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1.
Eur J Endocrinol ; 190(4): 338-346, 2024 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-38554392

RESUMEN

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%; >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.


Asunto(s)
Adiposidad , Fluorocarburos , Lactante , Femenino , Humanos , Preescolar , Obesidad/metabolismo , Composición Corporal , Antropometría
2.
Clin Chem Lab Med ; 62(5): 911-918, 2024 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-38063179

RESUMEN

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, 11ß-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.


Asunto(s)
Hidroxiprogesteronas , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Sensibilidad y Especificidad , 17-alfa-Hidroxiprogesterona , Esteroides
4.
JCO Precis Oncol ; 7: e2200678, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37343203

RESUMEN

PURPOSE: Dexamethasone, the preferred corticosteroid in most treatment protocols for pediatric acute lymphoblastic leukemia (ALL), can induce undesirable side effects. Neurobehavioral and sleep problems are frequently reported, but the interpatient variability is high. We therefore aimed to identify determinants for parent-reported dexamethasone-induced neurobehavioral and sleep problems in pediatric ALL. METHODS: Our prospective study included patients with medium-risk ALL and their parents during maintenance treatment. Patients were assessed before and after one 5-day dexamethasone course. Primary end points were parent-reported dexamethasone-induced neurobehavioral and sleep problems, measured with the Strengths and Difficulties Questionnaire and Sleep Disturbance Scale for Children, respectively. Analyzed determinants included patient and parent demographics, disease and treatment characteristics, parenting stress (Parenting Stress Index and Distress Thermometer for Parents), dexamethasone pharmacokinetics, and genetic variation (candidate single-nucleotide polymorphisms rs41423247 and rs4918). Statistically significant determinants identified in univariable logistic regression analyses were incorporated in a multivariable model. RESULTS: We included 105 patients: median age was 5.4 years (range, 3.0-18.8) and 61% were boys. Clinically relevant dexamethasone-induced neurobehavioral and sleep problems were reported by parents in 70 (67%) and 61 (59%) patients, respectively. In our multivariable regression models, we identified parenting stress as a significant determinant for parent-reported neurobehavioral (odds ratio [OR], 1.16; 95% CI, 1.07 to 1.26) and sleep problems (OR, 1.06; 95% CI, 1.02 to 1.10). Furthermore, parents who experienced more stress before start of a dexamethasone course reported more sleep problems in their child (OR, 1.16; 95% CI, 1.02 to 1.32). CONCLUSION: We identified parenting stress, and not dexamethasone pharmacokinetics, genetic variation, patient/parent demographics, or disease/treatment characteristics, as a significant determinant for parent-reported dexamethasone-induced neurobehavioral and sleep problems. Parenting stress may be a modifiable target to reduce these problems.


Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Trastornos del Sueño-Vigilia , Masculino , Humanos , Niño , Preescolar , Femenino , Estudios Prospectivos , Padres , Trastornos del Sueño-Vigilia/inducido químicamente , Trastornos del Sueño-Vigilia/epidemiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico
5.
Environ Int ; 164: 107274, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35524998

RESUMEN

BACKGROUND AND AIMS: Per- and polyfluoroalkyl substances (PFAS) are non-degradable, man-made-chemicals with an elimination half-life of multiple years, causing accumulation in the environment and humans with potential harmful effects. However, longitudinal PFAS levels in human milk, daily PFAS intake and the association with infant plasma PFAS levels have never been reported. We investigated longitudinal PFOA and PFOS levels in human milk and the daily PFAS intake through infant feeding in the first 3 months of life, the most important determinants and the correlation with PFAS plasma levels at age 3 months and 2 years. METHODS: In 372 healthy term-born Dutch infants, we determined PFOA and PFOS levels in human milk given at age 1 and 3 months, in 6 infant formula brands and in infant plasma at 3 months and 2 years, using liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry(LC-ESI-MS/MS). We studied the associations between daily PFAS intake and predictive characteristics by multiple regression models. RESULTS: PFOA and PFOS levels in human milk decreased between 1 and 3 months after delivery, regardless whether breastfeeding was given exclusively(EBF) or in combination with formula feeding. PFOA and PFOS could not be detected in any formula feeding. Daily PFAS intake(ng/kg) was highest in EBF-infants. Higher amount of human milk, older maternal age, lower parity and first-time breastfeeding were associated with higher daily intake. Daily PFAS intake in early life was strongly correlated with PFAS plasma levels at age 3 months and 2 years(R = 0.642-0.875, p < 0.001). CONCLUSIONS: Human milk contains PFOA and PFOS, in contrast to formula feeding. Daily PFOA and PFOS intake in early life is highest in exclusively breastfed infants and it is highly correlated with infant's plasma levels throughout infancy. Our findings show that breastfeeding is an important PFAS exposure pathway in the first months of life, with unknown but potential adverse effects. Knowing the important health benefits of breastfeeding, our findings warrant more research about the health outcomes in later life.


