whereas a one-sided perinuclear Golgi associated localization of the dye was observed in malignant cells. The uptake of p-HTIm was temperature dependent and the intracellular target was reached within 1 h after staining. The ability of p-HTIm to stain cells was reduced when the imidazole side chain was chemically altered, verifying that specific imidazole side-chain functionalities are necessary for achieving the observed cellular staining. Our findings confirm that properly functionalized oligothiophenes can be utilized as fluorescent tools for vital staining of cells and that the selectivity towards distinct intracellular targets are highly dependent on the side-chain functionalities along the conjugated thiophene backbone.

  • 39.
    Nezirevic Dernroth, Dzeneta
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Rundström, Annica
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Gas chromatography-mass spectrometry analysis of pheomelanin degradation products2009Ingår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, nr 30, s. 5730-5739Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Melanoma is most rapidly increasing in the white population and people with pheomelanin skin type are at high risk to develop melanoma. However, little is known about the pheomelanin structure and function, and further elucidation of this melanin is therefore an important task. A GC/MS method was developed based on hydriodic acid hydrolysis of pheomelanin in the urine. Derivatization was performed with ethyl chloroformate and ethanol:pyridine (4:1, v/v). N,O-Ethoxycarbonyl-ethyl esters were extracted with chloroform and analyzed by GC/MS. 4-Amino-3-hydroxyphenylaianine and 3-amino4-hydroxyphenylaianine together with one benzothiazinone and two benzothiazole compounds were detected and identified in hydrolyzed samples of synthetic pheomelanin and melanin from the urine of a patient with melanoma. These findings strongly suggest that heterocyclic pheomelanin-type units are incorporated in the pigment structures.

  • 40.
    Nezirevic Dernroth, Dzeneta
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Årstrand, Kerstin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Greco, Giorgia
    Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cyntia 4 I-80126, Naples Italy.
    Panzella, Lucia
    Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cyntia 4 I-80126, Naples Italy.
    Napolitano, Alessandra
    Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cyntia 4 I-80126, Naples Italy.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Pheomelanin-related benzothiazole isomers in the urine of patients with diffuse melanosis of melanoma2010Ingår i: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 411, nr 17-18, s. 1195-1203Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Currently used as structural markers for pheomelanin identification and quantitation, benzothiazole compounds derived from isomeric cysteinyldopas have been indicated by recent in vitro studies as new potential pheomelanogenesis intermediates. Prompted by previous reports on the occurrence of large amounts of 5-S-cysteinyldopa (5-S-CD) and trichochromes in urine of patient with diffuse melanosis of melanoma we investigated the presence of benzothiazole compounds in the urine of these patients.

    Hydrophilic interaction liquid chromatography on zwitterionic stationary phase (ZIC-HILIC) and photo-diode array (PDA) detection was used for analysis of 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BTCA-5), and 7-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BTCA-2), derived from 5-S-CD and 2-S-cysteinyldopa (2-S-CD) isomers, respectively. Isocratic mobile phase with minimal sample preparation allowed efficient separation of the compounds, which were safely identified by their typical absorption features.

    Among 21 melanoma patients examined three showed diffuse melanosis. The levels of urinary BTCAs were found to be highly associated with melanosis but more loosely to excreted 5-S-CD. Analysis of the pigmented fraction of urine following alkaline hydrogen peroxide degradation and quantitation of BTCAs provided evidence for the presence of pheomelanins at higher levels in patients with melanosis.

