Novel LC-MS/MS Targeted Analysis Shows that Retinol to RBP4 and TTR to RBP4 Ratio Decreases in Individuals with Diabetic Kidney Disease

Abstract ID 90343 Poster Board 028 Retinol binding protein 4 (RBP4) is critical in transport of retinol (vitamin A). RBP4 is also an adipokine. Elevated circulating RBP4 has been proposed as a biomarker of chronic kidney disease (CKD) and diabetes. However, CKD and diabetes are often comorbid conditions, and it is not clear which condition is causal for elevated RBP4 levels. RBP4 binds its ligand, retinol, with high affinity and this holo-RBP4 forms a complex with transthyretin (TTR) at a stoichiometry of two RBP4 to one TTR homotetramer. Unliganded (apo-) RBP4 has been suggested to have different physiological functions than holo-RBP4 beyond the delivery of retinol. Apo-RBP4 is rapidly cleared by renal filtration due to its small size (21 kDa). Binding of holo-RBP4 to TTR prevents renal elimination of RBP4 and the holo-RBP4-TTR complex has a 6-fold longer half-life than apo-RBP4. Hence apo- to holo-RBP4 ratio reflects overall RBP4 signaling and homeostasis and has been proposed as a biomarker of metabolic disease. We have shown that circulating RBP4 and retinol concentrations are higher in patients with CKD when compared to healthy subjects, whereas TTR concentrations are unaffected. However, apo- to holo-RBP4-TTR ratios could not be determined due to limitations of immunoassays used to quantify RBP4 and TTR and LC-UV methods used for retinol quantification. To assess retinol:RBP4 and TTR:RBP4 ratios in different patient populations, a novel LC-MS/MS based method was developed for absolute quantification of RBP4 and TTR. This method was applied to quantify RBP4 and TTR in plasma samples from patients (n = 49, 63% female, 24–61 years old) with diabetes and a range of kidney function (eGFR: 30–150 mL/min/1.73m2). We hypothesized that RBP4, retinol, and holo-RBP4 concentrations will increase with decreasing eGFR. We expect TTR will remain unchanged and the TTR:RBP4 ratio will decrease with decreasing eGFR. LC-MS/MS method development included optimization of a surrogate matrix for standard curves, digest conditions including reducing agents, source of trypsin, and trypsin digest time. Surrogate peptides were selected from in silico analysis, and peptides with reproducible, linear response were used for quantification with synthetic heavy labelled internal standards. The assay was validated in accordance with FDA Bioanalytical Guidance and had <10% inter-assay variability. The concentrations (mean and interquartile range (IQR)) in this patient population were as follows: retinol 3.35 mM (IQR 2.71, 4.28), RBP4 2.93 mM (2.36, 3.46), and TTR 5.09 mM (4.25, 5.60). The mean retinol:RBP4 ratio was 1.16 suggesting that retinol and RBP4 are present in circulation as the tight holo-RBP4 complex. RBP4 concentrations were significantly increased with decreasing eGFR (p = 0.024), while TTR was unchanged. The mean TTR:RBP4 ratio decreased from 2.34 in healthy eGFR to 1.74 with decreasing eGFR (p = 0.001) indicating that in healthy kidney function holo-RBP4 is entirely complexed with TTR tetramer while in CKD excess holo-RBP4 exists. The mean retinol:RBP4 ratio decreased from 1.64 in healthy eGFR to 1.14 with decreasing eGFR (p = 0.01). This suggests that in healthy individuals 40% of plasma retinol is bound to other binding partners besides RBP4 but this binding is lost in CKD. Taken together, these findings indicate RBP4 synthesis and holo-RBP4 secretion from the liver are increased in CKD while RBP4 and TTR expression are not coregulated in the liver. This highlights that complex multi-organ nature of CKD and diabetes.