BACKGROUND: Hyperbilirubinemia in jaundiced neonates is routinely assessed by use of total serum bilirubin. However, the unbound or free form (B f ), not total bilirubin, crosses the blood-brain barrier and can be neurotoxic. Although the peroxidase-mediated oxidation of bilirubin can be used to measure plasma concentrations of B f , this measurement is relatively complex and the assay is not routinely used. We describe a fluorescence sensor for quantifying B f in plasma.

We report the first measurements to profile mixtures of unbound free fatty acids. Measurements utilized fluorescent probes with distinctly different response profiles for different free fatty acids (FFA). These probes were constructed by labeling site-specific mutants of the rat intestinal fatty acid binding protein (rI-FABP) with acrylodan. The probes were produced and screened by high throughput methods and from more than 30,000 such probes we selected 6 that together have sufficient specificity and sensitivity to resolve the profile of unbound FFA (FFA u ) in mixtures of different FFA u . We developed analytical methods to determine the FFA u profile from the fluorescence (ratio) response of the different probes and used these methods to determine FFA u profiles for mixtures of arachidonate, linoleate, oleate, palmitate and stearate in equilibrium with bovine serum albumin (BSA). Measurements were performed using mixtures with a range of total FFA u concentrations, including 0.9 nM, which is similar to normal plasma levels. We also measured single FFA binding isotherms for BSA and found that binding was well described by 6-7 sites with the same binding constants (K d ). The K d values for the FFA (4 to 38 nM) were inversely related to the aqueous solubility of the FFA. We constructed a model with these parameters to predict the FFA u profile in equilibrium with BSA and found excellent agreement between the profiles measured using the FFA probes and those calculated with this model. These results should lead to a better understanding of albumin's role in buffering FFA u and to profiling FFA u in intra and extracellular biological fluids. Keywordsunbound free fatty acids; fatty acid binding proteins; fluorescent probes; site-specific mutants; high throughput screening; fatty acid profiling; binding affinities; bovine serum albumin Metabolomics is advancing rapidly as a result of new technologies and the expanding interest in systems biology (1). This advance is being driven by the recognition that physiologic phenotype is essentially a reflection of the metabolic profile, and therefore, metabolic profiling should provide an accurate representation of the states of health and disease. The activity of a given metabolite is frequently dictated by its solubility as a "free" or unbound molecule in aqueous bodily fluids. For many metabolites the unbound concentration represents a small fraction of the total, with most of the total metabolite bound in carrier complexes. Total metabolite concentrations are typically measured, but it is the unbound metabolite that interacts

Background Persistent acute kidney injury (AKI) portends worse clinical outcomes and remains a therapeutic challenge for clinicians. A recent study found that urinary C–C motif chemokine ligand 14 (CCL14) can predict the development of persistent AKI. We aimed to externally validate urinary CCL14 for the prediction of persistent AKI in critically ill patients. Methods This was a secondary analysis of the prospective multi-center SAPPHIRE study. We evaluated critically ill patients with cardiac and/or respiratory dysfunction who developed Kidney Disease: Improving Global Outcomes (KDIGO) stage 2–3 AKI within one week of enrollment. The main exposure was the urinary concentration of CCL14 measured at the onset of AKI stage 2–3. The primary endpoint was the development of persistent severe AKI, defined as  ≥ 72 h of KDIGO stage 3 AKI or death or renal-replacement therapy (RRT) prior to 72 h. The secondary endpoint was a composite of RRT and/or death by 90 days. We used receiver operating characteristic (ROC) curve analysis to assess discriminative ability of urinary CCL14 for the development of persistent severe AKI and multivariate analysis to compare tertiles of urinary CCL14 and outcomes. Results We included 195 patients who developed KDIGO stage 2–3 AKI. Of these, 28 (14%) developed persistent severe AKI, of whom 15 had AKI  ≥ 72 h, 12 received RRT and 1 died prior to  ≥ 72 h of KDIGO stage 3 AKI. Persistent severe AKI was associated with chronic kidney disease, diabetes mellitus, higher non-renal APACHE III score, greater fluid balance, vasopressor use, and greater change in baseline serum creatinine. The AUC for urinary CCL14 to predict persistent severe AKI was 0.81 (95% CI, 0.72–0.89). The risk of persistent severe AKI increased with higher values of urinary CCL14. RRT and/or death at 90 days increased within tertiles of urinary CCL14 concentration. Conclusions This secondary analysis externally validates urinary CCL14 to predict persistent severe AKI in critically ill patients.

Background: Clinical use of biomarkers requires the development of standardized assays and establishment of cutoffs. Urinary C-C-motif chemokine ligand 14 (CCL14) has been validated to predict persistent severe AKI in critically ill patients with established AKI. We now report on the performance of standardized cutoffs using a clinical assay. Methods: A second aim of the multicenter Ruby study was to establish two cutoffs for the prediction of persistent severe AKI (defined as KDIGO stage 3 AKI for at least 72 consecutive hours). Patients who received kidney replacement therapy (KRT) or died prior to achieving 72 hours in stage 3 AKI were also considered to have reached the endpoint. Results: A cutoff value for urinary CCL14 of 1.3 ng/ml was determined to achieve high sensitivity (91%, (95%CI 84%-96%)) and 13 ng/ml achieved high specificity (93% (89%-96%)). The cutoff of 1.3 ng/mL identifies the majority (91%) of patients who developed persistent severe AKI with a negative predictive value of 92%. The cutoff at 13 ng/ml had a positive predictive value of 72% (with a negative predictive value of 75%). In multivariable adjusted analyses, a CCL14 concentration between 1.3 and 13 ng/mL had an adjusted odds ratio (aOR) of 3.82 (95%CI 1.73-9.12) p=0.001 for the development of persistent severe AKI compared to those with CCL14 ≤ 1.3, while a CCL14 > 13 ng/ml had an aOR of 10.4 (3.89-29.9); p<0.001. Conclusions: Using a clinical assay, these standardized cutoffs (1.3 and 13 ng/mL) allow for the identification of patients at high risk for the development of severe persistent AKI. These results have immediate utility in helping to guide AKI patient care and may facilitate future clinical trials.