Duality of Tocopherol Isoforms and Novel Associations with Vitamins Involved in One-Carbon Metabolism: Results from an Elderly Sample of the LifeLines Cohort Study.

Whether the affinity of serum vitamin E with total lipids hampers the appropriate assessment of its association with age-related risk factors has not been investigated in epidemiological studies. We aimed to compare linear regression-derived coefficients of the association of non-indexed and total lipids-indexed vitamin E isoforms with clinical and laboratory characteristics pertaining to the lipid, metabolic syndrome, and one-carbon metabolism biological domains. We studied 1429 elderly subjects (non-vitamin supplement users, 60-75 years old, with low and high socioeconomic status) from the population-based LifeLines Cohort and Biobank Study. We found that the associations of tocopherol isoforms with lipids were inverted in total lipids-indexed analyses, which may be indicative of overcorrection. Irrespective of the methods of standardization, we consistently found positive associations of α-tocopherol with vitamins of the one-carbon metabolism pathway and inverse associations with characteristics related to glucose metabolism. The associations of γ-tocopherol were often opposite to those of α-tocopherol. These data suggest that tocopherol isoforms and one-carbon metabolism are related, with beneficial and adverse associations for α-tocopherol and γ-tocopherol, respectively. Whether tocopherol isoforms, or their interplay, truly affect the one-carbon metabolism pathway remains to be further studied.

Measurement of plasma vitamin K1 (phylloquinone) and K2 (menaquinones-4 and -7) using HPLC-tandem mass spectrometry.

BACKGROUND: Given the growing interest in the health benefits of vitamin K, there is great need for development of new high-throughput methods for quantitative determination of vitamin K in plasma. We describe a simple and rapid method for measurement of plasma vitamin K1 (phylloquinone [PK]) and K2 (menaquinones [MK]-4 and -7). Furthermore, we investigated the association of fasting plasma vitamin K with functional vitamin K insufficiency in renal transplant recipients (RTR). METHODS: We used HPLC-tandem mass spectrometry with atmospheric pressure chemical ionization for measurement of plasma PK, MK-4, and MK-7. Solid-phase extraction was used for sample clean-up. Mass spectrometric detection was performed in multiple reaction monitoring mode. Functional vitamin K insufficiency was defined as plasma desphospho-uncarboxylated matrix Gla protein (dp-ucMGP) >500 pmol/L. RESULTS: Lower limits of quantitation were 0.14 nmol/L for PK and MK-4 and 4.40 nmol/L for MK-7. Linearity up to 15 nmol/L was excellent. Mean recoveries were >92%. Fasting plasma PK concentration was associated with recent PK intake (ρ=0.41, p=0.002) and with plasma MK-4 (ρ=0.49, p<0.001). Plasma PK (ρ=0.38, p=0.003) and MK-4 (ρ=0.46, p<0.001) were strongly correlated with plasma triglyceride concentrations. Furthermore, we found that MK-4-triglyceride ratio, but not PK-triglyceride ratio, was significantly associated with functional vitamin K insufficiency (OR 0.22 [0.07-0.70], p=0.01) in RTR. CONCLUSIONS: The developed rapid and easy-to-use LC-MS/MS method for quantitative determination of PK, MK-4, and MK-7 in human plasma may be a good alternative for the labor-intensive and time-consuming LC-MS/MS methods and enables a higher sample throughput.

Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma.

BACKGROUND: Plasma free metanephrines have proved a highly sensitive biochemical test for the diagnosis of pheochromocytoma. We have developed and validated a simple, LC-MS/MS method to determine plasma metanephrines and compared the diagnostic efficacy of the method with an enzyme immunoassay procedure in 151 patients, 38 with histologically confirmed pheochromocytoma. METHODS: Off-line solid phase extraction in a 96-well plate format was used to isolate metanephrines from 100-microL of plasma, followed by rapid separation with hydrophilic interaction chromatography. Mass spectrometry detection was performed in multiple-reaction monitoring mode using a tandem quadrupole mass spectrometer with positive electrospray ionization. RESULTS: Detection limits were <0.1nmol/l with method linearity up to 23.0nmol/L for normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT). Method comparison with an automated LC-MS/MS yielded Deming regression slopes of r=0.94 for NMN, r=0.98 for MN and r=0.94 for 3-MT. Method comparison with enzyme immunoassay revealed regression slope of r=1.28 (NMN) and 1.25 (MN) with values approximately 25% lower than LC-MS/MS. Plasma metanephrines by LC-MS/MS identified all 38 patients with phaeochromocytoma compared with 36 cases by immunoassay. CONCLUSIONS: Plasma metanephrines measured by LC-MS/MS are a reliable and sensitive test for the biochemical detection of pheochromocytoma.

