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Relation between retinol, retinol-binding protein 4, transthyretin and carotid intima media thickness
Atherosclerosis 213 (2010) 549–551
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Relation between retinol, retinol-binding protein 4, transthyretin and carotid intima media thickness Thomas Bobbert a , Jens Raila c , Franziska Schwarz a , Knut Mai a , Andrea Henze c , Andreas F.H. Pfeiffer a,b , Florian J. Schweigert c , Joachim Spranger a,b,∗ a b c
Department of Endocrinology, Diabetes and Nutrition, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany Department of Clinical Nutrition, German Institute of Human Nutrition, Nuthetal, Germany Department of Physiology and Pathophysiology of Nutrition, Institute of Nutritional Science, University of Potsdam, Potsdam, Germany
a r t i c l e
i n f o
Article history: Received 17 June 2010 Received in revised form 20 July 2010 Accepted 31 July 2010 Available online 19 August 2010 Keywords: RBP4 Retinol IMT
a b s t r a c t Objective: Retinol is transported in a complex with retinol-binding protein 4 (RBP4) and transthyretin (TTR) in the circulation. While retinol is associated with various cardiovascular risk factors, the relation between retinol, RBP4, TTR and carotid intima media thickness (IMT) has not been analysed yet. Methods: Retinol, RBP4 and TTR were measured in 96 individuals and their relation to mean and maximal IMT was determined. Results: Mean IMT correlated with RBP4 (r = 0.335, p < 0.001), retinol (r = −0.241, p = 0.043), RBP/TTR ratio (r = 0.254, p = 0.025) and retinol/RBP4 ratio (r = −0.549, p < 0.001). Adjustment for age, sex, BMI, blood pressure, HDL/total cholesterol ratio, triglyceride, diabetes and smoking revealed that the retinol/RBP4 ratio was strongly and independently associated with mean IMT. Similar results were found for maximal IMT, which included the measurement of plaques. Conclusion: The data support that the transport complex of vitamin A is associated with the IMT, an established parameter of atherosclerosis. Changes in RBP4 saturation with retinol may link renal dysfunction and insulin resistance to atherosclerosis. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Objective
2. Methods
RBP4 is the only specific carrier protein for retinol in the circulation. Recent data suggested that RBP4 itself may induce insulin resistance, since elevated RBP4 levels have been found in patients with type 2 diabetes (T2DM) [1,2]. Additionally, elevated RBP4 levels have also been described in individuals with impaired renal function [3]. Thus, circulating RBP4 seems to be a marker for cardiovascular disease especially in insulin resistance or T2DM. In line with these findings, a correlation between RBP4 and IMT has been recently described [4,5]. However, these studies did not consider the impact of retinol or transthyretin (TTR), although these components are known to strongly interact with RBP4 in the transport complex of vitamin A and may themselves affect cardiovascular risk [6]. We therefore investigated the association of retinol, RBP4, and TTR with intima media thickness (IMT).
