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K Arstrand

Publications and source records attributed to K Arstrand.

13 recordsLinked to original sources
PubMed2004

Improved method for analysis of cysteinyldopa in human serum.

5-S-L-Cysteinyl-L-dopa is a well-known pigment intermediate and analysis of its serum concentration is well suited for evaluation of treatment and follow-up of stage III and IV malignant melanoma. A simplified analytical method is described using organic extraction followed by clean-up on a boronate gel to capture the compound containing vicinal hydroxyls. Weak acid solution elutes the 5-S-cysteinyldopa suitable for high-performance liquid chromatography (HPLC). The absolute recoveries of cysteinyldopa and its diastereomer 5-S-D-cysteinyl-L-dopa (used as an internal standard) were 81.5 +/- 2.8% and 81.3 +/- 2.7%, respectively, and use of the internal standard for the whole procedure gave an analytical recovery of 101 +/- 0.8%. The limit of quantitation was 1.5 nmol/L and the imprecision of the method was < 5.0% over the analytical range 1.5-500 nmol/L. The method is cheap and easy to perform and compares well with other described techniques. The use of the method is illustrated by results obtained during treatment of a patient with metastatic malignant melanoma.

Aged↗
PubMed2002

Quantitative relationships between pigment-related mRNA and biochemical melanoma markers in melanoma cell lines.

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-100 beta 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 10(3) transcripts/cell, i.e. by a factor > 10(7) in the different cell lines. S-100 beta 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-100 beta 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 10(7) must have great impact on the diagnostic sensitivity. Measurement of S-100 beta mRNA would be more sensitive, but the use of this transcript is hampered by its presence in the blood cells.

Antigens, Neoplasm↗
PubMed2000

The value of cysteinyldopa in the follow-up of disseminated malignant melanoma.

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 micromol/mol creatinine) were seen for 83% of the occasions when clinical progression was recorded, and decrements in 5SCD excretion (median 145 micromol/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 micromol/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.

Adult↗
PubMed2000

Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction.

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.

Evaluation Studies as Topic↗
PubMed2000

Quantitative analysis of tyrosinase transcripts in blood.

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: Ultraspec(TM)-II RNA isolation system, FastRNA(TM) 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.

Cell Line↗
PubMed1997

Effects on interstitial glutathione, cysteine and 5-S-cysteinyldopa of buthionine sulphoximine in human melanoma transplants.

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.

Acetylcysteine↗
PubMed1997

S100B protein, 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid as biochemical markers for survival prognosis in patients with malignant melanoma.

Elevated levels of the phaeomelanin metabolite 5-S-cysteinyldopa and the eumelanin metabolite 6-hydroxy-5-methoxyindole-2-carboxylic acid in urine and serum have been shown in previous studies to correlate with disseminated malignant melanoma. Immunohistochemical detection of S100B protein is an acknowledged method for the diagnosis of malignant melanoma, and it has been suggested that rising serum levels of S100B protein are associated with the survival rate of patients with malignant melanoma. In the present study serum levels of S100B protein and urinary concentrations of 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid were measured in 91 patients with histopathologically verified malignant melanoma. At the time of sampling 13 patients were in clinical stage I, 13 in stage II and 65 in stage III. The urinary levels of the melanin metabolites were determined by automated high performance liquid chromatography, and the serum levels of S100B protein by an immunoradiometric assay with two monoclonal antibodies. The overall survival rate was most strongly associated with the serum levels of S100B protein (P < 0.001), but there was also a significant correlation to urinary levels of 5-S-cysteinyldopa (P < 0.001). A corresponding association with urinary levels of 6-hydroxy-5-methoxyindole-2-carboxylic acid was found in only a very few patients with extremely high urinary concentrations. A statistically significant increase in relative hazard was found for S100B protein levels exceeding 0.6 microgram/l (P < 0.001), and predictably for patients in clinical stage III (P < 0.001). An analysis of S100B protein levels in patients in clinical stage III showed a significant correlation to survival (P = 0.005). Our study suggests that of the three biochemical tumour markers, S100B and to a lesser extent 5-S-cysteinyldopa have the greatest potential to be used as predictors of survival prognosis in patients with malignant melanoma.

Adult↗
PubMed1996

Altered glutathione levels in ischemic and postischemic skeletal muscle: difference between severe and moderate ischemic insult.

