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Afamin Serum Concentrations are Associated with Insulin Resistance and Metabolic Syndrome in PCOS

Seeber B, Morandell E, Lunger F, Wildt L, Dieplinger H. Reprod Biol Endocrinol 2014. doi: 10.1186/1477-7827-12-88.

Background: High plasma concentrations of the vitamin E-binding protein afamin have been previously shown to be associated with insulin resistance and metabolic syndrome. We set out to determine whether the concentration of afamin in the serum of women with polycystic ovarian syndrome (PCOS) is elevated in relation to the presence and severity of insulin resistance (IR).

Methods: This cross-sectional study looked at 53 patients with PCOS and 49 non-PCOS patients. IR was diagnosed as a HOMA Index >2.4 and confirmed with a three-hour glucose tolerance test. Serum concentrations of afamin were determined using enzyme-linked immunosorbent assay (ELISA). Clinical characteristics, hormone and metabolic parameters were correlated to afamin concentrations.

Results: Serum concentrations of afamin did not differ between women with PCOS and controls. When separated according to the presence of IR a significant difference in median afamin levels was seen between PCOS with IR and PCOS without IR (73.06+/-27.36 mg/L and 64.25+/-17.41 mg/L, p = 0.033). No difference in afamin levels was detected when comparing the few controls with IR and the controls without IR (76.20+/-27.96 mg/L and 60.44+/-21.03 mg/L, p = 0.235). On univariate analyses, afamin serum concentrations significantly correlated with BMI, triglycerides, HOMA Index, and AUC-Insulin. On multivariate linear regression analysis, only triglyceride concentration was seen to be an independent predictor of afamin. Subjects with metabolic syndrome had higher median afamin concentrations than did those without metabolic syndrome (77.43+/-28.60 mg/L and 65.08+/-18.03 mg/L, p = 0.010).

Conclusions: Elevated afamin concentrations are associated with the presence of metabolic syndrome in young women and may potentially serve as an independent predictor for the development of metabolic syndrome in at-risk women, especially those with IR.


Plasma Concentrations of Afamin Are Associated with Prevalence and Development of Metabolic Syndrome

Kronenberg F, Kollerits B, Kiechl S, Lamina C, Kedenko L, Meisinger C, Willeit J, Huth C, Wietzorrek G, Altmann ME, Thorand B, Melmer A, Dähnhardt D, Santer P, Rathmann W, Paulweber B, Koenig W, Peters A, Adham IM, Dieplinger H. Circ Cardiovasc Genet 2014. doi: CIRCGENETICS.113.000654.

Background: Afamin is a human plasma vitamin E-binding glycoprotein primarily expressed in the liver and secreted into the bloodstream. Since little is known about (patho)-physiological functions of afamin, we decided to identify phenotypes associated with afamin by investigating transgenic mice overexpressing the human afamin gene and performing large-scale human epidemiological studies.

Methods and Results: Transgenic mice overexpressing afamin revealed increased body weight and serum concentrations of lipids and glucose. We applied a random-effects meta-analysis using age- and sex-adjusted baseline and follow-up investigations in the population-based Bruneck (n=826), SAPHIR (n=1499), and KORA F4 studies (n=3060). Mean afamin concentrations were 62.5±15.3, 66.2±14.3, and 70.6±17.2 mg/L in Bruneck, SAPHIR and KORA F4, respectively. Per 10 mg/L increment in afamin measured at baseline, the number of metabolic syndrome components increased by 19% (incidence rate ratio (IRR)=1.19 (95%CI 1.16-1.21), p=5.62×10-64). With the same afamin increment used at baseline we observed an 8% gain in metabolic syndrome components between baseline and follow-up (IRR=1.08 (95%CI 1.06-1.10), p=8.87×10-16). Afamin concentrations at baseline were highly significantly related to all individual metabolic syndrome components at baseline and follow-up. This observation was most pronounced for elevated waist circumference (OR=1.79 (95%CI 1.54-2.09), p=4.15×10-14 at baseline and OR=1.46 (95%CI 1.31-1.63), p=2.84×10-11 for change during follow-up) and for elevated fasting glucose concentrations (OR=1.46 (95%CI 1.40-1.52), p=1.87×10-69, and OR=1.46 95%CI 1.24-1.71, p=5.13×10-6, respectively).