96 individuals (44 males, 52 females), including 34 individuals with T2DM and 8 smokers, were randomly drawn from the MeSyBePo-study cohort (Metabolic-Syndrome-Berlin–Potsdam). Mean age was 55 ± 1.3 years with a body mass index of 30.8 ± 0.7 kg/m2 . Mean blood pressure was 131/80 ± 2/1 mm Hg. The following anti-diabetic, anti-hypertensive and lipid lowering drugs were used by study participants: metformin (n = 8), sulfonylureas (n = 5), acarbose (n = 1), glinides (n = 2), pioglitazone (n = 1), calcium antagonists (n = 6), beta blockers (n = 8), ACE inhibitors (n = 13), AT1-receptor antagonists (n = 2), diuretics (n = 7) and statins (n = 8). Renal function was estimated by the abbreviated MDRD study equation with individuals having a mean eGFR of 82.1 ± 1.7 ml/min/1.73 m2 . Lipids (mean LDL cholesterol 1.89 ± 0.05 mmol/l, mean HDL cholesterol 1.37 ± 0.04 mmol/l, mean triglycerides 1.51 ± 0.07 mmol/l) and glucose metabolism (mean fasting glucose 107 ± 2.3 mg/dl, mean fasting insulin 11.5 ± 1.0 mU/l, mean HbA1c 5.8 ± 0.1%) were measured by standard techniques. A description of the detailed phenotyping and laboratory methods was previously published [3,7,8]. Measurement of RBP4 (mean 1.89 ± 0.05 mol/l) and TTR (mean 4.63 ± 0.17 mol/l) was also performed as previously described [3]. Retinol (1.78 ± 0.04 g/ml) was determined by rpHPLC. Carotid
∗ Corresponding author at: Charité, Department of Endocrinology, Diabetes and Nutrition, Hindenburgdamm 30, 12200 Berlin, Germany, Tel.: +49 30 8445 2287; fax: +49 30 8445 4204. E-mail address: [email protected] (J. Spranger). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.07.063
550
T. Bobbert et al. / Atherosclerosis 213 (2010) 549–551
Table 1 Coefficients of correlation between parameters of the retinoid system and other cardiovascular risk factors, *p < 0.05 or † p < 0.001. RBP4 Age BMI IMT IMTMAX Systolic BP Fasting glucose HbA1c HDL cholesterol LDL cholesterol Triglycerides MDRD
†
0.298 0.130 0.335† 0.290* −0.026 0.308† 0.341† −0.192 0.107 0.319† −0.117
Retinol
TTR
RBP4/TTR ratio
Retinol/RBP4 ratio
−0.084 −0.075 −0.241* −0.198 0.000 −0.005 −0.193 0.021 −0.027 0.172 0.152
0.129 −0.174 0.084 0.063 −0.015 0.158 0.124 0.037 0.109 0.070 0.067
0.141 0.380† 0.254* 0.229* 0.025 0.092 0.194 −0.230* −0.058 0.255* −0.171
−0.311† −0.274† −0.549† −0.476† −0.096 −0.362† −0.523† 0.345† −0.098 −0.236* 0.177
arterial IMT was measured at the posterior wall of the common carotid artery at three different positions using a high resolution ultrasound (Kretz AG, Germany). Mean and maximum values for IMT, which included possible plaques (IMTMAX ), were calculated (IMT = 0.719 ± 0.016 mm) and compared with the concentrations retinol, RBP4 and TTR in plasma. The experimental protocol of the study was approved by the Institutional Review Board; all subjects gave written informed consent. Statistical calculations were performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA).
smoking (yes/no), eGFR and T2DM (yes/no)) the retinol/RBP4 ratio remained significantly associated with mean IMT and explained a considerable proportion of the IMT variation (19.4%) (Table 2). Adjustment for current medication did also not substantially modify our findings. Similar results were found for IMTMAX . Thus, only BMI, age and the retinol/RBP4 ratio were independently and significantly associated with IMTMAX and explained in total about 44% of the variability of IMTMAX .
4. Discussion 3. Results RBP4 was higher in diabetic compared to non-diabetic individuals (2.0 ± 0.07 mol/l vs. 1.6 ± 0.08 mol/l, p < 0.001), while retinol and TTR were not significantly different. Mean IMT was positively correlated with RBP4 (r = 0.335; p < 0.001) and negatively to retinol (r = −0.241; p < 0.05) concentrations, but no significant correlation was found for IMT and TTR. Mean IMT also correlated significantly to the molar retinol/RBP4 (Fig. 1), an index for RBP4 saturation with retinol (Table 1). After adjustment for established cardiovascular risk factors (age, gender, BMI, systolic blood pressure, HDL/cholesterol ratio, triglyceride,
Fig. 1. Correlation between IMT and retinol/RBP4 ratio.
