Development of a LC-MS/MS method to measure serum 3-sulfate and 3-glucuronide 25-hydroxyvitamin D3 metabolites; comparisons to unconjugated 25OHD in pregnancy and polycystic ovary syndrome.

Vitamin D status is routinely assessed by measuring circulating concentrations of 25-hydroxyvitamin D (25OHD2 or 25OHD3). However as deconjugation is not routinely incorporated into sample treatment prior to analysis, conjugated forms of 25OHD (particularly the more abundant 25OHD3) are often not considered in determining serum concentrations of total 25OHD. Two major circulating conjugated forms of 25OHD3 are 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25OHD3-G). Incorporating these two conjugated metabolites into the measurement of vitamin D status could improve our understanding of vitamin D status in health, particularly if there are changes in sulfation and glucuronidation activities. The aim of this study was to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) targeted method for measurement of 25OHD3-S and 25OHD3-G in serum to enable comparisons with circulating levels of the free 25OHD3 form. We developed and validated a new LC-MS/MS method that measured both 25OHD3-S and 25OHD3-G following a solid phase extraction sample preparation method. Partial separation of analytes by LC, and the separation of analytes by the optimized multiple reaction monitoring transitions enabled the quantitation of both 25OHD3-S and 25OHD3-G in the single method. Serum concentrations of 25OHD3-S (24.7 ± 11.8 ng/mL) and 25OHD3-G (2.4 ± 1.2 ng/mL) were shown to be a significant proportion of circulating vitamin D metabolites in healthy donor serums. These levels of 25OHD3-S and 25OHD3-G closely associated with 25OHD3 concentrations, r = 0.728, p = 0.001 and r = 0.632, p = 0.006 respectively. However in serum from pregnant women and non-pregnant women with polycystic ovary syndrome (PCOS) significant differences in the ratios between conjugated and free 25OHD3 were observed between pregnancy groups (25OHD3/25OHD3-S and 25OHD3/25OHD3-G p < 0.001), and between healthy and PCOS subjects (25OHD3/25OHD3-G p < 0.050). Development of this novel high-throughput LC-MS/MS method indicates that 25OHD3-S and 25OHD3-G are substantial components of circulating vitamin D metabolites. The concentrations of these metabolites relative to conventional 25OHD3 may vary in different physiological and pathophysiological settings, and may therefore play an unrecognized but important role in the actions of vitamin D.

Clinico-microbiological profile of urinary tract infection in south India.

The knowledge of etiology and antibiotic resistance pattern of the organisms causing urinary tract infection is essential. This study was taken up to determine the presentation and risk factors associated with community-acquired urinary tract infection (CA-UTI). The distribution of bacterial strains isolated from these patients and their resistance pattern were also studied. This multidisciplinary prospective observational study was conducted in M. S. Ramaiah Hospital, Bangalore, between January and December, 2008. Patients who had CA-UTI confirmed by positive urine culture reports were included in the study. Statistical analysis was done using the SPSS version 16. Symptomatology and others risk factors for CA-UTI were studied in these patients and the causative organisms and their resistance patterns were recorded. Of the total 510 patients included, 57% belonged to the elderly age group (50-79 years). Fever and dysuria were the most common clinical presentation, but were not specific in predicting CA-UTI. Escherichia coli (66.9%) was the most common organism causing CA-UTIs with extended spectrum beta lactamase (ESBL) resistance seen in nearly two-thirds of these cases (42.2%). The organisms recorded least resistance against carbapenems (3.9%). A high resistance rate was seen for fluoroquinolones (74.1%). In conclusion, a high rate of ESBL-positive organisms and their resistance to commonly used antibiotics brings a concern for future options in treating these conditions.