Plasma Metabolite Markers of Parkinson's Disease and Atypical Parkinsonism.

Differentiating between Parkinson's disease (PD) and the atypical Parkinsonian disorders of multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) is difficult clinically due to overlapping symptomatology, especially at early disease stages. Consequently, there is a need to identify metabolic markers for these diseases and to develop them into viable biomarkers. In the present investigation, solution nuclear magnetic resonance and mass spectrometry metabolomics were used to quantitatively characterize the plasma metabolomes (a total of 167 metabolites) of a cohort of 94 individuals comprising 34 PD, 12 MSA, and 17 PSP patients, as well as 31 control subjects. The distinct and statistically significant differences observed in the metabolite concentrations of the different disease and control groups enabled the identification of potential plasma metabolite markers of each disorder and enabled the differentiation between the disorders. These group-specific differences further implicate disturbances in specific metabolic pathways. The two metabolites, formic acid and succinate, were altered similarly in all three disease groups when compared to the control group, where a reduced level of formic acid suggested an effect on pyruvate metabolism, methane metabolism, and/or the kynurenine pathway, and an increased succinate level suggested an effect on the citric acid cycle and mitochondrial dysfunction.

Long-term stability of the alcohol consumption biomarker phosphatidylethanol in erythrocytes at -80 °C.

Phosphatidylethanol (PEth) is a recently introduced biomarker with high specificity, high sensitivity, and response correlating with alcohol consumption. It has the potential to be a valuable biomarker in population studies on the health effects of alcohol, however its stability in long-term stored blood is not known. We used LC-MS/MS to assess the stability of PEth-16:0/18:1 in blood samples (packed erythrocytes) that were stored between 1 and 19 years at -80 °C in a biobank from a large population survey. The participants answered a life-style questionnaire that included questions on alcohol consumption. For analysis, we selected blood samples from seven homogenous ethanol consumption cohorts collected at intervals from 1997 to 2015. Despite the narrow stated alcohol consumption range, 10-15 g/day, there were large differences in PEth values between individuals in the cohorts, from below the limit of detection of 0.005 µmol/L to 1.40 µmol/L. The median was 0.08 µmol/L. Neither generalized linear modeling, nor principal component analysis revealed a statistically significant association between time of storage and PEth levels. The PEth results indicate that the participants had, on average, under-reported their alcohol consumption several-fold. The findings suggest that PEth in blood has a sufficient long-term stability for use as an alcohol biomarker in prospective case-control studies. Analysis of blood stored in biobanks could significantly improve the validity of assessments exploring the health effects of alcohol.

Early pregnancy sex steroids during primiparous pregnancies and maternal breast cancer: a nested case-control study in the Northern Sweden Maternity Cohort.

BACKGROUND: Pregnancy and parity are associated with subsequent breast cancer risk. Experimental and epidemiologic data suggest a role for pregnancy sex steroid hormones. METHODS: We conducted a nested case-control study in the Northern Sweden Maternity Cohort (1975-2007). Eligible women had provided a blood sample in the first 20 weeks of gestation during a primiparous pregnancy leading to a term delivery. The current study includes 223 cases and 417 matched controls (matching factors: age at and date of blood collection). Estrogen receptor (ER) and progesterone receptor (PR) status was available for all cases; androgen receptor (AR) data were available for 41% of cases (n = 92). Sex steroids were quantified by high-performance liquid chromatography tandem mass spectrometry. Odds ratios (ORs) and 95% confidence intervals were estimated using conditional logistic regression. RESULTS: Higher concentrations of circulating progesterone in early pregnancy were inversely associated with ER+/PR+ breast cancer risk (ORlog2: 0.64 (0.41-1.00)). Higher testosterone was positively associated with ER+/PR+ disease risk (ORlog2: 1.57 (1.13-2.18)). Early pregnancy estrogens were not associated with risk, except for relatively high estradiol in the context of low progesterone (split at median, relative to low concentrations of both; OR: 1.87 (1.11-3.16)). None of the investigated hormones were associated with ER-/PR- disease, or with AR+ or AR+/ER+/PR+ disease. CONCLUSIONS: Consistent with experimental models, high progesterone in early pregnancy was associated with lower risk of ER+/PR+ breast cancer in the mother. High circulating testosterone in early pregnancy, which likely reflects nonpregnant premenopausal exposure, was associated with higher risk of ER+/PR+ disease.