  • 41.
    Nezirevic Dernroth, Dzeneta
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi.
    Årstrand, Kerstin
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi.
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi.
    Hydrophilic interaction liquid chromatographic analysis of aminohydroxyphenylalanines from melanin pigments2007Ingår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1163, nr 1-2, s. 70-79Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Malignant melanomas are more often seen in subjects with light colored skin who tan poorly than in persons who tan more rapidly. This has been attributed to the structure of their pigment, pheomelanin, which differs markedly from the eumelanin of persons with darker skin. To study the hydrolysis products of pheomelanin pigments a new method was developed for analysis of 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP). Pheomelanin samples were hydrolyzed and extracted with solid-phase extraction columns using strong cation-exchange (SCX) cartridges. Separation of 4-AHP and 3-AHP was achieved on a ZIC-HILIC column (150 mm × 2.1 mm I.D.) with a mobile phase consisting of acetonitrile:0.1 M ammonium acetate buffer, pH 4.5 (82:18, v/v). Detection was performed with an electrochemical detector at +400 mV. Run time was 30 min. The limits of detection were 73 pg and 51 pg for 4-AHP and 3-AHP respectively, using 2 μl injections. Good linearity was found within the range 0.05-5.0 μg/ml. Absolute recovery was 70% and relative recovery was 100%. The AHPs were stable for 1 year in the hydrolyzed samples, for 4 days in the eluates from solid-phase sorbents stored in the refrigerator, and for 2 days diluted with mobile phase and stored in the autosampler at 10 °C. The within-day imprecision was <5% and the between-day imprecision was <7% for the two analytes. The method, applied to the analysis of pheomelanin in urine from human melanoma patients, allows the analysis of 30 samples in one set and is suitable for routine work with human hair and melanoma cells. By using the ZIC-HILIC stationary phase, ion-pairing reagents could be avoided, which makes the method suitable to further analysis of degradation products from pheomelanins using mass spectrometric detection. © 2007 Elsevier B.V. All rights reserved.

  • 42.
    Rosdahl, Inger
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Dermatologi. Linköpings universitet, Hälsouniversitetet.
    Andersson, Eva
    Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Törma, H.
    Department of Dermatology, University of Uppsala, Sweden.
    Vitamin A metabolism and mRNA expression of retinoid-binding protein and receptor genes in human epidermal melanocytes and melanoma cells1997Ingår i: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 7, nr 4, s. 267-274Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Retinoids inhibit proliferation of melanocytes and melanoma cells and affect disorders of hypo- and hyperpigmentation. Such effects might involve retinoid-binding proteins, retinoid metabolites and nuclear retinoid receptors for transcriptional activation. We detected messenger RNA transcripts for the cellular retinol- and retinoic acid-binding proteins (CRBP, CRABP I and II) in cultured epidermal melanocytes. In the melanoma cell lines the major transcript was CRABP II. Nuclear retinoic acid (RA) receptor transcripts and the 9-cis-retinoic acid receptor transcript were detected in all cells. The endogenous concentrations of retinol (ROH) and its metabolite 3,4-didehydroretinol (ddROH) in melanocytes were five times those in melanoma cells. When cells were incubated with [3H]ROH the main metabolites in the melanocytes were [3H]ddROH (4%) and [3H]RA (0.4%). Formation of [3H]RA was only detected in one melanoma cell line. Both melanocytes and melanoma cells produced an unidentified metabolite when incubated with [3H]ROH and [3H]RA. Dissimilarities in the metabolism and endogenous concentration of retinoids between benign and malignant melanocytes might play a key role in differentiation and growth regulation.

