| 1. |
- Håkansson, Annika, 1962-, et al.
(författare)
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Bcl-2 monitoring in malignant melanoma
- 2004
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Ingår i: Hospital Pharmacy. - 0018-5787 .- 1945-1253. ; , s. 46-47
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 2. |
- Kågedal, Bertil, 1943-, et al.
(författare)
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How useful are housekeeping genes? Variable expression in melanoma metastases
- 2007
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Ingår i: Clinical Chemistry and Laboratory Medicine. - 1434-6621 .- 1437-4331. ; 45:11, s. 1481-1487
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Tidskriftsartikel (refereegranskat)abstract
- 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, beta(2)-glucuronidase and beta(2)-microglobulin, 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 alpha 2b. 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 metastases, 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.
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| 3. |
- Baryawno, Ninib, et al.
(författare)
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Tumor-growth-promoting cyclooxygenase-2 prostaglandin E2 pathway provides medulloblastoma therapeutic targets
- 2008
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Ingår i: Neuro-Oncology. - : Oxford University Press (OUP). - 1522-8517 .- 1523-5866. ; 10:5, s. 661-674
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Berois, Nora, et al.
(författare)
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ppGalNAc-TI3 : A new molecular marker of bone marrow involvement in neuroblastoma
- 2006
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 52:9, s. 1701-1712
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Dahlin, Lars-Göran, 1956-, et al.
(författare)
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Early Identification of Permanent Myocardial Damage after Coronary Surgery is Aided by Repeated Measurements of CK-MB
- 2002
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Ingår i: Scandinavian Cardiovascular Journal. - : Informa UK Limited. - 1401-7431 .- 1651-2006. ; 36:1, s. 35-40
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Dahlin, Lars-Göran, 1956-, et al.
(författare)
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Unspecific elevation of plasma troponin-T and CK-MB after coronary surgery
- 2003
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Ingår i: Scandinavian Cardiovascular Journal. - : Informa UK Limited. - 1401-7431 .- 1651-2006. ; 37:5, s. 283-287
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Dizdar (Dizdar Segrell), Nil, 1960-, et al.
(författare)
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Analysis of L-dopa in human serum
- 2002
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Ingår i: BioTechniques. - 0736-6205 .- 1940-9818. ; 33:5, s. 1000-1002
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Tidskriftsartikel (refereegranskat)
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| 8. |
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| 9. |
- Johansson, Malin, 1972-, et al.
(författare)
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Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction
- 2000
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Ingår i: Melanoma research. - 0960-8931 .- 1473-5636. ; 10:3, s. 213-222
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Johansson, Malin, 1972-, et al.
(författare)
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Quantitative analysis of tyrosinase transcripts in blood
- 2000
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 46:7, s. 921-927
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 11. |
- Johansson, Malin, 1972-, et al.
(författare)
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Quantitative relationships between pigment-related mRNA and biochemical melanoma markers in melanoma cell lines
- 2002
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Ingår i: Melanoma research. - : Ovid Technologies (Wolters Kluwer Health). - 0960-8931 .- 1473-5636. ; 12:3, s. 193-200
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 12. |
- Johnsen, J I, et al.
(författare)
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Inhibitors of mammalian target of rapamycin downregulate MYCN protein expression and inhibit neuroblastoma growth in vitro and in vivo
- 2008
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Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 27:20, s. 2910-2922
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 13. |
- Kronstrand, Robert, et al.
(författare)
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Codeine Concentration in Hair after Oral Administration Is Dependent on Melanin Content
- 1999
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 45:9, s. 1485-1494
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 14. |
- Kågedal, Bertil, 1943-, et al.
