Gry Kvalheim - Academia.edu (original) (raw)

Papers by Gry Kvalheim

Research paper thumbnail of Cohort profile: Biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer recurrence and survival – the FOCUS Consortium

BMJ Open

PurposeThe overarching goal of the FOCUS (biomarkers related to folate-dependent one-carbon metab... more PurposeThe overarching goal of the FOCUS (biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer (CRC) recurrence and survival) Consortium is to unravel the effect of folate and folate-mediated one-carbon metabolism (FOCM) biomarkers on CRC prognosis to provide clinically relevant advice on folate intake to cancer patients and define future tertiary prevention strategies.ParticipantsThe FOCUS Consortium is an international, prospective cohort of 2401 women and men above 18 years of age who were diagnosed with a primary invasive non-metastatic (stages I–III) CRC. The consortium comprises patients from Austria, two sites from the Netherlands, Germany and two sites from the USA. Patients are recruited after CRC diagnosis and followed at 6 and 12 months after enrolment. At each time point, sociodemographic data, data on health behaviour and clinical data are collected, blood samples are drawn.Findings to dateAn increased risk of cancer recurrences was observed...

Research paper thumbnail of Homocysteine and other thiols in plasma and urine: automated determination and sample stability

Clinical Chemistry, 1993

We have developed a modified version of our fully automated column-switching HPLC method for dete... more We have developed a modified version of our fully automated column-switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid-precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid-precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV < 5%). The sampl...

Research paper thumbnail of Plasma B vitamins and LINE-1 DNA methylation in leukocytes of patients with a history of colorectal adenomas

Molecular Nutrition & Food Research, 2012

Scope: Low concentrations of folate, other B vitamins, and methionine are associated with colorec... more Scope: Low concentrations of folate, other B vitamins, and methionine are associated with colorectal cancer risk, possibly by changing DNA methylation patterns. Here, we examine whether plasma concentrations of B vitamins and methionine are associated with methylation of long interspersed nuclear element-1 (LINE-1) among those at high risk of colorectal cancer, i.e. patients with at least one histologically confirmed colorectal adenoma (CRA) in their life. Methods and results: We used LINE-1 bisulfite pyrosequencing to measure global DNA methylation levels in leukocytes of 281 CRA patients. Multivariable linear regression was used to assess associations between plasma B vitamin concentrations and LINE-1 methylation levels. Plasma folate was inversely associated with LINE-1 methylation in CRA patients, while plasma methionine was positively associated with LINE-1 methylation. Conclusion: This study does not provide evidence that in CRA patients, plasma folate concentrations are positively related to LINE-1 methylation in leukocytes but does suggest a direct association between plasma methionine and LINE-1 methylation in leukocytes.

Research paper thumbnail of Higher vitamin B6 status is associated with improved survival among patients with stage I-III colorectal cancer

The American Journal of Clinical Nutrition

Background Folate-mediated one-carbon metabolism requires several nutrients, including vitamin B6... more Background Folate-mediated one-carbon metabolism requires several nutrients, including vitamin B6. Circulating levels of biomarkers indicating high vitamin B6 status are associated with a reduced risk of colorectal cancer (CRC). However, little is known about the effect of B6 status in relation to clinical outcomes in CRC patients. Objectives We investigated survival outcomes in relation to vitamin B6 status in prospectively followed CRC patients. Methods A total of 2031 patients with stage I-III CRC participated in six prospective patient cohorts in the international FOCUS Consortium. Preoperative blood samples were used to measure vitamin B6 status by the direct marker pyridoxal 5′-phosphate, PLP, as well as functional marker the HK-ratio (3′-hydroxykynurenine: [kynurenic acid + xanthurenic acid + 3′-hydroxy anthranilic acid + anthranilic acid]). Using Cox proportional hazards regression, we examined associations of vitamin B6 status with overall survival (OS), disease-free surviv...

Research paper thumbnail of High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS

Analytical and Bioanalytical Chemistry, Dec 13, 2012

High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-car... more High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS Øivind Midttun, Gry Kvalheim & Per Magne Ueland 1 2 3 Your article is protected by copyright and all rights are held exclusively by Springer-Verlag Berlin Heidelberg. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author's version for posting to your own website or your institution's repository. You may further deposit the accepted author's version on a funder's repository at a funder's request, provided it is not made publicly available until 12 months after publication.

