Early Pregnancy Markers in the Serum of Ewes Identified via Proteomic and Metabolomic Analyses.

The diagnosis of ewes' pregnancy status at an early stage is an efficient way to enhance the reproductive output of sheep and allow producers to optimize production and management. The techniques of proteomics and metabolomics have been widely used to detect regulatory factors in various physiological processes of animals. The aim of this study is to explore the differential metabolites and proteins in the serum of pregnant and non-pregnant ewes by proteomics and metabolomics. The serum of ewes at 21, 28 and 33 days after artificial insemination (AI) were collected. The pregnancy stratus of the ewes was finally determined through ultrasound examination and then the ewes were grouped as Pregnant (n = 21) or N on-pregnant (n = 9). First, the serum samples from pregnant or non-pregnant ewes at 21 days after AI were selected for metabolomic analysis. It was found that the level of nine metabolites were upregulated and 20 metabolites were downregulated in the pregnant animals (p < 0.05). None of these differential metabolomes are suitable as markers of pregnancy due to their small foldchange. Next, the proteomes of serum from pregnant or non-pregnant ewes were evaluated. At 21 days after AI, the presence of 321 proteins were detected, and we found that the level of three proteins were upregulated and 11 proteins were downregulated in the serum of pregnant ewes (p < 0.05). The levels of serum amyloid A (SAA), afamin (AFM), serpin family A member 6 (SERPINA6) and immunoglobulin-like domain-containing protein between pregnant and non-pregnant ewes at 21-, 28- and 33-days post-AI were also analyzed via enzyme-linked immunosorbent assay (ELISA). The levels of SAA and AFM were significantly higher in pregnant ewes than in non-pregnant ewes, and could be used as markers for early pregnancy detection. Overall, our results show that SAA and AFM are potential biomarkers to determine the early pregnancy status of ewes.

Impaired Organokine Regulation in Non-Diabetic Obese Subjects: Halfway to the Cardiometabolic Danger Zone.

Altered organokine expression contributes to increased cardiometabolic risk in obesity. Our aim was to evaluate the associations of serum afamin with glucose homeostasis, atherogenic dyslipidemia, and other adipokines in severe obesity to clarify the early metabolic alterations. 106 non-diabetic obese (NDO) subjects and 62 obese patients with type 2 diabetes matched for age, gender, and body mass index (BMI) were enrolled in this study. We compared their data with 49 healthy, lean controls. Serum afamin and retinol-binding protein 4 (RBP4), as well as plasma plasminogen activator inhibitor-1 (PAI-1), were measured with ELISA, and lipoprotein subfractions were analyzed using Lipoprint gel electrophoresis. Afamin and PAI-1 found to be significantly higher in the NDO and T2M group (p < 0.001 and p < 0.001, respectively) than in the controls. In contrast, RBP4 was unexpectedly lower in the NDO and T2DM group compared to controls (p < 0.001). Afamin showed negative correlations with mean LDL size and RBP4, but positive correlations with anthropometric, glucose/lipid parameters, and PAI-1 in both the overall patients and the in NDO + T2DM groups. BMI, glucose, intermediate HDL, and small HDL were predictors of afamin. Afamin may serve as a biomarker for the severity of cardiometabolic disturbances in obesity. The complexity of organokine patterns in NDO subjects draws attention to the diverse spectrum of obesity-related comorbidities.

Afamin predicts the prevalence and incidence of nonalcoholic fatty liver disease.

