Development and validation of a liquid chromatography-tandem mass spectrometry assay to quantify plasma 24(S)-hydroxycholesterol and 27-hydroxycholesterol: A new approach integrating the concept of ion ratio.

Accurate quantification of 24(S)-hydroxycholesterol and 27-hydroxycholesterol holds substantial biological significance due to their involvement in pivotal cellular processes, encompassing cholesterol homeostasis, inflammatory responses, neuronal signaling, and their potential as disease biomarkers. The plasma determination of these oxysterols is challenging considering their low concentrations and similarities in terms of empirical formulae, molecular structure, and physicochemical properties across all human endogenous plasma oxysterols. To overcome these sensitivity and specificity issues, we developed and validated a quantification method using liquid chromatography coupled to a tandem mass spectrometry instrument. Validation studies were designed inspired by Clinical and Laboratory Standards Institute (CLSI) C62-A Guidelines. The linearity ranged between 20 and 300 nM for both oxysterols with limits of quantification at 20 nM and 30 nM for 24(S)-OHC and 27-OHC, respectively. Inter-day precision coefficient variations (CV) were lower than 10% for both oxysterols. An optimal separation of 25-OHC was obtained from 24(S)-OHC and 27-OHC with a resolution (Rs) > 1.25. The determination and validation of ion ratios for 24(S)-OHC and 27-OHC enabled another quality check in identifying interferents that could impact the quantification. Our developed and validated LC-MS/MS method allows consistent and reliable quantification of human plasmatic 24(S)-OHC and 27-OHC that is warranted in fundamental and clinical research projects.

Associations of maternal glucose markers in pregnancy with cord blood glucocorticoids and child hair cortisol levels.

Exposure to maternal hyperglycemia in utero has been associated with adverse metabolic outcomes in offspring. However, few studies have investigated the relationship between maternal hyperglycemia and offspring cortisol levels. We assessed associations of gestational diabetes mellitus (GDM) with cortisol biomarkers in two longitudinal prebirth cohorts: Project Viva included 928 mother-child pairs and Gen3G included 313 mother-child pairs. In Project Viva, GDM was diagnosed in N = 48 (5.2%) women using a two-step procedure (50 g glucose challenge test, if abnormal followed by 100 g oral glucose tolerance test [OGTT]), and in N = 29 (9.3%) women participating in Gen3G using one-step 75 g OGTT. In Project Viva, we measured cord blood glucocorticoids and child hair cortisol levels during mid-childhood (mean (SD) age: 7.8 (0.8) years) and early adolescence (mean (SD) age: 13.2 (0.9) years). In Gen3G, we measured hair cortisol at 5.4 (0.3) years. We used multivariable linear regression to examine associations of GDM with offspring cortisol, adjusting for child age and sex, maternal prepregnancy body mass index, education, and socioeconomic status. We additionally adjusted for child race/ethnicity in the cord blood analyses. In both Project Viva and Gen3G, we observed null associations of GDM and maternal glucose markers in pregnancy with cortisol biomarkers in cord blood at birth (ß = 16.6 nmol/L, 95% CI -60.7, 94.0 in Project Viva) and in hair samples during childhood (ß = -0.56 pg/mg, 95% CI -1.16, 0.04 in Project Viva; ß = 0.09 pg/mg, 95% CI -0.38, 0.57 in Gen3G). Our findings do not support the hypothesis that maternal hyperglycemia is related to hypothalamic-pituitary-adrenal axis activity.

Establishment of reference intervals for the salivary cortisol circadian cycle, by electrochemiluminescence (ECLIA), in healthy adults.