Recent studies suggested that RBP4, an integral part of the circulating vitamin A transport complex, may link insulin resistance, T2DM, renal dysfunction and atherosclerosis. A positive association between RBP4 and carotid IMT was (recently) found in a group of hypertensive women [5], which is in agreement with our results. Interestingly this relation was not found in a cohort of elderly people [9]. The reason for this discrepancy is unclear, although age-dependent effects may explain some of the differences. These studies, however, did not consider the interaction of RBP4 with retinol or TTR, which might be important to understand the influence of the circulating vitamin A transport complex in the pathogenesis of cardiovascular disease. In our study retinol inversely correlated with IMT, which are in agreement with a previous publication investigating the association between retinol and atherosclerosis [10,11]. Notably, the retinol/RBP4 ratio was highly associated with IMT, even after adjustment for established cardiovascular risk factors. Given that the retinol/RBP4 ratio indicates the saturation of RBP4 with retinol, this result suggests that the amount of retinol-free RBP4 (apo-RBP4) may have a specific role in the development of atherosclerosis. Indeed another study recently demonstrated that the association between T2DM and the RBP4/retinol ratio is higher than that of RBP4 itself [12]. Some limitations need to be considered. Our study was rather small and therefore no stratified analyses were performed. Thus, i.e. genderor age-specific effects may modify the relation between the vitamin A transport system and IMT. Our study was cross-sectional and no information about time-dependence between the described changes and IMT are possible. In summary, our data support that the vitamin A transport system, especially the saturation of RBP4 with retinol is involved in the pathogenesis of atherosclerosis. Further studies should consider
Table 2 Multivariate linear regression models for mean IMT. The multiplicative term (Correlation × Standardized  × 100) explains the variation of mean IMT explained by the respective parameter in percent. Parameters
Correlation
Standardized 
Correlation × Standardized  × 100 (%)
p-value
Age BMI Retinol/RBP4 Total
0.582 0.317 −0.495
0.489 0.217 −0.391
28.5 6.9 19.4 54.8
<0.001 0.052 0.002 <0.001
T. Bobbert et al. / Atherosclerosis 213 (2010) 549–551
retinol and RBP4 to investigate the relation between the vitamin A transport complex and the risk of atherosclerosis. Acknowledgements JS was supported by a Heisenberg-Professorship and a clinical research group of the Deutsche Forschungsgemeinschaft (SP716/2-1 and KFO218/1) and a research group and the “Kompetenznetz Adipositas” of the German Ministry for Education and Research. References [1] Yang Q, Graham TE, Mody N, et al. Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 2005;436:356–62. [2] Graham TE, Yang Q, Bluher M, et al. Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. N Engl J Med 2006;354:2552–63. [3] Henze A, Frey SK, Raila J, et al. Evidence that kidney function but not type 2 diabetes determines retinol-binding protein 4 serum levels. Diabetes 2008;57:3323–6.
551
[4] Ingelsson E, Sundstrom J, Melhus H, et al. Circulating retinol-binding protein 4, cardiovascular risk factors and prevalent cardiovascular disease in elderly. Atherosclerosis 2009;206:239–44. [5] Solini A, Santini E, Madec S, Rossi C, Muscelli E. Retinol-binding protein4 in women with untreated essential hypertension. Am J Hypertens 2009;22:1001–6. [6] Zanotti G, Berni R. Plasma retinol-binding protein: structure and interactions with retinol, retinoids, and transthyretin. Vitam Horm 2004;69:271–95. [7] Bobbert T, Mai K, Fischer-Rosinsky A, Pfeiffer AF, Spranger J. HbA1c is associated with intima media thickness in individuals with normal glucose tolerance. Diabetes Care 2009. [8] Raila J, Henze A, Spranger J, et al. Microalbuminuria is a major