(författare)
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Determination of glomerular filtration rate, a spin off aftercontrast-enhanced computed tomography among criticallyill patients − proof of concept
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background Recently, Gong et al. (Gong et al. 2022) showed, in nine heathy subjects, that plasma clearance of high doses of iohexol given as contrast enhanced computed tomography (CT) could be used for determination of glomerular filtration rate (GFR). We utilized high doses of iohexol from angiographic or other contrast enhanced CT given to critical ill patients for calculation of GFRiohexol and compared these data with standard low dose iohexol GFR determinations.Method Patients at intensive care units (ICUs) in Southeast Sweden intended for radiographic investigations that included injection of 45-120 ml of iohexol (Omnipaque) were included, and the concentration of iohexol in plasma was measured by HPLC. Iohexol clearance was calculated by the method of Bröchner-Mortensen. The following days was iohexol clearance determined using the standard low dose of 5 mL of iohexol. Sixteen patients admitted to ICUs were included in this pilot study.Results GFR after high dosing of iohexol at contrast enhanced CT could be measured for all sixteen critically ill patients. Patients with normal or increased renal function had neglectable iohexol concentrations the day following the CT scan. There was excellent correlation between GFR determination with high and standard low iohexol dosing among these 6 patients. Ten patients had decreased renal function and delayed elimination of iohexol, thus was not GFR measurement with low dose iohexol possible to analyse the day after CT scan with high dose iohexol.Conclusion This pilot study showed that GFR can be measured after high doses of iohexol at enhanced CT and compare well with the standard low dose of iohexol clearance determinations.
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| 15. |
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| 16. |
- Kågedal, Bertil, 1943-, et al.
(författare)
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Pterin-dependent tyrosine hydroxylase mRNA is not expressed in human melanocytes or melanoma cells
- 2004
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Ingår i: Pigment Cell Research. - : Wiley. - 0893-5785 .- 1600-0749. ; 17:4, s. 346-351
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 17. |
- Kärnell, R, et al.
(författare)
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The value of cysteinyldopa in the follow-up of disseminated malignant melanoma
- 2000
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Ingår i: Melanoma research. - : Ovid Technologies (Wolters Kluwer Health). - 0960-8931 .- 1473-5636. ; 10:4, s. 363-369
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 18. |
- Nezirevic Dernroth, Dzeneta, 1969-, et al.
(författare)
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Hydrophilic interaction liquid chromatographic analysis of aminohydroxyphenylalanines from melanin pigments
- 2007
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1163:1-2, s. 70-79
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 19. |
- Nezirevic Dernroth, Dzeneta, 1969-, et al.
(författare)
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Pheomelanin-related benzothiazole isomers in the urine of patients with diffuse melanosis of melanoma
- 2010
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Ingår i: Clinica Chimica Acta. - : lsevier Science B.V., Amsterdam. - 0009-8981 .- 1873-3492. ; 411:17-18, s. 1195-1203
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 20. |
- Träger, Chatarina, 1962-, et al.
(författare)
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Quantitative analysis of tyrosine hydroxylase mrna for sensitive detection of neuroblastoma cells in blood and bone marrow
- 2003
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 49:1, s. 104-112
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 21. |
- Viprey, Virginie F, et al.
(författare)
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Standardisation of operating procedures for the detection of minimal disease by QRT-PCR in children with neuroblastoma : Quality assurance on behalf of SIOPEN-R-NET
- 2007
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Ingår i: European Journal of Cancer. - : Elsevier BV. - 0959-8049 .- 1879-0852. ; 43:2, s. 341-350
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 22. |
- Vrethem, Magnus, 1955-, et al.
(författare)
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Increased plasma homocysteine levels without signs of vitamin B12 deficiency in patients with multiple sclerosis assessed by blood and cerebrospinal fluid homocysteine and methylmalonic acid
- 2003
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Ingår i: Multiple Sclerosis Journal. - : SAGE Publications. - 1352-4585 .- 1477-0970. ; 9:3, s. 239-245
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 23. |
- Wakamatsu, Kazumasa, et al.
(författare)
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Determination of eumelanin in human urine
- 2006
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Ingår i: Pigment Cell Research. - : Wiley. - 0893-5785 .- 1600-0749. ; 19:2, s. 163-169
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 24. |
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