Changes in intracellular folate metabolism during high-dose methotrexate and Leucovorin rescue therapy in children with acute lymphoblastic leukemia

Background Methotrexate (MTX) is an important anti-folate agent in pediatric acute lymphoblastic leukemia (ALL) treatment. Folinic acid rescue therapy (Leucovorin) is administered after MTX to reduce toxicity. Previous studies hypothesized that Leucovorin could ‘rescue’ both normal healthy cells and leukemic blasts from cell death. We assessed whether Leucovorin is able to restore red blood cell folate levels after MTX. Methods We prospectively determined erythrocyte folate levels (5-methyltetrahydrofolate (THF) and non-methyl THF) and serum folate levels in 67 children with ALL before start (T0) and after stop (T1) of HD-MTX and Leucovorin courses. Results Erythrocyte folate levels increased between T0 and T1 (mean ± SD: 416.7 ± 145.5 nmol/L and 641.2 ± 196.3 nmol/L respectively, pT genotype. Conclusion Intracellular folate levels accumulate after HD-MTX and Leucovorin therapy in children with ALL, suggesting that Leucovorin restores the intracellular folate pool. Future studies are necessary to assess concomitant lower uptake of MTX

Ensuring quality in 17OHP mass spectrometry measurement:an international study assessing isomeric steroid interference

Objectives: Interference from isomeric steroids is a potential cause of disparity between mass spectrometry-based 17-hydroxyprogesterone (17OHP) results. We aimed to assess the proficiency of mass spectrometry laboratories to report 17OHP in the presence of known isomeric steroids. Methods:A series of five samples were prepared using a previously demonstrated commutable approach. These samples included a control (spiked to 15.0 nmol/L 17OHP) and four challenge samples further enriched with equimolar concentrations of 17OHP isomers (11α-hydroxyprogesterone, 11β-hydroxyprogesterone, 16α-hydroxyprogesterone or 21-hydroxyprogesterone). These samples were distributed to 38 participating laboratories that reported serum 17OHP results using mass spectrometry in two external quality assurance programs. The result for each challenge sample was compared to the control sample submitted by each participant. Results: Twenty-six laboratories (68 % of distribution) across three continents returned results. Twenty-five laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS), and one used gas chromatography-tandem mass spectrometry to measure 17OHP. The all-method median of the control sample was 14.3 nmol/L, ranging from 12.4 to 17.6 nmol/L. One laboratory had results that approached the lower limit of tolerance (minus 17.7 % of the control sample), suggesting the isomeric steroid caused an irregular result. Conclusions: Most participating laboratories demonstrated their ability to reliably measure 17OHP in the presence of the four clinically relevant isomeric steroids. The performance of the 12 (32 %) laboratories that did not engage in this activity remains unclear. We recommend that all laboratories offering LC-MS/MS analysis of 17OHP in serum, plasma, or dried bloodspots determine that the isomeric steroids are appropriately separated.</p

Ensuring quality in 17OHP mass spectrometry measurement:an international study assessing isomeric steroid interference

Objectives: Interference from isomeric steroids is a potential cause of disparity between mass spectrometry-based 17-hydroxyprogesterone (17OHP) results. We aimed to assess the proficiency of mass spectrometry laboratories to report 17OHP in the presence of known isomeric steroids. Methods:A series of five samples were prepared using a previously demonstrated commutable approach. These samples included a control (spiked to 15.0 nmol/L 17OHP) and four challenge samples further enriched with equimolar concentrations of 17OHP isomers (11α-hydroxyprogesterone, 11β-hydroxyprogesterone, 16α-hydroxyprogesterone or 21-hydroxyprogesterone). These samples were distributed to 38 participating laboratories that reported serum 17OHP results using mass spectrometry in two external quality assurance programs. The result for each challenge sample was compared to the control sample submitted by each participant. Results: Twenty-six laboratories (68 % of distribution) across three continents returned results. Twenty-five laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS), and one used gas chromatography-tandem mass spectrometry to measure 17OHP. The all-method median of the control sample was 14.3 nmol/L, ranging from 12.4 to 17.6 nmol/L. One laboratory had results that approached the lower limit of tolerance (minus 17.7 % of the control sample), suggesting the isomeric steroid caused an irregular result. Conclusions: Most participating laboratories demonstrated their ability to reliably measure 17OHP in the presence of the four clinically relevant isomeric steroids. The performance of the 12 (32 %) laboratories that did not engage in this activity remains unclear. We recommend that all laboratories offering LC-MS/MS analysis of 17OHP in serum, plasma, or dried bloodspots determine that the isomeric steroids are appropriately separated.</p

Early life poly- and perfluoroalkyl substance levels and adiposity in the first 2 years of life

