Simone K. Frey

Evidence That Kidney Function but Not Type 2 Diabetes Determines Retinol-Binding Protein 4 Serum Levels

OBJECTIVE— It has been suggested that retinol-binding protein 4 (RBP4) links adiposity, insulin resistance, and type 2 diabetes. However, circulating RBP4 levels are also affected by kidney function. Therefore, the aim of this study was to test whether RBP4 serum levels are primarily associated with kidney function or type 2 diabetes. RESEARCH DESIGN AND METHODS— RBP4 serum concentration was determined by enzyme-linked immunosorbent assay in 126 nondiabetic and 104 type 2 diabetic subjects. The study population was divided according to estimated glomerular filtration rate (eGFR) into the following groups: eGFR >90 ml/min per 1.73 m2 (n = 53), 60–90 ml/min per 1.73 m2 (n = 90), 30–60 ml/min per 1.73 m2 (n = 38), and <30 ml/min per 1.73 m2 (n = 49). Each group was subdivided into nondiabetic and type 2 diabetic subjects. RESULTS— RBP4 serum concentration was elevated (2.65 vs. 2.01 μmol/l; P < 0.001) and eGFR was reduced (56 vs. 74 ml/min per 1.73 m2; P < 0.001) in type 2 diabetic vs. nondiabetic subjects, respectively. By stratifying for eGFR, no more differences in RBP4 serum concentration were detectable between type 2 diabetic and nondiabetic subjects. A linear regression analysis revealed an influence of eGFR (r = −0.477; P < 0.001) but not A1C (r = 0.093; P = 0.185) on RBP4 serum concentration. CONCLUSIONS— Existing human data showing elevated RBP4 levels in type 2 diabetic patients may be the result of moderate renal insufficiency rather than support for the suggestion that RBP4 links obesity to type 2 diabetes.

Isoforms of Retinol binding protein 4 (RBP4) are increased in chronic diseases of the kidney but not of the liver

BackgroundThe levels of retinol-binding protein 4 (RBP4) – the carrier protein for Vitamin A in plasma – are tightly regulated under healthy circumstances. The kidney, the main site of RBP4 catabolism, contributes to an elevation of RBP4 levels during chronic kidney disease (CKD) whereas during chronic liver disease (CLD) RBP4 levels decrease. Little is known about RBP4 isoforms including apo-RBP4, holo-RBP4 as well as RBP4 truncated at the C-terminus (RBP4-L and RBP4-LL) except that RBP4 isoforms have been reported to be increased in hemodialysis patients. Since it is not known whether CLD influence RBP4 isoforms, we investigated RBP4 levels, apo- and holo-RBP4 as well as RBP4-L and RBP4-LL in plasma of 36 patients suffering from CKD, in 55 CLD patients and in 50 control subjects. RBP4 was determined by ELISA and apo- and holo-RBP4 by native polyacrylamide gel electrophoresis (PAGE). RBP4-L and RBP4-LL were analyzed after immunoprecipitation by mass spectrometry (MALDI-TOF-MS).ResultsRBP4 isoforms and levels were highly increased in CKD patients compared to controls (P < 0.05) whereas in CLD patients RBP4 isoforms were not different from controls. In addition, in hepatic dysfunction RBP4 levels were decreased whereas the amount of isoforms was not affected.ConclusionThe occurrence of RBP4 isoforms is not influenced by liver function but seems to be strongly related to kidney function and may therefore be important in investigating kidney function and related disorders.

Alterations of retinol-binding protein 4 species in patients with different stages of chronic kidney disease and their relation to lipid parameters

Retinol-binding protein 4 (RBP4) is elevated in patients with chronic kidney disease (CKD) and has been discussed as marker of kidney function. In addition to an elevated concentration, the existence of truncated RBP4 species, RBP4-L (truncated at last C-terminal leucine) and RBP4-LL (truncated at both C-terminal leucines), has been reported in serum of hemodialysis patients. Since little is known about the occurrence of RBP4 species during the progression of CKD it was the aim of this study to analyse this possible association. The presence of RBP4, RBP4-L, RBP4-LL and transthyretin (TTR) was assessed in serum of 45 healthy controls and 52 patients with stage 2-5 of CKD using ELISA and RBP4 immunoprecipitation with subsequent MALDI-TOF-MS analysis. A reduction of glomerular filtration rate was accompanied by a gradual elevation of RBP4 serum levels and relative amounts of RBP4-LL. Correlation analysis revealed a strong association of the RBP4-TTR ratio with parameters of lipid metabolism and with diabetes-related factors. In conclusion, RBP4 serum concentration and the appearance of RBP4-LL seem to be influenced by kidney function. Furthermore, the RBP4-TTR ratio may provide diagnostic potential with regard to metabolic complications in CKD patients.