Asunto(s)
Ácidos Alcanesulfónicos , Contaminantes Ambientales , Fluorocarburos , Ácidos Alcanesulfónicos/análisis , Lactancia Materna , Femenino , Humanos , Lactante , Leche Humana/química , Embarazo , Espectrometría de Masas en Tándem
6.
Environ Int ; 160: 107068, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34968992

RESUMEN

BACKGROUND AND AIMS: Per- and polyfluoroalkyl substances (PFAS) are a potential hazard for public health. These man-made-chemicals are non-degradable with an elimination half-life of multiple years, causing accumulation in the environment and humans. Rodent studies demonstrated that PFAS are harmful, especially when present during the critical window in the first months of life. Because longitudinal data during infancy are limited, we investigated longitudinal plasma levels in infants aged 3 months and 2 years and its most important determinants. METHODS: In 369 healthy term-born Dutch infants, we determined plasma PFOS, PFOA, PFHxS, PFNA and PFDA levels at age 3 months and 2 years, using liquid chromatography-electrospray-ionization-tandem-mass-spectrometry (LC-ESI-MS/MS). We studied the associations with maternal and child characteristics by multiple regression models. RESULTS: At age 3 months, median plasma levels of PFOS, PFOA, PFHxS, PFNA and PFDA were 1.48, 2.40, 0.43, 0.23 and 0.07 ng/mL, resp. Levels decreased slightly until age 2 years to 1.30, 1.81, 0.40, 0.21 and 0.08 ng/mL, resp. Maternal age, first born, Caucasian ethnicity and exclusive breastfeeding were associated with higher infant's plasma levels at age 3 months. Levels at 3 months were the most important predictor for PFAS levels at age 2 years. Infants with exclusive breastfeeding during the first 3 months of life (EBF) had 2-3 fold higher levels throughout infancy compared to infants with exclusive formula feeding (EFF), with PFOA levels at 3 months 3.72 ng/mL versus 1.26 ng/mL and at 2 years 3.15 ng/mL versus 1.22 ng/mL, respectively. CONCLUSION: Plasma PFAS levels decreased only slightly during infancy. Higher levels at age 3 months were found in Caucasian, first-born infants from older mothers and throughout infancy in EBF-infants. Our findings indicate that trans-placental transmission and breastfeeding are the most important determinants of PFAS exposure in early life.


Asunto(s)
Ácidos Alcanesulfónicos , Contaminantes Ambientales , Fluorocarburos , Ácidos Alcanesulfónicos/análisis , Etnicidad , Femenino , Humanos , Placenta/química , Embarazo , Espectrometría de Masas en Tándem
7.
Epigenetics ; 16(1): 45-53, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32614650

RESUMEN

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.