Importance: Poly- and perfluoroalkyl substances (PFASs) are nondegradable, man-made chemicals. They accumulate in humans with potential harmful effects, especially in susceptible periods of human development, such as the first months of life. We found that, in our cohort, exclusively breastfed (EBF) infants had 3 times higher PFAS plasma levels compared with exclusively formula-fed (EFF) infants at the age of 3 months. Thus, PFASs could potentially reduce the health benefits of breastfeeding. Objective: We investigated the associations between PFAS levels at the age of 3 months and accelerated gain in fat mass during the first 6 months of life, body composition at 2 years, and whether these associations differ between EBF and EFF infants. Setting: In 372 healthy term-born infants, we longitudinally assessed anthropometrics, body composition (by air-displacement plethysmography and dual-energy X-ray absorptiometry), and visceral and subcutaneous fat (by abdominal ultrasound) until the age of 2 years. Measures: The plasma levels of 5 individual PFASs were determined by liquid chromatography–electrospray ionization–tandem mass spectrometry at the age of 3 months. Main outcomes: We studied associations between PFAS levels and outcomes using multiple regression analyses. Results: Higher early life plasma perfluorooctanoic acid and total PFAS levels were associated with an accelerated gain in fat mass percentage [FM%; &gt;0.67 SD score (SDS)] during the first 6 months of life. Higher early life PFAS levels were associated with lower fat-free mass (FFM) SDS at the age of 2 years, but not with total FM% SDS at 2 years. Furthermore, we found opposite effects of PFAS levels (negative) and exclusive breastfeeding (positive) at the age of 3 months on FFM SDS at 2 years. Conclusion: Higher PFAS levels in early life are associated with accelerated gains in FM% during the first 6 months of life and with lower FFM SDS at the age of 2 years, which have been associated with an unfavorable body composition and metabolic profile later in life. Our findings warrant further research with longer follow-up times.</p

Association between methylation potential and nutrient metabolism throughout the reproductive cycle of sows

DNA methylation is an important epigenetic strategy for embryo development and survival. The one-carbon metabolism can be disturbed by inadequate provision of dietary methyl donors. Because of the continuous selection for larger litters, it is relevant to explore if highly prolific sows might encounter periods of methyl donor deficiency throughout their reproductive cycles. This study, therefore, assesses the fluctuation(s) in methylation potential (MP) and aims to link possible methyl donor deficiencies to nutrient metabolism. In total, 15 hybrid sows were followed from weaning of the previous reproductive cycle (d-5) to weaning of the present cycle. Blood samples were taken at d-5, 0, 21, 42, 63, 84 and d108 of gestation, the day of parturition (d115), two weeks of lactation (d129) and at weaning (d143). Blood plasma samples were analysed for S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), free methionine, free glycine, acetylcarnitine and 3-hydroxybutyrylcarnitine. Serum samples were analysed for urea and creatinine. Generally, MP (i.e. ratio SAM:SAH) increased throughout gestation (p = 0.009), but strongly fluctuated in the period around parturition and weaning. From d108 to parturition, absolute plasma levels of SAM (p < 0.001), SAH (p = 0.031) and methionine (p = 0.001) increased. The first two weeks of lactation were characterised by an increase in MP (p = 0.039) due to a remaining high value of SAM and a distinct decrease in SAH (p = 0.008). During the last two weeks of lactation, MP decreased (p = 0.038) due to a decrease in SAM (p < 0.001) and a stable value for SAH. The methylation reactions seem to continue after weaning, a period crucial for the follicular and embryonic development of the subsequent litter. This study thus demonstrates that the methylation status fluctuates substantially throughout a sow's reproductive cycle, and further research is needed to identify the factors affecting methylation status

New developments in the standardization of total prostate-specific antigen

Objective : Analytical evaluation of the calibration of three recently launched assays for the measurement of total prostate-specific antigen, i.e., IMx Total PSA (Abbott), Elecsys PSA (Roche), and IMMULITE 3rd Generation PSA (DPC). Design and methods : For accuracy assessment two reference materials were applied namely, Stanford 90:10 PSA Calibrator and Certified Reference Material 613 Prostate-Specific Antigen. Dilutions of these preparations were analyzed with all assays. In addition, clinical specimens from known prostate cancer or benign prostate hyperplasia patients and samples taken from an ongoing prostate cancer screening study were used for comparison. Results : Application of the Stanford Calibrator revealed results well within 10% of the calculated values for all assays. Regarding the CRM Calibrator only the IMx Total PSA proved to approach the line of identity. The IMMULITE results differed about 40% and the Elecsys about 18% from the calculated values. The comparison with clinical specimens showed statistically different results for the combination IMMULITE-IMx and for IMMULITE-Elecsys. The regression lines for both collections were: y(IMx) = 0.86x (IMMULITE) + 0.12 (n = 104, r = 0.970, S(y/x) = 0.883 μg/L) and y(Elecsys) = 0.98x(IMMULITE) + 0.38 (n = 97, r = 0.976, S(y/x) = 0.733 μg/L). In the lower measuring range (PSA < 5.0 μg/L) as measured with the screening samples (n = 43), these differences were less pronounced. Conclusion : In analytical sense a difference was found for both reference preparations in the assays studied. Clinically, despite improvements in methodology, results for total prostate-specific antigen are still not interchangeable. The possible consequences need to be elaborated

Global DNA (hydroxy)methylation is stable over time under several storage conditions and temperatures

Background: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation. Methods: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4°C, −20°C and −80°C for 0, 1 (endpoint blood 4°C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean ± SD) were quantified using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models. Results: global DNA methylation was stable over 18 months in blood at −20°C and −80°C and DNA at 4°C and −80°C. However, at 18 months DNA methylation from DNA stored at −20°C relatively decreased −6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze–thaw cycles. Conclusion: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of  small differences occuring during storage depend on the expected effect size and research question

Global DNA (hydroxy)methylation is stable over time under several storage conditions and temperatures

Background: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation. Methods: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4°C, −20°C and −80°C for 0, 1 (endpoint blood 4°C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean ± SD) were quantified using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models. Results: global DNA methylation was stable over 18 months in blood at −20°C and −80°C and DNA at 4°C and −80°C. However, at 18 months DNA methylation from DNA stored at −20°C relatively decreased −6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze–thaw cycles. Conclusion: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of  small differences occuring during storage depend on the expected effect size and research question