OBJECTIVES: In the general population, increased afamin concentrations are associated with the prevalence and incidence of metabolic syndrome as well as type 2 diabetes. Although metabolic syndrome is commonly associated with nonalcoholic fatty liver disease (NAFLD), there exist no information on afamin and NAFLD. METHODS: Afamin concentrations were cross-sectionally measured in 146 Austrian patients with NAFLD, in 45 patients without NAFLD, and in 292 age- and sex-matched healthy controls. Furthermore, the feasibility of afamin to predict incident NAFLD was evaluated in 1,434 adult participants in the population-based Cardiovascular Risk in Young Finns Study during a 10-year follow-up. RESULTS: Median afamin concentrations were significantly higher in NAFLD patients (83.6 mg/L) than in patients without NAFLD (61.6 mg/L, p<0.0001) or in healthy controls (63.9 mg/L, p<0.0001). In age- and sex-adjusted logistic regression analyses a 10 mg/L increase of afamin was associated with a 1.5-fold increase of having NAFLD as compared with patients without NAFLD and the risk was even two-fold when compared with healthy controls. In the population-based cohort, afamin concentrations at baseline were significantly lower in participants without NAFLD (n=1,195) than in 239 participants who developed NAFLD (56.5 vs. 66.9 mg/L, p<0.0001) during the 10-year follow up, with highest afamin values observed in individuals developing severe forms of NAFLD. After adjustment for several potentially confounding parameters, afamin remained an independent predictor for the development of NAFLD (OR=1.37 [95% CI 1.23-1.54] per 10 mg/L increase, p<0.0001). CONCLUSIONS: Afamin concentrations are increased in patients with NAFLD and independently predict the development of NAFLD in a population-based cohort.

Novel Biomolecules in the Pathogenesis of Gestational Diabetes Mellitus 2.0.

Gestational diabetes mellitus (GDM) has become a major public health problem and one of the most discussed issues in modern obstetrics. GDM is associated with serious adverse perinatal outcomes and long-term health consequences for both the mother and child. Currently, the importance and purposefulness of finding a biopredictor that will enable the identification of women with an increased risk of developing GDM as early as the beginning of pregnancy are highly emphasized. Both "older" molecules, such as adiponectin and leptin, and "newer" adipokines, including fatty acid-binding protein 4 (FABP4), have proven to be of pathophysiological importance in GDM. Therefore, in our previous review, we presented 13 novel biomolecules, i.e., galectins, growth differentiation factor-15, chemerin, omentin-1, osteocalcin, resistin, visfatin, vaspin, irisin, apelin, FABP4, fibroblast growth factor 21, and lipocalin-2. The purpose of this review is to present the potential and importance of another nine lesser known molecules in the pathogenesis of GDM, i.e., 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), angiopoietin-like protein-8 (ANGPTL-8), nesfatin-1, afamin, adropin, fetuin-A, zonulin, secreted frizzled-related proteins (SFRPs), and amylin. It seems that two of them, fetuin-A and zonulin in high serum levels, may be applied as biopredictors of GDM.

Evaluation of maternal serum afamin and vitamin E levels in pregnant women with COVID-19 and its association with composite adverse perinatal outcomes.

To evaluate the maternal serum afamin and vitamin E levels in pregnant women with coronavirus disease 2019 (COVID-19) and to investigate their association with composite adverse perinatal outcomes. This prospective, case-control study consisted of 60 pregnant women with COVID-19 infection and 36 age-matched pregnant women without any defined risk factors. Demographic features, laboratory test results, afamin and vitamin E levels were compared between the groups. A receiver operating characteristic (ROC) curve was used to assess the relationship of afamin and vitamin E levels in predicting composite adverse perinatal outcomes. A correlation analysis was performed between afamin and C-reactive protein (CRP) levels in pregnant women with COVID-19. The obstetric complication rate was higher in the COVID-19 group (13.3% vs. 2.8%) (p = .01). Afamin levels were higher and vitamin E levels were lower in the COVID-19 group (p = .02 and p < .001, respectively). Vitamin E levels were lower in the COVID-19 group for the all trimesters (p < .001, p < .001, and p = .004, respectively). Afamin levels were higher in the COVID-19 group for the all trimesters without reaching statistical significance (p > .05). The values in the ROC curves with the best balance of sensitivity/specificity for afamin and vitamin E were 0.424 mg/l (70.6% sensitivity, 44.3% specificity) and 3.150 µg/ml (76.5% sensitivity, 58.2% specificity), respectively. A positive moderate statistically significant correlation was found between afamin and CRP levels (r = .264, p = .009). Higher afamin and lower vitamin E levels may support the elevated oxidative stress in the etiopathogenesis of COVID-19 and the relationship with composite adverse perinatal outcomes.

