Estimation of plasma afamin levels in patients with periodontitis and Type II diabetes mellitus: A clinico-biochemical study.

Periodontitis is a chronic inflammatory disease, caused by a specific group of microorganisms, resulting in progressive destruction of periodontal ligament and alveolar bone. Polymorphonuclear neutrophils perform an innate cellular response against the bacterial virulence factors and release proteolytic enzymes, which are known to increase oxidative stress (OS).[ 1 ] Periodontitis has been associated with diabetes mellitus (DM), nutritional deficiencies, obesity, atherosclerosis, adverse pregnancy outcomes, rheumatoid arthritis, and chronic obstructive pulmonary disease. Löe reported periodontitis as the 6 th complication of DM after retinopathy, nephropathy, neuropathy, microvascular diseases, and altered wound healing.[ 2 ] Hyperglycemia is characterized by nonenzymatic reaction of glucose with amino acids that cause the accumulation of irreversible advanced glycated end products (AGE). Receptors for AGE (RAGE) are present on endothelial cells, fibroblasts, monocytes, macrophages, and neutrophils. AGE-RAGE interactions on endothelial cells cause an increased cell permeability and adhesion. On fibroblasts, it decreases collagen production and increases susceptibility to attachment loss, leading to apical migration of microorganisms. On macrophages, it causes secretion of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and IL-6.[ 3 ] On the other hand, uncontrolled hyperglycemia also provides a suitable environment for the growth of subgingival microflora. Periodontitis is characterized by increased secretion of inflammatory cytokines (TNF-α, IL-6, and IL-1β), which has a negative impact on glucose metabolism, leading to increased IR. TNF-α enhances adipocyte lipolysis, stimulating Jun N-terminal kinase and Nuclear Factor Kappa B (NF-κB) signaling pathways, which in turn increase serine/threonine phosphorylation of insulin receptor substrate– 1 (IRS-1) and cause the development of IR. Simultaneously, IL-6, being a proinflammatory cytokine, induces toll-like receptor-4 (TLR4) gene expression through activation of signal transducer and activator of transcription-3. TLR4 also activates NF-κB which in turn initiates osteoclastogenesis. IL-6 also induces IR by impairing glycogen synthesis through downregulation of miR-200 gene expression.[ 4 ] Type II DM (TIIDM) is associated with increased OS, due to excessive production of reactive oxygen species (ROS), which increase the production of transcription factors such as NF-κB and activating protein-1, which play a crucial role in periodontal disease pathogenesis.[ 5 ] ROS are scavenged by antioxidants like Vitamin E, which is a fat-soluble molecule that requires a binding protein for its transport across the cell membrane, to protect against lipid peroxidation and maintain cell membrane integrity. Afamin (AFM) is a Vitamin E-binding glycoprotein, mainly expressed in the liver and partly by tissues of the brain, kidney, ovaries, and testes, with 15% carbohydrate and 55% amino acid sequencing, and was first described by Lichtenstein in 1994, as the 4 th member of albumin gene family.[ 6 ] As an antioxidant binding protein, it plays a crucial role in antiapoptotic cellular processes in conditions with increased OS. Studies have also reported a linear correlation between increased OS and AFM concentrations.[ 7 8 9 10 ] Kronenberg et al . reported a 19% increase in the metabolic component with every 10 mg/L increase in AFM concentration.[ 11 ] Other authors have reported an increase in AFM concentration by approximately 10 units in individuals with TIIDM.[ 12 13 ] Various factors associated with TIIDM, such as high glucose, adipokines, and free fatty acids, trigger highly sensitive C-reactive protein (hs-CRP) production by endothelial cells, monocytes, and macrophages. Seeber et al . have reported that AFM concentration is directly proportional to the levels of hs-CRP.[ 8 ] CRP is reported as an inflammatory protein that is elevated as a part of the acute phase response in chronic periodontitis. Concerning the relationship between IR and increased AFM concentration, this biomarker appears to be a promising indicator for IR in periodontitis also. Hence, the objective of this study was to assess and compare plasma AFM concentrations in periodontitis subjects with and without TIIDM.

Mean plasma AFM levels in Group I individuals were 30.64 ng/ml. A significant increase was seen in Group II and III, with highest concentrations in diabetic individuals with periodontitis, compared to systemically healthy individuals with periodontitis alone, i.e. 86.44 ng/ml versus 50.76 ng/ml, respectively [ Table 1 ]. Demographic details of all three Groups PI – Plaque index; GI – Gingival index; PPD – Probing pocket depth; AFM – Afamin On comparing AFM concentration and PPD, Group II showed concentrations of 60.33 ng/ml, 43.80 ng/ml, and 51.12 ng/ml in mild, moderate, and deep pockets, respectively, and Group III showed 89 ng/ml, 83.6 ng/ml, and 87.05 ng/ml in mild, moderate, and deep pockets, respectively. Plasma AFM levels were significantly increased in moderate and deep pockets in Groups II and III [ Table 2 ]. Correlation of mild, moderate, and deep pockets with plasma afamin levels in Group II and III * P <0.05 – Significant; ** P <0.001 – Highly significant. PPD – Probing pocket depth; AFM – Afamin; P – Probability value On comparing AFM concentration with glycemic control group III individuals, it was found that mean AFM concentration increased with worsening of glycemic control of the patients, i.e. 80.25 ng/ml, 84.25 ng/ml, and 90.92 ng/ml in good, fair, and poor control patients, respectively [ Table 3 ]. Comparison of glycated hemoglobin controls with plasma afamin levels, Group III ** P <0.001 – Highly significant. HbA1c – Glycated hemoglobin; AFM – Afamin; P – Probability value