Asunto(s)
Conservación de la Sangre/efectos adversos , Metilación de ADN , Células Sanguíneas/metabolismo , Conservación de la Sangre/métodos , Criopreservación/métodos , Humanos
8.
Nutrients ; 12(8)2020 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-32717800

RESUMEN

Hyperhomocysteinemia (HHcy) is a risk factor for atherosclerosis through mechanisms which are still incompletely defined. One possible mechanism involves the hypomethylation of the nuclear histone proteins to favor the progression of atherosclerosis. In previous cell studies, hypomethylating stress decreased a specific epigenetic tag (the trimethylation of lysine 27 on histone H3, H3K27me3) to promote endothelial dysfunction and activation, i.e., an atherogenic phenotype. Here, we conducted a pilot study to investigate the impact of mild HHcy on vascular methylating index, atherosclerosis progression and H3K27me3 aortic content in apolipoprotein E-deficient (ApoE -/-) mice. In two different sets of experiments, male mice were fed high-fat, low in methyl donors (HFLM), or control (HF) diets for 16 (Study A) or 12 (Study B) weeks. At multiple time points, plasma was collected for (1) quantification of total homocysteine (tHcy) by high-performance liquid chromatography; or (2) the methylation index of S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH ratio) by liquid chromatography tandem-mass spectrometry; or (3) a panel of inflammatory cytokines previously implicated in atherosclerosis by a multiplex assay. At the end point, aortas were collected and used to assess (1) the methylating index (SAM:SAH ratio); (2) the volume of aortic atherosclerotic plaque assessed by high field magnetic resonance imaging; and (3) the vascular content of H3K27me3 by immunohistochemistry. The results showed that, in both studies, HFLM-fed mice, but not those mice fed control diets, accumulated mildly elevated tHcy plasmatic concentrations. However, the pattern of changes in the inflammatory cytokines did not support a major difference in systemic inflammation between these groups. Accordingly, in both studies, no significant differences were detected for the aortic methylating index, plaque burden, and H3K27me3 vascular content between HF and HFLM-fed mice. Surprisingly however, a decreased plasma SAM: SAH was also observed, suggesting that the plasma compartment does not always reflect the vascular concentrations of these two metabolites, at least in this model. Mild HHcy in vivo was not be sufficient to induce vascular hypomethylating stress or the progression of atherosclerosis, suggesting that only higher accumulations of plasma tHcy will exhibit vascular toxicity and promote specific epigenetic dysregulation.


Asunto(s)
Aterosclerosis , Dieta/efectos adversos , Progresión de la Enfermedad , Histonas/metabolismo , Hiperhomocisteinemia/metabolismo , Animales , Aorta/diagnóstico por imagen , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/genética , Citocinas , Metilación de ADN , Epigénesis Genética , Hiperhomocisteinemia/genética , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Proyectos Piloto , Placa Aterosclerótica , S-Adenosilmetionina/metabolismo
9.
PLoS One ; 14(12): e0226969, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31887212

RESUMEN

INTRODUCTION: The pathophysiology of preeclampsia is largely unknown. Serum placental induced growth factor (PlGF) levels are decreased during second trimester pregnancy. Aberrant DNA methylation is suggested to be involved in the etiology of preeclampsia (PE). We hypothesize that DNA methylation is altered in PE placentas determined the methylation index by measuring placental S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels. In addition, we assessed global DNA methylation status by long-interspersed nuclear element-1 (LINE-1) and DNA methylation status of the PlGF gene. METHODS: Placental tissue of 11 early onset PE (EOPE), 11 late onset PE (LOPE) and 60 controls consisting of 25 uncomplicated controls 20 fetal growth restriction (FGR) and 15 preterm births (PTB) controls was collected from a nested case-control study of The Rotterdam Periconceptional Cohort. RNA and DNA was isolated from placental tissue and DNA was treated with sodium bisulfite. SAM and SAH levels were measured by LC-ESI-MS/MS. Methylation of LINE-1 and PlGF genes was analyzed by Sequenom Epityper and. mRNA expression of PlGF was assessed with qPCR. Differences were assessed by analysis of covariance (ANCOVA) corrected for gestational age and birth weight. RESULTS: Placental SAM levels were significantly lower in placental tissue of EOPE pregnancies compared to PTB controls (mean difference -240 ± 71.4 nmol/g protein, P = 0.01). PlGF DNA methylation was decreased in placental tissue of EOPE cases versus LOPE (mean difference -17.4 ± 5.1%, P = 0.01), uncomplicated controls (mean difference -23.4 ± 5.4%%, P <0.001), FGR controls (mean difference -17.9 ± 4.6%, P = 0.002) and PTB controls (mean difference -11.3 ± 3.8% P = 0.04). No significant differences were observed in SAH, SAM:SAH ratio, LINE-1 DNA methylation and PlGF mRNA expression between groups. DISCUSSION: The hypomethylation state of the placenta in EOPE, which is reflected by lower SAM and PlGF DNA hypomethylation underlines the possible role of placental DNA hypomethylation in the pathophysiology of EOPE, which needs further investigation.