Identification and Validation of Combination Plasma Biomarker of Afamin, Fibronectin and Sex Hormone-Binding Globulin to Predict Pre-eclampsia.

The purpose of the present study was to identify a plasma protein biomarker able to predict pre-eclampsia (PE). Comprehensive quantitative proteomics using mass spectrometry with sequential window acquisition of all theoretical fragment ion spectra (SWATH-MS) was applied to plasma samples of 7 PE and 14 healthy pregnant women (for PE subjects, plasma samples were taken before onset of PE), and 11 proteins were selected as candidates potentially able to differentiate the two groups. Plasmas collected at gestational weeks 14-24 from 36 PE and 120 healthy pregnant women (for PE subjects, plasma samples were taken before onset of PE) were used to conduct selected reaction monitoring quantification analysis, optimize protein combinations and conduct internal validation, which consisted of 30 iterations of 10-fold cross-validation using multivariate logistic regression and receiver operating characteristic (ROC) analysis. The combination of afamin, fibronectin, and sex-hormone-binding globulin was selected as the best candidate. The 3-protein combination predictive model (predictive equation and cut-off value) generated using the internal validation subjects was successfully validated in another group of validation subjects (36 PE and 54 healthy (for PE subjects, plasma samples were taken before onset of PE)) and showed good predictive performance, with the area under the curve (AUC) 0.835 and odds ratio 13.43. In conclusion, we newly identified a 3-protein combination biomarker and established a predictive equation and cut-off value that can predict the onset of PE based on analysis of plasma samples collected during gestational weeks 14-24.

Expression, Purification, and Biochemical Characterization of Human Afamin.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.

Urine proteomics of primary membranous nephropathy using nanoscale liquid chromatography tandem mass spectrometry analysis.

BACKGROUND: Primary membranous nephropathy (PMN) is an important cause of nephrotic syndrome in adults. Urine proteome may provide important clues of pathophysiological mechanisms in PMN. In the current study, we analyzed and compared the proteome of urine from patients with PMN and normal controls. METHODS: We performed two technical replicates (TMT1 and TMT2) to analyze and compare the urine proteome from patients with PMN and normal controls by tandem mass tag (TMT) technology coupled with nanoscale liquid chromatography tandem mass spectrometry analysis (LC-MS/MS). Gene ontology (GO) enrichment analysis was performed to analyse general characterization of the proteins. The proteins were also matched against the database of Kyoto Encyclopedia of Genes and Genomes (KEGG). For validation, Western blot was used to analyze the selected proteins. RESULTS: A total of 509 proteins and 411 proteins were identified in TMT1 and TMT2, respectively. 249 proteins were both identified in two technical replicates. GO analysis and KEGG analysis revealed immunization and coagulation were predominantly involved. Among the differential protein, the overexcretion of alpha-1-antitrypsin (A1AT) and afamin (AFM) were validated by Western blot analysis. CONCLUSIONS: Our data showed the important role of immunologic mechanism in the development of PMN, and the value of urinary A1AT and AFM in biomarker discovery of patients with PMN. The discovery of the overexcretion of A1AT and AFM in the urine can help to further elucidate pathogenetic mechanisms involved in PMN.

Is Afamin a novel biomarker for gestational diabetes mellitus? A pilot study.