In this study, plasma AFM concentration was assessed in periodontitis subjects, with and without TIIDM. Our study reported a significant increase in PI and GI scores in both the test groups. This can be explained by poor oral hygiene being the root cause for initiation of periodontal disease, and bleeding on probing is the initial clinical indicator for the presence of periodontal disease.[ 15 ] Several environmental and genetic factors are responsible for differences in the severity of this condition, and TIIDM emerges to be most prominently associated with periodontitis. Several biomarkers have been previously reported to increase in patients with periodontitis and TIIDM (IL-1β, TLR-2, TLR-4, and TNF-α).[ 16 ] AFM is one such biomarker, previously associated with IR. It is an antioxidant (Vitamin E) carrier protein.[ 9 ] Kratzer et al . reported AFM to have 18 binding sites for α-tocopherol (a member of Vitamin E family).[ 17 ] Qualitative and quantitative analyses revealed the presence of AFM in plasma (59.8 ± 13.3 μg/mL), follicular fluid (34.4 ± 12.7 μg/mL), and cerebrospinal fluid (0.28 ± 0.16 μg/mL).[ 18 ] Saadoun et al . reported 45.93 ± 2.26 ng/ml of AFM in healthy patients.[ 19 ] Another author reported 34.50 ± 26.97 ng/ml of AFM to be present under physiological conditions.[ 10 ] Findings of these studies are in accordance with our findings, wherein the mean plasma AFM concentration in healthy controls was 30.64 ± 5.88 ng/ml. This AFM is readily available in plasma for binding to Vitamin E for its transport to the target tissues. Hirtz et al . quantified AFM in the saliva of healthy individuals and reported a mean concentration of 60.1 pg/ml.[ 20 ] Orti et al . reported the presence of AFM in the saliva of patients with periodontitis, within a clinical range of 0.269–19.472 μg/ml.[ 21 ] AFM plays a role in antiapoptotic cellular processes related to increased OS. The underlying mechanism involves the binding of AFM to a redox protein, thioredoxin, which mediates redox signaling.[ 22 ] One study reported that Vitamin E binds with AFM and neutralizes ROS by giving out a hydrogen ion from its chromanol ring, under the influence of protein kinase C, and there exists an inverse relationship between lipid peroxidation and Vitamin E levels, indicating consumption of the antioxidant molecules whenever OS increases.[ 23 ] Evidence shows a compromised antioxidant capacity total antioxidant capacity (TAOC) in periodontitis this is due to excessive demand and consumption of antioxidant molecules, which leads to Vitamin E exhaustion and free unbound AFM left to circulate in the bloodstream. This explains the higher plasma AFM concentration in Group II and III (50.76 ± 9.653 ng/ml and 86.44 ± 12.101 ng/ml, respectively), when compared to Group I (30.64 ± 5.880) in our study. The current study reported a higher mean AFM concentration in patients with TIIDM, when compared to systemically healthy individuals (86.44 ± 12.101 ng/ml vs. 50.76 ± 9.653 ng/ml, respectively). Many authors have reported plasma AFM concentrations to be significantly associated with metabolic risk factors like obesity and TIIDM, both of which are relevant for the development of metabolic syndromes (MSs).[ 11 24 25 ] A study reported that every 10 mg/L increase in AFM concentration was associated with an increase in MS component by 19%.[ 16 ] Dieplinger and Mueller reported that every 10 mg/L increase in AFM was associated with a 20% increase in prevalence for TIIDM.[ 26 ] In a pooled analysis of more than 20,000 individuals, Kollerits et al . reported that every 10 mg/L increase in AFM concentration was associated with a higher prevalence of TIIDM.[ 13 ] Periodontitis has been established as a risk factor for TIIDM. OS plays a major role in the pathophysiology of this two-way relationship. Significantly increased AFM levels in Group III can be explained by either a pro-oxidative state in individuals with TIIDM and periodontitis, when compared to systemically healthy individuals with periodontitis in Group II. Another reasoning could be increased OS in periodontal tissues of patients with TIIDM, which makes their oral cavity suitable for the survival of periodontal pathogens, which further invade gingival tissues and increase OS. Our study showed that individuals with poor glycemic index (HbA1c >8.9%) showed a higher mean AFM concentration, when compared to those with good glycemic index (HbA1c <6.3%–7.2%) (i.e., 90.92 ng/ml vs. 84.25 ng/ml, respectively). Ali and Ahmed reported similar findings of lower mean AFM concentration in prediabetic individuals, when compared to previously diagnosed TIIDM patients (i.e. 13.10 ng/ml vs. 20.30 ng/ml, respectively).[ 12 ] Studies on other phenotypes of IR reported increased AFM concentration in gestational DM (GDM) when compared to systemically healthy counterparts.[ 27 ] On exploring other pregnancy complications involving conditions with increased OS, Hubalek et al . found higher AFM levels in patients with preeclampsia when compared to healthy controls (70 mg/l vs. 55.4 mg/l, respectively).[ 9 ] Endometriosis was reported to be associated with increased AFM concentration and decreased Vitamin E levels in the peritoneal fluid.[ 7 ] Underlying reasoning is the increased OS in endometriosis, which is responsible for decreasing the TAOC and increasing the amount of free circulating unbound AFM molecules. Concerning the relationship between OS and glucose metabolism, authors have reported AFM to be a good indicator of IR in PCOS patients, as increased AFM concentrations were found in the first- and mid-trimester pregnant females with GDM, when compared to those without GDM.[ 8 28 ] All these findings indicate AFM concentrations to be an independent predictor for the development of TIIDM. The strength of our study is that it is one of the first to evaluate plasma AFM concentration in patients with periodontitis. One of the limitations of our study could be that changes in AFM concentration were not assessed after periodontal therapy. Further studies with a larger sample size are required to confirm those findings. This study paves the way for further research to confirm AFM concentration in other secretory fluids, such as saliva and gingival crevicular fluid.