  • 43.
    Svedjeholm, Rolf
    et al.
    Östergötlands Läns Landsting, Hjärtcentrum, Kardiologiska kliniken.
    Dahlin, Lars-Göran
    Östergötlands Läns Landsting, Hjärtcentrum, Kardiologiska kliniken.
    Lundberg, Claes
    Östergötlands Läns Landsting, Hjärtcentrum, Kardiologiska kliniken.
    Szabo, Zoltan
    Department of Cardiothoracic Surgery, Debrecen, Hungary.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Nylander, Eva
    Östergötlands Läns Landsting, Hjärtcentrum, Fysiologiska kliniken.
    Olin, Christian
    Östergötlands Läns Landsting, Hjärtcentrum, Kardiologiska kliniken.
    Rutberg, Hans
    Östergötlands Läns Landsting, Hjärtcentrum, Kardiologiska kliniken.
    Are electrocardiographic Q-wave criteria reliable for diagnosis of perioperative myocardial infarction after coronary surgery?1998Ingår i: European Journal of Cardio-Thoracic Surgery, ISSN 1010-7940, E-ISSN 1873-734X, Vol. 13, nr 6, s. 655-661Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Objective: A major assumption in cardiovascular medicine is that Q-waves on the electrocardiogram indicate major myocardial tissue damage. The appearance of a new Q-wave has therefore been considered the most reliable criterion for diagnosis of perioperative myocardial infarction (PMI) in cardiac surgery. In a study, originally intended to evaluate troponin-T as a marker of PMI, analysis of our data aroused the need to address the reliability of Q-wave criteria for diagnosis of PMI.

    Methods: In 302 consecutive patients undergoing coronary surgery, Q-wave and other electrocardiogram (ECG) criteria were compared with biochemical markers of myocardial injury and the postoperative course. All ECGs were analysed by a cardiologist blinded to the biochemical analyses and the clinical course.

    Results: The incidence of positive Q-wave criteria was 8.1%. Combined biochemical (CK-MB≥70 μg/l) and Q-wave criteria were found in 1.0%. Patients with new Q-waves did not have CK-MB or troponin-T levels significantly different from those without Q-waves. More than 25% of the Q-waves were associated with plasma troponin-T below the reference level (<0.2 μg/l) on the fourth postoperative day. Q-wave criteria alone did not influence the postoperative course. In contrast, biochemical markers correlated with clinical outcome.

    Conclusions: The majority of Q-waves appearing after coronary surgery were not associated with major myocardial tissue damage, and according to troponin-T one-fourth of the Q-waves were not associated with myocardial necrosis. Furthermore, the appearance of Q-waves had little influence on short term clinical outcome. Therefore, the use of Q-wave criteria as the gold standard for diagnosis of PMI may have to be questioned.

  • 44.
    Takasaki, Akihiko
    et al.
    Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan.
    Nezirevic Dernroth, Dzeneta
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Årstrand, Kerstin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Wakamatsu, Kazumasa
    Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan.
    Ito, Shosuke
    Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    HPLC analysis of pheomelanin degradation products in human urine2003Ingår i: Pigment Cell Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 16, nr 5, s. 480-486Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A sensitive and specific high performance liquid chromatography (HPLC) method was developed to quantify 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP) in urine. In degradation studies of melanin pigment, 4-AHP and 3-AHP are derived from benzothiazine units of pheomelanin and pheomelanin-related metabolites such as trichochromes. 5-S-Cysteinyldopa-derived benzothiazine products give 4-AHP while 2-S-cysteinyldopa-derived benzothiazine products give 3-AHP. 3-AHP is also derived from nitrotyrosine formed by nitration of tyrosine with reactive nitrogen species. For this reason, the influence of this biological process on the amount of 3-AHP found in biological material have been investigated. The method is based on hydriodic acid hydrolysis of the melanin polymer and reversed-phase HPLC with electrochemical detection of the degradation products 4-AHP and 3-AHP. The mobile phase consists of 25 mM ammonium acetate and sodium octanesulfonate as an ion-pairing reagent. The 4-AHP and 3-AHP peaks were well separated and the detector response was linear within the range 0-2 ng injected for both compounds. With the developed chromatographic system, 4-AHP and 3-AHP showed good separation in the biological samples. There was a strong correlation between 4-AHP and 3-AHP in the urine of 50 malignant melanoma patients and two healthy subjects (R0.977). The two compounds were also strongly correlated with 5-S-cysteinyldopa in urine, the correlation coefficients being 0.862 and 0.907, respectively. The method described is sensitive enough for analysis of pheomelanin in urine and in several other biological samples. The results indicate that 3-AHP in urine is not influenced by excreted 3-nitrotyrosine and the data indicate that pheomelanins are excreted in the urine of melanoma patients.