BACKGROUND: In search of potential early biomarkers for timely prediction of gestational diabetes mellitus (GDM), we focused on afamin, a vitamin E-binding protein in human plasma.. Afamin plays a role in anti-apoptotic cellular processes related to oxidative stress and is associated with insulin resistance and other features of metabolic syndrome. During uncomplicated pregnancy its serum concentrations increase linearly. The aim of this study was to investigate the suitability of afamin as early marker for predicting GDM. METHODS: In a first-trimester cohort from a prospective observational study of adverse pregnancy outcomes we secondarily analyzed afamin concentrations in 59 patients diagnosed with GDM and 51 controls. Additionally, afamin concentrations were cross-sectionally examined in a mid-trimester cohort of 105 women and compared with results from a simultaneously performed oral glucose tolerance test (OGTT). Subgroup analysis comparing patients treated with either insulin (iGDM) or dietary intervention (dGDM) was performed in both cohorts. Patients were recruited at the University Hospital Essen, Germany, between 2003 and 2016. RESULTS: Results were adjusted for body-mass-index (BMI) and gestational age. First and mid-trimester cohorts yielded significantly elevated afamin concentrations in patients with pathological OGTT compared to patients without GDM (first trimester cohort: mean, 113.4 mg/l; 95% CI, 106.4-120.5 mg/l and 87.2 mg/l; 95% CI, 79.7-94.7 mg/l; mid-trimester cohort: mean, 182.9 mg/l; 95% CI, 169.6-196.2 mg/l and 157.3 mg/l; 95% CI, 149.1-165.4 mg/l, respectively). In the first-trimester cohort, patients developing iGDM later in pregnancy presented with significantly higher afamin concentrations compared to patients developing dGDM and compared to patients without GDM. In the mid-trimester cohort, mean concentrations of afamin differed significantly between patients with dGDM compared to controls and between patients with iGDM and controls. Patients with iGDM showed only slightly higher afamin levels compared to patients with dGDM. CONCLUSION: Afamin may serve as a new early biomarker for pathological glucose metabolism during pregnancy. Further research is needed to determine afamin's concentrations during pregnancy, its predictive value for early detection of pregnancies at high risk to develop GDM and its diagnostic role during the second trimester.

Changes in serum afamin and vitamin E levels after selective LDL apheresis.

BACKGROUND: Afamin is a plasma vitamin E-binding glycoprotein partially associated with ApoA1-containing high-density lipoprotein (HDL) subfractions. In a previous study, the serum vitamin E decreased after low-density lipoprotein (LDL) apheresis, while vitamin E/cholesterol ratio increased. We aimed to study the effect of LDL apheresis on serum afamin level. METHODS: The serum level of afamin and oxidized LDL were measured by enzyme-linked immunosorbent assay in six severe heterozygous FH patients before and after their first LDL apheresis treatments and in seven healthy controls. We also investigated the changes in total cholesterol, LDL-C, HDL-C, ApoB, ApoA1, HDL subfractions, and α- and γ-tocopherol levels during the treatment. HDL subfractions were detected by an electrophoretic method on polyacrylamide gel (Lipoprint). Serum α- and γ-tocopherol levels were detected by gas chromatography-mass spectrometry. RESULTS: The first treatment sessions decreased serum afamin levels by an average of 9.4%. Total cholesterol, LDL-C, HDL-C and ApoA1 levels decreased by 52.6; 61.8; 10.5; and 14.1%, respectively. We found that α- and γ-tocopherol levels markedly decreased (by 34.1 and 32.9%, respectively), while α- tocopherol/cholesterol and γ-tocopherol/cholesterol ratios significantly increased (by 41.4 and 40.3%, respectively). Oxidized LDL levels significantly decreased. There was a shift toward the larger HDL subfractions. CONCLUSION: LDL apheresis moderately decreases the circulating levels of afamin parallel to lowering HDL-C and ApoA1 levels. Tocopherol levels decreases markedly compared to afamin levels, however, beneficial changes in vitamin E/cholesterol ratios, oxidized LDL levels and HDL subfraction distribution were detected. These additional effects of LDL apheresis may result in further cardiovascular risk reduction in FH patients.

Afamin: an early predictor of preeclampsia.