Research paper thumbnail of Automated Assay for the Determination of Methylmalonic Acid, Total Homocysteine, and Related Amino Acids in Human Serum or Plasma

lonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and dis... more lonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and distin-guishing between cobalamin and folate deficiencies. We developed and validated an isotope-dilution gas chro-matography–mass spectrometry (GC-MS) method with automated sample workup for the determination of MMA, tHcy, and the related amino acids Met, total cysteine (tCys), Ser, and Gly in serum or plasma. Methods: Serum or plasma samples (100 L) were treated with a reductant (dithioerythritol), deprotein-ized with ethanol, and derivatized and extracted in a single step by the addition of methylchloroformate and toluene. All liquid handling was performed in 96-well (1 mL) microtiter plates by a robotic workstation. The N(S)-methoxycarbonyl ethyl ester derivatives were ana-

Research paper thumbnail of Determination of Choline, Betaine, and Dimethylglycine in Plasma by a High-Throughput Method Based on Normal-Phase Chromatography–Tandem Mass Spectrometry

choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the syn... more choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate sta-tus. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases. Methods: Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that con-tained d9-choline and d9-betaine as internal standards. We used a normal-phase silica column for the separa-tion of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectros-

Research paper thumbnail of Nutrition Determination of Choline , Betaine , and Dimethylglycine in Plasma by a High-Throughput Method Based on Normal-Phase Chromatography – Tandem Mass Spectrometry

Background: The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) res... more Background: The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate sta­ tus. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases. Methods: Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that con­ tained d9-choline and d9-betaine as internal standards. We used a normal-phase silica column for the separa­ tion of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectros­ copy in the multiple-reaction monitoring mode, using the molecular transitions m/z 104360 (choline), m/z 113369 (d9-choline), m/z 118359 (betaine)...

Research paper thumbnail of Capillary zone electrophoresis with laser-induced fluorescence detection for analysis of methylmalonic acid and other short-chain dicarboxylic acids derivatized with 1-pyrenyldiazomethane J &

Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiaxomethan... more Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiaxomethane (PDAM) in aqueous matrices and form stable, highly fluorescent 1-pyrenylmethyl monoesters [Schneede and Ueland, Anal. Chem., 64 (1992) 315-3191. We investigated the migration behaviour of these derivatives in capillary zone electrophoresis in fused-silica capillaries. The 1-pyrenyldiaxomethane derivatives were detected with a laserinduced fluorescence detector that was connected to a helium-cadmium laser, delivering light at a wavelength of 325 nm, which exactly matched an excitation maximum of the 1-pyrenylmethyl monoesters. These esters have one free carboxylic acid group, which carries negative charge at pH >4.0. The electrophoretic mobility varied according to pH, and the pH effect was most pronounced at values close to the pK, value (5.2-5.8) of the esters. The effects of the composition of the running buffer (pH, organic modifier concentration, ionic strength) and some operati...

Research paper thumbnail of Circulating Folate and Folic Acid Concentrations: Associations With Colorectal Cancer Recurrence and Survival

JNCI Cancer Spectrum

Background Folates, including folic acid, may play a dual role in colorectal cancer development. ... more Background Folates, including folic acid, may play a dual role in colorectal cancer development. Folate is suggested to be protective in early carcinogenesis but could accelerate growth of premalignant lesions or micrometastases. Whether circulating concentrations of folate and folic acid, measured around time of diagnosis, are associated with recurrence and survival in colorectal cancer patients is largely unknown. Methods Circulating concentrations of folate, folic acid, and folate catabolites p-aminobenzoylglutamate and p-acetamidobenzoylglutamate were measured by liquid chromatography-tandem mass spectrometry at diagnosis in 2024 stage I-III colorectal cancer patients from European and US patient cohort studies. Multivariable-adjusted Cox proportional hazard models were used to assess associations between folate, folic acid, and folate catabolites concentrations with recurrence, overall survival, and disease-free survival. Results No statistically significant associations were o...