Human Chorionic Gonadotropin Does Not Correlate with Risk for Maternal Breast Cancer: Results from the Finnish Maternity Cohort.

Human chorionic gonadotropin (hCG) is necessary for the maintenance of early pregnancy and promotes normal breast cell differentiation. Administered hCG reduces risk of carcinogen-induced breast cancer in animal models, and higher circulating hCG concentrations were associated with significantly lower long-term risk of breast cancer in a prior nested case-control study. In this study, we investigated early-pregnancy hCG concentrations and subsequent breast cancer risk. We conducted a nested case-control study with 1,191 cases and 2,257 controls (matched on age and date at blood collection) in the Finnish Maternity Cohort, a cohort with serum samples from 98% of pregnancies registered in Finland since 1983. This study included women with a serum sample collected early (<140 days gestation) in their first pregnancy resulting in a live, term birth. Breast cancer cases were identified via the Finnish Cancer Registry. Age at breast cancer diagnosis ranged from 22 to 58 years (mean: 41 years). hCG was measured using a solid-phase competitive chemiluminescence assay. Odds ratios (OR) were calculated using conditional logistic regression. We observed no association between hCG and breast cancer risk, overall [Quartile 4 vs. 1, OR, 1.14; 95% confidence interval (CI), 0.94-1.39], by estrogen and progesterone receptor status, or by ages at first-term birth or diagnosis. Associations did not differ by time between pregnancy and diagnosis (e.g., <5 years, ORQ4 vs. Q1, 1.10; 95% CI, 0.64-1.89; ≥15 years, ORQ4 vs. Q1, 1.36; 95% CI, 0.86-2.13; pheterogeneity = 0.62). This large prospective study does not support an inverse relationship between early pregnancy serum hCG concentrations and breast cancer risk. Cancer Res; 77(1); 134-41. ©2016 AACR.

Hormone concentrations throughout uncomplicated pregnancies: a longitudinal study.

BACKGROUND: Evidence suggests that the hormonal milieu of pregnancy is an important determinant of subsequent cancer and other chronic diseases in both the mother and the offspring. Many of the existing maternity and birth cohorts include specimens drawn only once during pregnancy. How well a single blood specimen collected during a pregnancy characterizes exposure to these hormones throughout gestation, and also in subsequent pregnancies, is not well understood. METHODS: We used serial serum samples from 71 pregnant women (25 primiparous, 25 multiparous, and 21 with two consecutive pregnancies) with natural, complication-free pregnancies and a healthy offspring at term who participated in a population-based screening trial for congenital infections in Finland between January 1st, 1988 and June 30, 1989 and provided a blood sample in each trimester. RESULTS: Hormone levels were more strongly correlated between consecutive trimesters of a pregnancy than between the 1st and 3rd trimester (e.g., estradiol, rT1 vs. T2 = 0.51 and rT2 vs. T3 = 0.60, p < 0.01; rT1 vs. T3 = 0.32, p < 0.05). Concentrations of sRANKL remained stable throughout gestation, whereas estradiol, estrone, progesterone, testosterone, prolactin, and osteoprotegerin increased throughout pregnancy. First trimester hormone concentrations explained less of the variation in the third trimester on their own than second trimester hormone levels (e.g. estradiol R(2) T1 = 16 % and R(2) T2 = 42 %). Addition of maternal (e.g., smoking) and/or child characteristics (e.g., sex) improved the accuracy of the 3rd trimester estimates for some of the hormones. CONCLUSIONS: One hormone measurement in early pregnancy, in conjunction with maternal and fetal characteristics, permits estimation of 3rd trimester hormone concentrations. Therefore, single hormone measurements available from maternity cohorts are suitable to quantify hormone exposure during pregnancy. To our knowledge, we provide the first data on correlations between hormone concentrations both across trimesters of a single pregnancy, as well as between two subsequent pregnancies.

Early pregnancy sex steroids and maternal risk of epithelial ovarian cancer.