Concludions: This study in transgenic mice and more than 5,000 participants in epidemiological studies shows that afamin is strongly associated with the prevalence and development of metabolic syndrome and all its components.


Serum concentrations of afamin are elevated in patients with polycystic ovary syndrome

Köninger A, Edimiris P, Koch L, Enekwe A, Lamina C,  Kasimir-Bauer S, Kimmig R, Dieplinger H. Endocr Connect 2014;3:120-6.

Oxidative stress seems to be present in patients with polycystic ovary syndrome (PCOS). The aim of this study was to evaluate the correlation between characteristics of PCOS and serum concentrations of afamin, a novel binding protein for the antioxidant vitamin E. A total of 85 patients with PCOS and 76 control subjects were investigated in a pilot cross-sectional study design between 2009 and 2013 in the University Hospital of Essen, Germany. Patients with PCOS were diagnosed according to the Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group. Afamin and diagnostic parameters of PCOS were determined at early follicular phase. Afamin concentrations were significantly higher in patients with PCOS than in controls (odds ratio (OR) for a 10 mg/ml increase in afamin=1.3, 95% CI=1.08-1.58). This difference vanished in a model adjusting for age, BMI, free testosterone index (FTI), and sex hormone-binding globulin (SHBG) (OR=1.05, 95% CI=0.80-1.38). In patients with PCOS, afamin correlated significantly with homeostatic model assessment-insulin resistance (HOMA-IR), fasting glucose, BMI, FTI, and SHBG (P<0.001), but in a multivariate linear model, only HOMA-IR remained significantly associated with afamin (P=0.001). No correlation was observed between afamin and androgens, LH, FSH, LH/FSH ratio, antral follicle count, ovarian volume, or anti-Müllerian hormone. In conclusion, elevated afamin values may indicate a state of oxidative stress and inflammation, strongly associated with IR and offering an indicator of impaired glucose tolerance in patients with PCOS irrespective of obesity.

The Vitamin E-binding Protein Afamin increases in Maternal Serum during Pregnancy

Hubalek M, Buchner H, Mörtl MG, Schlembach D, Huppertz B, Firulovic B, Köhler W, Hafner E, Dieplinger B, Wildt L, Dieplinger H. Clin Chim Acta 2014;434:41-7.

Background: Afamin is a liver-derived plasma glycoprotein with vitamin E-binding properties and a putative function in fertility. This study evaluated serum afamin concentrations during and postpartum to uncomplicated pregnancies and investigated a potential association between afamin concentrations and pregnancy outcome.

Methods: Afamin serum concentrations were measured in women with uncomplicated pregnancies in a retrospective cohort (n=466) at different gestational ages and a prospective observational study (n=76) in the first, second and third trimester. Furthermore, afamin was determined in the first trimester in a cross-sectional pilot study including women with preeclampsia (PE), pregnancy-induced hypertension (PIH) and women without pregnancy complications (n=13 each). Finally, expression of afamin was investigated in human placental tissue by RT-PCR and immunohistochemistry.

Results: Afamin concentrations increased linearly almost two-fold during pregnancy in both retrospective and prospective studies in women without pregnancy complications with median afamin serum concentrations of 61.9 mg/l, 79.6 mg/l, and 98.6 mg/l in the first, second, and third trimester, respectively. After delivery, median afamin concentrations decreased to baseline values of 54.6 mg/l. In the pilot study with pregnancy complications, women with PE displayed significantly higher median afamin concentrations than did women with uncomplicated pregnancy (70.0 mg/l vs. 55.4 mg/l, P=0.007). Expression analyses revealed no placental afamin expression at either mRNA or protein level in uncomplicated pregnancy.