Research paper thumbnail of Combined Measurement of 6 Fat-Soluble Vitamins and 26 Water-Soluble Functional Vitamin Markers and Amino Acids in 50 μL of Serum or Plasma by High-Throughput Mass Spectrometry

Analytical Chemistry, 2016

Targeted metabolic profiling characterized by complementary platforms, multiplexing and low volum... more Targeted metabolic profiling characterized by complementary platforms, multiplexing and low volume consumption are increasingly used for studies using biobank material. Using liquid-liquid extraction, we developed a sample workup suitable for quantification of 6 fat- and 26 water-soluble biomarkers. 50 μL of serum/plasma was mixed with dithioerythritol, ethanol, and isooctane/chloroform. The organic layer was used for analysis of the fat-soluble vitamins all-trans retinol (A), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, α-tocopherol (E), γ-tocopherol (E), and phylloquinone (K1) by LC-MS/MS. The remaining aqueous fraction was mixed with ethanol, water, pyridine, and methylchloroformate (in toluene) to derivatize the water-soluble biomarkers. The resulting toluene layer was used for GC-MS/MS analysis of alanine, α-ketoglutarate, asparagine, aspartic acid, cystathionine, total cysteine, glutamic acid, glutamine, glycine, histidine, total homocysteine, isoleucine, kynurenine, leucine, lysine, methionine, methylmalonic acid, ornithine, phenylalanine, proline, sarcosine, serine, threonine, tryptophan, tyrosine, and valine. Isotope-labeled internal standards were used for all analytes. Chromatographic run times for the LC-MS/MS and GC-MS/MS were 4.5 and 11 min, respectively. The limits of detection (LOD) for the low-concentration analytes (25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and phylloquinone) were 25, 17, and 0.33 nM, respectively, while all other analytes demonstrated sensitivity significantly lower than endogenous concentrations. Recoveries ranged from 85.5-109.9% and within- and between-day coefficients of variance (CVs) were 0.7-9.4% and 1.1-17.5%, respectively. This low-volume, high-throughput multianalyte assay is currently in use in our laboratory for quantification of 32 serum/plasma biomarkers in epidemiological studies.

Research paper thumbnail of Determination of Warfarin in Human Plasma by High Performance Liquid Chromatography and Photodiode Array Detector

Therapeutic Drug Monitoring, 1985

Research paper thumbnail of Capillary zone electrophoresis with laser-induced fluorescence detection for analysis of methylmalonic acid and other short-chain dicarboxylic acids derivatized with 1-pyrenyldiazomethane

Journal of Chromatography A, 1994

Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiaxomethan... more Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiaxomethane (PDAM) in aqueous matrices and form stable, highly fluorescent 1-pyrenylmethyl monoesters [Schneede and Ueland, Anal. Chem., 64 (1992) 315-3191. We investigated the migration behaviour of these derivatives in capillary zone electrophoresis in fused-silica capillaries. The 1-pyrenyldiaxomethane derivatives were detected with a laserinduced fluorescence detector that was connected to a helium-cadmium laser, delivering light at a wavelength of 325 nm, which exactly matched an excitation maximum of the 1-pyrenylmethyl monoesters. These esters have one free carboxylic acid group, which carries negative charge at pH >4.0. The electrophoretic mobility varied according to pH, and the pH effect was most pronounced at values close to the pK, value (5.2-5.8) of the esters. The effects of the composition of the running buffer (pH, organic modifier concentration, ionic strength) and some operational parameters (voltage, temperature and capillary length) were tested, and were largely found to comply with validated theoretical models for capillary zone electrophoresis. In an optimal system, as judged by high number of theoretical plates, high resolution and short run times, the MMA derivative was separated from related 1-pyrenylmethyl monoesters. The laser-induced fluorescence detection afforded a limit of detection of about 40 nmol/l (signal-to-noise ratio of 5) for the MMA derivative. Under optimal conditions, we were able to detect ~1 pmol/l endogenous MMA in human serum. . Among the dicarboxylic acids, methylmalonic acid * Corresponding author.

Research paper thumbnail of Determination of Choline, Betaine, and Dimethylglycine in Plasma by a High-Throughput Method Based on Normal-Phase Chromatography-Tandem Mass Spectrometry