Asunto(s)
Metilación de ADN , Factor de Crecimiento Placentario/metabolismo , Preeclampsia/etiología , S-Adenosilmetionina/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Placenta/metabolismo , Factor de Crecimiento Placentario/genética , Preeclampsia/metabolismo , Embarazo , S-Adenosilhomocisteína/metabolismo
10.
PLoS One ; 14(9): e0221591, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31527879

RESUMEN

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, p<0.001). This was due to an increase in 5-methyl THF levels (mean increase: 217.7 ± 209.5 nmol/L, p<0.001), whereas non-methyl THF levels did not change (median increase: 0.6 nmol/L [-9.9-11.1], p = 0.676). Serum folate levels increased between T0 and T1 (median increase: 29.2 nmol/L [32.9-74.0], p<0.001). Results were not significantly affected by age, sex, ALL immunophenotype and MTHFR c.677C>T 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.


Asunto(s)
Ácido Fólico/sangre , Leucovorina/administración & dosificación , Metotrexato/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Adolescente , Antídotos/administración & dosificación , Niño , Preescolar , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Femenino , Antagonistas del Ácido Fólico/administración & dosificación , Antagonistas del Ácido Fólico/efectos adversos , Homocisteína/sangre , Humanos , Lactante , Masculino , Redes y Vías Metabólicas , Metotrexato/efectos adversos , Estudios Prospectivos , Vitamina B 12/sangre
11.
Arthritis Res Ther ; 21(1): 157, 2019 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-31242943

RESUMEN

BACKGROUND: Low-dose methotrexate (MTX) is the first-line therapy in early rheumatoid arthritis (eRA). Up to 40% of eRA patients do not benefit from MTX therapy. MTX has been shown to inhibit one-carbon metabolism, which is involved in the donation of methyl groups. In this study, we investigate baseline global DNA methylation and changes in DNA methylation during treatment in relation to clinical non-response after 3 months of MTX treatment. METHODS: Two hundred ninety-four blood samples were collected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, ISRCTN26791028), a multicenter, stratified single-blind clinical trial of eRA patients. Global DNA (hydroxy)methylation was quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and validated with a global DNA LINE-1 methylation technique. MTX response was determined as ΔDAS28. Additionally, patients were stratified into two response groups according to the European League Against Rheumatism (EULAR) response criteria. Associations between global DNA methylation and response were examined using univariate regression models adjusted for baseline DAS28, baseline erythrocyte folate levels, and body mass index (BMI). RESULTS: Higher baseline global DNA methylation was associated with less decrease of DAS28 (ß = 0.15, p = 0.013) and with MTX non-response (OR = 0.010, 95% CI = 0.001-0.188). This result was validated in LINE-1 elements (ß = 0.22, p = 0.026). Changes in global DNA (hydroxy)methylation were not associated with MTX response over 3 months. CONCLUSIONS: These results show that higher baseline global DNA methylation in treatment naïve eRA patients is associated with decreased clinical response after 3 months of treatment of eRA patients and can be further evaluated as a predictor for MTX therapy non-response. TRIAL REGISTRATION: ISRCTN, ISRCTN26791028 , registered 23 August 2007-retrospectively registered.