Plasma free metanephrine measurement using automated online solid-phase extraction HPLC tandem mass spectrometry.

BACKGROUND: Quantification of plasma free metanephrine (MN) and normetanephrine (NMN) is considered to be the most accurate test for the clinical chemical diagnosis of pheochromocytoma and follow-up of pheochromocytoma patients. Current methods involve laborious, time-consuming, offline sample preparation, coupled with relatively nonspecific detection. Our aim was to develop a rapid, sensitive, and highly selective automated method for plasma free MNs in the nanomole per liter range. METHODS: We used online solid-phase extraction coupled with HPLC-tandem mass spectrometric detection (XLC-MS/MS). Fifty microliters plasma equivalent was prepurified by automated online solid-phase extraction, using weak cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogs was achieved by hydrophilic interaction chromatography. Mass spectrometric detection was performed in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in positive electrospray ionization mode. RESULTS: Total run-time including sample cleanup was 8 min. Intra- and interassay analytical variation (CV) varied from 2.0% to 4.7% and 1.6% to 13.5%, respectively, whereas biological intra- and interday variation ranged from 9.4% to 45.0% and 8.4% to 23.2%. Linearity in the 0 to 20 nmol/L calibration range was excellent (R(2) > 0.99). For all compounds, recoveries ranged from 74.5% to 99.6%, and detection limits were <0.10 nmol/L. Reference intervals for 120 healthy adults were 0.07 to 0.33 nmol/L (MN), 0.23 to 1.07 nmol/L (NMN), and <0.17 nmol/L (3-methoxytyramine). CONCLUSIONS: This automated high-throughput XLC-MS/MS method for the measurement of plasma free MNs is precise and linear, with short analysis time and low variable costs. The method is attractive for routine diagnosis of pheochromocytoma because of its high analytical sensitivity, the analytical power of MS/MS, and the high diagnostic accuracy of free MNs.

Angiotensin administration stimulates renal 11 beta-hydroxysteroid dehydrogenase activity in healthy men.

BACKGROUND: We examined whether acute administration of angiotensin modulates the activity of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD), the intracellular enzyme catalyzing the interconversion between the hormonally active cortisol and inactive cortisone. METHODS: Twenty-one male healthy subjects were examined after 1 week of a low- and high-salt diet (50 and 200 mmol/day, respectively). Separate infusions of angiotensin I (Ang I) and II (Ang II) were administered, both at rates of 4 and 8 ng/kg/min. The ratios of tetrahydrocortisol + allotetrahydrocortisol/tetrahydrocortisone (THF + allo-THF/THE) and of free cortisol/free cortisone (UFF/UFE) in urine were measured as indices of overall 11 beta HSD set point and activity of renal 11 beta HSD type 2, respectively. Glomerular filtration rate (GFR) was measured by constant infusion of (125)I-iothalamate. RESULTS: Ang I and Ang II infusion dose-dependently increased mean arterial blood pressure (MAP) and plasma aldosterone, and decreased plasma renin activity (PRA) and GFR at both diets. Ang I and Ang II infusion resulted in a dose-dependent decrease in the excretion of UFF, UFE, and of the UFF/UFE ratio at both diets, without changing the urinary (THF + allo-THF)/THE ratio. Salt restriction did not affect these 11 beta HSD variables, but was accompanied by a decrease in UFF and UFE excretion. CONCLUSION: This study suggests that acute angiotensin administration stimulates the activity of 11 beta HSD type 2 in human kidney. Angiotensin might therefore exert a dual effect on the mineralocorticoid receptor (i.e., an indirect agonistic effect by increasing aldosterone availability and a direct or indirect antagonistic effect by stimulation of renal 11 beta HSD type 2 activity).