PURPOSE: Oxidative stress is involved in the pathogenesis of hypertensive disorders such as preeclampsia (PE) and associated with the human vitamin E-binding protein afamin. The aim of this study was, therefore, to analyse afamin in the first trimester of patients developing PE later in pregnancy and in control subjects without pregnancy complications. METHODS: In this retrospective study, 137 serum samples from the first trimester of pregnancy were analysed in a case-control study design. 39 patients developed PE (10 patients with early-onset and 29 patients with late onset disease) and 98 women had an uncomplicated pregnancy. Mann-Whitney U test, t test, logistic regression and ROC analyses were performed for statistical evaluation. RESULTS: Pregnant women developing PE presented with higher afamin concentrations in the first trimester [median 101.81 mg/L; interquartile range (IQR) 88.94-113.26] compared to subjects with uncomplicated pregnancy (median 86.40; IQR 75.26-96.92; p < 0.001). After adjusting for confounders, the odds ratio per afamin standard deviation was 1.60 (95% CI: 1.04-2.58; p = 0.04). An afamin threshold concentration of 87.8 mg/L exhibited the best sensitivity (79.5%) and specificity (57.1%) in predicting PE. Subgroup analysis of early- and late-onset disease resulted in substantially higher afamin concentrations in women with developing late-onset PE compared to controls (p < 0.001) with an odds ratio per afamin standard deviation of 1.62 (95% CI: 0.98-2.70; p = 0.06). CONCLUSIONS: Serum afamin concentrations are elevated in the first trimester among patients developing PE compared to controls. Substantial differences were observed mainly among patients with late-onset PE.

Label-free mass spectrometric profiling of urinary proteins and metabolites from paediatric idiopathic nephrotic syndrome.

Idiopathic nephrotic syndrome (INS) is caused by renal diseases that increase the permeability of the glomerular filtration barrier without evidence of a specific systemic cause. The aim of the present work was to reveal inherent molecular features of INS in children using combined urinary proteomics and metabolomics profiling. In this study, label-free mass spectrometric analysis of urinary proteins and small molecule metabolites was carried out in 12 patients with INS versus 12 sex- and age-matched control subjects with normal renal function. Integration and biological interpretation of obtained results were carried out by Ingenuity IPA software. Validation of obtained proteomics data was carried out by Western blot method. Proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000765. This study indicates for the first time that paediatric INS is associated with up-regulation of afamin, hydroxyphenylacetate and uridine, and concomitant down-regulation in glutamine and phenylalanine levels, and many of these molecular species were previously shown to be involved in oxidative stress. Further studies in larger patient population are underway to investigate the role of oxidative stress in renal injury in paediatric INS.

Circulating afamin positively correlated with the miR-122 expression and type 2 diabetes mellitus-related phenotype according to the duration of diabetes.

Background: Afamin is a hepatokine that involves in glucose and lipids metabolism. miR-122 is mainly expressed in liver and involves in lipid and carbohydrate metabolism. This study aimed at investigating the circulating afamin, its correlation with type 2 diabetes mellitus (T2DM) and miR-122 gene expression in T2DM patients and healthy control subjects according to the duration of diabetes. Methods: This case-control study included 220 participants, with 100 individuals serving as controls and 120 individuals diagnosed with type 2 diabetes mellitus (T2DM). The miR-122 gene expression was assessed using real-time PCR. The serum concentration of biochemical parameters such as glucose levels, lipid profile, and small-dense low-density lipoprotein (sdLDL) were measured using colorimetric kits. Circulating afamin and insulin levels were assayed using an ELISA kit. Glycated hemoglobin (HbA1c) was measured using capillary electrophoresis. Results: Circulating afamin level was significantly higher in T2DM patients compared to the control group, (73.8 ± 10.8 vs. 65.9 ± 8.7, respectively; P < 0.001). Similarly, miR122 expression was significantly increased in T2DM patients compared to healthy control subjects (4.24 ± 2.01 vs. 1.00 ± 0.85, respectively; P < 0.001). Among patients diagnosed with T2DM, those with longstanding diabetes (>5 years) exhibited significantly higher levels of circulating afamin and miR-122 expression compared to individuals with a shorter duration of diabetes (≤5 years) (P < 0.05). Circulating afamin levels were significantly correlated with waist circumference, small-dense low-density lipoprotein (sdLDL), fasting blood sugar (FBS), insulin, resistance to insulin, and miR-122 expression, depending on the duration of the disease (P < 0.05). Furthermore, the performance of afamin as a diagnostic marker for T2DM was confirmed through receiver operating characteristic (ROC) analysis, yielding an area under the curve (AUC) of 0.7 (P < 0.001). Conclusions: Circulating afamin involved in the T2DM-related complications and its concentration is positively correlated to the miR-122 expression, especially in patient with longstanding diabetes.