determinant of elevated plasma retinol-binding protein 4 in type 2 diabetic patients. Kidney Int 2007;72:505–11. [9] Ingelsson E, Lind L. Circulating retinol-binding protein 4 and subclinical cardiovascular disease in the elderly. Diabetes Care 2009;32:733–5. [10] Polidori MC, Pratico D, Parente B, et al. Elevated lipid peroxidation biomarkers and low antioxidant status in atherosclerotic patients with increased carotid or iliofemoral intima media thickness. J Investig Med 2007;55:163–7. [11] Gey KF, Ducimetiere P, Evans A, et al. Low plasma retinol predicts coronary events in healthy middle-aged men: the PRIME Study. Atherosclerosis 2009. [12] Erikstrup C, Mortensen OH, Nielsen AR, et al. RBP-to-retinol ratio, but not total RBP, is elevated in patients with type 2 diabetes. Diabetes Obes Metab 2009;11:204–12.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Relation between retinol, retinol-binding protein 4, transthyretin and carotid intima media thickness Thomas Bobbert a , Jens Raila c , Franziska Schwarz a , Knut Mai a , Andrea Henze c , Andreas F.H. Pfeiffer a,b , Florian J. Schweigert c , Joachim Spranger a,b,∗ a b c
Department of Endocrinology, Diabetes and Nutrition, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany Department of Clinical Nutrition, German Institute of Human Nutrition, Nuthetal, Germany Department of Physiology and Pathophysiology of Nutrition, Institute of Nutritional Science, University of Potsdam, Potsdam, Germany
a r t i c l e
i n f o
Article history: Received 17 June 2010 Received in revised form 20 July 2010 Accepted 31 July 2010 Available online 19 August 2010 Keywords: RBP4 Retinol IMT
a b s t r a c t Objective: Retinol is transported in a complex with retinol-binding protein 4 (RBP4) and transthyretin (TTR) in the circulation. While retinol is associated with various cardiovascular risk factors, the relation between retinol, RBP4, TTR and carotid intima media thickness (IMT) has not been analysed yet. Methods: Retinol, RBP4 and TTR were measured in 96 individuals and their relation to mean and maximal IMT was determined. Results: Mean IMT correlated with RBP4 (r = 0.335, p < 0.001), retinol (r = −0.241, p = 0.043), RBP/TTR ratio (r = 0.254, p = 0.025) and retinol/RBP4 ratio (r = −0.549, p < 0.001). Adjustment for age, sex, BMI, blood pressure, HDL/total cholesterol ratio, triglyceride, diabetes and smoking revealed that the retinol/RBP4 ratio was strongly and independently associated with mean IMT. Similar results were found for maximal IMT, which included the measurement of plaques. Conclusion: The data support that the transport complex of vitamin A is associated with the IMT, an established parameter of atherosclerosis. Changes in RBP4 saturation with retinol may link renal dysfunction and insulin resistance to atherosclerosis. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Objective
2. Methods
RBP4 is the only specific carrier protein for retinol in the circulation. Recent data suggested that RBP4 itself may induce insulin resistance, since elevated RBP4 levels have been found in patients with type 2 diabetes (T2DM) [1,2]. Additionally, elevated RBP4 levels have also been described in individuals with impaired renal function [3]. Thus, circulating RBP4 seems to be a marker for cardiovascular disease especially in insulin resistance or T2DM. In line with these findings, a correlation between RBP4 and IMT has been recently described [4,5]. However, these studies did not consider the impact of retinol or transthyretin (TTR), although these components are known to strongly interact with RBP4 in the transport complex of vitamin A and may themselves affect cardiovascular risk [6]. We therefore investigated the association of retinol, RBP4, and TTR with intima media thickness (IMT).