Well-established associations between reproductive characteristics and epithelial ovarian cancer (EOC) support an involvement of sex steroid hormones in the etiology of EOC. Limited previous studies have evaluated circulating androgens and the risk of EOC, and estrogens and progesterone have been investigated in only one of the previous studies. Furthermore, there is little data on potential heterogeneity in the association between circulating hormones and EOC by histological subgroup. Therefore, we conducted a nested case-control study within the Finnish Maternity Cohort and the Northern Sweden Maternity Cohort to investigate the associations between circulating pre-diagnostic sex steroid concentrations and the histological subtypes of EOC. We identified 1052 EOC cases among cohort members diagnosed after recruitment (1975-2008) and before March 2011. Up to three controls were individually matched to each case (n=2694). Testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP), progesterone, estradiol (E2), and sex hormone-binding globulin levels were measured in serum samples collected during the last pregnancy before EOC diagnosis. We used conditional logistic regression to estimate odds ratios (ORs) and 95% CIs. Associations between hormones and EOC differed with respect to tumor histology and invasiveness. Sex steroid concentrations were not associated with invasive serous tumors; however, doubling of testosterone and 17-OHP concentration was associated with approximately 40% increased risk of borderline serous tumors. A doubling of androgen concentrations was associated with a 50% increased risk of mucinous tumors. The risk of endometrioid tumors increased with higher E2 concentrations (OR: 1.89 (1.20-2.98)). This large prospective study in pregnant women supports a role of sex steroid hormones in the etiology of EOC arising in the ovaries.

Early pregnancy sex steroids and maternal breast cancer: a nested case-control study.

Pregnancy, parity, and circulating steroid hormone levels are associated with risk of breast cancer, but little is known about hormone concentrations during pregnancy and subsequent breast cancer risk. We evaluated early pregnancy (<140 days gestation) serum estradiol, estrone, progesterone, and testosterone and breast cancer risk in a nested case-control study in the Finnish Maternity Cohort. The cohort includes 98% of pregnancies registered in Finland since 1983. Individuals with samples collected in the first pregnancy leading to a live birth were eligible. Breast cancer cases (n = 1,199) were identified through linkage with the Finnish Cancer Registry; 2,281 matched controls were selected using incidence density sampling. ORs were calculated using conditional logistic regression. Hormone concentrations were not associated with breast cancer overall. Estradiol was positively associated with risk of breast cancer diagnosed age <40 [4th vs. 1st quartile OR 1.60 (1.07-2.39); Ptrend = 0.01], and inversely associated with breast cancer diagnosed at age ≥40 [4th vs. 1st quartile OR 0.71 (0.51-1.00); Ptrend = 0.02]. Elevated concentrations of the steroid hormones were associated with increased risk of estrogen receptor (ER)- and progesterone receptor (PR)-negative tumors in women age <40 at diagnosis. We observed no association between steroid hormones and ER(+)/PR(+) disease. These data suggest a positive association between high concentrations of early pregnancy steroid hormones and risk of ER(-)/PR(-) breast cancer in women diagnosed age <40, and an inverse association for overall breast cancer diagnosed age ≥40. Further research on pregnancy hormones and risk of steroid receptor-negative cancers is needed to further characterize this association.

Circulating estrogens and progesterone during primiparous pregnancies and risk of maternal breast cancer.

Pregnancy reduces maternal risk of breast cancer in the long term, but the biological determinants of the protection are unknown. Animal experiments suggest that estrogens and progesterone could be involved, but direct human evidence is scant. A case-control study (536 cases and 1,049 controls) was nested within the Finnish Maternity Cohort. Eligible were primiparous women who delivered at term a singleton offspring before age 40. For each case, two individually matched controls by age (± 6 months) and date of sampling (± 3 months) were selected. Estradiol, estrone and progesterone in first-trimester serum were measured by high-performance liquid chromatography tandem mass spectrometry and sex-hormone binding globulin (SHBG) by immunoassay. Odds ratios (OR) and 95% confidence intervals (CI) were estimated through conditional logistic regression. In the whole study population there was no association of breast cancer with any of the studied hormones. In analyses stratified by age at diagnosis, however, estradiol concentrations were positively associated with risk of breast cancer before age 40 (upper quartile OR, 1.81; CI, 1.08-3.06), but inversely associated with risk in women who were diagnosed ≥ age 40 (upper quartile OR, 0.64; CI, 0.40-1.04), p(interaction) 0.004. Risk estimates for estrone mirrored those for estradiol but were less pronounced. Progesterone was not associated with risk of subsequent breast cancer. Our results provide initial evidence that concentrations of estrogens during the early parts of a primiparous pregnancy are associated with maternal risk of breast cancer and suggest that the effect may differ for tumors diagnosed before and after age 40.