Conclusion: A linear increase in the maternally expressed glycoprotein afamin during pregnancy may serve as basic reference for subsequent investigations of afamin in pregnancy-related disorders.


Analytical Characterization and Clinical Evaluation of an ELISA for Measurement of Afamin in Plasma

Dieplinger B, Egger M, Gabriel C, Poelz W, Morandell E, Seeber B, Kronenberg F, Haltmayer M, Mueller T, Dieplinger H. Clin Chim Acta 2013;425:236-41.

Background: Comparative proteomics has recently identified afamin, the newest member of the albumin gene family, as a potential biomarker for ovarian cancer. The aim of this study was the analytical and clinical evaluation of a sandwich enzyme-linked immunosorbent assay for the determination of afamin in human plasma.

Methods: We evaluated precision, linearity, and detection limit of the assay, analyte stability and biological variability, determined reference values and quantified afamin concentrations in various diseases.

Results: Within-run and total coefficients of variation were <10%. The method was linear across the tested measurement range. Detection limit was 7 mg/L for the assay. The analyte was stable for 24 h at room temperature, for 48 h at 4°C, and for at least one year at -20°C and -80°C. The reference change value for healthy individuals was 24%. Age- and sex-independent reference values in healthy blood donors were 45-99 mg/L (median 68 mg/L). In the clinical assay evaluation afamin plasma concentrations were modestly decreased in patients with heart failure. Patients with pneumonia or sepsis exhibited markedly decreased afamin plasma concentrations. However, patients with chronic renal disease or chronic obstructive pulmonary disease showed no difference in afamin plasma concentrations as compared to healthy individuals. Correlation analyses revealed an inverse association between afamin and inflammatory biomarkers.

Conclusions: The afamin assay meets quality specifications for laboratory medicine. The results of the clinical assay evaluation revealed novel insights with respect to afamin as a potential negative acute phase protein and should encourage further studies.


Plasma Concentrations of the Vitamin E-Binding Protein Afamin are Associated with Overall and Progre

Melmer A, Fineder L, Lamina C, Kollerits B, Dieplinger B, Braicu I, Sehouli J, Cadron I, Vergote I, Mahner S, Zeimet AG, Castillo-Tong DC, Ebenbichler CF, Zeillinger R, Dieplinger H. Gynecol Oncol 2013;128:38-43.

Objective: Comparative proteomics identified the plasma protein afamin as potential biomarker for ovarian cancer (OC). Significantly decreased afamin plasma concentrations in pre-therapeutic OC patients reconstituted to control values after successful tumor surgery. This study evaluates the association of afamin with survival and response to therapy in serous OC patients within the OVCAD consortium project.

Methods: We measured afamin in 215 pre-therapeutic plasma samples, 246 tumor lysates and 109 plasma samples taken 6months after finish
ing platinum-based chemotherapy. Differences in afamin plasma concentrations among FIGO stages were tested by Kruskal-Wallis test; association of afamin concentrations with overall and progression-free survival was evaluated using Kaplan-Meier survival plots and multivariate adjusted COX regression analysis.

Results: Pre-therapeutic afamin correlated significantly with FIGO stages (p=0.012) and was lower in the presence of metastases (p=0.013) and poorly differentiated OC in patients responding to therapy (p=0.016). Afamin ≥48.0mg/L was also associated with a lower hazard ratio for recurrent disease as compared to afamin <48.0mg/L (p=0.007). Post-therapeutic afamin ≥48mg/L was positively correlated with overall (p<0.001) and progression-free (p=0.012) survival and was lower in non-responders than in responders (p=0.048). Thus, afamin returned post-therapeutically to values of healthy controls in responders (p<0.001) but not in non-responders (p=0.114). Afamin in tumor lysates was lower in poorly differentiated OC than in G 1+2 tumors (p=0.041). Higher afamin concentrations in tumor lysates were associated with increased overall survival (p=0.003).

