Bacterial overgrowth is one of the major concerns in collection and storage of biofluids, particularly 24-h urine. However, there is no previous systematic analysis of effects of bacterial overgrowth on urinary proteome analysis, and necessity, type, and appropriate concentration of preservatives to prevent bacterial overgrowth in the urine remain unclear. We, therefore, performed such systematic evaluation. Pooled normal urine was either centrifuged at 1500 g (to remove cell debris) or uncentrifuged. The samples were then added with either sodium azide (NaN3) or boric acid with various concentrations, and kept at room temperature (RT) or at 4 degrees C. Bacterial overgrowth was determined by UV-visible spectrophotometry (lambda620 nm) and Gram staining. At both temperatures, centrifugation to remove cell debris could effectively delay the bacterial overgrowth. At RT, both centrifuged and uncentrifuged samples without any preservative had the detectable overgrowth of Gram-positive and Gram-negative cocci and bacilli as early as 12 and 8 h, respectively, whereas 0.1-1 mM NaN3 and 2-20 mM boric acid could delay bacterial overgrowth, which started at 16-20 h in the centrifuged urine and 12-16 h in the uncentrifuged urine. Greater delay (for at least 48 h) was achieved with 10 mM NaN3 and 200 mM boric acid. At 4 degrees C, no bacterial overgrowth was detected in all centrifuged samples. However, it was observed at 20 h in the uncentrifuged urine without preservative, and at 48 h for the uncentrifuged urine with 0.1 mM NaN3 or 2 mM boric acid. There was no bacterial overgrowth detectable in the uncentrifuged urine preserved with higher concentrations of NaN3 or boric acid. 2-DE showed obvious changes in the urinary proteome profile of the sample with bacterial contamination, and the bacterial proteins could be identified by MALDI-TOF MS. Our data suggest that the urine should be centrifuged to remove cell debris and kept at 4 degrees C, rather than at RT, during the collection interval prior to long-term storage in the freezer. Moreover, the addition of 200 mM boric acid or 10 mM NaN3 is highly recommended for the prevention of bacterial overgrowth in the urine.

Beta-amyloid (A beta) plays a key role in the pathogenesis of Alzheimer's disease (AD) by inducing neurotoxicity and cell death mainly through production of reactive oxygen species (ROS). Garcinia mangostana L. (mangosteen) has been recognized as a major source of natural antioxidants that could decrease ROS. However, its role in protection of A beta-induced cytotoxicity and apoptosis in neuronal cells remains unclear. We therefore examined such a protective effect of mangosteen extract (ME) by evaluating cell viability using MTT test, ROS level, caspase-3 activity, and cellular proteome. Treating SK-N-SH cells with 5-20 microM A beta((1-42)) for 24 h caused morphologically cytotoxic changes, decreased cell viability and increased ROS level, whereas preincubation with 50-400 microg/mL ME 30 min before the induction by A beta((1-42)) successfully prevented such cytotoxic effects in a dose-dependent manner (completely at 400 microg/mL). The A beta-induced increase in caspase-3 activity was also preventable by 400 microg/mL ME. Proteomic analysis using 2-D gel electrophoresis (n = 5 gels/group) followed by mass spectrometry revealed 63 proteins whose levels were significantly altered by A beta((1-42)) induction. Interestingly, changes in 10 proteins were successfully prevented by the ME pretreatment. In summary, we report herein the significant protective effects of ME against A beta-induced cytotoxicity, increased ROS, and increased caspase activity in SK-N-SH cells. Moreover, proteomic analysis revealed some proteins that might be responsible for these protective effects by ME. Further characterizations of these proteins may lead to identification of novel therapeutic targets for successful prevention and/or decreasing the severity of AD.

One of the obstacles in analyzing frozen urine samples is the formation of uncharacterized precipitates. Frequently, these precipitates are discarded before analysis. Some laboratory data may be erroneous if these precipitates contain important compounds. In the present study, we examined urinary sediments precipitated after overnight storage at -20 degrees C. Although cells and debris were removed before freezing, the precipitates remained, whereas storing the centrifuged urine overnight at 4 degrees C did not result in precipitate formation. There were no significant differences observed among 10 healthy individuals (5 men and 5 women). EDTA (5 mM) could efficiently reduce the amount of precipitates to approximately 25% of the initial amount. The addition of exogenous CaCl2, but not sodium oxalate and NaCl, significantly increased the amount of precipitates in a dose-dependent manner. Linear regression analysis revealed a significant correlation between endogenous urinary calcium level and the amount of precipitates (r = 0.894; P < 0.001). Urine pH also had some effects on the type and amount of precipitates. These precipitates were composed mainly of calcium oxalate dihydrate and amorphous calcium crystals. The results also showed that these precipitates could deplete urinary proteins and calcium ions (23.6 +/- 1.1% decrease). Therefore, these freezer-induced urinary sediments significantly affect protein analysis and measurement of calcium levels in the urine. However, vigorous shaking of the sample at room temperature could redissolve these precipitates. Our data strongly indicate that these freezer-induced precipitates must be taken into account when the frozen urine samples are analyzed.