  • 45.
    Trager, Catarina
    et al.
    Karolinska University Hospital, Sweden.
    Vernby, Asa
    Karolinska University Hospital, Sweden.
    Kullman, Anita
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Ora, Ingrid
    Lund University Hospital, Sweden.
    Kogner, Per
    Karolinska University Hospital, Sweden.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    mRNAs of tyrosine hydroxylase and dopa decarboxylase but not of GD2 synthase are specific for neuroblastoma minimal disease and predicts outcome for children with high-risk disease when measured at diagnosis2008Ingår i: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 123, nr 12, s. 2849-2855Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several transcripts have been claimed to be clinically valuable for detecting minimal disease in neuroblastoma, but they have not been prospectively compared in a standardized manner. Tyrosine hydroxylase (TH), dopa decarboxylase (DDC) and GD2 synthase (GD2S) mRNAs were analyzed in 554 blood (PB) and bone marrow (BM) samples from 58 children with neuroblastoma. Samples from 44 children with other diseases served as controls. High transcript concentrations of TH, GD2S or DDC in PB or BM at diagnosis were associated with poor prognosis. TH in BM above median indicated worse outcome for a homogenous cohort with high-risk neuroblastoma (survival probability 91% for TH below median versus 33% for TH above median, p = 0.009). The number of children with localized neuroblastoma with increased results in PB did not differ between the three transcripts. In these children, all without morphologically detectable neuroblastoma in BM, the number of patients with elevated GD2S in BM at diagnosis was significantly higher than for the other transcripts (10/16 elevated, P = 0.012). GD2S was elevated in PB from 10/28 controls without neuroblastoma compared to 1/28 for TH and DDC (p < 0.001). In BM from these children GD2S was significantly elevated. We conclude that high expression of TH and DDC both in Ill and BM corresponds to metastatic neuroblastoma at diagnosis, residual disease, and poor outcome. Children with high-risk neuroblastoma and low levels of TH in BM at diagnosis may be cured by current therapy. GD2S is less specific than TH and DDC mRNA for neuroblastoma detection in PB and BM.

  • 46.
    Träger, Chatarina
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi.
    Kogner, Per
    Lindskog, Magnus
    Ponthan, Frida
    Kullman, Anita
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi.
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Quantitative analysis of tyrosine hydroxylase mrna for sensitive detection of neuroblastoma cells in blood and bone marrow2003Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 49, nr 1, s. 104-112Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Sensitive monitoring of minimal residual disease may improve the treatment of neuroblastoma in children. To detect and monitor neuroblastoma cells in blood and bone marrow, we developed a quantitative method for the analysis of tyrosine hydroxylase mRNA. Methods: We used real-time reverse transcription-PCR. The calibrator was constructed from a segment of tyrosine hydroxylase mRNA that included the target. Blood and bone marrow samples from 24 children with neuroblastoma and 1 child with ganglioneuroma were analyzed. Controls were blood samples from the cords of 40 babies, from 58 children 6 months to 15 years of age, and from 34 healthy adults, as well as from 12 children with other diseases. Results: The detection limit was ~70 transcripts/mL. All 144 blood controls were below this limit. At diagnosis, blood tyrosine hydroxylase mRNA was higher in children with widespread disease (stage 4/4S, n = 6, range, 203-46 000 transcripts/mL) than in patients with localized disease (stages 1-3, n = 6, =83 transcripts/mL, P = 0.002). Bone marrow from all five children with localized disease had concentrations <72 transcripts/mL, whereas five of six stage 4 patients had increased concentrations (6000-8 000 000 transcripts/mL, P <0.05). In nine children in whom tyrosine hydroxylase mRNA was measured repeatedly, the results corresponded to the clinical course. Conclusion: Quantitative analysis of tyrosine hydroxylase mRNA in blood and bone marrow is reliable and easy to perform and may be used for upfront staging, prognostic assessment, and treatment monitoring of neuroblastoma.