Asunto(s)
Artritis Reumatoide/genética , ADN/genética , Leucocitos/metabolismo , Metotrexato/administración & dosificación , Antirreumáticos/administración & dosificación , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Metilación de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Método Simple Ciego , Espectrometría de Masas en Tándem , Resultado del Tratamiento
12.
J Anim Physiol Anim Nutr (Berl) ; 103(3): 858-867, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30900324

RESUMEN

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.


Asunto(s)
Alimentación Animal/análisis , Metilación de ADN/fisiología , Dieta/veterinaria , Nutrientes/metabolismo , Porcinos/fisiología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Composición Corporal , Peso Corporal , Femenino , Nutrientes/sangre , Embarazo , Porcinos/sangre , Porcinos/embriología
13.
Clin Chem Lab Med ; 57(5): 659-667, 2019 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-30496132

RESUMEN

Background Failure to report a creatinine concentration, especially in icteric patients who are eligible for a liver transplant, can result in a life-threatening situation. We assessed the influence of bilirubin interference on several creatinine assays and investigated ways to circumvent icteric interference without interfering with our normal automated sample logistics. Methods Using icteric patient sera (total bilirubin >255 µmol/L) we determined creatinine concentrations using an enzymatic and Jaffé assay (Roche Diagnostics) in both normal (i.e. undiluted) and decreased mode (i.e. diluted) as well as an enzyme-coupled amperometric assay on a Radiometer ABL837 FLEX analyzer. Creatinine concentrations from the five methods were compared with an in-house developed LC-MS/MS method. Passing and Bablok (proportional and constant bias) as well as difference plot parameters (bias and 95% limits of agreement [LoA]) were calculated. Interferograph-based regression analysis of the enzymatic and Jaffé results was used to investigate if such an approach could be used to report corrected creatinine concentrations in icteric patient sera. Results In icteric patient sera the enzyme-coupled amperometric assay was hardly influenced by icteric interference as shown by a difference plot bias of -1.5% (95% LoA -11.6 to +8.5%). The undiluted Jaffé method had a bias of -1.4% with a very broad 95% LoA (-35.1 to +32.2%) emphasizing the poor specificity of this method. The undiluted enzymatic method had the largest bias (-13.4%, 95% LoA -35.8 to +9.0%). Diluting sera in the enzymatic method did not improve the bias (-10.5%, 95% LoA -25.4 to 4.4%), while diluting the Jaffé method resulted in a bias increase (+11.4%, 95% LoA -14.7 to 37.5%). Using interferograph-based regression analysis we were able to reliably correct enzymatic creatinine concentrations in 97 out of 100 icteric patient sera. Conclusions Analytically, quantifying creatinine in icteric sera using the Radiometer ABL837 FLEX analyzer is the method of choice within our laboratory. However, not all laboratories are equipped with this method and even if available, the limited number of highly icteric patient sera makes this method costly. For those laboratories using the Roche enzymatic method, mathematically correcting an icteric creatinine concentration using an interferograph based on an LC-MS/MS reference method is a suitable alternative to report reliable creatinine results in icteric patients.


Asunto(s)
Creatinina/sangre , Hiperbilirrubinemia/sangre , Bilirrubina/sangre , Análisis Químico de la Sangre/estadística & datos numéricos , Cromatografía Liquida/estadística & datos numéricos , Humanos , Espectrometría de Masas en Tándem/estadística & datos numéricos
14.
J Invest Dermatol ; 139(2): 360-368, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30130617

RESUMEN

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6-/- mouse model of PXE. Thus, we bred Abcc6-/- mice to heterozygous Alpl+/- mice that display approximately 50% plasma TNAP activity. The Abcc6-/-Alpl+/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6-/-Alpl+/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6-/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease.


Asunto(s)
Niacinamida/análogos & derivados , Seudoxantoma Elástico/tratamiento farmacológico , Pirofosfatasas/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Difosfatos/sangre , Difosfatos/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Niacinamida/administración & dosificación , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Seudoxantoma Elástico/sangre , Seudoxantoma Elástico/genética , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Piel/metabolismo , Piel/patología , Calcificación Vascular/sangre , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/genética
16.
Mol Pharm ; 14(6): 2147-2157, 2017 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-28493713

RESUMEN

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.


