Androsterone glucuronide to dehydroepiandrosterone sulphate ratio is discriminatory for obese Caucasian women with polycystic ovary syndrome.

BACKGROUND: Androsterone glucuronide (ADTG) concentrations have been suggested as a marker of the effects of androgens at the target tissue level. As the mechanism for hyperandrogenemia in obese and nonobese polycystic ovary syndrome (PCOS) may differ, this study compared the different androgen parameters in non-obese compared to obese women with PCOS, and in normal subjects. METHODS: Eleven non-obese and 14 obese women with PCOS were recruited and compared to 11 control women without PCOS. Total testosterone, dehydroepiandrosterone sulphate (DHEAS), ADTG, and androstenedione were analysed using gold standard tandem mass spectrometry, and the free androgen index (FAI) was calculated. RESULTS: Total testosterone, ADTG and androstendione levels did not differ between non-obese (body mass index (BMI) ≤25 kg/m2) and obese PCOS (BMI >25 kg/m2) but all were significantly higher than for controls (p < 0.01). The ADTG to DHEAS ratio was significantly elevated 39 ± 6 (p < 0.01) in obese PCOS in comparison to non-obese PCOS and controls (28 ± 5 and 29 ± 4, respectively). The free androgen index (FAI) and insulin resistance (HOMA-IR) were significantly higher in obese PCOS compared to non-obese PCOS and controls (p < 0.01). DHEAS was significantly higher in the non-obese versus obese PCOS (p < 0.01). All androgen parameters were significantly lower and sex hormone binding globulin (SHBG) significantly higher in normal subjects compared to those with obese and non-obese PCOS. CONCLUSIONS: The ADTG:DHEAS ratio was significantly elevated in obese PCOS compared to non-obese PCOS and controls suggesting that this may be a novel biomarker discriminatory for obese PCOS subjects, perhaps being driven by higher hepatic 5α reductase activity increasing ADTG formation in these women.

Measurement of plasma 5-hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry--comparison with HPLC methodology.

In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 micorL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters Quattro Premier XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/z 191.9>145.6 and 193.9>147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97-113%) and the method showed good linearity to 10,000 nmol/L (r(2)=0.999). Within-batch and between-batch imprecision was <10% and bias <15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC-MS/MS and HPLC showed good agreement between the two methods but this LC-MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.

Adenovirally mediated expression of insulin-like growth factors enhances the function of first trimester placental fibroblasts.

IGFs are critical in fetal growth because of their role in placental development and function. In this study, we used adenovirus (Ad-IGF) to deliver sense or antisense IGF-I or IGF-II cDNA to human primary placental fibroblasts (PPF) in vitro to determine whether this could lead to enhanced placental cell function. PPFs virally transfected with Ad-IGF-I or Ad-IGF-II showed 7-fold (P < 0.01) and 3-fold (P < 0.01) increases in [(3)H]thymidine incorporation at 48 h post infection compared with nontransfected controls. In a coculture system designed to assess cell migration, nontransfected PPF cells positioned over a monolayer transfected by Ad-IGF-I or Ad-IGF-II showed a more than 10-fold (P < 0.01) and a 7-fold (P < 0.01) increase in migration compared with cells positioned above a nontransfected monolayer. After 96 h in culture, PPFs transfected with sense Ad-IGF-I or Ad-IGF-II showed 2% apoptosis compared with 16% of nontransfected cells, whereas 37% and 25% of cells transfected with antisense Ad-IGF-I or Ad-IGF-II were apoptotic. This work has established that cells of placental origin are amenable to adenoviral transfection and that IGFs exert autocrine and paracrine effects on proliferation, migration, and survival, suggesting that enhancement of IGF levels in the placenta may augment placental function and increase fetal growth.