Clinical Chemistry, 2003

The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a ... more The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate status. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases. Methods: Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that contained d 9 -choline and d 9 -betaine as internal standards. We used a normal-phase silica column for the separation of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectroscopy in the multiple-reaction monitoring mode, using the molecular transitions m/z 104360 (choline), m/z 113369 (d 9 -choline), m/z 118359 (betaine), m/z 127368 (d 9 -betaine), and m/z 104358 (DMG). Results: For all three metabolites, the assay was linear in the range 0.4 -400 mol/L, and the lower limit of the detection (signal-to-noise ratio ‫؍‬ 5) was <0.3 mol/L. The within-and between-day imprecision (CVs) was 2.1-7.2% and 3.5-8.8%, respectively. The analytical recovery was 87-105%. The fasting plasma concentrations (median, 25th-75th percentiles) were 8.0 (7.0 -9.3) mol/L for choline, 31.7 (27.0 -41.1) mol/L for betaine, and 1.66 (1.30 -2.02) mol/L for DMG in 60 healthy blood donors. In individuals who had eaten a light breakfast, plasma concentrations of all three metabolites were significantly (25-30%) higher than in fasting individuals. Conclusion: This is the first method for the combined measurement of choline, betaine, and DMG in human plasma or serum. The assay is characterized by simple sample preparation, no derivatization, high throughput, imprecision (CV) <10%, detection limits below the values seen in volunteers, and the high specificity provided by tandem mass spectroscopy.

Research paper thumbnail of Automated Assay for the Determination of Methylmalonic Acid, Total Homocysteine, and Related Amino Acids in Human Serum or Plasma by Means of Methylchloroformate Derivatization and Gas Chromatography-Mass Spectrometry

Clinical Chemistry, 2005

The combined measurement of methylmalonic acid (MMA) and total homocysteine (tHcy) in serum or pl... more The combined measurement of methylmalonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and distinguishing between cobalamin and folate deficiencies. We developed and validated an isotope-dilution gas chromatography-mass spectrometry (GC-MS) method with automated sample workup for the determination of MMA, tHcy, and the related amino acids Met, total cysteine (tCys), Ser, and Gly in serum or plasma. Methods: Serum or plasma samples (100 L) were treated with a reductant (dithioerythritol), deproteinized with ethanol, and derivatized and extracted in a single step by the addition of methylchloroformate and toluene. All liquid handling was performed in 96-well (1 mL) microtiter plates by a robotic workstation. The

Research paper thumbnail of High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS

Analytical and Bioanalytical Chemistry, 2013

Risk of chronic diseases, like cardiovascular disease and cancer, has been associated with biomar... more Risk of chronic diseases, like cardiovascular disease and cancer, has been associated with biomarkers related to one-carbon metabolism, which comprises a metabolic network of cross-talking pathways. To address this complexity in epidemiological studies, we have established an isotope dilution HPLC-MS/MS method for quantification of 12 biomarkers and metabolites. All sample handling is performed by a robotic workstation. The assay uses 45 μL of plasma, and sample treatment consists of protein precipitation by trichloroacetic acid. The analytes were separated on a Fortis Phenyl column using an isocratic mobile phase that contained water, methanol and acetic acid. Methionine, methionine sulfoxide, choline, betaine, dimethylglycine, arginine, asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine, creatinine, cystathionine and trimethyllysine all showed limits of detection well below the 5th percentile of plasma distributions in healthy humans, coefficients of variation were in the range 2.2-12.3 %, and recoveries were 80-131 %. Simple sample processing, low-volume consumption, multiplexing and high capacity/short run time of this method make it suitable for large-scale metabolic profiling of precious biobank samples.

Research paper thumbnail of Plasma B vitamins and LINE-1 DNA methylation in leukocytes of patients with a history of colorectal adenomas

Molecular Nutrition & Food Research, 2013

Low concentrations of folate, other B vitamins, and methionine are associated with colorectal can... more Low concentrations of folate, other B vitamins, and methionine are associated with colorectal cancer risk, possibly by changing DNA methylation patterns. Here, we examine whether plasma concentrations of B vitamins and methionine are associated with methylation of long interspersed nuclear element-1 (LINE-1) among those at high risk of colorectal cancer, i.e. patients with at least one histologically confirmed colorectal adenoma (CRA) in their life. Methods and results: We used LINE-1 bisulfite pyrosequencing to measure global DNA methylation levels in leukocytes of 281 CRA patients. Multivariable linear regression was used to assess associations between plasma B vitamin concentrations and LINE-1 methylation levels. Plasma folate was inversely associated with LINE-1 methylation in CRA patients, while plasma methionine was positively associated with LINE-1 methylation. Conclusion: This study does not provide evidence that in CRA patients, plasma folate concentrations are positively related to LINE-1 methylation in leukocytes but does suggest a direct association between plasma methionine and LINE-1 methylation in leukocytes.