OBJECTIVES: To determine salivary cortisol reference intervals in a healthy adult population, at 6 different time points during a 24-hour (h) period. METHODS: In a prospective study, salivary cortisol concentrations were measured upon waking, one-hour post-waking and at specific times of the day: at 12 h00, 16 h00, 20 h00 and midnight. Samples were analyzed by the first and second-generation electrochemiluminescence assays (ECLIA) from Roche Cobas Cortisol®. RESULTS: Salivary cortisol values were obtained from 134 healthy volunteers. Reference intervals for the first-generation assay were 6.14-33.19 nmol/L (95% prediction interval) at waking, 5.42-28.06 nmol/L one-hour post-waking, 3.62-16.23 nmol/L at 12 h00, 2.78-15.27 nmol/L at 16 h00, 2.08-14.90 nmol/L at 20 h00 and 2.09-16.92 nmol/L at midnight. Mean salivary cortisol values were 14.63 nmol/L at waking and 6.44 nmol/L at midnight. Reference intervals for the second-generation assay were 1.50-22.02 nmol/L (2.5th to 97.5th percentiles) at waking, 1.50-20.87 nmol/L one-hour post-waking, 1.50-12.51 nmol/L at 12 h00, 1.50-13.03 nmol/L at 16 h00, 1.50-9.52 nmol/L at 20 h00 and 1.50-6.28 nmol/L at midnight. Values for the second-generation assay at all 6 different time points were almost half of the first-generation assay. The second-generation assay showed a better correlation with LC-MS/MS (r = 0,97). CONCLUSION: Our study confirms that reference intervals for salivary cortisol are not comparable across first and second-generation Roche Cobas Cortisol® assays. Furthermore, the second-generation assay has a better correlation with LC-MS/MS and a better analytical performance (accuracy and precision).

Maternal Vitamin D Insufficiency Early in Pregnancy Is Associated with Increased Risk of Preterm Birth in Ethnic Minority Women in Canada.

Background: Maternal vitamin D insufficiency (plasma 25-hydroxyvitamin D [25(OH)D] <75 nmol/L) may play a role in ethnic disparities in rates of preterm and spontaneous preterm births.Objective: We explored the relation between maternal plasma 25(OH)D concentration in the first trimester (8-14 wk of gestation) and the risk of preterm and spontaneous preterm births (<37 wk of gestation) by ethnicity.Methods: We designed a case-control study that included 120 cases of preterm birth (<37 wk of gestation) and 360 term controls (≥37 wk of gestation) of singleton pregnancies from the 3D cohort, a multicenter study in 2456 pregnant women in Quebec, Canada. Plasma 25(OH)D was measured by LC-mass spectrometry. We compared the distribution of vitamin D status between cases and controls for 8 ethnic minority subgroups. We explored the association between maternal plasma 25(OH)D concentration and preterm and spontaneous preterm births with the use of splines in logistic regression by ethnicity.Results: The distributions of maternal vitamin D status (<50, 50-75, and >75 nmol/L) were different in preterm and spontaneous preterm birth cases compared with controls but only in women of ethnic minority (P-trend = 0.003 and 0.024, respectively). Among ethnic subgroups, sub-Saharan Africans (P-trend = 0.030) and Arab-West Asians (P-trend = 0.045) showed an inverse relation between maternal vitamin D status and the risk of preterm birth. Maternal plasma 25(OH)D concentrations of 30 nmol/L were associated with 4.05 times the risk of preterm birth in the total ethnic minority population (95% CI: 1.16, 14.12; P = 0.028) relative to participants with a concentration of 75 nmol/L. In contrast, there was no such association among nonethnic women (OR: 0.94; 95% CI: 0.48, 1.82; P = 0.85). There was no association when we considered only spontaneous preterm births in the total ethnic minority population (OR: 1.75; 95% CI: 0.39, 7.79; P = 0.46).Conclusion: Vitamin D insufficiency is associated with an increased risk of preterm birth in ethnic minority women in Canada.

Comparison of two methods for measuring 25-OH vitamin D in the follow-up of patients after bilio-pancreatic diversion bariatric surgery.