Asunto(s)
Cetuximab/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Metotrexato/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Bencimidazoles/metabolismo , Supervivencia Celular/efectos de los fármacos , Glutatión/análogos & derivados , Glutatión/metabolismo , Células HEK293 , Humanos , Compuestos de Metilmercurio/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo
17.
Clin Chem Lab Med ; 54(4): 609-16, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26457777

RESUMEN

BACKGROUND: The aim of this study was to compare different analytical methods that are currently in use in the Netherlands for the measurement of whole blood vitamin B6. METHODS: This method comparison study consisted of two separate parts. (1) Four laboratories participated in a pilot study in which the commercial Chromsystems and INstruchemie method, and a laboratory developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and HPLC method were compared. Sixty-nine frozen whole blood samples and six lyophilized whole blood samples were used for comparison. (2) In the nationwide part of the study, 49 laboratories participated in the analysis of three identical sets of two whole blood samples of which one set was freshly analyzed, one set was analyzed after storage at -20 °C and one set was analyzed after lyophilization. RESULTS: In both parts of the study, the HPLC and LC-MS/MS methods showed equivalent results for all sample types tested. The Chromsystems method showed a positive bias of 45% (pilot study) and 30% (nationwide study) towards the LC-MS/MS method when fresh or frozen samples were used. The measurement of lyophilized samples showed no differences between the methods. The results of the INstruchemie method were inconclusive due to the low number of participants. CONCLUSIONS: The different analytical methods for measuring vitamin B6 produce different results when whole blood patient samples are measured. The recognition of a reference method or the development of suitable reference materials and quality control materials might serve as a first step towards improved standardization or harmonization of the whole blood vitamin B6 assay.


Asunto(s)
Análisis Químico de la Sangre , Técnicas de Laboratorio Clínico , Vitamina B 6/sangre , Cromatografía Liquida , Humanos , Estudios Multicéntricos como Asunto , Proyectos Piloto , Espectrometría de Masas en Tándem
18.
Artículo en Inglés | MEDLINE | ID: mdl-22857864

RESUMEN

Vitamin B6 is a cofactor in numerous biologic processes that include gluconeogenesis, neurotransmitter synthesis and amino acid metabolism. The aim of this study was to develop a method to measure the concentration of the biologically active form of vitamin B6 (pyridoxal-5'-phosphate, PLP) in whole blood with stable isotope dilution LC-ESI-MS/MS and compare this new procedure with an established HPLC method based on derivatization of pyridoxal-5'-phosphate. 50 µl of stable isotope (PLP-d3) was added to 250 µl of sample, followed by deproteinization with 10% trichloroacetic acid. After centrifugation, 20 µl of the supernatant was injected into the LC-ESI-MS/MS. Reversed phase chromatography was performed on a UPLC system, using a Waters™ Symmetry C18 column, with a gradient of 0.1% formic acid in methanol. PLP was measured on a tandem MS with a mass transition of 247.8>149.8 in the positive ion mode with a collision energy of 14 eV. The chromatographic run lasted 4 min. The method was linear from 4 to 8000 nmol/l. The intra-day and inter-day precision ranged between 1.7-2.8% and 3.0-4.1%, respectively. The mean absolute matrix-effect was 99.3% [97-102%]. The relative matrix-effect was 98.8%. The mean recovery was 98% [89-103%]. The lower limit of quantification was 4 nmol/l. The comparison of the LC-ESI-MS/MS method with our current HPLC method yielded the following equation: LC-ESI-MS/MS=1.11 [confidence interval, CI: 1.03-1.20] × HPLC+4.6 [CI: -1.3 to 11.0] (r²=0.94). This LC-ESI-MS/MS based method is characterized by simple sample processing and a short run time. The comparison with the current HPLC method is excellent although a significant proportional bias was detected. To conclude, the LC-ESI-MS/MS method is an appropriate method to determine PLP in whole blood.