Research paper thumbnail of Cohort profile: Biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer recurrence and survival – the FOCUS Consortium

BMJ Open

PurposeThe overarching goal of the FOCUS (biomarkers related to folate-dependent one-carbon metab... more PurposeThe overarching goal of the FOCUS (biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer (CRC) recurrence and survival) Consortium is to unravel the effect of folate and folate-mediated one-carbon metabolism (FOCM) biomarkers on CRC prognosis to provide clinically relevant advice on folate intake to cancer patients and define future tertiary prevention strategies.ParticipantsThe FOCUS Consortium is an international, prospective cohort of 2401 women and men above 18 years of age who were diagnosed with a primary invasive non-metastatic (stages I–III) CRC. The consortium comprises patients from Austria, two sites from the Netherlands, Germany and two sites from the USA. Patients are recruited after CRC diagnosis and followed at 6 and 12 months after enrolment. At each time point, sociodemographic data, data on health behaviour and clinical data are collected, blood samples are drawn.Findings to dateAn increased risk of cancer recurrences was observed...

Research paper thumbnail of Homocysteine and other thiols in plasma and urine: automated determination and sample stability

Clinical Chemistry, 1993

We have developed a modified version of our fully automated column-switching HPLC method for dete... more We have developed a modified version of our fully automated column-switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid-precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid-precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV < 5%). The sampl...

Research paper thumbnail of Plasma B vitamins and LINE-1 DNA methylation in leukocytes of patients with a history of colorectal adenomas

Molecular Nutrition & Food Research, 2012

Scope: Low concentrations of folate, other B vitamins, and methionine are associated with colorec... more Scope: Low concentrations of folate, other B vitamins, and methionine are associated with colorectal cancer risk, possibly by changing DNA methylation patterns. Here, we examine whether plasma concentrations of B vitamins and methionine are associated with methylation of long interspersed nuclear element-1 (LINE-1) among those at high risk of colorectal cancer, i.e. patients with at least one histologically confirmed colorectal adenoma (CRA) in their life. Methods and results: We used LINE-1 bisulfite pyrosequencing to measure global DNA methylation levels in leukocytes of 281 CRA patients. Multivariable linear regression was used to assess associations between plasma B vitamin concentrations and LINE-1 methylation levels. Plasma folate was inversely associated with LINE-1 methylation in CRA patients, while plasma methionine was positively associated with LINE-1 methylation. Conclusion: This study does not provide evidence that in CRA patients, plasma folate concentrations are positively related to LINE-1 methylation in leukocytes but does suggest a direct association between plasma methionine and LINE-1 methylation in leukocytes.

Research paper thumbnail of Higher vitamin B6 status is associated with improved survival among patients with stage I-III colorectal cancer

The American Journal of Clinical Nutrition

Background Folate-mediated one-carbon metabolism requires several nutrients, including vitamin B6... more Background Folate-mediated one-carbon metabolism requires several nutrients, including vitamin B6. Circulating levels of biomarkers indicating high vitamin B6 status are associated with a reduced risk of colorectal cancer (CRC). However, little is known about the effect of B6 status in relation to clinical outcomes in CRC patients. Objectives We investigated survival outcomes in relation to vitamin B6 status in prospectively followed CRC patients. Methods A total of 2031 patients with stage I-III CRC participated in six prospective patient cohorts in the international FOCUS Consortium. Preoperative blood samples were used to measure vitamin B6 status by the direct marker pyridoxal 5′-phosphate, PLP, as well as functional marker the HK-ratio (3′-hydroxykynurenine: [kynurenic acid + xanthurenic acid + 3′-hydroxy anthranilic acid + anthranilic acid]). Using Cox proportional hazards regression, we examined associations of vitamin B6 status with overall survival (OS), disease-free surviv...

Research paper thumbnail of High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS

Analytical and Bioanalytical Chemistry, Dec 13, 2012

High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-car... more High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS Øivind Midttun, Gry Kvalheim & Per Magne Ueland 1 2 3 Your article is protected by copyright and all rights are held exclusively by Springer-Verlag Berlin Heidelberg. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author's version for posting to your own website or your institution's repository. You may further deposit the accepted author's version on a funder's repository at a funder's request, provided it is not made publicly available until 12 months after publication.