Serum afamin and its implications in adult growth hormone deficiency: a prospective GH-withdrawal study.

Introduction: Adult growth hormone deficiency (AGHD) is associated with a high prevalence of metabolic syndrome (MS), which contributes to the unfavorable cardiovascular risk profile in these patients. Insulin like growth factor-1 (IGF-1) is a widely used biomarker, however it does not always reflect the cardiometabolic risk and has a poor relationship with clinical efficacy endpoints. Consequently, there is an unmet need for biomarkers to monitor responses to GH-replacement. Afamin is a hormone-like glycoprotein, expressed in the liver. Higher afamin levels are strongly associated with MS and insulin resistance (IR). Although both MS and IR are very common in AGHD, afamin has not been investigated in these patients. Purpose: To investigate afamin as a potential biomarker in patients with AGHD. Materials and methods: Participants included 20 AGHD patients (11 GH-substituted and 9 GH-unsubstituted) and 37 healthy controls. Subjects underwent routine laboratory examinations, anthropometric measurements, body composition analysis using multi-frequency bioelectrical impedance analysis (InBody720) and measurement of serum afamin concentrations. In GH-substituted subjects, GH-substitution was withdrawn for 2 months. Measurements were carried out right before GH-withdrawal, at the end of the 2-month withdrawal period, and 1 month after reinstituting GH-replacement therapy (GHRT). Results: GH-unsubstituted patients demonstrated higher afamin levels compared to controls (p=0.03). Afamin positively correlated with skeletal muscle mass, bone mineral content, total body water, extracellular- and intracellular water content, insulin (all, p<0.01), HOMA-IR (p=0.01) and C-peptide (p=0.03) levels in AGHD but not in healthy controls. In GH-substituted patients 2-month of GH-withdrawal caused significant changes in body composition, including decreased fat-free mass, skeletal muscle mass, total body water, and intracellular water content (all, p<0.01); but these changes almost fully recovered 1 month after reinstituting GHRT. Unexpectedly, afamin levels decreased after GH-withdrawal (p=0.03) and increased with reinstitution (p<0.01). Changes of afamin levels during GH-withdrawal positively correlated with changes of HOMA-IR (r=0.80; p<0.01) and changes of insulin (r=0.71; p=0.02). Conclusion: Higher afamin levels in unsubstituted AGHD patients might indicate severe metabolic dysregulation. Significant changes accompanying GH-withdrawal and reinstitution, along with strong correlations with measures of IR, suggest that afamin could be a promising biomarker to monitor GHRT-associated changes of insulin sensitivity.

Molecular phylogenetic analyses of albuminoids reveal the molecular evolution of allosteric properties.

Serum albumin, α-fetoprotein, afamin (also named α-albumin and vitamin E binding protein), and vitamin D binding protein are members of the albuminoid superfamily. Albuminoids are plasma proteins characterized by a marked ability for ligand binding and transport. Here, a focused phylogenetic analysis of sequence evolution by maximum likelihood of fatty acid binding sites FA1-FA7 of mammalian albuminoids reveals that the FA1, FA2, and FA3+FA4 sites in serum albumins have evolved from the most recent common ancestor through an intermediate that has originated the α-fetoprotein and afamin clades. The same topology has been observed for the whole protein sequences, for the sequences of all the fatty acid binding sites (FA1-FA7) taken together, and for the allosteric core corresponding to residues 1-303 of human serum albumin. The quantitative divergence analysis indicates that the ligand binding cleft corresponding to the FA2 site could be the main determinant of allosteric properties of serum albumins only. In fact, this binding cleft is structurally not effective in vitamin D binding proteins, whereas key residues that serve to allocate the allosteric effectors are not present in afamins and α-fetoproteins.

Clinical Significance of Adropin and Afamin in Evaluating Renal Function and Cardiovascular Health in the Presence of CKD-MBD Biomarkers in Chronic Kidney Disease.