The purpose of the present study was to investigate how the duration of ischemia and reperfusion affect the glutathione (GSH) levels in skeletal muscle and to assess the presence of oxidative stress by quantitating oxidized glutathione (GSSG) and the ratio of GSSG/GSH. The amounts of GSH and GSSG were quantitated in the tibialis anterior muscle of the rat hind limb after 2 and 4 hours of tourniquet ischemia and after 1 and 5 hours of reperfusion, and the levels were compared to those in nonischemic control tibialis anterior muscles. In muscles subjected to 2 hours of ischemia, the levels of GSH, GSSG, and the ratio GSSG/GSH did not differ significantly from those of nonischemic controls. After 4 hours of ischemia without reperfusion, the GSH levels were slightly increased, compared to controls (p < 0.05). After 1 hour of reperfusion following 4 hours of ischemia, the levels of GSH decreased by 50% compared to control (p < 0.01), and still after 5 hours of reperfusion the levels of GSH were 50% lower than control levels. The GSSG/ GSH ratio did not change during 1 and 5 hours of reperfusion compared to control. A major finding in this study was that, during reperfusion after severe ischemia of 4 hours, there was a marked depletion of glutathione, which was not seen after a moderate ischemic insult of 2 hours.

Animals↗
PubMed1996

In situ microdialysis for monitoring of extracellular glutathione levels in normal, ischemic and post-ischemic skeletal muscle.

Microdialysis probes were inserted into the tibialis anterior muscle and into the femoral vein of anaesthetised Sprague-Dawley rats for monitoring of reduced (GSH) and oxidized (GSSG) extracellular glutathione. The dialysates were analysed using HPLC. The levels of GSH and GSSG were high immediately after implantation in the skeletal muscle and declined to steady state levels after 90 minutes into the same range as that found in the venous dialysate. Total ischemia was induced two hours after implantation of the dialysis probe after steady state levels had been reached. The extracellular levels of GSH increased during total ischemia and had doubled at the end of the ischemic period compared to preischemic values. During the following initial 30 minutes of reperfusion the levels increased further to four-fold the preischemic levels. The levels of GSSG also increased (100%) during the initial 30 minutes of reperfusion. The extracellular GSH levels remained elevated for 1 hour of reperfusion, but the GSSG levels returned to preischemic levels. The results indicate that intermittent hypoxia or anoxia in muscle tissue through hypoperfusion or ischemia decreases intracellular GSH stores by leakage, reducing the intracellular antioxidative capacity and increasing the risk for oxidative reperfusion injury upon final normalization of tissue blood supply.

Animals↗
PubMed1992

The stability of 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid in human urine.

5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid are important intermediate metabolites in the formation of cutaneous melanin pigment. Since they both are serious candidates as markers of melanoma progression, their stability in urine has been investigated during storage at various conditions. The results show that storage at -20 degrees C is necessary. Both compounds are nonstable at room temperature, particularly if the urine was not acidified to pH 4-5. Reference levels were obtained from analysis of urine from 31 men and 40 women. The mean (SD) excretion of 5-S-cysteinyldopa was 32 (12.5) mumol/mol creatinine (women). Corresponding figures for 6H5MI2C were 23 (10.3) and 24 (8.1) mumol/mol creatinine for men and women respectively.

Adult↗
PubMed1989

Automated high-performance liquid chromatographic determination of 5-S-cysteinyl-3,4-dihydroxyphenylalanine in urine.

An automated high-performance liquid chromatographic (HPLC) method has been developed for measurement of 5-S-cysteinyl-DOPA in urine (DOPA = 3,4-dihydroxyphenylalanine). The urinary sample was injected into an HPLC boronate column. With a mobile phase of 0.1 M phosphate buffer containing 0.2 mM disodium ethylenediaminetetraacetate (Na2EDTA) (pH 6.0) mixed with methanol (9:1), 5-S-cysteinyl-DOPA was adsorbed while most other compounds were washed away. By column switching, the column flow was reversed and 5-S-cysteinyl-DOPA was desorbed by a mobile phase of 0.1 M formic acid and 0.2 mM Na2EDTA at pH 3.0 and chromatographed on a reversed-phase column. The precision, as estimated from repeated analysis of an urinary sample and from duplicate analysis of a number of samples, ranged from 1.4 to 5.2% (coefficient of variation), and the analytical recovery was 93 +/- 4.1%. The method is suitable for use in the clinical laboratory.

Catecholamines↗
PubMedDate not supplied

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.

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.

Chromatography, High Pressure Liquid↗