Asunto(s)
Cromatografía de Fase Inversa/métodos , Fosfato de Piridoxal/sangre , Espectrometría de Masas en Tándem/métodos , Humanos , Fosfato de Piridoxal/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodos
19.
Ther Drug Monit ; 34(4): 432-9, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22689188

RESUMEN

INTRODUCTION: High-dose methotrexate (MTX) is used in the treatment of proliferative diseases such as acute lymphoblastic leukemia. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed to develop a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS)-based method to determine MTX in plasma for therapeutic drug monitoring and pharmacokinetic studies. METHODS: Samples were analyzed using a Waters Acquity UPLC and Quattro Premier XE. A Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm) was used running an isocratic mobile phase of 21% methanol and 10 mM ammonium bicarbonate. The electrospray was operated in the positive ionization mode monitoring the following mass transitions: m/z 455.2 > 308.2 for MTX and m/z 458.2 > 311.2 for MTXd3. The analysis combined straightforward sample preparation, consisting of dilution and protein precipitation, with a 3-minute run time. RESULTS: The method was linear up to 50 µM (r > 0.99), and the coefficient of variation was <6% for intraday and <10% for interday precision. Average recovery was 99%. There were no significant matrix effects. The lower limit of quantitation, defined as the lowest concentration at which the coefficient of variation <20% and S/N > 1:10, was 5 nM. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement, and a small but significant negative constant bias was detected (LC-MS/MS = 0.98 × FPIA - 7.3). CONCLUSIONS: [corrected] The authors developed a specific and sensitive stable isotope dilution LC-ESI-MS/MS method to monitor MTX concentrations in plasma within the clinically relevant range. The method can be easily applied in clinical laboratories because it combines straightforward sample pretreatment with LC-MS/MS.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Monitoreo de Drogas/métodos , Metotrexato/sangre , Metotrexato/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Antimetabolitos Antineoplásicos/sangre , Antimetabolitos Antineoplásicos/química , Humanos , Técnica de Dilución de Radioisótopos
20.
J Infect ; 62(1): 93-100, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20970450

RESUMEN

OBJECTIVE: To prospectively assess the diagnostic utility of S-adenosylmethionine (AdoMet) and (1→3)-ß-D-glucan (ß-D-glucan) serum markers for Pneumocystis pneumonia (PCP) in HIV-negative patients. METHODS: HIV-negative, immunocompromised patients suspected of PCP based on clinical presentation and chest imaging were included. PCP was confirmed or rejected by results of direct microscopy and/or real-time PCR on broncho-alveolar lavage (BAL) fluid. Measurement of serum ß-D-glucan and AdoMet was performed on serum samples collected at enrollment and during follow-up. Both serum ß-D-glucan and AdoMet were assessed for diagnostic accuracy and correlation with clinical and laboratory parameters. RESULTS: In 31 patients enrolled (21 PCP-positive, 10 PCP-negative), AdoMet levels did not discriminate between patients with and without PCP. Elevated serum ß-D-glucan was a reliable indicator for PCP with a sensitivity of 0.90 and specificity of 0.89 at the 60 pg/ml cut-off. In PCP-positive patients ß-D-glucan serum levels decreased during treatment and inversely correlated with Pneumocystis PCR cycle threshold values in BAL fluid. CONCLUSIONS: The level of ß-D-glucan--but not AdoMet--was diagnostic for PCP within the clinical context and may serve as marker for pulmonary fungal load and treatment monitoring.


Asunto(s)
Neumonía por Pneumocystis/diagnóstico , S-Adenosilmetionina/sangre , beta-Glucanos/sangre , Anciano , Biomarcadores/sangre , Femenino , Seronegatividad para VIH , Humanos , Huésped Inmunocomprometido , Masculino , Persona de Mediana Edad , Pneumocystis carinii/aislamiento & purificación , Neumonía por Pneumocystis/sangre , Neumonía por Pneumocystis/inmunología , Estudios Prospectivos , Proteoglicanos , Sensibilidad y Especificidad
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