Determinants of maternal sex steroids during the first half of pregnancy.

OBJECTIVE: To examine the associations of maternal and child characteristics with early pregnancy maternal concentrations of testosterone, androstenedione, progesterone, 17-hydroxyprogesterone, and estradiol (E2). METHODS: We analyzed these hormones among 1,343 women with singleton pregnancies who donated serum samples to the Finnish Maternity Cohort from 1986 to 2006 during the first half of pregnancy (median 11 weeks). The associations of maternal and child characteristics with hormone concentrations were investigated by correlation and multivariable regression. RESULTS: Women older than age 30 years had lower androgen and E2 but higher progesterone concentrations than women younger than that age. Multiparous women had 14% lower testosterone, 11% lower androstenedione and 17-hydroxyprogesterone, 9% lower progesterone, and 16% lower E2 concentrations compared with nulliparous women (all P<.05). Smoking mothers had 11%, 18%, and 8% higher testosterone, androstenedione, and 17-hydroxyprogesterone levels, respectively, but 10% lower progesterone compared with nonsmoking women (all P<.05). E2 concentrations were 9% higher (P<.05) among women with a female fetus compared with those with a male fetus. CONCLUSION: Parity, smoking, and, to a lesser extent, maternal age and child sex are associated with sex steroid levels during the first half of a singleton pregnancy. The effects of smoking on the maternal hormonal environment and the possible long-term deleterious consequences on the fetus deserve further evaluation.

Association of serum 25-hydroxyvitamin D (25-OHD) concentrations with maternal sex steroids and IGF-1 hormones during pregnancy.

BACKGROUND: Vitamin D may influence circulating levels of sex steroid hormones in women during reproductive life, but associations in pregnant women have not been explored. METHODS: Correlation and linear regression models were used to assess the association between sex steroids, (estradiol, progesterone, 17-hydroxyprogesterone, testosterone, and androstenedione), IGF-1, and serum 25-hydroxyvitamin D (25-OHD) concentrations during the first trimester of pregnancy in 106 cancer-free women from the Finnish Maternity Cohort. RESULTS: There was no significant association of serum 25-OHD with any of the hormones measured. One-unit increase in serum 25-OHD concentration was associated with a non-significant 6% increase in estradiol concentrations. Multiparous women had higher levels of vitamin D (40.4 vs. 32.9 nmol/L, p-value = 0.01) than primiparous women. CONCLUSION: Our study does not support an association between maternal serum 25-OHD levels and sex steroids or IGF-I concentrations during the first trimester of pregnancy.

Circulating sex steroids during pregnancy and maternal risk of non-epithelial ovarian cancer.

BACKGROUND: Sex steroid hormones have been proposed to play a role in the development of non-epithelial ovarian cancers (NEOC) but so far no direct epidemiologic data are available. METHODS: A case-control study was nested within the Finnish Maternity Cohort, the world's largest biorepository of serum specimens from pregnant women. Study subjects were selected among women who donated a blood sample during a singleton pregnancy that led to the birth of their last child preceding diagnosis of NEOC. Case subjects were 41 women with sex cord stromal tumors (SCST) and 21 with germ cell tumors (GCT). Three controls, matching the index case for age, parity at the index pregnancy, and date at blood donation were selected (n = 171). OR and 95% CI associated with concentrations of testosterone, androstenedione, 17-OH-progesterone, progesterone, estradiol, and sex hormone-binding globulin (SHBG) were estimated through conditional logistic regression. RESULTS: For SCST, doubling of testosterone, androstenedione, and 17-OH-progesterone concentrations were associated with about 2-fold higher risk of SCST [ORs and 95% CI of 2.16 (1.25-3.74), 2.16 (1.20-3.87), and 2.62 (1.27-5.38), respectively]. These associations remained largely unchanged after excluding women within 2-, 4-, or 6-year lag time between blood donation and cancer diagnosis. Sex steroid hormones concentrations were not related to maternal risk of GCT. CONCLUSIONS: This is the first prospective study providing initial evidence that elevated androgens play a role in the pathogenesis of SCST. IMPACT: Our study may note a particular need for larger confirmatory investigations on sex steroids and NEOC.

Quantification of methylmalonic acid in human plasma with hydrophilic interaction liquid chromatography separation and mass spectrometric detection.