  • 47. Viprey, Virginie F
    et al.
    Corrias, Maria V
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Oltra, Silvestre
    Swerts, Katrien
    Vicha, Ales
    Ladenstein, Ruth
    Burchill, Susan A
    Standardisation of operating procedures for the detection of minimal disease by QRT-PCR in children with neuroblastoma: Quality assurance on behalf of SIOPEN-R-NET2007Ingår i: European Journal of Cancer, ISSN 0959-8049, E-ISSN 1879-0852, Vol. 43, nr 2, s. 341-350Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The clinical utility of detecting minimal residual disease (MRD) in children with neuroblastoma (NB) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) is not clear. This in part reflects the lack of uniform methodology for analysis and reporting. Reference laboratories across Europe have therefore established standard operating procedures (SOPs) for the detection of NB cells by QRT-PCR. Haemopoietic samples are collected into PAXgene™ blood RNA tubes, which stabilise mRNA for 48 h at room temperature and more than 6 months at -80 °C. Tyrosine hydroxylase (TH) was selected as the target for NB cell detection, expression is normalised to β2-microglobulin and reported using the ΔΔCt method. The sensitivity of QRT-PCR increased from 58% to 90% following the development of SOPs. A robust, transferable, objective method for the detection of NB cells by QRT-PCR has been defined to improve the power and consistency of studies on MRD in children with NB. © 2006 Elsevier Ltd. All rights reserved.

  • 48.
    Vrethem, Magnus
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan. Östergötlands Läns Landsting, Närsjukvården i centrala Östergötland, Neurologiska kliniken.
    Mattsson, E
    Hebelka, H
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan.
    Leerbeck, K
    Österberg, A
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan. Östergötlands Läns Landsting, Närsjukvården i centrala Östergötland, Neurologiska kliniken.
    Landtblom, Anne-Marie
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan. Östergötlands Läns Landsting, Närsjukvården i centrala Östergötland, Neurologiska kliniken.
    Balla, B
    Motala.
    Nilsson, H
    Norrköping.
    Hultgren, M
    Jönköping.
    Brattström, L
    Kalmar.
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Increased plasma homocysteine levels without signs of vitamin B12 deficiency in patients with multiple sclerosis assessed by blood and cerebrospinal fluid homocysteine and methylmalonic acid2003Ingår i: Multiple Sclerosis, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 9, nr 3, s. 239-245Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Objective: The aim of this study was to evaluate if multiple sclerosis (MS) is associated with vitamin B12 (cobalamin) deficiency. Methods: We measured serum vitamin B12, plasma folate, serum methylmalonic acid (MMA), plasma homocysteine (tHcy) and also cerebrospinal fluid (CSF) MMA and tHcy in 72 patients with MS and 23 controls. Results: The mean plasma tHcy level was significantly increased in MS patients (11.6 ╡mol/L) compared with controls (7.4╡mol/L) (P = 0.002). Seven patients showed low serum vitamin B12 levels but only one of them had concomitant high plasma tHcy. None of them showed high serum MMA. Plasma or blood folate levels did not differ between MS patients and controls. We found no significant differences in mean values or frequency of pathological tests of serum B12, serum MMA, mean corpuscular volume (MCV), haemoglobin concentration, CSF tHcy or CSF MMA between patients and healthy subjects. There were no correlations between CSF and serum/plasma levels of MMA or tHcy. Serum vitamin B12, serum MMA, plasma tHcy, CSF Hey or CSF MMA were not correlated to disability status, activity of disease, duration of disease or age. Conclusions: The relevance of the increased mean value of plasma tHcy thus seems uncertain and does not indicate functional vitamin B12 deficiency. We can not, however, exclude the possibility of a genetically induced dysfunction of the homocysteine metabolism relevant for the development of neuroinflammation/degeneration. Our findings indicate that, regardless of a significant increase in plasma tHcy in MS patients, the MS disease is not generally associated with vitamin B12 deficiency since we did not find any other factors indicating vitamin B12 deficiency. Analysis of CSF MMA and CSF tHcy, which probably reflects the brain vitamin B12 status better than serum, are not warranted in MS. We conclude that B12 deficiency, in general, is not associated with MS.