96 individuals (44 males, 52 females), including 34 individuals with T2DM and 8 smokers, were randomly drawn from the MeSyBePo-study cohort (Metabolic-Syndrome-Berlin–Potsdam). Mean age was 55 ± 1.3 years with a body mass index of 30.8 ± 0.7 kg/m2 . Mean blood pressure was 131/80 ± 2/1 mm Hg. The following anti-diabetic, anti-hypertensive and lipid lowering drugs were used by study participants: metformin (n = 8), sulfonylureas (n = 5), acarbose (n = 1), glinides (n = 2), pioglitazone (n = 1), calcium antagonists (n = 6), beta blockers (n = 8), ACE inhibitors (n = 13), AT1-receptor antagonists (n = 2), diuretics (n = 7) and statins (n = 8). Renal function was estimated by the abbreviated MDRD study equation with individuals having a mean eGFR of 82.1 ± 1.7 ml/min/1.73 m2 . Lipids (mean LDL cholesterol 1.89 ± 0.05 mmol/l, mean HDL cholesterol 1.37 ± 0.04 mmol/l, mean triglycerides 1.51 ± 0.07 mmol/l) and glucose metabolism (mean fasting glucose 107 ± 2.3 mg/dl, mean fasting insulin 11.5 ± 1.0 mU/l, mean HbA1c 5.8 ± 0.1%) were measured by standard techniques. A description of the detailed phenotyping and laboratory methods was previously published [3,7,8]. Measurement of RBP4 (mean 1.89 ± 0.05 mol/l) and TTR (mean 4.63 ± 0.17 mol/l) was also performed as previously described [3]. Retinol (1.78 ± 0.04 g/ml) was determined by rpHPLC. Carotid
∗ Corresponding author at: Charité, Department of Endocrinology, Diabetes and Nutrition, Hindenburgdamm 30, 12200 Berlin, Germany, Tel.: +49 30 8445 2287; fax: +49 30 8445 4204. E-mail address: [email protected] (J. Spranger). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.07.063
550
T. Bobbert et al. / Atherosclerosis 213 (2010) 549–551
Table 1 Coefficients of correlation between parameters of the retinoid system and other cardiovascular risk factors, *p < 0.05 or † p < 0.001. RBP4 Age BMI IMT IMTMAX Systolic BP Fasting glucose HbA1c HDL cholesterol LDL cholesterol Triglycerides MDRD
†
0.298 0.130 0.335† 0.290* −0.026 0.308† 0.341† −0.192 0.107 0.319† −0.117
Retinol
TTR
RBP4/TTR ratio
Retinol/RBP4 ratio
−0.084 −0.075 −0.241* −0.198 0.000 −0.005 −0.193 0.021 −0.027 0.172 0.152
0.129 −0.174 0.084 0.063 −0.015 0.158 0.124 0.037 0.109 0.070 0.067
0.141 0.380† 0.254* 0.229* 0.025 0.092 0.194 −0.230* −0.058 0.255* −0.171
−0.311† −0.274† −0.549† −0.476† −0.096 −0.362† −0.523† 0.345† −0.098 −0.236* 0.177
arterial IMT was measured at the posterior wall of the common carotid artery at three different positions using a high resolution ultrasound (Kretz AG, Germany). Mean and maximum values for IMT, which included possible plaques (IMTMAX ), were calculated (IMT = 0.719 ± 0.016 mm) and compared with the concentrations retinol, RBP4 and TTR in plasma. The experimental protocol of the study was approved by the Institutional Review Board; all subjects gave written informed consent. Statistical calculations were performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA).
smoking (yes/no), eGFR and T2DM (yes/no)) the retinol/RBP4 ratio remained significantly associated with mean IMT and explained a considerable proportion of the IMT variation (19.4%) (Table 2). Adjustment for current medication did also not substantially modify our findings. Similar results were found for IMTMAX . Thus, only BMI, age and the retinol/RBP4 ratio were independently and significantly associated with IMTMAX and explained in total about 44% of the variability of IMTMAX .
4. Discussion 3. Results RBP4 was higher in diabetic compared to non-diabetic individuals (2.0 ± 0.07 mol/l vs. 1.6 ± 0.08 mol/l, p < 0.001), while retinol and TTR were not significantly different. Mean IMT was positively correlated with RBP4 (r = 0.335; p < 0.001) and negatively to retinol (r = −0.241; p < 0.05) concentrations, but no significant correlation was found for IMT and TTR. Mean IMT also correlated significantly to the molar retinol/RBP4 (Fig. 1), an index for RBP4 saturation with retinol (Table 1). After adjustment for established cardiovascular risk factors (age, gender, BMI, systolic blood pressure, HDL/cholesterol ratio, triglyceride,
Fig. 1. Correlation between IMT and retinol/RBP4 ratio.