AIM: The study aims to test the hypothesis that concentrations of adropin and afamin differ between patients in various stages of chronic kidney disease when compared with healthy controls. The study also investigates the association of the biomarkers (adropin and afamin) with CKD-MBD and traditional cardiovascular risk parameters in CKD patients. METHODOLOGY: The cross-sectional study includes the subjects divided into four groups comprising the control group (healthy volunteers = 50), CKD stages 1-2 patients (n = 50), CKD stages 3-4 patients (n = 50), CKD stage 5 patients (n = 50). Serum concentrations of adropin and afamin were determined using ELISA. Clinical variables (renal, lipid, and CKD-MBD parameters) were correlated to adropin and afamin concentrations. RESULTS: Afamin concentration was found to be higher in group IV, followed by groups III and II when compared to the control group, i.e., (83.243 ± 1.46, 64.233 ± 0.99, and 28.948 ± 0.72 vs. 14.476 ± 0.5) mg/L (p < 0.001), and adropin concentration was found to be lower in group IV as compared to groups III, II, and I (200.342 ± 8.37 vs. 284.682 ± 9.89 vs. 413.208 ± 12.32 vs. 706.542 ± 11.32) pg/mL (p < 0.001), respectively. Pearson correlation analysis showed that afamin was positively correlated with traditional cardiovascular risk biomarkers, while adropin showed a negative correlation. CONCLUSIONS: Adropin and afamin may potentially serve as futuristic predictors for the deterioration of renal function and may be involved in the pathological mechanisms of CKD and its associated complications such as CKD-MBD and high lipid levels.

Investigation of serum afamin concentration in pregnant women diagnosed with late fetal growth restriction or small for gestational age fetus.

OBJECTIVE: To investigate the afamin concentration in the serum of pregnant women diagnosed with late fetal growth restriction (FGR) or small for gestational age (SGA) in the third trimester. METHODS: This prospective case-control study was conducted on 126 pregnant women, 42 of whom were diagnosed with late FGR in the third trimester, 43 were SGA, and 41 were healthy controls. The groups were compared in terms of maternal serum afamin concentrations. RESULTS: Three groups were similar in terms of demographic characteristics and gestational age at blood sampling for afamin (p < .05). The median afamin concentration was determined as 199 ng/mL in the late FGR group, 153 ng/mL in the SGA group, and 108 ng/mL in the control group (p = .000). In the post-hoc analysis, while maternal serum afamin concentrations were found to be significantly higher in the late FGR group and SGA group compared to the control group but, this significance could not be shown between the FGR group and the SGA group (p = .00001, p = .005, p = .137, respectively). In the ROC analysis, the optimal cutoff value of serum afamin concentration to predict late FGR was determined as 141 ng/mL, with a sensitivity of 66.6% and a specificity of 85.3%. CONCLUSIONS: The serum afamin concentration in the third trimester was found to be higher in pregnant women with late FGR compared to the SGA and control groups. Although afamin is seen as a promising molecule in the clinical prediction of late FGR, this needs to be supported by large series of studies.

Evaluation of Afamin Level after Bariatric Surgery in Patient with Obesity.

BACKGROUND: The aim of this study is to evaluate afamin levels after weight loss in obese patients who underwent laparoscopic sleeve gastrectomy (LSG) and to investigate the relationship between them. In addition, after bariatric surgery, thyroid stimulating hormone (TSH), thyroxine (T4), low-density lipoprotein (LDL), very low-density protein (VLDL), total cholesterol, triglyceride (TG), high-density lipoprotein (HDL), insulin, and hemoglobin A1c (HgbA1c) levels were evaluated. METHODS: Preoperative and postoperative 6th month venous blood samples were obtained from 43 patients included in this study. The preoperative and postoperative 6th month body mass index (BMI), TG, total cholesterol, VLDL, HDL, insulin, HgbA1c, TSH, T4, and afamin levels of the patients who underwent bariatric surgery with obesity were compared. RESULTS: Serum afamin levels of patients decreased at 6 months postoperatively; however, it was not statistically significant. We observed a statistically significant decrease in patients' BMI, HDL, VLDL, TG, total cholesterol, TSH, T4, HgbA1c, and insulin values (p < 0.05). There were significant increases in HDL and T4 values. The change in LDL value was statistically insignificant. CONCLUSIONS: Recent studies have shown that there may be a cause-effect relationship between afamin and obesity. In our study, we observed a decrease in serum afamin levels after weight loss following bariatric surgery. In addition, we think that afamin may be used as a potential marker of metabolic syndrome in the future and may lead to improvements in the medical treatment of obesity.