Factors that influence retinol-binding protein 4–transthyretin interaction are not altered in overweight subjects and overweight subjects with type 2 diabetes mellitus

Retinol-binding protein 4 (RBP4) is an adipokine bound in plasma to transthyretin (TTR), which prevents its glomerular filtration and subsequent catabolism in the kidney. Alterations of this interaction have been suggested to be implicated in the elevation of RBP4 that are thought to contribute to the development of insulin resistance associated with obesity and type 2 diabetes mellitus (T2DM). However, the factors linking RBP4 to TTR in humans are not clear. Therefore, this study evaluated parameters influencing the RBP4-TTR interaction and their relation to obesity and T2DM. The RBP4 and TTR levels were quantified in plasma of 16 lean controls, 28 overweight controls, and 14 overweight T2DM patients by enzyme-linked immunosorbent assay. Transthyretin isoforms involved in RBP4 binding were determined by linear matrix-assisted laser desorption/ionization-time of flight-mass spectrometry after RBP4 coimmunoprecipitation. Holo-RBP4 (retinol-bound) and apo-RBP4 (retinol-free) were assessed by immunoblotting using nondenaturating polyacrylamide gel electrophoresis. Plasma levels of both RBP4 and TTR did not differ among the groups of lean controls, overweight controls, and overweight T2DM subjects. Using RBP4 immunoprecipitation, 4 mass signals were observed for TTR representing native, S-cysteinylated, S-cysteinglycinylated, and S-glutathionylated TTR. No differences in peak intensity of TTR isoforms were observed among the groups. Moreover, no differences in the ratio of holo- and apo-RBP4 were evident. The results suggest that circulating RBP4 and TTR were not affected by human obesity or T2DM, which might be attributed to the absence of alterations of TTR isoforms and the ratio of holo- and apo-RBP4 that might modify the TTR-RBP4 interaction.

Quantification of Vitamin A in Palm Oil Using a Fast and Simple Portable Device: Method Validation and Comparison to High-Performance Liquid Chromatography

Vitamin A deficiency continues to be a global public health problem. Fortification of oil with vitamin A is considered a cost-effective, feasible strategy to prevent this problem but quality control poses a challenge to program implementation. To overcome this, we have validated a newly developed device that quantitatively measures the content of retinyl palmitate in refined palm oil, is simple to use, and yields immediate results.Linearity of analysis ranged from 2.5 - 30 mg retinol equivalents (RE)/ kg of palm oil, with 2.5 mg RE/kg being the determination limit; inter- and intra-assay precision ranged from 1.4 - 7.1 %. Comparison with a high-performance liquid chromatography method showed high agreement between the methods (R(2) = 0.92; Limits of Agreement: -1.24 mg to 2.53 mg RE/kg), and further comparisons illustrate that the new device is useful in low-resource settings. This device offers a field- and user-friendly solution to quantifying the vitamin A content in refined palm oil.

Effect of renal replacement therapy on retinol-binding protein 4 isoforms

BACKGROUND Retinol-binding protein 4 (RBP4) levels are elevated in the serum of patients with kidney dysfunction. We recently showed that RBP4 isoforms including apo-RBP4 (RBP4 not bound to retinol) and RBP4 truncated at the C-terminus (RBP4-L, RBP4-LL) are increased in the serum of patients with kidney diseases but not in serum of patients with various liver diseases. The aim of this study was to investigate the effect of renal replacement therapy on RBP4 isoforms. METHODS We investigated serum levels of RBP4, apo-RBP4, holo-RBP4, RBP4-L, RBP4-LL, retinol and transthyretin (TTR) in 18 hemodialysis (HD) patients, 30 patients after renal transplantation (RTx) and in 35 healthy controls. RBP4 and TTR levels were measured by enzyme-linked immunosorbent assay, apo- and holo-RBP4 by native electrophoresis, retinol by high performance liquid chromatography and RBP4-L and RBP4-LL were analyzed by mass spectrometry. RESULTS HD and RTx patients had elevated RBP4, apo-RBP4 and RBP4-LL levels compared to controls. RTx patients had elevated amounts of RBP4-L compared to controls and elevated RBP4 and apo-RBP4 levels compared to HD patients. CONCLUSION The results demonstrate a strong correlation between kidney function and RBP4 isoforms and provide data for investigating the relation of RBP4 and insulin resistance in these patients.

Validation of a User-Friendly and Rapid Method for Quantifying Iodine Content of Salt

Background Despite considerable progress made in the past decade through salt iodization programs, over 2 billion people worldwide still have inadequate iodine intake, with devastating consequences for brain development and intellectual capacity. To optimize these programs with regard to salt iodine content, careful monitoring of salt iodine content is essential, but few methods are available to quantitatively measure iodine concentration in a simple, fast, and safe way. Objective We have validated a newly developed device that quantitatively measures the content of potassium iodate in salt in a simple, safe, and rapid way. Methods The linearity, determination and detection limit, and inter- and intra-assay variability of this colorimetric method were assessed and the method was compared with iodometric titration, using salt samples from several countries. Results Linearity of analysis ranged from 5 to 75 mg/kg iodine, with 1 mg/kg being the determination limit; the intra- and interassay imprecision was 0.9%, 0.5%, and 0.7% and 1.5%, 1.7%, and 2.5% for salt samples with iodine contents of 17, 30, and 55 mg/kg, respectively; the interoperator imprecision for the same samples was 1.2%, 4.9%, and 4.7%, respectively. Comparison with the iodometric method showed high agreement between the methods (R2 = 0.978; limits of agreement, −10.5 to 10.0 mg/kg). Conclusions The device offers a field- and user-friendly solution to quantifying potassium iodate salt content reliably. For countries that use potassium iodide in salt iodization programs, further validation is required.