OBJECTIVE: Our objective was to compare the ECLIA from Roche versus the LC-MS/MS method for quantitation of serum 25-hydroxy-vitamin D in patients who have undergone bariatric surgery. DESIGN AND METHODS: Cross-sectional and correlational studies were performed on three different groups for the 25-OH-D levels quantitated by both methods. The control group of apparently healthy subjects was randomly selected in a clinical chemistry laboratory. Test groups were patients who had undergone bilio-pancreatic diversion (BPD) and were supplemented either with vitamin D2 or with vitamin D3. The number of samples per group was established according to the CLSI recommendation protocol (EPO9-A2-IR). RESULTS: The agreement of LC-MS/MS with the Roche method was acceptable in the apparently healthy subjects group and in the post-BPD D3-supplemented group with an average bias of -1.7% and -9.2%, respectively. However, this agreement was unacceptable in the post-BPD D2-supplemented group with an average bias of -45.3%. The LC-MS/MS enabled us to detect four patients who had excess vitamin D or poisoning with vitamin D for which it was necessary to stop the supplementation with vitamin D in the D2 -supplemented group. CONCLUSION: Despite the apparent good agreement between the Roche method and LC-MS/MS in the healthy subjects group and in the post-DBP D3-supplemented patient group, a considerable bias seems to exist, particularly in the presence of D2. The LC-MS/MS method is therefore the most accurate method to follow the vitamin D2 -supplemented bariatric population.

Saturated fatty acid exposure induces androgen overproduction in bovine adrenal cells.

BACKGROUND: Polycystic ovary syndrome (PCOS) is mainly defined by hyperandrogenemia, from ovarian and adrenal origin, and is characterized by insulin resistance (IR). Studies found that raising in vivo non-esterified fatty acid (NEFA) levels, which induces lipotoxicity, increases androgen levels and IR. The aim of this study was therefore to determine the effects of in vitro over-exposure to NEFA on androgen synthesis in a bovine adrenocortical cell model. METHODS: Bovine fasciculata/reticularis cells were cultured for 2days in the absence or presence of ACTH (10nmol/L) or Forskolin (fsk, 10µmol/L), alone or in combination with the saturated fatty acid (FA) palmitate (100µmol/L). Steroid production was measured in medium and corrected for initial cell seeding count. CYP17 protein expression and ERK1/2 phosphorylation were assessed by Western blotting. RESULTS: Under unstimulated conditions, dehydroepiandrosterone (DHEA) levels were barely detected and no difference was observed after palmitate exposure, which was also the case for CYP17 expression and ERK1/2 phosphorylation. Under stimulation, palmitate exposure increased DHEA production by 38% and 69%, for ACTH and fsk, respectively, as compared to untreated conditions (Ps⩽0.05). In palmitate-treated vs untreated cells, fsk-stimulated ERK1/2 phosphorylation was reduced by 46% (P=0.0047), but stimulated CYP17 expression was not significantly affected. CONCLUSION: In a model of androgen-producing cells, under stimulated conditions, overexposure to saturated FAs significantly increases androgen production and reduces MEK/ERK activation. Therefore, this study is the first to demonstrate that lipotoxicity can directly trigger androgen overproduction in vitro, in addition to its well-described impact on IR, which strongly supports a central role of lipotoxicity in PCOS pathophysiology.

Reference ranges for total and calculated free and bioavailable testosterone in a young healthy women population with normal menstrual cycles or using oral contraception.

OBJECTIVE: Determine reference values for testosterone in women. DESIGN AND METHODS: 18-40 Year-old women with regular menses or using oral contraception were assessed by questionnaire and blood sample. RESULTS: In 155 women, reference values were different between women with and without hyperandrogenism for total testosterone (0.48-3.42 vs. 0.54-2.72 nmol/L) and calculated free (3-65 vs. 3-39 pmol/L) and bioavailable (0.06-1.35 vs. 0.06-0.81 nmol/L) testosterone. CONCLUSION: Upper reference values for testosterone in women must be established from a well-characterized population.

Prevalence and predictors of vitamin D insufficiency in women of reproductive age living in northern latitude.