Serum afamin levels in predicting gestational diabetes mellitus and preeclampsia: A systematic review and meta-analysis.

Objectives: The association between biomarkers and the risk of gestational diabetes mellitus (GDM) or preeclampsia (PE) has been extensively studied. However, there is still a lack of convenient, specific, and sensitive indicators for early identification of GMD and PE. Therefore, we conducted a meta-analysis of published articles to investigate the value of afamin circulating levels in the early diagnosis of GDM and PE. Methods: We searched the PubMed, Embase, Cochrane Library, and Web of Science databases for English studies published before November 16, 2022, that examined the association between afamin and GDM or PE. In addition, we searched Clinicaltrials.gov for the relevant completed and ongoing clinical trials. Pooled standard mean differences (SMDs) and weighted mean differences (WMDs) with 95% confidence intervals (CIs) were used to compare the levels of afamin in different groups. Results: Eleven studies were included in our analysis (N = 3047 participants: 1195 GDM, 1407 non-GDM, 195 PE, and 250 non-PE). Subgroup analysis based on different blood collection periods found that the plasma afamin levels in pregnant women with GDM in the first trimester were significantly higher than those in healthy pregnant women (SMD = 0.481, 95% CI: 0.280-0.682), but the analysis showed the opposite results in the second and late stages (SMD = 0.292, 95% CI: -0.092-0.676). The plasma afamin levels of pregnant women with PE in the first trimester (SMD = 0.808, 95% CI: 0.558-1.059) and second/third trimesters (SMD = 0.904, 95% CI: 0.570-1.239) were significantly higher than those in healthy pregnant women. Conclusion: The plasma afamin levels in pregnant women with GDM in the first trimester were significantly higher than those in healthy pregnant women, but the analysis showed the opposite results in the second and third trimesters. The plasma afamin levels in pregnant women with PE in the first, second, and third trimesters were significantly higher than those in healthy pregnant women. Additional large-scale prospective studies are desired to verify these findings, and it is recommended that afamin should be included as a routine diagnostic test for women with GDM and PE. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=339171, identifier CRD42022339171.

Afamin Levels and Their Correlation with Oxidative and Lipid Parameters in Non-diabetic, Obese Patients.

Background: Afamin is a liver-produced bioactive protein and features α- and γ-tocopherol binding sites. Afamin levels are elevated in metabolic syndrome and obesity and correlate well with components of metabolic syndrome. Afamin concentrations, correlations between afamin and vitamin E, afamin and lipoprotein subfractions in non-diabetic, obese patients have not been fully examined. Methods: Fifty non-diabetic, morbidly obese patients and thirty-two healthy, normal-weight individuals were involved in our study. The afamin concentrations were measured by ELISA. Lipoprotein subfractions were determined with gel electrophoresis. Gas chromatography-mass spectrometry was used to measure α- and γ tocopherol levels. Results: Afamin concentrations were significantly higher in the obese patients compared to the healthy control (70.4 ± 12.8 vs. 47.6 ± 8.5 µg/mL, p < 0.001). Positive correlations were found between afamin and fasting glucose, HbA1c, hsCRP, triglyceride, and oxidized LDL level, as well as the amount and ratio of small HDL subfractions. Negative correlations were observed between afamin and mean LDL size, as well as the amount and ratio of large HDL subfractions. After multiple regression analysis, HbA1c levels and small HDL turned out to be independent predictors of afamin. Conclusions: Afamin may be involved in the development of obesity-related oxidative stress via the development of insulin resistance and not by affecting α- and γ-tocopherol levels.