Recent studies suggested that RBP4, an integral part of the circulating vitamin A transport complex, may link insulin resistance, T2DM, renal dysfunction and atherosclerosis. A positive association between RBP4 and carotid IMT was (recently) found in a group of hypertensive women [5], which is in agreement with our results. Interestingly this relation was not found in a cohort of elderly people [9]. The reason for this discrepancy is unclear, although age-dependent effects may explain some of the differences. These studies, however, did not consider the interaction of RBP4 with retinol or TTR, which might be important to understand the influence of the circulating vitamin A transport complex in the pathogenesis of cardiovascular disease. In our study retinol inversely correlated with IMT, which are in agreement with a previous publication investigating the association between retinol and atherosclerosis [10,11]. Notably, the retinol/RBP4 ratio was highly associated with IMT, even after adjustment for established cardiovascular risk factors. Given that the retinol/RBP4 ratio indicates the saturation of RBP4 with retinol, this result suggests that the amount of retinol-free RBP4 (apo-RBP4) may have a specific role in the development of atherosclerosis. Indeed another study recently demonstrated that the association between T2DM and the RBP4/retinol ratio is higher than that of RBP4 itself [12]. Some limitations need to be considered. Our study was rather small and therefore no stratified analyses were performed. Thus, i.e. genderor age-specific effects may modify the relation between the vitamin A transport system and IMT. Our study was cross-sectional and no information about time-dependence between the described changes and IMT are possible. In summary, our data support that the vitamin A transport system, especially the saturation of RBP4 with retinol is involved in the pathogenesis of atherosclerosis. Further studies should consider
Table 2 Multivariate linear regression models for mean IMT. The multiplicative term (Correlation × Standardized  × 100) explains the variation of mean IMT explained by the respective parameter in percent. Parameters
Correlation
Standardized 
Correlation × Standardized  × 100 (%)
p-value
Age BMI Retinol/RBP4 Total
0.582 0.317 −0.495
0.489 0.217 −0.391
28.5 6.9 19.4 54.8
<0.001 0.052 0.002 <0.001
T. Bobbert et al. / Atherosclerosis 213 (2010) 549–551
retinol and RBP4 to investigate the relation between the vitamin A transport complex and the risk of atherosclerosis. Acknowledgements JS was supported by a Heisenberg-Professorship and a clinical research group of the Deutsche Forschungsgemeinschaft (SP716/2-1 and KFO218/1) and a research group and the “Kompetenznetz Adipositas” of the German Ministry for Education and Research. References [1] Yang Q, Graham TE, Mody N, et al. Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 2005;436:356–62. [2] Graham TE, Yang Q, Bluher M, et al. Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. N Engl J Med 2006;354:2552–63. [3] Henze A, Frey SK, Raila J, et al. Evidence that kidney function but not type 2 diabetes determines retinol-binding protein 4 serum levels. Diabetes 2008;57:3323–6.
551
[4] Ingelsson E, Sundstrom J, Melhus H, et al. Circulating retinol-binding protein 4, cardiovascular risk factors and prevalent cardiovascular disease in elderly. Atherosclerosis 2009;206:239–44. [5] Solini A, Santini E, Madec S, Rossi C, Muscelli E. Retinol-binding protein4 in women with untreated essential hypertension. Am J Hypertens 2009;22:1001–6. [6] Zanotti G, Berni R. Plasma retinol-binding protein: structure and interactions with retinol, retinoids, and transthyretin. Vitam Horm 2004;69:271–95. [7] Bobbert T, Mai K, Fischer-Rosinsky A, Pfeiffer AF, Spranger J. HbA1c is associated with intima media thickness in individuals with normal glucose tolerance. Diabetes Care 2009. [8] Raila J, Henze A, Spranger J, et al. Microalbuminuria is a major determinant of elevated plasma retinol-binding protein 4 in type 2 diabetic patients. Kidney Int 2007;72:505–11. [9] Ingelsson E, Lind L. Circulating retinol-binding protein 4 and subclinical cardiovascular disease in the elderly. Diabetes Care 2009;32:733–5. [10] Polidori MC, Pratico D, Parente B, et al. Elevated lipid peroxidation biomarkers and low antioxidant status in atherosclerotic patients with increased carotid or iliofemoral intima media thickness. J Investig Med 2007;55:163–7. [11] Gey KF, Ducimetiere P, Evans A, et al. Low plasma retinol predicts coronary events in healthy middle-aged men: the PRIME Study. Atherosclerosis 2009. [12] Erikstrup C, Mortensen OH, Nielsen AR, et al. RBP-to-retinol ratio, but not total RBP, is elevated in patients with type 2 diabetes. Diabetes Obes Metab 2009;11:204–12.