Research paper thumbnail of Automated Assay for the Determination of Methylmalonic Acid, Total Homocysteine, and Related Amino Acids in Human Serum or Plasma

lonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and dis... more lonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and distin-guishing between cobalamin and folate deficiencies. We developed and validated an isotope-dilution gas chro-matography–mass spectrometry (GC-MS) method with automated sample workup for the determination of MMA, tHcy, and the related amino acids Met, total cysteine (tCys), Ser, and Gly in serum or plasma. Methods: Serum or plasma samples (100 L) were treated with a reductant (dithioerythritol), deprotein-ized with ethanol, and derivatized and extracted in a single step by the addition of methylchloroformate and toluene. All liquid handling was performed in 96-well (1 mL) microtiter plates by a robotic workstation. The N(S)-methoxycarbonyl ethyl ester derivatives were ana-

Research paper thumbnail of Determination of Choline, Betaine, and Dimethylglycine in Plasma by a High-Throughput Method Based on Normal-Phase Chromatography–Tandem Mass Spectrometry

choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the syn... more choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate sta-tus. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases. Methods: Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that con-tained d9-choline and d9-betaine as internal standards. We used a normal-phase silica column for the separa-tion of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectros-

Research paper thumbnail of Nutrition Determination of Choline , Betaine , and Dimethylglycine in Plasma by a High-Throughput Method Based on Normal-Phase Chromatography – Tandem Mass Spectrometry

Background: The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) res... more Background: The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate sta­ tus. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases. Methods: Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that con­ tained d9-choline and d9-betaine as internal standards. We used a normal-phase silica column for the separa­ tion of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectros­ copy in the multiple-reaction monitoring mode, using the molecular transitions m/z 104360 (choline), m/z 113369 (d9-choline), m/z 118359 (betaine)...

Research paper thumbnail of Capillary zone electrophoresis with laser-induced fluorescence detection for analysis of methylmalonic acid and other short-chain dicarboxylic acids derivatized with 1-pyrenyldiazomethane J &

Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiaxomethan... more Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiaxomethane (PDAM) in aqueous matrices and form stable, highly fluorescent 1-pyrenylmethyl monoesters [Schneede and Ueland, Anal. Chem., 64 (1992) 315-3191. We investigated the migration behaviour of these derivatives in capillary zone electrophoresis in fused-silica capillaries. The 1-pyrenyldiaxomethane derivatives were detected with a laserinduced fluorescence detector that was connected to a helium-cadmium laser, delivering light at a wavelength of 325 nm, which exactly matched an excitation maximum of the 1-pyrenylmethyl monoesters. These esters have one free carboxylic acid group, which carries negative charge at pH >4.0. The electrophoretic mobility varied according to pH, and the pH effect was most pronounced at values close to the pK, value (5.2-5.8) of the esters. The effects of the composition of the running buffer (pH, organic modifier concentration, ionic strength) and some operati...

Research paper thumbnail of Circulating Folate and Folic Acid Concentrations: Associations With Colorectal Cancer Recurrence and Survival

JNCI Cancer Spectrum

Background Folates, including folic acid, may play a dual role in colorectal cancer development. ... more Background Folates, including folic acid, may play a dual role in colorectal cancer development. Folate is suggested to be protective in early carcinogenesis but could accelerate growth of premalignant lesions or micrometastases. Whether circulating concentrations of folate and folic acid, measured around time of diagnosis, are associated with recurrence and survival in colorectal cancer patients is largely unknown. Methods Circulating concentrations of folate, folic acid, and folate catabolites p-aminobenzoylglutamate and p-acetamidobenzoylglutamate were measured by liquid chromatography-tandem mass spectrometry at diagnosis in 2024 stage I-III colorectal cancer patients from European and US patient cohort studies. Multivariable-adjusted Cox proportional hazard models were used to assess associations between folate, folic acid, and folate catabolites concentrations with recurrence, overall survival, and disease-free survival. Results No statistically significant associations were o...