Conclusion: These data indicate that afamin is associated with therapy response and survival rate in advanced OC patients.

The Vitamin E-Binding Protein Afamin is Altered in the Peritoneal Fluid of Women with Endometriosis

Seeber BE, Czech T, Buchner H, Barnhart KT, Seger C, Daxenbichler G, Wildt L, Dieplinger H. Fertil Steril 2010;94:2923-6.

The objective of this case-control study of 242 reproductive-age women was to determine the concentration of afamin in the serum and peritoneal fluid of women with and without endometriosis and to test afamin as a diagnostic marker of endometriosis. Afamin levels were altered significantly in the peritoneal fluid of women with endometriosis compared with disease-free controls, correlated with vitamin E levels, and are consistent with increased oxidative stress in the peritoneal cavity of women with endometriosis.

Afamin and Apolipoprotein A-IV: Novel Protein Markers for Ovarian Cancer

Dieplinger H, Ankerst DP, Burges A, Lenhard M, Lingenhel A, Fineder L, Buchner H, Stieber P. Cancer Epidemiol Biomarkers Prev 2009;18:1127-33.

Comparative proteomics identified the vitamin E-binding plasma protein afamin as a potential novel tumor marker for ovarian cancer. In addition, we observed in a previous small study decreased plasma concentrations of apolipoprotein A-IV (apoA-IV) in preoperative patients with kidney cancer. The aim of this study was therefore to analyze afamin and apoA-IV in a large case-control study to evaluate the diagnostic utility of the two potential novel tumor markers in ovarian cancer patients. We measured plasma concentrations of afamin and apoA-IV by means of a specific sandwich-type ELISA using affinity-purified polyclonal and monoclonal antibodies in 181 ovarian cancer patients of various clinical stages, 399 patients with benign gynecologic diseases, including endometriosis, and 177 controls and compared results with those for the conventional ovarian cancer tumor marker cancer antigen 125 (CA125). Afamin concentrations decreased from a median of 70.7 mg/L (range, 34.6-116.1 mg/L) in healthy controls to 65.2 mg/L (range, 20.2-206.6 mg/L) in patients with benign gynecologic diseases to 56.0 mg/L (range, 4.7-96.0 mg/L) in ovarian cancer patients (P < 0.001 for all pairwise comparisons). Similar results were obtained with apoA-IV concentrations decreasing from 13.0 mg/dL (range, 5.5-34.0 mg/dL) in controls to 11.7 mg/dL (range, 2.0-32.3 mg/dL) in benign conditions to 9.4 mg/dL (range, 0.3-29.5 mg/dL) in ovarian cancer (all P < 0.001). Receiver operating characteristic analysis for differentiating ovarian cancer patients from healthy controls revealed for a specificity of 90% sensitivity values of 92.4%, 42.4%, and 40.8% for CA125, afamin, and apoA-IV, respectively. Afamin, but not apoA-IV, added independent diagnostic information to CA125 and age for differentiating ovarian cancer from benign and healthy samples; the odds ratio of ovarian cancer was reduced by 44% for each doubling of afamin (P = 0.032). The relatively low sensitivity, however, clearly indicates that afamin and apoA-IV alone are not sufficiently suitable as diagnostic markers for ovarian cancer. Afamin contributes, however, independent diagnostic information to CA125, thus establishing its potential as an adjunct marker to CA125.

Afamin is Synthesized by Cerebrovascular Endothelial Cells and Mediates Alpha-Tocopherol Transport A

Kratzer I, Bernhart E, Wintersperger A, Hammer A, Waltl S, Malle E, Sperk G, Wietzorrek G, Dieplinger H, Sattler W. J Neurochem 2009;108:707-18.

Alpha-tocopherol (alphaTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of alphaTocH transport into the CNS, transfer mechanisms across the blood-brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known alphaTocH-binding protein, contributes to alphaTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, alphaTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating alphaTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to alphaTocH homeostasis at the BBB in vivo.