BACKGROUND: Measurement of methylmalonic acid (MMA) in serum or plasma is useful for diagnosing cobalamin deficiency. We developed a method for quantifying MMA in plasma based on hydrophilic interaction liquid chromatography (HILIC) and single-stage negative electrospray ionization (ESI) mass spectrometry. METHODS: We deproteinized plasma samples (200 microL) with 800 microL acidified acetonitrile containing 0.17 micromol/L deuterated MMA (D(3)-MMA) internal standard, centrifuged the samples, and injected 4 microL of the supernatant into the LC-MS instrument. Separation was achieved within 3 min on a Merck SeQuant ZIC-HILIC column with a mobile phase consisting of 4 volumes acetonitrile plus 1 volume 100 mmol/L ammonium acetate buffer, pH 4.5, at a flow rate of 400 microL/min. Subsequent column washing and reconditioning contributed to a total run time of 10 min. MMA and D(3)-MMA were quantified by single-ion monitoring (m/z 117.2 and 120.2, respectively) in negative ESI mode at a drying-gas flow rate of 10 L/min, 300 degrees C, and a capillary voltage of 3.0 kV. RESULTS: The estimated limits of MMA quantification and detection were 0.09 micromol/L and 0.03 micromol/L, respectively, in plasma. The assay was linear to 200 micromol/L. Interassay and intraassay CVs were < or = 5% at all tested concentrations. Recoveries were 90%-93%. CONCLUSIONS: This robust assay allows analysis of MMA in human plasma without derivatization. Sample preparation is simple and suitable for automation.

Urine monitoring of diazepam abuse- new intake or not?

Testing for drugs-of-abuse in urine is requested for multiple reasons, including legal and workplace policies. Two cases were studied in which there was a suspicion that the patients continued to abuse diazepam, because of repeatedly positive urine samples. In these cases, diazepam metabolites were measured in urine samples by gas or liquid chromatography coupled to mass spectrometry. The concentrations of diazepam metabolites were subsequently creatinine correlated. Very long elimination times were found in the described cases. None of them had in fact ingested diazepam again during the study period. By the use of pharmacogenetic typing, one of the subjects was found to have a slow metabolism for CYP2C9 as well as for CYP2C19. In the second case, there was a possible drug interaction between diazepam and zolpidem.

Serum levels of olanzapine in a non-fatal overdose.

Olanzapine is a widely used second generation antipsychotic drug. Case reports of intoxications have been published, but reports in the literature of non-fatal intoxications of olanzapine containing repeated measurements of serum levels are scarce. Therefore, this case of non-fatal olanzapine intoxication is presented, in which 19 blood samples were drawn during 2 weeks. The highest (initial) measured value was estimated at 800 pg/L. This patient ingested 550 mg of olanzapine resulting in clinical signs of intoxication, including seizures. Because the patient was found the day after the intoxication, the initial concentration had probably been higher. The pharmacokinetics of olanzapine has been described as linear and dose-proportional throughout the therapeutic dosing range. Large overdoses, however, have been described to show non-linear pharmacokinetics. In this study's series of serum concentrations, a two-phase elimination was seen, with an initial elimination half-life of about 24 h during the first 3 days, followed by a second phase with a half-life of about 2.5 days. The patient in this case recovered completely. Because the elimination time after intoxication can be considerably longer than expected, it is recommended that the patient's serum concentrations after intoxication be monitored.

Determination of dextropropoxyphene and nordextropropoxyphene in urine by liquid chromatography-electrospray ionization mass spectrometry.

Dextropropoxyphene and nordextropropoxyphene were extracted from urine samples with mixed mode solid-phase extraction cartridges. After elution and evaporation to dryness, the eluate was dissolved in mobile phase and each sample was injected in a LC-ESI-MS system. Quantification was carried out in the selected ion monitoring mode. This article shows the possibility to analyse drugs of abuse substances in urine with a single quadrupole mass spectrometer if only a thorough work-up procedure and a sufficient chromatographic separation is accomplished. In order to enhance the fragmentation of the analytes, in-source fragmentation was carried out. One fragment and the pseudomolecular ion per analyte together with chromatographic retention times were sufficient to verify that the sought compound was found in the samples. In- and between day variation was lower than 10% and the recovery was well above 90%. The analytes were quantified in the range 100-10000 ng/ml urine.