Research paper thumbnail of Combined Measurement of 6 Fat-Soluble Vitamins and 26 Water-Soluble Functional Vitamin Markers and Amino Acids in 50 μL of Serum or Plasma by High-Throughput Mass Spectrometry

Analytical Chemistry, 2016

Targeted metabolic profiling characterized by complementary platforms, multiplexing and low volum... more Targeted metabolic profiling characterized by complementary platforms, multiplexing and low volume consumption are increasingly used for studies using biobank material. Using liquid-liquid extraction, we developed a sample workup suitable for quantification of 6 fat- and 26 water-soluble biomarkers. 50 μL of serum/plasma was mixed with dithioerythritol, ethanol, and isooctane/chloroform. The organic layer was used for analysis of the fat-soluble vitamins all-trans retinol (A), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, α-tocopherol (E), γ-tocopherol (E), and phylloquinone (K1) by LC-MS/MS. The remaining aqueous fraction was mixed with ethanol, water, pyridine, and methylchloroformate (in toluene) to derivatize the water-soluble biomarkers. The resulting toluene layer was used for GC-MS/MS analysis of alanine, α-ketoglutarate, asparagine, aspartic acid, cystathionine, total cysteine, glutamic acid, glutamine, glycine, histidine, total homocysteine, isoleucine, kynurenine, leucine, lysine, methionine, methylmalonic acid, ornithine, phenylalanine, proline, sarcosine, serine, threonine, tryptophan, tyrosine, and valine. Isotope-labeled internal standards were used for all analytes. Chromatographic run times for the LC-MS/MS and GC-MS/MS were 4.5 and 11 min, respectively. The limits of detection (LOD) for the low-concentration analytes (25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and phylloquinone) were 25, 17, and 0.33 nM, respectively, while all other analytes demonstrated sensitivity significantly lower than endogenous concentrations. Recoveries ranged from 85.5-109.9% and within- and between-day coefficients of variance (CVs) were 0.7-9.4% and 1.1-17.5%, respectively. This low-volume, high-throughput multianalyte assay is currently in use in our laboratory for quantification of 32 serum/plasma biomarkers in epidemiological studies.

Research paper thumbnail of Determination of Warfarin in Human Plasma by High Performance Liquid Chromatography and Photodiode Array Detector

Therapeutic Drug Monitoring, 1985

Research paper thumbnail of Capillary zone electrophoresis with laser-induced fluorescence detection for analysis of methylmalonic acid and other short-chain dicarboxylic acids derivatized with 1-pyrenyldiazomethane

Journal of Chromatography A, 1994

Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiaxomethan... more Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiaxomethane (PDAM) in aqueous matrices and form stable, highly fluorescent 1-pyrenylmethyl monoesters [Schneede and Ueland, Anal. Chem., 64 (1992) 315-3191. We investigated the migration behaviour of these derivatives in capillary zone electrophoresis in fused-silica capillaries. The 1-pyrenyldiaxomethane derivatives were detected with a laserinduced fluorescence detector that was connected to a helium-cadmium laser, delivering light at a wavelength of 325 nm, which exactly matched an excitation maximum of the 1-pyrenylmethyl monoesters. These esters have one free carboxylic acid group, which carries negative charge at pH >4.0. The electrophoretic mobility varied according to pH, and the pH effect was most pronounced at values close to the pK, value (5.2-5.8) of the esters. The effects of the composition of the running buffer (pH, organic modifier concentration, ionic strength) and some operational parameters (voltage, temperature and capillary length) were tested, and were largely found to comply with validated theoretical models for capillary zone electrophoresis. In an optimal system, as judged by high number of theoretical plates, high resolution and short run times, the MMA derivative was separated from related 1-pyrenylmethyl monoesters. The laser-induced fluorescence detection afforded a limit of detection of about 40 nmol/l (signal-to-noise ratio of 5) for the MMA derivative. Under optimal conditions, we were able to detect ~1 pmol/l endogenous MMA in human serum. . Among the dicarboxylic acids, methylmalonic acid * Corresponding author.

Research paper thumbnail of Determination of Choline, Betaine, and Dimethylglycine in Plasma by a High-Throughput Method Based on Normal-Phase Chromatography-Tandem Mass Spectrometry