Proteomic Profiling Identifies Afamin as a Potential Biomarker for Ovarian Cancer

Jackson D, Craven RA, Hutson RC, Graze I, Lueth P, Tonge RP, Hartley JL, Nickson JA, Rayner SJ, Johnston C, Dieplinger B, Hubalek M, Wilkinson N, Perren TJ, Kehoe S, Hall GD, Daxenbichler G, Dieplinger H, Selby PJ, Banks RE. Clin Cancer Res 2007;13:7370-9.

Purpose: To discover and validate serumglycoprotein biomarkers in ovarian cancer usingproteomic-based approaches.
Experimental Design: Serum samples froma‘‘discovery set’’of 20 patients with ovarian canceror benign ovarian cysts or healthy volunteers were compared by fluorescence two-dimensional differential in-gel electrophoresis and parallel lectin-based two-dimensional profiling.Validation ofa candidate biomarker was carried out withWestern blotting and immunoassay (n = 424).

Results: Twenty-six proteins that changed significantly were identified by mass spectrometric sequencing.One of these, confirmedbyWestern blotting,was afamin, a vitamin E binding protein, with two isoforms decreasing in patients with ovarian cancer.Validation using cross-sectional samples from 303 individuals (healthy controls and patientswith benign, borderline,  rmalignant ovarian conditions and other cancers) assayed by ELISA showed significantly decreased total afamin concentrations in patients with ovarian cancer compared with healthy controls (P = 0.002) and patients with benign disease (P = 0.046). However, the receiver operating characteristic areas for total afamin for the comparison of ovarian cancer with healthy controls or benign controls were only 0.67 and 0.60, respectively, with comparable figures for CA-125 being 0.92 and 0.88 although corresponding figures for a subgroup of samples analyzed by isoelectric focusing for afamin isoform 2 were 0.85 and 0.79. Analysis of a further 121samples collected prospectively from 9 patients pretreatment through to relapse indicated complementarity of afamin with CA-125, including two cases inwhom CA-125 was noninformative.

Conclusions: Afamin shows potential complementarity with CA-125 in longitudinal monitoring of patients with ovarian cancer, justifying prospective larger-scale investigation. Changes in specific isoforms may provide further information.

Afamin is a Novel Human Vitamin E-Binding Glycoprotein Characterization and In Vitro Expression

Jerkovic L, Voegele AF, Chwatal S, Kronenberg F, Radcliffe CM, Wormald MR, Lobentanz EM, Ezeh B, Eller P, Dejori N, Dieplinger B, Lottspeich F, Sattler W, Uhr M, Mechtler K, Dwek RA, Rudd PM, Baier G, Dieplinger H. J Proteome Res 2005;4:889-99.

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.

Characterization of the Vitamin E-Binding Properties of Human Plasma Afamin

Voegele AF, Jerković L, Wellenzohn B, Eller P, Kronenberg F, Liedl KR, Dieplinger H. Biochemistry 2002;41:14532-8.

Human plasma afamin, the fourth member of the albumin gene family, is shown to be a specific binding protein for vitamin E. A radio ligand-binding assay followed by Scatchard and Hill analysis are used to show that afamin has a binding affinity for both α-tocopherol and γ-tocopherol, two of the most important forms of vitamin E, in vitro. The binding-dissociation constant was determined to be 18 μM, indicating that afamin plays a role as vitamin E carrier in body fluids such as human plasma and follicular fluid under physiological conditions. Additionally, we demonstrate that afamin has multiple binding sites for both α- and γ-tocopherol. Due to the large binding capacity of afamin for vitamin E, it might take over the role of vitamin E transport in body fluids under conditions where the lipoprotein system is not sufficient for vitamin E transport. To confirm the experimental results, we performed homology modeling and docking calculations on the predicted tertiary structure, which showed coincidence between calculated and in vitro results.