  • 49. Wakamatsu, Kazumasa
    et al.
    Takasaki, Akihiko
    Kågedal, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Kageshita, Toshiro
    Ito, Shosuke
    Determination of eumelanin in human urine2006Ingår i: Pigment Cell Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 19, nr 2, s. 163-169Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Normal and malignant melanocytes produce melanins and melanin-related metabolites, most of which are retained in the cells but some are secreted into the blood and then excreted in the urine. In this study, we developed a method to measure levels of eumelanin in urine samples and evaluated its clinical significance in comparison with the melanin-related metabolites 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) and 5-S-cysteinyldopa (5-S-CD), and with pheomelanin, measured after degradation as 4-amino-3-hydroxyphenylalanine (4-AHP). The method is based on the production of pyrrole-2,3,5-tricarboxylic acid (PTCA) on permanganate oxidation of eumelanin, followed by quantification by liquid chromatography. For 118 urine samples from 10 control subjects, mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD and 4-AHP were 19, 67, 37 and 59 μmol/mol creatinine respectively. In melanoma patients (n = 45), the mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD, and 4-AHP were 91, 926, 4070 and 3530 μmol/mol creatinine respectively. Median level of PTCA in melanoma patients was elevated 2.1-fold compared with control subjects. The degrees of elevation for 6H5MI2C, 5-S-CD, and 4-AHP were 1.8-, 22- and 6.2-fold respectively. Thus, although urinary PTCA is of little clinical value in following the progression of melanoma, urinary 4-AHP appears to be of considerable value in this respect. © 2006 Blackwell Munksgaard.

  • 50.
    Zdolsek, Joachim
    et al.
    Östergötlands Läns Landsting, Anestesi- och operationscentrum, Intensivvårdskliniken US.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Lisander, Björn
    Linköpings universitet, Institutionen för medicin och hälsa, Anestesiologi med intensivvård. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Anestesi- och operationscentrum, Intensivvårdskliniken US.
    Hahn, Robert
    Linköpings universitet, Institutionen för medicin och hälsa, Anestesiologi med intensivvård. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Anestesi- och operationscentrum, Intensivvårdskliniken US.
    Glomerular filtration rate is increased in burn patients2010Ingår i: Burns, ISSN 0305-4179, E-ISSN 1879-1409, Vol. 36, nr 8, s. 1271-1276Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Urinary output a key parameter guiding fluid resuscitation in burn trauma is an inadequate measure of renal function In this study the clearance of iohexol (CL) was used to follow the glomerular filtration rate during the first week after burn Nineteen adults with major burns received an intravenous bolus injection of iohexol every other day Plasma concentration of iohexol was measured over 4 h and CL was calculated by a one compartment kinetic model The results were compared to the CL as obtained by a two compartment model and also to the CL measured in 10 healthy controls The results show that CL values for burn patients were high The first day after burn median CL was 155 mL/min/1 73 m(2) (range 46-237) which exceeded that for the controls (mean 117 mL/min/1 73 m(2) P less than 0 01) However on day 7 the CL approached the expected baseline (mean 122 mL/min/1 73 m(2)) CL was 10% lower when calculated from two compartment kinetics and a correction factor of 0 9 was applied to all results obtained by the one compartment calculations to give results comparable to those from the two compartment kinetics In conclusion CL is increased early after burn The mechanism is unclear but it parallels the period of vascular dysfunction and increased cardiac output

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