The findings of our study indicate that increased AFM concentrations in TIIDM indicate its role in the regulation of glucose metabolism and its potential to predict future metabolic disorders. Keeping in mind the linear correlation of OS and development of IR, AFM can be used as a marker to assess the incidence of IR in patients with periodontitis or to identify individuals who are at higher risk of developing TIIDM. Assessment of HbA1c, RBS, FBS, and OGGT indicates the prevalence and severity of IR in previously diagnosed diabetic individuals. AFM levels can be used to assess insulin sensitivity in diabetic, nondiabetic, and prediabetic individuals with/without conditions such as MS, obesity, PCOS, GDM, endometriosis, and complicated pregnancies. The findings of this study confirm elevated AFM concentrations in patients with both periodontitis and TIIDM and demonstrate a positive correlation between increasing AFM levels and the severity of periodontitis. There are no conflicts of interest.

A total of 75 subjects, above 35 years of age, were selected for the study and divided into 3 groups: Group I – 25 systemically healthy subjects without periodontitis, Group II – 25 systemically healthy subjects with periodontitis, and Group III – 25 subjects with periodontitis and TIIDM. Each subject was explained regarding the study, and a signed informed consent was obtained. Patients were diagnosed with periodontitis, according to the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.[ 14 ] Individuals who had received any form of periodontal therapy or had taken antibiotics or corticosteroids within the previous 6 months were excluded from the study. Participants with a history of chronic inflammatory conditions other than periodontitis, or systemic diseases other than TIIDM, were also not included. In addition, pregnant or lactating women, those using oral contraceptives, and individuals who smoked or consumed alcohol were excluded from participation. All periodontal parameters (plaque index [PI], gingival index [GI], and probing pocket depth [PPD]) were recorded, and radiographic evaluation was done using an orthopantomogram for all the participants. Group III individuals were analyzed for glycated hemoglobin % (HbA1c%). 2 ml of venous blood was collected and immediately transferred to ethylene diamine tetra acetic acid-coated vacutainers. Plasma was separated within 30 min and stored at −20°C in prelabeled sterile plastic vials. All the stored plasma samples were analyzed for AFM in one single batch, to avoid analysis bias. Standard working buffer was added to each well and incubated at 37°C for 80 min. The liquid was then discarded, and the plate was washed 3 times in a LISA wash machine. Plasma samples were added to the microtiter plate wells, followed by adding AFM-specific biotin-conjugated antibody, and incubated at 37°C for 50 min. Liquid was then discarded, and plates were washed 3 times again. Avidin-conjugated horseradish peroxidase was then added to each well and incubated at 37°C for 50 min. Liquid was then discarded, and the plate was washed 5 times with the wash buffer solution. TMB substrate solution was added only in wells that contain AFM, biotin-conjugated antibody, and enzyme-conjugated Avidin and incubated at 37°C for 20 min. This exhibited a color change. Enzyme-substrate reaction was then terminated by adding sulfuric acid solution. Colour change was measured spectrophotometrically at a wavelength of 450 nm ± 10 nm. The concentration of AFM was determined by comparing the optical density of samples to the standard curve. Normality of data was assessed using Kolmogorov–Smirnov test. Results were presented as mean ± standard deviation; Chi-square test was done to compare plasma AFM concentration between the groups. Analysis of variance test was done to assess the association between the variables and plasma AFM concentration. Comparison of parameters among the groups was done using Kruskal–Wallis test. The level of significance was fixed at 0.05. Data were analyzed using the IBM SPSS Statistics 20.0 (IBM Corporation, Armonk, NY, USA)