Clinical Chemistry, 2003

The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a ... more The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate status. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases. Methods: Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that contained d 9 -choline and d 9 -betaine as internal standards. We used a normal-phase silica column for the separation of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectroscopy in the multiple-reaction monitoring mode, using the molecular transitions m/z 104360 (choline), m/z 113369 (d 9 -choline), m/z 118359 (betaine), m/z 127368 (d 9 -betaine), and m/z 104358 (DMG). Results: For all three metabolites, the assay was linear in the range 0.4 -400 mol/L, and the lower limit of the detection (signal-to-noise ratio ‫؍‬ 5) was <0.3 mol/L. The within-and between-day imprecision (CVs) was 2.1-7.2% and 3.5-8.8%, respectively. The analytical recovery was 87-105%. The fasting plasma concentrations (median, 25th-75th percentiles) were 8.0 (7.0 -9.3) mol/L for choline, 31.7 (27.0 -41.1) mol/L for betaine, and 1.66 (1.30 -2.02) mol/L for DMG in 60 healthy blood donors. In individuals who had eaten a light breakfast, plasma concentrations of all three metabolites were significantly (25-30%) higher than in fasting individuals. Conclusion: This is the first method for the combined measurement of choline, betaine, and DMG in human plasma or serum. The assay is characterized by simple sample preparation, no derivatization, high throughput, imprecision (CV) <10%, detection limits below the values seen in volunteers, and the high specificity provided by tandem mass spectroscopy.

Research paper thumbnail of Automated Assay for the Determination of Methylmalonic Acid, Total Homocysteine, and Related Amino Acids in Human Serum or Plasma by Means of Methylchloroformate Derivatization and Gas Chromatography-Mass Spectrometry

Clinical Chemistry, 2005

The combined measurement of methylmalonic acid (MMA) and total homocysteine (tHcy) in serum or pl... more The combined measurement of methylmalonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and distinguishing between cobalamin and folate deficiencies. We developed and validated an isotope-dilution gas chromatography-mass spectrometry (GC-MS) method with automated sample workup for the determination of MMA, tHcy, and the related amino acids Met, total cysteine (tCys), Ser, and Gly in serum or plasma. Methods: Serum or plasma samples (100 L) were treated with a reductant (dithioerythritol), deproteinized with ethanol, and derivatized and extracted in a single step by the addition of methylchloroformate and toluene. All liquid handling was performed in 96-well (1 mL) microtiter plates by a robotic workstation. The

Research paper thumbnail of High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS

Analytical and Bioanalytical Chemistry, 2013

Risk of chronic diseases, like cardiovascular disease and cancer, has been associated with biomar... more Risk of chronic diseases, like cardiovascular disease and cancer, has been associated with biomarkers related to one-carbon metabolism, which comprises a metabolic network of cross-talking pathways. To address this complexity in epidemiological studies, we have established an isotope dilution HPLC-MS/MS method for quantification of 12 biomarkers and metabolites. All sample handling is performed by a robotic workstation. The assay uses 45 μL of plasma, and sample treatment consists of protein precipitation by trichloroacetic acid. The analytes were separated on a Fortis Phenyl column using an isocratic mobile phase that contained water, methanol and acetic acid. Methionine, methionine sulfoxide, choline, betaine, dimethylglycine, arginine, asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine, creatinine, cystathionine and trimethyllysine all showed limits of detection well below the 5th percentile of plasma distributions in healthy humans, coefficients of variation were in the range 2.2-12.3 %, and recoveries were 80-131 %. Simple sample processing, low-volume consumption, multiplexing and high capacity/short run time of this method make it suitable for large-scale metabolic profiling of precious biobank samples.

Research paper thumbnail of Plasma B vitamins and LINE-1 DNA methylation in leukocytes of patients with a history of colorectal adenomas

Molecular Nutrition & Food Research, 2013

Low concentrations of folate, other B vitamins, and methionine are associated with colorectal can... more Low concentrations of folate, other B vitamins, and methionine are associated with colorectal cancer risk, possibly by changing DNA methylation patterns. Here, we examine whether plasma concentrations of B vitamins and methionine are associated with methylation of long interspersed nuclear element-1 (LINE-1) among those at high risk of colorectal cancer, i.e. patients with at least one histologically confirmed colorectal adenoma (CRA) in their life. Methods and results: We used LINE-1 bisulfite pyrosequencing to measure global DNA methylation levels in leukocytes of 281 CRA patients. Multivariable linear regression was used to assess associations between plasma B vitamin concentrations and LINE-1 methylation levels. Plasma folate was inversely associated with LINE-1 methylation in CRA patients, while plasma methionine was positively associated with LINE-1 methylation. Conclusion: This study does not provide evidence that in CRA patients, plasma folate concentrations are positively related to LINE-1 methylation in leukocytes but does suggest a direct association between plasma methionine and LINE-1 methylation in leukocytes.