The determination of nicotinic acid in plasma by mixed-mode liquid chromatography-tandem mass spectrometry following ion exchange solid phase extraction

An assay for nicotinic acid in plasma samples has been developed using ion exchange solid phase extraction in 96-well format followed by mixed-mode ion exchange/reversed-phase liquid chromatography with positive ion tandem mass spectrometry detection. The assay avoids the need for time-consuming derivatisation procedures or involatile ion-pair chromatography reagents. The assay is linear over the wide range 0.05-20 microg/mL, based on a 100 microL sample (correlation coefficient>0.99). The assay is accurate and precise (bias and coefficient of variation<18%) over this calibration range.     

Access to drinking-water and arsenicosis in Bangladesh

The discovery of arsenic contamination in groundwater has challenged efforts to provide safe drinking-water to households in rural Bangladesh. Two nationally-representative surveys in 2000 and 2002 investigated water-usage patterns, water-testing, knowledge of arsenic poisoning, and behavioural responses to arsenic contamination. Knowledge of arsenicosis rose between the two surveys among women from 42% to 64% but awareness of consequences of arsenic remained limited; only 13% knew that it could lead to death. Behavioural responses to arsenic have been limited, probably in part because of the lack of concern but also because households are uncertain of how best to respond and have a strong preference for tubewell water even when wells are known to be contaminated. Further work conducted by the survey team highlighted the difficulties in providing alternative sources of water, with many households switching back to their original sources of water.     

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.     

Biomarker and drug-target discovery using proteomics in a new rat model of sepsis-induced acute renal failure

Sepsis is one of the common causes of acute renal failure (ARF). The objective of this study was to identify new biomarkers and therapeutic targets. We present a new rat model of sepsis-induced ARF based on cecal ligation and puncture (CLP). We used this model to find urinary proteins which may be potential biomarkers and/or drug targets. Aged rats were treated with fluids and antibiotics after CLP. Urinary proteins from septic rats without ARF and urinary proteins from septic rats with ARF were compared by difference in-gel electrophoresis (DIGE). CLP surgery elevated interleukin (IL)-6 and IL-10 serum cytokines and blood nitrite compared with sham-operated rats. However, there was a range of serum creatinine values at 24 h (0.4-2.3 mg/dl) and only 24% developed ARF. Histology confirmed renal injury in these rats. Forty-nine percent of rats did not develop ARF. Rats without ARF also had less liver injury. The mortality rate at 24 h was 27% but was increased by housing the post-surgery rats in metabolic cages. Creatinine clearance and urine output 2-8 h after CLP was significantly reduced in rats which died within 24 h. Using DIGE we identified changes in a number of urinary proteins including albumin, brush-border enzymes (e.g., meprin-1-alpha) and serine protease inhibitors. The meprin-1-alpha inhibitor actinonin prevented ARF in aged mice. In summary, we describe a new rat model of sepsis-induced ARF which has a heterogeneous response similar to humans. This model allowed us to use DIGE to find changes in urinary proteins and this approach identified a potential biomarker and drug target - meprin-1-alpha.