OBJECTIVE: This study assessed the prevalence of vitamin D deficiency (serum 25-hydroxyvitamin D (25OHD) ≤ 50 nmol/l) and insufficiency (serum 25OHD 51-74 nmol/l) during summer and the predictors of serum 25OHD in young women of reproductive age. DESIGN: Cross-sectional study. METHODS: Between May and September 2006, 153 healthy, ambulatory and essentially Caucasian women, aged 18-41 years, were recruited. Serum 25OHD and parathyroid hormone (PTH) levels were measured, and questionnaires were evaluated. RESULTS: About 3.9% of women had serum 25OHD ≤ 50 nmol/l with an additional 26.8% in the insufficient range. Most women (56.9%) had their blood sampled in September. Month of blood collection significantly influenced serum 25OHD. Body mass index (BMI) was inversely associated with serum 25OHD, while traveling to a warmer climate during winter/spring and using oral contraceptive pills (OCP) were associated with higher serum 25OHD. Sunscreen was used by 77.8% of women, but only 3.3% reported consuming vitamin D supplements. BMI, serum PTH, travel to a warmer climate, and OCP use were independently and significantly associated with serum 25OHD, after adjustment for the month of sampling, and explained 40% of the variance in serum 25OHD. CONCLUSIONS: In Canada, the prevalence of vitamin D insufficiency is relatively high (30%) during summer in healthy women of reproductive age. Given the expected decrease in serum 25OHD during winter and the low consumption of vitamin D supplements, a high prevalence of vitamin D deficiency and insufficiency is to be anticipated during winter, except maybe for those traveling to a warmer climate.

Improving second-generation surveillance: the biological measure of unprotected intercourse using prostate-specific antigen in vaginal secretions of West African women.

BACKGROUND: Second-generation surveillance for HIV includes measures of high-risk behaviors among the general adult population and sex workers (SW). Questionnaires are prone to social desirability biases because individuals minimize the frequency of behaviors not expected from them. OBJECTIVE: Determine whether the prostate-specific antigen (PSA) could be used as a biological marker of unprotected intercourse. METHODS: We measured the presence of PSA in vaginal secretions of women who were (n = 508) or were not (n = 658) SW presenting with vaginal discharge in health facilities of Ghana, Togo, Guinea, and Mali. The cutoff for a positive assay was determined as > or =0.4 microg/L based on a subsample of 95 non-SW claiming abstinence for 3 months. RESULTS: A positive PSA assay was correlated with infections with Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium. Among non-SW, a positive PSA was more common among those with HIV, but less frequent in those better educated. Among SW and non-SW, women from Ghana were less likely to have a positive PSA and had a lower prevalence of sexually transmitted infections than those from elsewhere. CONCLUSIONS: PSA can be used as a biological marker of unprotected intercourse, allowing interventions to target efforts on those at highest risk.

"Hook effect" in calcitonin immunoradiometric assay in patients with metastatic medullary thyroid carcinoma: case report and review of the literature.

CONTEXT: Calcitonin is a well-established tumor marker for medullary thyroid carcinoma (MTC). Because surgery is the only effective treatment for patients with MTC, the postoperative level of serum calcitonin will dictate whether residual disease was left behind and whether reintervention is necessary. RESULTS: We describe here the case of a 41-yr-old man with metastatic MTC. Despite extensive disease in the neck as well as metastatic lesions in the liver, his serum calcitonin, measured with a commercial one-step immunoradiometric assay, was only minimally elevated (244 ng/liter). After serial dilutions, a nonlinear relationship became evident, suggesting the presence of a "hook effect." Treatment of the serum with heterophilic blocking reagent revealed no change. Calcitonin was then measured with a different immunoradiometric assay and revealed a much higher level. Similar discrepancies were found in different samples from various patients when analyzed with different calcitonin immunoassays. CONCLUSION: To our knowledge, this is the first reported case of a phenomenon such as the hook effect in a calcitonin immunoradiometric assay in patients with MTC. Being aware of this phenomenon is important, because a low calcitonin result could give false reassurance to both the patient and the clinician and could dramatically change the prognosis of the patient.